Capillary Electrophoresis Guidebook: Principles, Operation, and Applications (Methods in Molecular Biology Vol 52)

CHAPTER1

Fundamentals of Capillary Electrophoresis

Theory

Kevin D. Altria 1. Introduction This section will describe the fundamental theory, equations, and definitions necessary to comprehend the concepts involved in capillary electrophoresis (CE). This is not an exhaustive treatment, but is considered sufficient to comprehend and appreciate the principles of CE. More detailed theoretical background can be obtained from a number of reference books (I-6). Developments in the field of CE are reviewed in detail annually in the journal Analytical Chemistry. For example, the 80 1 papers published in 1992-l 993 were recently reviewed (7). CE can be broadly described as high-efficiency separations of sample ions in a narrow bore (25-100 pm) capillary tube that is filled with an electrolyte solution. A typical schematic of an instrument setup is shown in Fig. 1. The principal components are a high-voltage power supply, a capillary that passes through the optical center of a detection system connected to a data acquisition device, a sample introduction system, and an autosampler. Typically, the CE instrument is controlled by a personal computer. The capillary is first filled with the required buffer solution. Sample solution (typically l-20 nL) is then introduced at the end of the capillary away from the detector (usually the anode). The capillary ends are then dipped into reservoirs containing high-voltage electrodes and the required buffer solution. One electrode is connected to a cable leading to From

Methods m Molecular Bology, Vol 52 Capdary Electrophoresrs Ed&d by K Altrla CopyrIght Humana Press Inc , Totowa, NJ

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4 n

High voltage supply

Empty vial

0

Fig. 1. Typical instrumental setup.

the high-voltage output, whereasthe other (situatedat the detector end of the capillary) is connectedto an earthing cable. Electrodesare composed of an inert material, such as platinum. Application of a voltage (for example, 10-30 kV) across the capillary causes electrophoretic and electroendosmoticmovements(discussedlater in this chapter) resulting in the ionic speciesin the samplemoving along the capillary and passing through the on-line detector. A plot of detector response(usually UV absorbance)with time is generated,which is termedan electropherogram. 1.1. Electrophoresis

This processis the movementof sampleions under the influence of an applied voltage. The ion will move toward the appropriateelectrodeand passthrough the detector. The migration rate, or mobility, of the solute ion is governed largely by its size and number of ionic charges. For instance, a smaller ion will move faster than a larger ion with the same number of charges.Similarly, an ion with two chargeswill move faster than an ion with only one charge and similar size. The ionic mobility (pE) is therefore related to the charge/massratio (Eq. [ 11). (1)

Fundamentals

of CE Theory

5

Detector response

Fig. 2. Theoretical separation of a range of catrons. where PE = electrophoretic mobility, CJ= number of charges, IJ = solution viscosity, and r = radius of the ion. Therefore, when we separate a hypothetical mixture of ions havmg different charges and sizes, the smaller, more highly charged ions will be detected first (Fig. 2). The actual electrophoretic velocity, or speedof the solute ions, is related to their mobilities and the magnitude of the applied voltage (Eq. [2]). v=pEE

(2)

where v = velocity of the ion and E = applied voltage (volts/cm). 1.2. Electra-Osmotic Flow (EOF) Application of voltage across a capillary filled with electrolyte causes a flow of solution along the capillary. This flow effectively pumps solute ions along the capillary toward the detector. This flow occurs because of ionization of the acidic silanol groups on the inside of the capillary when m contact with the buffer solution. At high pH, these groups are dissociated resulting in a negative charged surface. To maintain electroneutrality, cations build up near the surface. When a voltage is applied, these cations migrate to the cathode (Fig. 3). The water molecules solvating the cations also move, causing a net solution flow along the capillary (Fig. 3). This effect could be considered an “electric pump.” The extent of the flow is related (Eq. [3]) to the charge on the capillary, the buffer viscosity, and dielectric constant of the buffer: pEOF=(&&/q)

(3)

where pEOF = “EOF mobility,” IJ = viscosity, and 6 = Zeta potential (charge on capillary surface).

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Ftg. 3. Schematicof electroendosmoticflow. The level of EOF is highly dependent on electrolyte pH, since the &, potential is largely governed by the ionization of the acidic silanols. Below pH 4, the ionization is small (8), and the EOF flow rate is therefore not significant. Above -pH 9, the silanols are fully ionized and EOF is strong. The pH dependence of EOF is shown in Fig. 4. The level of EOF decreases with increased electrolyte concentration as the 6 potential is reduced. The presence of EOF allows the separation and detection of both cations and anions within a single analysis, smce EOF is sufficiently strong at pH 7, and above, to sweep anions to the cathode regardless of their charge. Analysis of a mixture of cations, neutral compounds, and anions would result in the electropherogram shown in Fig. 5. The migration times correspond to the time the individual peaks pass through the detector. The smaller anions fight more strongly against the EOF and are therefore detected later than anions with a lower mobility. Multiply charged anions will migrate more strongly against the EOF and will be detected later. Therefore, pH is clearly identified as the major operating parameter affecting the separation of ionic species, smce it governs both the solute charged state and the level of EOF. The overall migration time of a solute is therefore related to both the mobility of the solute and EOF. The term apparent mobility @A) is measured from the migration time, and is a sum of both yE and pEOF: PA = pE + JJEOF= (ZL/ tV) (4) where I= length along the capillary (cm) to detector, V = Voltage, and L = total length (cm) of the capillary.

Fundamentals

of CE Theory

7

15

1

05

0 3

4

5

6

I

9

PH

Fig. 4. Varlatron of EOF with pH. Mobility values can be calculated from migration times when both ionic and neutral components are measured. For instance, in the separation of a five-component mixture shown in Fig. 5, the mobility values for the peaks are calculated and given in Table 1. Example peak 2 = IA = (1L/ Vt) = (50 x 57 / 30,000 x 500) = 1.9 x lOA vEOF (from peak 3) = (IL / Vt) = (50 x 57 / 30,000 x 600) = 1.58 x 10q j~E=pA-pEOF=0.32x IO4 The negative values of PE for peaks 4 and 5 indicate that they are anions. The separation of ions is the simplest form of CE and is often termed Free Solution Capillary Electrophoresrs (FSCE). The separations rely

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8

Frg. 5 Theoretical

separation

Calculated Mobility

of a range of ionic and neutral solutes. Table 1 Values for the Peaks m Fig. 5

Peak no

Mlgratlon time, s

PA cm2/Vs

1 2 3 4 5

400 500 600 750 900

2.38 x lo” 190x1@ 1.58 x 1W’ 1.27 x lo” 1.06 x lti

PE 080x lOA 0 32 x lOA 0 -031 x lOA -052 x 10“

I = 50 cm, L = 57 cm, and V = 30,000 V

principally on the pH-controlled dissociation of acidic groups on the solute or the protonation of basic functions on the solute. In FSCE, all neutral compounds are swept, unresolved, through the detector together (Fig. 5). Separation of neutrals is generally achieved by incorporation of anionic surfactant, at sufficient concentration to form micelles. These anionic micelles migrate against the EOF and can chromatographically interact with neutral solutes. Solutes having a large interaction will migrate later than those having little or no interaction. Use of micellar solutions is known as micellar electrokinetic capillary chromatography (also called micellar electrokinetic chromatography) and is covered in depth in Chapter 12, which is coauthored by the originator of the technique. When dealing with large biomolecules, such as nucleic acids, their electrophoretic mobilities may be very similar, and FSCE is often insufficient for adequate resolution. In this case, separations are performed in

Fundamentals

of CE Theory

capillaries filled with gel solutions. In Capillary Gel Electrophoresis (CGE), a sieving effect occurs as solutes of various sizes migrate through the gel filled capillary toward the detector. Chapter 13 describes the exceptional, efficient separations that can be obtained in gel filled capillaries. The separation and quantitation of chiral samples are an important area in many industries. Highly efficient chiral CE separations (Chapter 14) can be obtained by the addition of chirally selective substances, such as cyclodextrins, into the electrolyte. Capillary electrochromatography (CEC), which is a hybrid between CE and HPLC, has been developed. In this technique, CE equipment is used to generate HPLC-type separations. Capillaries are filled with HPLC packing material, and the application of a voltage results in the EOF pumping the mobile phase through the capillary. The full details of this technology and some applications are given in Chapter 15, which is written by one of the initial developers of the technique. 1.3. Sample

Introduction

Sample can be introduced into the capillary by three techniques, all of which involve immersing the capillary end into the sample solution and exerting a force to inject sample into the capillary. The three mechanisms for introduction of sample solution into the capillary are hydrodynamic, gravity, and electrokinetic. All these methods are quantitative, and equations describing the volumes injected have been derived. Figure 6 shows the principles of operation for the three methods. 1.3.1.

Pressure Differential

In this method, the sampling end of the capillary is immersed in the sample solution and a pressure difference applied (positive pressure or vacuum). The volume of sample solution injected onto the column can be calculated: Volume = AP d411t / 128 q L (5) where AP = pressure difference (mbar), q = buffer viscosity, L = total capillary length, and d = capillary diameter (pm). Table 2 gives injection volumes (9) for l-s injections using 65-cm capillaries of varying bore, TJ= 1, and various AP values. These volumes generally correspond to sample plug lengths of
10

Al tria Hydrodynamic Pre

Siphoning

Electrokinetic

Fig. 6. Diagram

of the three sample mtroduction

methods.

Table 2 Inlectlon Volumes / s for Various Capillary Bores AP

50 pm

75 pm

100 pm

50 mbar 75 mbar 100 mbar

1 OnL 1 5 nL 2.0 nL

5 2 nL 7 8 nL 104nL

164nL 246nL 32.8 nL

cosity is inversely proportional to temperature. For example, raising the temperature from 20 to 25°C causesthe viscosity of water to change from 1.00 to 0.89 (IO). Therefore, it is essential to employ a constant temperature during routine operation to ensure reproducible injection volumes. The sampling pressure or vacuum setting is generally instrument-specific with the sampling variable being time. Lists are available from

Fundamentals

of CE Theory

instrument suppliers that equate a sampling time to the respective volume of sample (on the order of l-20 nL) introduced into the capillary. 1.3.2. Gravity Injection In this method (II), the capillary, while dipping into the sample vial, is mechanically raised above the height of the detector electrolyte vial. Typically, the sample vial may be raised 5 cm for 10 s. The volume injected (12) may be calculated: Volume = (pgAH d41YI t / 128 q 15) (6) where AH = height difference (cm), g = gravitational constant, and p = density of the liquid. An injection volume of 6.35 nL can be calculated for a 10-s injection at 5 cm using a capillary length of 67 cm and capillary diameter of 75 urn. The following values are employed in this calculation assuming water as the buffer at 20°C density is 0.99707g/mL, viscosity is 0.8904 x 1O-2 g/cm/s, g is 980 cm/s2. The sampling variables with this technique are time and the height the sample is raised. 1.3.3. Electrokinetic In this method, the sampling end of the capillary and the high-voltage electrode are inserted into the sample solution. A voltage is then applied, causing solute ions to enter the capillary by electrophoretic migration and EOF. A greater number of more mobile ions enter the capillary, which can lead to sample bias effects. This effect can be turned to advantage (1.3), especially when attempting to quantify trace levels of small ions. The amount introduced during electrokinetic sampling is related to a variety of factors (Eq. [6]). Q=[(~E+uEOF)VlY-Ir*Ct/L] (7) where Q = amount injected, C = concentration of sample, and r = capillary radius. The sampling variables are the level and polarity of voltage, and sampling time. Various modifications to these sample introductory procedures can be used to increase the volume of sample solution introduced onto the capillary dramatically. This has considerable benefit in terms of increased sensitivity. The various schemes reported are reviewed in Chapter 16 by the two most active developers of these techniques.

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1.4. Peak EfCciency The capillary format employed in CE minimizes most sources of band broadening that occur in conventional electrophoresis or m HPLC. l

l

l

Joule heating-the heat generated within the capillary during the voltage appllcatlon IS effectively dlsslpated through the capillary walls, which reduces convection-related band broadening encountered m conventional electrophoresrs; On-column detection-a portion of the capillary is used asthe detector, which ehmmates postseparation broadening effects owing to connections, and EOF flow dynamics-the flow profile of EOF is plug-like m nature, which mmlmlzes sample dispersion during solute transport along the capillary, compared to the lammar flow encountered m pumped systems, such as HPLC.

The major dispersive effect remaimng in CE is that of molecular diffusion of the solute as it passesalong the capillary. This diffusion IS lowest for large molecules, such as proteins, which have small diffusion coefficients. Therefore, it is possible to obtain theoretical plate counts (N) of several million for biomolecules, such as nucleotldes and proteins (Chapters 17 and 19, respectively). The theoretical plate count can be calculated: N= pEV/2D

(8)

where D = diffusion coefficient. References 1 Ll, S F. Y , ed (1992) Capillary Electrophoresls, Prmclples, Practice and Applecatzons Elsevler. 2 Kuhn, R and Hoffstetter-Kuhn, eds. (1993) Capillary Electrophoresls Prznclples and Practzce. Springer-Verlag, Berlin. 3. Wernberger, R , ed ( 1993) Practical Capdlary Electrophorew. Acadermc, London 4. Grossman, P. D. and Colburn, J C., eds. (1992) Capillary Electrophoreszs’ Theory and Practice Academic, London 5 Vmdevogel, J. and Sandra, P., eds, (1992) Introduction to Mlcellar Electrobnetrc Chromatography Huthlg, Heidelberg 6. Camllleri, P., ed. (1993) m Capillary Electrophoresls Theory and Practrce CRC, Boca Raton, FL 7. Monmg, C A. and Kennedy, R T. (1994) Capillary electrophoresls Anal Chem 66,28OR-3 14R 8 Altna, K. D. and Simpson,C. F (1987) High voltage capillary zoneelectrophoresis: operating parameter effects upon electroendosmotlc flows and electrophoretic mobllities. Chromatographla, 24, 527-530

Fundamentals

of CE Theory

9 Heiger, D N. (1994) m High Performance

10 11 I2 13

13

Capdaly Electrophoresu, Hewlett Packard, Waldbronn Rush, R S and Karger, B. L. (1990) Beckman Techmcal Bulletm TIBC 104 Rose, D. J and Jorgenson, J. W. (1988) Characterisation and automation of sample mtroduction methods for capillary electrophoresis. Anal Chem 60,642-648 Olechno, J D , Tso, J M Y , Thayer, J , and Wamwright, A (199 1) Znt Lab May, 42-48. Jackson, P E and Haddad, P R. (1993) Optimisatron of inJection technique m capillary electrophoresis for the determination of trace levels of anions m environmental samples J Chromatogr 640,48 l-487

CHAPTER2

Standard

Commercial Description Kevin

Instrument

D. Altria

1. Introduction Commercial instruments have been available since 1988, and systems are currently available from over 20 suppliers. Recent survey papers have considered the relative performance and features of these commercial systems (1,2). Systems are available in both single bench-top units or in modular form. Figure 1 shows a photograph of a commercial unit. Currently the CE equipment market is dominated by the major HPLC instrument supply companies, such as Beckman (Fullerton, CA), Hewlett Packard (Waldbronn, Germany), Perkin Elmer (Foster City, CA), and Waters (Milford, MA). 2. High-Voltage Supply Separations are normally performed employing voltages in the region of 5-30 kV. Electrolyte ionic strengths are generally selected during method development, such that application of these voltages generates currents of 10-100 p-IA. Operations with currents above this level may lead to unstable, irreproducible operating conditions. On many instruments, it is possible to operate by applying constant voltage (most common), constant current, or constant power across the capillary. However, constant voltage is the most commonly employed operational mode. 3. Capillaries Generally, these are composed of fused silica with typical dimensions 25-100 pm wrde and 25-100 cm long. The exact length and bore of the From

Methods III Molecular B/ology, Vol 52 CapNary Electrophoresls Edtted by K Altna Copyright Humana Press Inc , Totowa, NJ

15

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Fig. 1. Photographof commercial instrument.

capillary are optimized during method development.The capillaries are covered with a protective layer of polyimide, which is strongly UV absorbent.A sectionof this polyimide coatingis removedto allow on-capillary detection. Details regarding capillary treatment are given later in Section 7.4. of Chapter3. Capillaries can also have an internal coating to alter selectivity, and thesemay have specific handling requirements. Capillary volumes are on the order of a few microliters. For example, the approximate volume of a 50-pm wide, 50-cm long capillary is 1 pL. The volume can be calculated: Volume = (7cL S / 4)

(1)

4. Capillary Cartridge Many commercial instrumentshave a cartridge-like device in which the capillary is retained. This arrangementgives improved mechanical

Standard

Commercial

Instrument

17

support and guarantees consistent alignment of the capillary m the optical center of the detector. The ends of the capillary protrude from the cartridge and dip into the autosampler vials. The cartridge is manually inserted into the instrument and is then clamped into place to ensure optical alignment. Cartridges are used in conjunction with cooling systems. The cartridge may be either filled with a coolant fluid, or cooled air may be blown through the cartridge. In instruments with liquid coolant, the cartridge is filled with a fluorocarbon liquid that is maintained at the required temperature. Longer lengths of capillary are coiled on a spool within the cartridge. Capillaries are located and held by screws in noncartridge-based instruments. Particular attention should be paid to the alignment of the capillary in the optical center of the detector (see Section 7.4. of Chapter 3). If the capillary becomes blocked or broken, the cartridge should be fitted with a new capillary. Detailed specific instructions for this procedure are given in the instrument manuals. 5. Air Supply The majority of instruments employ a regulated air supply that is used to perform rinses and hydrodynamic sample injection. Individual instruments have specific requirements (detailed in the operating manuals) m terms of the type of gas required and the pressure settings. 6. Temperature Control Both the sample volume injected and solute migration times are strongly dependent on temperature. Therefore, it is important that a consistent temperature be maintained throughout an analytical sequence to ensure good migration time and peak area precision. Approaches to this are instrument-specific and involve the use of Peltier cooling devices, forced air cooling, and heating ovens. Temperature ranges typically employed are 20-50°C. Several instruments also offer the option of cooled autosamplers, which may be of benefit when analyzing temperature-sensitive samples. 7. Commercial Detection Systems All commercial instruments have W absorbancedetection as standard, and several now have diode array detection. The majority of CE methods employ UV absorbance detection. A few selected instruments also offer the possibility of fluorescence or laser-induced fluorescence detection.

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The UV absorbance detectors are srmilar in format to those routmely encountered in HPLC. Operating wavelengths range between 190 and 760 nm, depending on the mstrument. The UV lamps employed are similar to those employed in HPLC. Some instruments are fitted with filters, which limits the choice of wavelengths. The typical portion of capillary exposed for detection purposes is 200 x 50 pm. These tmy dimensions require excellent focusing of light from the UV lamp through the capillary. This focusing is achieved through appropriate use of high-quality lens and/or fiber optics. 8. Safety Given the possible hazards associated with voltages of this level, particular attention is paid to ensuring adequate safety precautions by electrical interlocks. Inadvertent attempts to access the autosampler during operation will cause shutdown of the instrument. 9. Data Acquisition Device The output from a standard CE instrument is similar to that of an HPLC detector, i.e., plot of UV absorbance with time. Peak integration is the preferred method of quantitatton. 10. Personal Computer Controller The majority of CE systems are controlled by an external PC that externally controls all functions of the instrument. The operating parameters for each injection are preprogrammed into the PC controller. For routine analysis, an identical set of parameters can be applied to a number of samples. The flexibility of a PC controller is of great benefit during method development, where the instrument can be preprogrammed to evaluate several operating parameters m an unattended sequence. Less elaborate systems are available that are manually programmed from a front panel on the instrument. 11. Autosampler Typical autosampler capacity ranges from 20-50 samples. Sample volume requirements range from as little as 10 pL to 5 mL. Numerous injections can be made from a single vial, since the injection volumes are on the order of a few nanoliters. Autosamplers can also be preprogrammed to perform mrcropreparative fraction collection (see Chapter 9).

Standard

Commercial

Instrument

19

12. Consumables Autosampler vials and caps are instrument-specific and may only be available from the equipment suppliers. However, suitable alternatives may be available from other sources. Several reagent suppliers, such as Fluka, and CE equipment suppliers are now developing reagent ranges specifically purified and certified for use as CE reagents. Reagents available include prepared buffers, SDS solutions, and electrolyte systems for performing inorganic anion and cation determinations. References 1 Oda, R P., Spelsberg, T C , and Landers, J P (1994) Commercial capillary electrophoresis mstrumentatlon. LC-GC 12, 50,5 1 2 Watzlg, H. and Dette, C (1993) Precise quantitatwe capillary electrophoresls. Methodological and instrumental aspects. J Chromatogr 636,3 l-38

CI-I.~PTER

Typical

Operating Kevin

3

Procedures

D. Altria

1. Introduction The CE instrument is controlled by a PC. The instrument settings are defined on the PC in method files. Settings, such as temperature, autosampler vial positions, injection parameters, and rinse cycles, are defined in the steps within the method. A typical method is given below. The exact settings for the parameters are determined during method development. A method can be run for a single sample. To analyze several samples, a sequence is created in which the method, number of samples, and injection repetitions are defined. A typical method listing is given below. Additional information concerning each process is then described. Set temperature:Select appropriatetemperature Rinse 1: 1-min 0. 1MNaOH (or equivalent to regenerate capillary surface) Rinse 2: 2-min operating electrolyte Set detector: autoselection of selected h and AUFS range Injection: selected time and mode of injection

Separate:selectedtime and applied voltage level, detectorautozero, and integrator autostart 2. Set Temperature It is important to set an operating temperature, since both injection volume and migration time are temperature-related. Typical operating ranges are 20-50°C. From

Methods m Molecular Brology, Vol 52 Capillary Electrophoresls Ed&d by K Altrla CopyrIght Humana Press Inc , Totowa, NJ

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22 3. Rinse

1 (Regenerating

Rinse)

In this step, the capillary is flushed with a solution to clean the capillary surface. This may be performed to remove unquantified components of the previous injection, adsorbed materials, or to rehydrate surface silanols on the capillary. The settings will be defined durmg the method development. Typically, a 2-min rmse with O.lMNaOH may be performed with the drops of liquid displaced from the capillary collected into an empty vial. The rinse time and autosampler positions for the empty vial and regenerating solution vial are entered into the method. 4. Rinse

2 (Electrolyte

Rinse)

The capillary is rinsed and filled with the run electrolyte. This allows the capillary to adjust to the pH of the electrolyte. A typical rinse time may be 3 min with the displaced drops from the capillary again being collected in an empty vial. The rinse time and autosampler posittons for the empty vial and electrolyte vial are entered. 5. Set Detector

The wavelength, absorbance range, detector rise time, and autozero details are set during this step. If indirect detection is employed, then this option is selected at this stage. 6. Injection

The time and mode of injectron are designated in this step along with the autosampler position of the sample vial. See Chapter 1 for a description of the various sample injection techniques available. Hydrodynamic injection is routinely employed, although electrokinetic injection may be selected for sample matrix or sensitivity reasons. Since different instruments have varied hydrodynamic sampling settings, it may be appropriate to define an mjection volume, in nanoliters, in addition to sampling time. 7. Separate

Details regarding analysis time, autosampler positions of electrolyte vials, and the operating voltage are specified in thts step. It is normal to operate with a constant voltage. However, it is possible to perform analysis employing constant power or current.

23

Typical Operating Procedures 8. Preanalysis

Procedures

1. Install capillary and turn lamp on to warm up for 20 min. 2. Filter the electrolyte, If appropriate, using a 0.45~pm filter or eqmvalent. 3. Fill autosampler vials with electrolyte, regeneration solution, and sample as required. Ensure vials are adequately filled and that air bubbles are avoided. 4. Place the vials m the positions designated in the method and/or sequence. 5. Assign integrator to collect detector signal. 6. Perform a test separation to ensure appropriate performance. This may involve reference to systemsuitability criteria detailed in a specific method.

9. Postanalysis

Procedures

1. Rinse the capillary, typically using a 2-min rinse from a vial contammg distilled water. 2. Flush the capillary with air, typically employmg a 2-min rinse from an empty vial. 3 Remove the capillary and store. If the instrument 1sto be used again with the same capillary, it 1sbest to leave the Instrument setup, since this avoids the possibility of damage to the capillary ends. 4. Turn off the lamp and instrument. 5. Remove vials and dispose of accordingly.

10. Good Working

Practices

The following points are noted, which, if adopted, will maximize the quality of the data. 1. Reagent quality-Use only AnalaR-grade reagents or better. Specific reagents are available in specially purified form, specifically for electrophoresis. Water should be doubly distilled or HPLC grade to minimize background UV absorbance and ionic strength. Prepared electrolytes are now available from suppliers (see Chapter 2, Section 12.). 2. Electrolyte preparation and storage--Electrolyte should be prepared and stored in plastic bottles. If pH adjustment of the electrolyte is reqmred, the pH should be checked prior to use. Alternatively, the pH-unadjusted electrolyte should be stored and pH adjusted immediately prior to use. Glass storage bottles should be avoided since material (principally metal ions) leaches from the glass with time, causing an increase m pH. A suitable shelf life may be assigned as 1 mo, unless specifically determined. 3. Capillary storag+Following use,the capillary should be rinsed with dlstllled water and flushed with air. To flush with air, an empty autosampler vial is placed on the autosampler and a rinse step performed from the empty vial.

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4. Conditionmg analyses-It is useful to conduct analyses of blanks or equtvalent at the start of a sequence to allow the system to settle. 5. Electrolyte filtration-Electrolyte should be filtered through a 0.45~pm filter or simrlar to remove particulates. Particulates would appear as apparent noise spikes on the detector baseline. Samples should also be filtered if particulate matter is presented. If sample filtration is performed, adequate recovery should be demonstrated during method validation. 6 Lamp warm-up-It is advisable to allow 15 mm for the lamp to achieve a steady baseline. If a filter mstrument 1s employed, the appropriate filter should be selected to allow the titter to reach the operating temperature. 7. Sample warm-up-The temperature within the autosampler tray can be significantly higher than room temperature. Therefore, it 1s advisable to place the samples on the instrument prior to the mltiation of the sequence This allows samples to reach a constant temperature, which is needed for consistent inlection volumes. This requirement can be avoided if conditioning analyses are conducted (see step 4). 8. Maintenance-To reduce the likelihood of instrument failure owing to voltage leakage, the instrument should be regularly cleaned with cotton wool or tissue dampened with water to remove traces of electrolyte. 9. Blanks-Blanks of dissolvmg solvent and/or matrix should be analyzed at the start of each sequence to allow the system to equilibrate. 10. Contammation-The cleanlmess of glassware and purity of dissolvmg solvent or water should be checked, especially if attempting to quantify trace levels of ions. 11. Buffer depletion-Electrolysts ofthe electrolyte during electrophorests can significantly alter the concentratton and nature of the electrolyte, This effect, termed “buffer depletton,” will be exhibited by shifts m migration time and peak area. Buffer replenishment, or the use of different buffer vials during a sequence, will mimmtze the effect of buffer depletion. Buffers should be used within their buffering pH range. 12. Capillary dedication-Separate capillaries should be reserved for each method to avoid cross contamination effects.

11. Capillary

Preparation

The following points should be considered during the preparation of a capillary prior to commencing use. These are essential to ensure that a repeatable separation is possible when switching between capillaries. 11 .l. Capillary Dimensions The internal and external diameter and the length (L) of the capillary should be specified in the method (see Fig. 1). A length of capillary

25

Typical Operating Procedures I L where I = length to detector L = total length

Fig. 1. Capillary lengths. should be measured and cut to a length slightly over that required. This allows some excess for trimrning to the exact length required. The length to the detector window (1) should also be marked prior to polyimide removal. Capillaries coated with a UV transparent material are available from Supelco (Bellefonte, PA) and do not require polyimide removal. However, these are unsuitable for use with liquid coolant-based cartridges. 11.2. Capillary

Coating

Removal

The fused silica capillaries generally used in CE are coated with polyimide to provide mechanical strength, because the exposed capillary is fragile. This coating strongly absorbs UV light and it is therefore necessary to remove the coating in the area of capillary that is to be used as the window for on-column detection. The exact distance along the capillary to the window (I) is instrument-specific and will be given in the instrument manual. The coating can be removed by several methods. The simplest method is to place the window area of the capillary briefly (ml s) in a low-heat flame from a burner or match. Avoid unnecessary time in the flame because this may cause the capillary to bend. The coating will char and the burned material can be gently removed employing a tissue dampened with methanol. If all the coating is not removed, further exposure to the flame is required. Alternatively, the coating can be removed by gently scraping the window area with a scalpel blade. An eyepiece or lens should be used to ensure that all the coating has been removed from the window, Commercial capillary window burning units have recently become available that employ a wire resistor. Application of a current across the resistor causes it to heat, which is sufficient to

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Fig. 2. Capillary cartridge assembly.

burn off the coating material. Use of these, or similar units, is recommended because these produce a reproducible capillary window with minimal effort. It is recommended that you carefully wipe the window area with a methanol-dampened tissue to remove any remaining burned polyimide. The capillary window is fragile following removal of the coating and should be handled as little as,possible to avoid breakage. In addition, the window area should not be touched, because grease will get onto the capillary window thereby reducing light throughput.

11.3. Capillary

Installation

The capillary housing is different for individual equipment manufacturers. In several instruments, the capillaries are held in cartridges whereas in others the capillary is held by retaining devices. The cartridges often incorporate a spool around which the capillary is spooled (Fig. 2). In all cases it is essential to align the window area of the capillary in the optical centre of the capillary housing device. Exact details of this aligning process will be given in the instrument manual.

27

Typical Operating Procedures

The alignment should be checked wherever possible using an eyepiece or lens. The ends of the capillary should be trimmed to the required length using a cutting stone to ensure a neat edge. The cutting stone should be used to lightly score the capillary and cut through the polyamide protection. Use of excessive force will result in the capillary end being crushed. If the capillary end is not cut correctly, peak tailing and poor precision may be obtained because sample solution can become irreproducibly trapped in the jagged end of the capillary. Depending on the instrument, this trimming may be needed prior to window alignment. Careful measurements of the capillary length are required to ensure correct alignment of the capillary during installation. Use of too long a length can result m the capillary becoming broken during operation. If the capillary is too short, it will not dip into the electrolyte reservoirs or sample solution. 11.4. Capillary

Surface

Rehydration

The majority of capillaries employed in CE are composed of uncoated fused silica. Fused silica is treated in a high-temperature oven and the surface therefore has no residual silanols. To rehydrate this surface, a rinse with a high pH solution is required (I). Following this rinse the surface silanols are restored and a consistent electroendosomotic flow (EOF) is possible. It is recommended that a rinse with O.lMNaOH for at least 20 min should be used to regenerate the capillary prior to its initial use. The capillary should then be filled with the method-specific electrolyte. A voltage should be applied across the capillary for 20-30 min prior to injection of the first sample. This procedure is to allow time for the capillary to stabilize and to achieve a consistent EOF level. This stabilization period could alternatively be accomplished by incorporation of appropriate blank injections at the beginning of a sequence. Use of rehydration steps for capillary coatmgs should be avoided unless prescribed in the instructions supplied with the capillary. Once the capillary is regenerated, it is not necessary to rehydrate it with an extended NaOH rinse before each subsequent use, providing that it is stored correctly. The capillary should be labeled and reserved for use solely for a selected electrolyte. Failure to do this may result in severe problems with irreproducible separations.

Al tria

28 11.5. Capillary

Storage

Prior to storage, capillaries should be flushed with water and then air to remove electrolyte from the capillary. Storage filled with air prevents formation of a gel layer inside the capillary that can form if the capillary is stored filled with solution (2). If the capillaries are stored filled with electrolyte, blockages can form as the electrolyte crystallizes out. References 1 Coufal, P., Stulik, K., Claessens, H A , and Cramers, C. A. (1994) The magmtude and reproducibllrty of the electroosmotic flow in sihca capillary tubes JHRCC 17, 325-334. 2. Schwer, C and Kenndler, E. (1991) Electrophoresls m fused-silica capillaries: the

influence of orgamc solvents on the electroosmotlc velocity and the zeta potential Anal. Chem. 63,180 l-1807.

CT~APTER

Method

4

Development/Optimization Kevin D. AZtria

1. Introduction This chapter provides some possible starting points for method development and further optimization. A later subsection in this chapter presents some starting points and guidelines for method development of chiral separations. A more extensive treatment of chiral separations is given in Chapter 14. A detailed background to the parameters affecting MECC separations is given in Chapter 12. Before proceeding with any CE experimental work, the following physicochemical information on the sample should be obtained if available. 1. The aqueoussolubihty of compound over a pH range; 2. Suitable dissolving solvent, preferably water; dtlute acid or base (> 10 rr&I), or water with the mimmum level of organic solvent required to solubke the compound; 3. pK, data;

4. UV spectralinformation. Many of the variables used to adjust selectivity are similar to those employed in HPLC method development. However, additional variables are also exploited (Table 1). The majority of these parameters must be optimized during method development. Resolution and analysis time are dependent on several factors (I). Therefore, it is necessary to follow a logical method development path, such as that given below: 1. Selectelectrolyte; 2. Select capillary and dimensions; From

Methods m Molecular Bology, Vol 52 Capillary Electrophoresls Edlted by K Altrla Copyright Humana Press Inc , Totowa, NJ

29

30

Al trta Table 1 Variables AvaIlable to Develop/Optlmlze

Variable

Typical range

Voltage Current Capillary length Capillary bore

5-30 kV 5-250 PA 20-100 cm 25-100 pm

Capillary coating PH

Various 1 5-11.5

Surfactant m MECC

1O-200 n-&l

Organic solvent

l-30% v/v

Urea

l-7M

Ion-pair reagent

l-20 M

Amme modifiers Cyclodextrms

l-50 mM l-100 n04

Vlscoslty Electrolyte cone

Various 5-200 mM

Catiomc surfactant Injection time

I-20 mA4 I-20 s

3. 4. 5. 6. 7. 8. 9. 10.

Separation

Effect of increasmg vanable Reduced analysts time and some loss m resolution Reduced analysis time and some loss m resolution Increased analysis time and gam m resolution Increased current, better sensltlvlty, and possible efficiency reduction Change of EOF and selectlvlty Increased EOF, Increased lomzatlon of acids, and reduced lomzatlon of bases Increased retention, lower EOF, and selectivity changes Increased solublllzatlon, reduced EOF (except acetomtnle), and alteration m selectivity Increased solublllzatlon of hydrophobic solutes and Increased migration times Can reduce or increase solute charge, and can alter selectivity Reduced surface charge and reduced peak tailing Increased vlscoslty, reduced EOF, and increased solute migration if complexation occurs Reduce EOF and longer migration times Increased current, EOF and solute lomzatlon, and reduced peak tailing Reversal of EOF dfrectlon and MECC condltlons Improved signal, some loss of resolution, and loss of peak symmetry

Optimize temperature; Optimize wavelength selection; Optimize sample concentration and compositlon; Determine voltage/current; Determme rinse cycle selection, Optimize injection selectlon; Optlmlze precision (see Chapter 6); Optlmlze sensitivity (see Chapter 7).

2. Select Electrolyte

Choice

There are three main separation mode options available in CE, and selection largely depends on the charge of the test solute in solution (Fig. 1).

Development /Optimization

YES

YES

0

u

No

0 u Low pH Uncoated +ve voltage

NO

Yes 0 Low pH Coated +ve voltage

0

MECC

FREE SOLUTION CE

No

NO

0

High pH Uncoated +ve voltage

Yes

0

High pH MECC Uncoated +ve voltage

Yes

0 Low pH MECC Coated -ve voltage

High pH Coated -ve voltage

Fig. 1. Method development options flowchart. Option 1: Low-pH free solution CE (FSCE): Cationic solutes separateby virtue of their differing mobilities. Option 2: High-pH FSCE: Anionic speciesseparateby virtue of their differing mobilities. Option 3: Micellar electrokinetic capillary chromatography: Soluteschromatographically interact with a migrating micellar phase; this mechanism allows separation of both charged and neutral species.

Capillaries can be internally coated to alter selectivity or to reduce EOF (see Section 3.). These coatings can be either permanent (chemically bonded) or temporary (use of electrolyte additives). Permanent coatings are usually achieved by derivatization of the capillary wall silanols followed by a covalent binding or material, such as polyacrylamide. Electrolyte additives, suchaspolyvinyl alcohol or ethylene gylcol, are used to reduce or eliminate EOF. Surfactant additives, such as tetradecyltrimethylammoniumbromide, can be employed as additives to reversethe EOF direction (this is usedespecially in the analysis of small anions--see Section 1. of Chapter 20). If p& data are available on the test compound,an informed selection of electrolyte can be made.If the compoundis basic,then option 1 would be preferred. If the compound is acidic, option 2 would be most appro-

Al tria

32

priate. Option 3 would be applicable in all cases. However, if no p& data are available, it may be appropriate to perform preliminary method scouting experiments to cover all of the above options. The test solution should be prepared in water or in water with the minimal amount of organic solvent to solubilize the analyte (see Section 6.). Sample concentration should be adequate to produce a measurable response; typically an initial sample concentration of -0.1 mg/mL may be appropriate. The sample solution must be soluble in the electrolyte, or it will precipitate out in the column, resulting in no peaks being detected. The test solution could then be analyzed under the following three starting conditions: Electrolyte A: 25 mMNaH,PO, adjusted to pH 2.5 with cont. H3P04 Electrolyte B: 25 mM borax Electrolyte C 20 mM Na2HP04, 10 mM borax, with 50 mM SDS Typically, set a run-time of 30 mm, and apply +25 kV across a 57 cm x 50 pm capillary for each separation. Set the detector to 200 nm or an appropriate UV absorbance maxima (if known) for the selected compound. If no peak is obtained using electrolyte A, then the compound is uncharged or only marginally ionized at low pH, which effectively eliminates option 1. When using electrolyte B, the electroendosmotic flow (EOF) in the system will result in a negative peak at about 2-4 min. This position is similar to the dead volume marker in HPLC. If the sample comigrates with the EOF, then it is neutral under these conditions, which would effectively eliminate option 2. Using option 3, a peak will be obtained between 3 and 30 mm. It may then be necessary to alter conditions to achieve the required selectivity. For the method scouting experiments, use typical method setup as given below: Step I: Rinse cycle 1: O.lMNaOH (2 mm). Step II: Rinse cycle 2: electrolyte A, B, or C (2 mm). Step III: Set detector: select desired iL. Step IV: Sampling. 1 s hydrodynamic. Step V: Operating voltage: + 25 kV. Run time: 30 mm. Capillary dimensions: 50 urn x 57 cm (50 cm to detector).

Development / Optimizatton

33

Table 2 Commonly Used Electrolytes Electrolyte Phosphate Citrate Acetate MES PIPES ACES MOPS0 MOPS HEPES Trls Borate CHES CAPS

2 12 pK,l 7.21 pK,2 12 32 pK,3 3.06 pK,l 5 A0 pK,2 4 75 6 15 6.80 6 90 6 90 7 20 7 55 8 30 9 24 9 50 1040

2.1. Free Solution CE A list of common buffers and pH ranges used in FSCE is given in Table 2. The inorganic electrolytes, such as phosphate and borate, are commonly used at concentrations in the area of 10-50 mM. Higher concentrations are not generally possible because of internal heating problems within the capillary, but can improve peak shape. The blological or “Goods buffers,” such as CAPS, TRIS (Tris-[hydroxymethyl]aminomethane), and so on, can be used at higher concentrations because of their low conductivities, but problems can occur owing to their high UV absorbances compared to inorganic electrolytes, such as phosphate or borate. 2.2. Low-pH FSCE (Option 1) The principal variables that can be employed to alter separation selectivity at low pH for the separation of cationic solutes are listed below: 1. Electrolyte

pH: This IS the most useful parameter

to vary since it can be

usedto alter the chargednatureof the solute.To ensurefull protonation of the compound, a pH of 1 or more units below its p& should be selected.

34

Altria

2. Cyclodextrm concentratton: These carbohydrate addmves are available m a range of native and derivatized forms and may differentially complex wtth the migrating solute, thereby increasing its migration time and separation selectivity. Typically l-1 00 rnA4hydroxy- propyl+-cyclodextrm (2) or similar are utilized. 3. Organic solvent. Solute pK,lpKbs are altered by the addition of orgamc solvents. Typically acetonitnle, methanol, or isopropanol at levels of l-30% v/v are employed often to improve the solubility of the solute (3,4). 4. Ion-pair reagents: These additives are used to alter the net charge of the solute. Typically, l-40 mA4sodium heptanesulfomc acid (2,5,6) or similar ton-pair reagent are added to the electrolyte. 5. Electrolyte nature and concentration: Higher ionic strength buffers improve peak shape, but generally necessitate a reduced applied voltage level since the current increases with buffer concentration. It 1srecommended that the voltage be reduced sufficiently to maintain current levels below 100 PA. The use of Goods buffers (7) at high concentrations can be advantageous, since these generate relatively low currents compared to inorganic electrolytes, such as phosphate or borate. The choice of electrolyte at a particular pH also affects both the selectivity and EOF level. For instance, the level of EOF decreases acetate > phosphate > citrate > borate (8). Also, the level of current generated by a specific electrolyte can be reduced by switching to an electrolyte counterton having a smaller ionic radius, 1.e, using lithium acetate instead of sodium acetate (lithium dodecyl sulfate is commercially available, which can be used at higher concentrations m MECC than SDS). Higher ionic strengths generally lead to improved peak efficiencies (Fig. 2), because sample “stacking” is improved. Figure 2 shows the analysts of a peptide mixture using various electrolyte concentrattons. 6. Zwitterionic additives: Compounds, such as betame, gylcine, and taurine, can be added to the electrolyte to reduce tailing effects. Given their zwttteriomc nature they can be used at lOO+ rnJ4 concentrations with no significant impact on overall current (9). 7. Flow reversal by hydrodynamic coating of the capillary-the direction of EOF can be reversed by addition of catronic surfactants (Z&I I) or polybrene (12). These additives form a double layer at the capillary wall, resulting in a net positive charge. Application of a voltage therefore results in a reversal of the conventional EOF direction. Therefore, a negative applied potential is employed to cause a flow m the direction of the detector. 8. Amine modifier: Excessive peak tailmg for highly basic solutes, such as peptides, can occur when they interact with the acidic silanols on the cap-

35

Development/Optimization

O.lOO M

0.060 M

4

II

Time

10 ( Minutes

la

14

)

Fig. 2. Effect of electrolyte concentration on resolution. illary surface. Additives, such as diaminopropane (13), can be employed to reduce this interaction by removing the active sites. 2.3. High-pH

FSCE

(Option

2)

This separation option is useful for the separation of anionic solutes. The variables are similar to those considered for low-pH FSCE. To ensure full ionization of the compound a pH, 1 or more units above its pK,, should be selected. EOF is more important at higher pH values and is decreased by decreases in pH, increases in organic solvent content (except acetonitrile), and the addition of cyclodextrins. Since the solutes

36

Altria

are now anionic, the nature of the ion-pair reagent should be changed to typically l-10 mM tetraethylamtnonium bromide (TEAB).

2.4. MECC

(Option

3)

This option is useful for the separation of charged and neutral species (a more detailed background to MECC [also known as micellar electrokinetic chromatography] is given in Chapter 12). The options available in optimization of MECC separation have been summarized (14,1.5). The selectivity variables and effects are identical to high-pH FSCE. However, there are also several additional parameters that greatly affect both selectivity and migration times: 1. Surfactant type: Altering the nature of the surfactant greatly affects the chromatographic mteractlons with the mlcelle. The prmclpal alternative to SDS 1sto employ bile salt surfactants (16,17), such as sodium cholate. If the solute is anionic under the separation conditions, it may be appropriate to employ a catlomc surfactant, such as cetyltrimethylammontum bromide, and to apply a negative potential (18). Noniomc surfactants, such as Brig-35, can also be added to SDS-based MECC electrolytes to alter selectivity (f 9). 2. Surfactant concentration: Increased concentration results m a higher number of micelles, and therefore, the solute 1smore retained, resulting in a longer migration time. There is an optimal surfactant concentration for each separation, and a range should be exammed during method development. 3. Cyclodextrins: These are useful as selectivity manipulators m MECC, especially when separating hydrophobic compounds. 4. Urea: This additive can be employed to increase solubihzation of hydrophobic compounds if they are poorly retained in the mlcelle (20). If the separation 1sgood, but analysis times are too long, increase pH and/or decrease SDS concentration. If the migration times are long with poor resolution, use an organic modifier or additive. If the migration times are short with poor resolution, increase SDS concentration. If the migration times are short with moderate resolution, decrease pH, and increase SDS concentration. l

l

l

l

Given that optimization can be achieved through adjustment of an assortment of variables, the appropriate use of experimental design procedures can significantly reduce the number of experiments required. A detailed section on experimental design is given in Chapter 20. Overall,

Development/Optimization

37

the electrolyte should be chosen that gives low UV absorbance at the detection wavelength, good buffering capacity at the pH required, and sufficiently low conductance to give stable operating currents. 3. Select Capillary and Dimensions Capillaries are almost exclusively composed of fused silica material, which is relatively cheap and readily available for a number of capillary suppliers, such as Polymicro (Phoenix, AZ), SGE (Ringwood, Victoria, Australia), and Supelco (Bellefonte, PA). Generally, these capillaries are not internally coated and have bare internal walls. However, there are a number of examples where capillaries are internally coated to modify the level of EOF and to alter selectivity. This area has recently been reviewed (21). Coatings are often with polymers, such as cellulose or dextran (22), which effectively suppress EOF and can reduce sample adsorptton onto the capillary wall. Alternatively, the capillaries can be internally coated with such substances as polyethylenimine (23), which can induce an effective positive charge on the capillary wall, resulting in a reversal of electroendosmotic flow direction. Alternatively, the capillary walls may be coated with long-chain (C6-Cl8) hydrocarbons (24,25). This is achieved following reaction of the capillary wall with appropriate silanes. A detailed procedure for preparation of bonded phase capillaries has been published (26). Alternatively, capillaries internally coated with bonded polyacrylamide are also employed. Coated capillaries are available from both a number of CE instrument suppliers and capillary manufacturers. The coated capillaries are generally supplied with handling/rinsing instructions and often with recommended electrolytes for particular applications. However, it is stressedthat the majority of CE applications are performed in bare fused silica capillaries. The choice of capillary bore and length largely governs the speed and sensitivity of the method. 3.1. Capillary Length Use of a longer capillary increases migration times for two reasons. First, the length of capillary to the detector area is increased, and in addition, the voltage gradient (V/cm) is decreased moving to a longer capillary while maintaining the same applied voltage. Therefore, a doubling in capillary length dramatically increases migration time, but also gives improved resolution. Figure 3 shows use of the same separation performed on both a 27- (Fig. 3A) and a 57-cm capillary (Fig. 3B). Limita-

Altria

38

ma

am Mtgrabon

7m

coo

aoa

xl00

bme (minutes)

c

I

I iso

I t4a Mgrabon

bme

I lm

I 2m

(minutes)

Fig. 3. Effect of capillary length reduction on resolution. (A) 57 cm; (B) 27 cm.

tions to the minimum capillary length possible are specific to individual commercial instruments. If attempting to employ a relatively high electrolyte concentration, it may be advisable to apply a low voltage (i.e., l-5 kV) across a short capillary to avoid problems of excessive current generation.

Development /Optimization

39

3.2. Capillary Bore The choice of capillary internal diameter largely governs the sensittvity of the method. If sensitivity is not an issue, then it is advisable to employ a 50 pm capillary to avoid any problems with excessive current generation. For maximum sensitivity, the bore may be extended to 100 pm if necessary, but at the expense of a reduced voltage and/or electrolyte concentration since internal heating problems increase with capillary diameter. 4. Optimize Temperature Temperature plays an important part in many separations, becauseboth solute mobility and the level of EOF are temperature-related. The majority of commercial instruments have capillary thermostatting facilities, and typical operating ranges are on the order of 25-6O”C. Temperature can have a marked effect on selectivity in MECC, where increased partitioning occurs at higher temperatures. On-column chelation, for example, interactions between borate ions and sugars, may also be increased at higher temperatures (2 7). 5. Optimize Wavelength Selection The maJority of electrolyte systems employed in CE have only limited U activity, and it is therefore possible to operate in the 190-220 nm UV region, which is generally inappropriate in HPLC. Many compounds have significantly greater UV activity in this region, and these UV wavelengths are widely employed in CE. Alternatively, it may be appropriate to employ indirect detection. 6. Optimize Sample Concentration and Composition This parameter should also be optimized during method development. Excessive sample concentration can lead to severely distorted peaks. Generally, peak shapebecomes more triangular as sample concentration is increased excessively. When attempting to determine trace impurities, these high concentrations may not necessarily be avoided. However, if possible, use of lower concentrations will result in more symmetrical peaks, which will result in improved resolutions. In addition, excessive sample concentrations may lead to distorted peaks if the sample has only a limited solubility in the run buffer. Operation at high temperatures can reduce on-column solubility problems, but will alter selectivity.

Altria The efficiency and performance of a CE separation can also be greatly affected by the presence of undesired sample components, such as high levels of salt or organic solvent. The most appropriate sample solutions are water or a 1: 10 dilution of the run buffer. If the ionic strength of the sample solution is lower than the run buffer, a focusing of the sample solution, known as “stacking,” occurs during the initial seconds of the separation. Stacking improves peak efficiency and can greatly improve resolution. A full treatment of sampling stacking and its limitations is given in Chapter 16. Samples having a high ionic strength present the most difficulties m CE. In these circumstances, the stacking process works against the technique, and results m band broadening and loss of resolution. This can result in severe difficulties, since many biological samples analyzed may contain high levels of salt. To minimize this disruption, the use of short mjection times is recommended. If sensitivity permits, dilution of the sample will also reduce the effect. If these approaches are inappropriate, then a sample pretreatment, such as solid-phase extraction, may be required to remove ionic interferences. When attemptmg longer injection ttmes of high-ionic-strength samples, run failures may occur. These events are caused by boiling of the sample solution during the initial secondsof separation, because most of the heat initially generated would be in the sample zone area. If this occurs, it IS necessary to reduce the sampling time and reinject. High levels of organic solvent in the sample solution can also have an undesired impact on the quality of a separation (28). Again, the best strategy is to minimize the sample injection time. Poor water solubility may require high levels of organic solvents. Therefore, higher sample concentrations may be appropriate. Alternatively, it may be appropriate to dissolve the sample m dilute acid or base (29) if possible. Sample stability in the drssolving solvent will need to be evaluated. If excessive sampling times are attempted with samples containing high organic solvents, run failure can occur. This failure is owing to out-gassing of dissolved gases in the solvent. Ultimately, sample solvent composition is dependent on the solubility of the sample. When performing quantitative analysis, it is important to match the viscosities of all samples with each other and the standards, because the volume injected is related directly to the sample viscosity. If

Development /Optimization

41

4 6

10 a Voltage

JI

c!ONDn-XON.9

l+!l!.L

CnpUhr&Ocm(Ld)x63Bcm~t~ x '76um (id) I 376um bd) FS Temperature= 26’C BuITer 0 03M SBS and O.OBM N&rate PA - 6 82 in 66% Waler/16% Me011 PEAK IDENTIF-ICATION l- NIACINAMIDE 2 - CYANOCOBALAhUN (812) 3 - PYRIDOXINFl HCL (BE)

26 kV

016

)(I 7s (kv)

4-NJACM 6 - TiaAMINE

01

tiCL

(B1)

16 kV

4

-A.-

1

00%

1

2

3

(4 10 kV

CI

I.

i 3

6

13

TIME

18 ( Minutes

\ 23

28

9

)

Fig. 4. Influence of voltage on resolution (From ref. Z). it is impossible to match viscosities, use of an internal standard will compensate for this problem. 7. Determine Voltage/Current This factor (I) largely affects the speed and quality of a separation. Application of a high voltage reduces analysis time, but may lead to significant losses of resolution and peak efficiencies if excessive heating occurs within the capillary. The choice of operating voltage should be optimized in conJunction with the choice of electrolyte concentration, capillary dimensions, and temperature to produce an acceptable level of current. Figure 4 shows that resolution is improved at lower voltages, but at the expense of increased analysis time.

42

Altria

Many instruments may be operatedin constant current mode, which may be an advantageif temperaturefluctuateswithm a separationsequence.However, it is advisable to operate generally in constant voltage mode, since slight mterday variations in electrolyte composition will have an impact on the conducttvity, which may alter the run current significantly. 8. Determine

Rinse

Cycle

Selection

It is important to maintain a consistent EOF run-to-run since any variation results in poor migration time precision (30). Sample components, such as proteins, can become adsorbed onto the capillary surface and change the effective charge on the wall, resulting in a reduction m EOF. The adsorped material can also chromatographically interact with the solute and cause tailing. To prevent difficulties owing to adsorption and to ensure a consistent EOF, the capillary 1s flushed between injections with a dilute sodium hydroxide solution that effectively strips the top surface of the capillary wall. Typically, a 1-min rinse with 0. IM NaOH is sufficient. The capillary is then flushed with the buffer prior to injection. Other rinsing regimens may involve use of a dilute acid solution or a strong buffer solution. For example, if the run buffer is 20 mA4 phosphate, it may be appropriate to rinse with 100 mk! phosphate.It is not obvious whether a rinse stepshould be included in a particular method. Generally, it is better to include one to prevent potential problems. If the sample is an uncomplicated matrix, then it may be possible to avoid a rinse step. During method development, the rinse step should be optimized to give a good migration time precision. Having established the optimum buffer conditions, ten replicate analyses should be conducted of a typical sample solution using the selected rinse step(s) between injections. If the migration time precision is poor, it may be necessary to extend the rinse times or include additional steps. Alternatively, if good migration time precision is obtained, the possibility of reducing the time or number of rinse steps should be evaluated. During robustness testing, the time and concentration of rinse solutions should be varied to assesstheir impact on the performance of the method. Following this robustness, testing limits can be put on the rinse time(s) and rinse solution concentrations. It is also important to allow a capillary to become adjusted to new buffer conditions. Therefore, it may be appropriate to employ an extended rinse (i.e., 5 min) with buffer at the start of a sequence or change in buffers during method development.

Development /Optimization

43

9. Optimize Injection Selection The means of sample injection should be selected and optimized for the particular application. Generally, electrokinetic sampling should only be conducted when quantifying trace levels of very mobile ions or when employing gel-filled capillaries where it is not possible to perform hydrodynamic injection. Injection times should be optimized together with sample concentration to give an acceptable peak height and shape. Generally, injection times of l-10 s may be possible. More elaborate mjection schemes employing sample stacking techniques may be employed to improve sample loading--see Chapter 16 for more details. 10. Chiral Separation Methods Development This important application areaof CE is covered in greater depth in Chapter 14.However, the following section representssomeinitial starting condition suggestionsbasedon the electrophoretic characteristics of the solute in electrolyte options l-3 (Le., low-pH FSCE, high-pH FSCE, or MECC). 10.1. LowpH FSCE This is the most commonly employed condition for chiral basic compounds. A useful starting point would be a pH 2.5 phosphate buffer containing 15 mM hydroxypropyl+cyclodextrin. The method should be developed and optimized as shown in Fig. 5. 10.2. High-pH FSCE Under high-pH conditions, the EOF sweeps along the cyclodextrin, while the anions migrate after the EOF flow. Interactions with the cyclodextrin reduce migration time. An initial electrolyte may be 15 mM borax (natural pH -9.3) containing 15 mM hydroxypropyl+cyclodextrin. Figure 6 shows a schematic for the possible optimization of a method. 10.3. MECC Combinations of SDS micellar electrolyte containing cyclodextrins have been shown to achieve chiral selectivity for both neutral and charged compounds. Optimization (Fig. 7) largely involves selection of the appropriate cyclodextrin and its concentration. 10.4. MECC for Hydrophobic Chiral Compounds For large hydrophobic compounds, it may be advisable to employ a micellar electrolyte containing bile salts, such as sodium cholate, which

44

Altria

r-l

Fig. 5. Optimization of the chiral separationof a basic compound.

Fig. 6. Optimization of the chiral separationof an acidic compound. are naturally occurring chirally selective compounds. The addition of organic solvents, such as isopropanol (IPA), can have a beneficial effect on chiral resolutions (Fig. 8).

45

Development /Optimization

Fig. 7. Optimization of the chiral MECC separation.

~~

0 ~~I~/1

0 7

&, ‘

LIzzl

~,o,~,

Fig. 8. Optimization of the chiral MECC separationfor hydrophobic compounds.

11. Method

Protocol

Figure 9 gives a detailed method protocol that, when fiAly completed, would contain all the information needed to fully document the details of a particular method.

Method no. Method purpose Method condltlons Rmse 1 Rmse 2

mm with mm with

seconds employmg

InJection parameters

Detector settmgs

. .

(equivalent to nl) nm AUFS rise time

Separate

nunutes at . ,, constant

Instrument

.

supplier

Capillary

mode model no

pm,

. cm(

. . cm todetector)

(Regenerate fresh captlkuy using 0 5M NaOH for 20 mmutes prior to use) Electrolyte composition

* .

. . .

. ... Sample composltlon

. .

. sample into

Typxal weight . Sample treatment

mgJml m .. .

.

* .

.

sonicate/filter/centfuge . .

Method development details reference

page

employmg

of

Fig. 9. Typical method protocol. 46

. ..

47

Development/Optimization References

1. McLaughlm, G. M., Nolan, J. A, Lmdahl, J. L., Morrrson, J A , and Bronze& T. J. (1992) Practical drug separations by HPCE. practical considerations. J Lzquzd Chrumatogr 15,961-102 1 2 Yeo, S K., Ong, C. P., and Li, S. F. Y (1991) Optimisation of high-performance capillary electrophoresrs of plant growth regulators usmg the overlappmg resolution mapping scheme Anal Chem. 63,2222-2225. 3. Chadwick, R. K. and Hsteh, J. C. (1991) Separation of CIS and trans double bond isomers using capillary zone electrophoresrs. Anal Chem 63,2377-2380. 4. Wemmann, W., Mater, C., Baumeister, K , Przybylski, M , Parker, C E., and Tomer K. B. (1994) Isolatton of hydrophobic hpoprotems in organic solvents by pressure assisted capdlary electrophoresis for subsequent mass spectrometrtc charactertzatton. J. Chromatogr 664,271-275 5. Sctacchitano, C. J , Mopper, B., and Specchio, J. J (1994) Identificatton and separatton of five cephalosporms by mtcellar electrokmettc capillary chromatography J Chromatogr. 657,395-399. 6. Rush, R. S., Derby, P. L., Strickland, T. W., and Rhode, F. (1993) Peptide mapping and evaluation of glycopepttde mtcroheterogenelty derived from endoprotemase digestion of erythroporetin by affimty high-performance capillary electrophorests Anal. Chem 65,1834-1842. 7. Nesi, M , Chrart, M., Carrea, G , Ottolma, G., and Rrghettt, P G (1994) Caprllary electrophorests of mcotmamtde-adenine dmucleotide and nicotmamtde-adenme dinucleottde phosphate derivatrves in coated tubular columns. J Chromatogr. 670, 215-221 8. Atamna, I. Z , Metral, C J , Muschik, G. M , and Issaq, J (1990) Factors which influence the mobility, resolutron and selectrvtty in capillary zone electrophoresls III The role of the buffer cation J Lzquzd Chromatogr 13,320 l-32 10 9. Bushey, M. M and Jorgenson, J W. (1989) Capillary electrophorests of proteins in buffers containing high concentrations of zwttteriomc salts. J Chromatogr 480, 301-3 10. 10. Altrra, K. D., Goodall D. M., and Rogan, M. M. (1994) Quantitative determinatron of drug counter-ton stolchtometry by capillary electrophoresls. Chromatographza 38,637-642. 11. Lm, Y.-M. and Sheu, S.-J. (1994) Separatron of aromatic acids by reversed electroosmottc flow capillary electrophoresis. J. Chromatogr 663,239-243. 12. Honda, S., Taga, A , Kakeht, K., Koda, S., and Okamoto, Y. (1992) Determination of cefixtme and its metabohtes by high-performance caprllary electrophoresis. J Chromatogr. 590,364-368. 13. Bullock, J. A. and Yuan, L.-C. (1991) Free solution capillary elecrophoresis of basic protems m uncoated fused-silica captllary tubing. J Micro Sep 3,241-248 14. Foly, J. P. (1990) Opttmtsatton of micellar electrokmetrc chromatography Anal Chem. 62, 1302-1308. 15. Strasters, J. K. and Khaledr, M. G. (1991) Migration behaviour of cationic solutes in micellar electrokmetic capillary chromatography. Anal Chem 63, 2502-2508.

Altria

48

16 Cole, R. 0 , Sepamak, M J , Hmze, W L., Gorse, J., and Oldlges, K. (1991) Bile salt structures III micellar electrokmetlc capillary chromatography Apphcation to hydrophobic molecule separations J Chromatogr 557, 113-123. 17 Ingvardsen, L , Mlchaelsen, S., and Sorensen, H. (1994) Analysis of individual phosphohplds by high performance capillary electrophorests J Am Od Chem Sot 71, 183-188 18 Crosby, D and El Rassl, Z (1993) Mlcellar electrokmetlc capillary chromatography wtth catlomc surfactants. J Liquid Chromatogr 16,2 16 l-2 187 19 Goebel, L. K and McNalr, H M (1991) Optlmlsatlon of resolution m mlcellar electrokinetic chromatography JHRCC 14,25 20 Terabe, S., Ishlhama, Y., Nlshl, H , Fukuyama, T , and Otsuka, K (199 1) Effect of urea addltlon m mlcellar electrokinetic chromatography J Chromatogr 545,359368 2 1 Wehr, T (1993) Recent advances in capillary electrophoresls columns LC-CG 11,

14-20. 22 HJerten, S. (1993) Electrophoreszs

14, 390

23 Smith, J. T. and Rassi, El (1993) Electrophoresrs 14,396 24 Chen, M and Cassldy, R. M (1992) Bonded-phase capdlarles and the separation of morgamc ions by capillary zone electrophoresis. J Chromatogr 602,227-234 25 Dougherty, A M , Wooley, C L , WIlllams, D L , Swaile, D F , Cole, R 0 , and Sepamak, M J. (1991) Stable phases for capillary electrophoresls J Llquld Chromatogr 14,907-912 26 Cobb, K. A , Dolmk, V , and Novotony, M (1990) Electrophoresls of protems m capillaries with hydrolytically stable surface structures Anal Chem 62, 24782483. 27 Hoffstetter-Kuhn, S , Paulus, A , Gassmann, E., and Wldmer, H M (1991) Influ-

ence of borate complexation on the electrophoretlc behavlour of carbohydrates m capillary electrophoresis. Anal Chem. 63, 1541-1547 28 Ackermans, M. T , Everaerts, F M., and Beckers, J. L (1991) Determination of some drugs by mlcellar electrokmetlc capillary chromatography The pseudoeffective moblhty as parameter for screening. J Chromatogr. 585, 123-l 3 1, 29. Altria, K. D and Chanter, Y (1993) Validation of a capillary electrophoresls method for the determination of a qumolme antlblotlc and its related impurities J Chromatogr

652,459-463

30 Coufal, P , Stulik, K , Claessens, H. A., and Cramers, C. A (1994) The magnitude and reproduclblhty of the electroosmotic flow in silica capillary tubes. JHRCC 17, 325-334.

CHAPTER5 Quantitation

Procedures

Kevin D. Altria 1. Introduction The options for conducting quantitative analysis are similar to those adopted in HPLC (I). The output format is similar to an HPLC chromatogram, i.e., a plot of UV absorbance vs time. Therefore, HPLC data handling and peak integration packages are generally applicable to CE. Main component assay may be performed using external and internal standardization or by standard addition. Impurity data can be calculated and reported as either % w/w or % area/area. Although all aspects of data handling are essentially the same as tn HPLC, it is important to normalize peak areas when calculating impurities (or enantiomers) as % area/area (2). This necessity arises smce all peaks do not pass through the detector at the same velocity. Therefore, the slower-moving peaks spend longer m the detector, giving rise to longer response times and larger peak areas.This is unlike HPLC, where all solutes are pumped through the detector at a constant flow rate. To obtain a time-independent peak area, the peak area of each peak (2,3) is divided by its corresponding migration time. The calculated areas are termed “normalized” areas. This simple manipulation is conducted by most of the commercial GE software packages on instrumentation. Calculation of results without using normalization can result in calculation of incorrect results (2). For example, a synthetic precursor to the antiulcer drug ranitidine was spiked into a solution of ranitldine at the 9.3% w/w level. The two compounds have identical UV response at the detection wavelength used for the CE separation shown in Fig. 1. From

Methods in Molecular hology, Vol 52 Cap/&y Nectrophoress Edtted by K Altna Copyright Humana Press Inc , Totowa, NJ

49

Altria

50

LlB

ClAcl AJ

40

1 l.40

Mtgratlon time (minutes)

I i6rJ

I 180

Fig. 1 Separation of a ramtldme sample spiked with a known amount of a synthetic impurity (from ref. 2)

However, the calculated % area/area indicated only 6.3% area/area impurity content. Use of normalized areas gave a result of 9.1% area/

area, confirming the splkmg level. In this example, the impurity 1sunderestimated. In the case of an impurity migrating after the main peak, it would be overestimated, 2. Main Peak Assay 2.1. External Standardization Calibration solutions of weighed amounts of the standard material are prepared and analyzed. The areas of the integrated peaks are used to calculate the response factor. Sample solutions are then analyzed, and the

recorded peak areas are multiplied by the response factor to calculate the concentration in the sample solution. Using a well-controlled validated method, it is reasonable to expect RSD values of better than l-2% for peak area (4). Typical steps are: 1. Accurately weigh and transfer a portion of the reference material and samplesto appropriatevolumetric flasks

Quantitation

Procedures

51

2. Dilute to volume with the dissolving solvent. 3 Shake or somcate as required to ensure dissolution of the maternal. 4. Falter or centrifuge the sample tf requu-ed, and transfer the sample solution to autosampler vials 5. Place the vials contammg the electrolyte, rinse solution(s), calrbrations, blank, and samples mto the approprtate positions on the autosampler. 6 Perform a test injection of a test mixture to confirm the system 1soperating correctly, 7. Analyze the samples under the conditions specified m the method. A typical sequence will contain analysis of a solution of the dissolving solvent, and a number of injections of the calibration solutions and sample solutions. An appropriate sequence for four samples may be: 1. 2 3. 4. 5 6 7. 8. 9. 10. Il.

Blank. Calibration Calibration Sample 1 Sample 2 Calibration Calibration Sample 3. Sample 4. Calibratton Calibratron

1. 2. 1. 2 1. 2.

Each solution should be injected in duplicate resulting in 22 injections for this example. The integrated peak areas for the calibrations are then used to calculate the response as given below: Response factor = [weight calibration (mg) x % purity / (1) peak area x dilution volume (mL) x 1001 The response factors for all the calibration solutions are calculated and averaged to give a Mean Response Factor (MRF). The concentration of analyte in the sample, if supplied as a solution, is calculated by: mg/mL = peak area x MRF

(2)

If a solid sample was sampled, then the % w/w purity is calculated as: % w/w = [MRF x peak area x dilution volume (mL) / wt (mg)] (3) An example of the use of an internal standard is shown in Fig. 2 (5) m the determination of the drug sumatriptan in injection solutions. The use of

Altria

52

3.00

Retention

5.00

7.00

8.00

time h mhutee

Fig. 2. Separation of sumatriptan employmg an internal standard (from ref. 5). an internal standard allowed an average RSD value of < 1.O%for response

factors to be obtained. 2.2. Internal

Standardization

Steps l-7 (see Section 1.1.) for external standardization are followed, replacing the dissolving solvent by a solution of an appropriate internal standard. The concentration of the internal standard in the final sample solution should approximately match that of the sample. Calculations are modified to incorporate the internal standard. Peak area ratio (PAR) = (peak area sample / peak area mtemal standard)

(4)

Response factor = [wt (mg) x % purity / PAR x dilution volume (mL) x 1001

(5)

Solution concentration = PAR x MRF % w/w = [PAR x MRF x dilution volume (mL) / wt (mg)] 2.3. Standard Addition

(6)

This calibration procedure involves addition of known amounts of standard to the sample solutions (6). Thesespiked samplesare then analyzed and

Quantitation

53

Procedures

the peak areas are plotted against spiking level. The sample content is then calculated from the intercept and slope of the line. The sample spiking can be performed manually or can be preprogrammed into the separation method. 2.4. Manual

Standard

Addition

This procedure (6) is of particular benefit when dealing with samples of varying viscosity, such as biofluids. Since injection volumes are related to sample viscosity, it is essential to match closely the viscosity of the samples and calibration solutions. 1. Takealiquotsof the samplesolutions,typically four, into appropriate vessels. 2. Prepare four calibratron solutions over the required calibration range. 3. Add a consistent volume of each of the standard solutions to the sample solution (for example, add 1.O mL of standard solution to 10.0 mL of sample ahquot). 4. Analyze, at least in duplicate, each of the spiked samples and an unspiked sample. 5. Plot the peak areas agamst spiking level (typlcally recorded m mg/mL or mg addition). 6. Using the slope and intercept values, calculate the sample concentration. A typical calculation is shown below where spiking level IS recorded as mg: mg/mL = [intercept x sample volume (mL) / slope] (7) This approach is demonstrated in Fig. 3 (6), where standard addition is

used to determine trace levels of fluoride. 2.5. Automated

Standard

Addition

In this approach (7), two injection steps are incorporated into the separation method as shown below: Rinse 1: Xmin Rinse 2: Y min Set detector: desired 1 InjectIon 1: x s from standard Injection 2: x s from sample

Separate:requiredmin at selectedvoltage To cover a calibration range, sampling times should be varied appropriately. For example, the time for injection 2 could be set at 5 s, and sampling times for injection 1 set at 0, 1,3, and 5 s. This approach is of use when quantifying a component in a complex mixture, as component identification is simultaneously confirmed. Fig. 4A shows separation of

Altria

54 1600. 1400

-

1200

-

lcaO800

-

600400

-

200

0,. 0.0

, 0 2

I‘

0.4

) 0.6

.

I 0.8

'

I 1.0

-

J 1.2

Fluoride Concentration Ftg. 3. Standard addition graph for fluoride deterrnmatton (from ref. 6). a 5-s injection of a solution of a pharmaceutical. Fig. 4B shows a 5-s injection of a solution of a pharmaceutical followed immediately by a 5-s mjection of a solution of a known impurity. It can be clearly seen which peak in Ftg. 4A is attributable to the specified impurity.

3. Impurity

Contents

Impurity levels may be quoted as % w/w (8) or % area/area (9,10) depending on the application. The impurity level may be calculated as % w/w if an appropriate standard of the impurity is available (8). More commonly, impurity results are calculated as % total area, since standards for all impurities may not be available.

3.1. Percent

(wlw) Impurity

Determination

Procedure

1 Prepare impurity caltbratton soluttons of approprtate concentratton to match likely levels present in samples. 2. Prepare sample soluttons rn the same dissolving solvent as the standards. 3. Analyze both samples and standards in an injection sequence. 4. Calculate impurity levels using response factors from calculatton soluttons. RF = [wt calibration (mg) x % purity / dilutton volume (mL) x 1001 (8)

Quantitation

3 c i5 m 88

Procedures

55

B.OO-

B.OoII 7.00-

pp

6 lO.OO-

B.OD-

I

8.at3-

6.0

Ill

II

1 8.00

7.00 Migrallon

time

8.00

(mmutes)

Fig. 4. Separation of salbutamol tmpurities (from ref. 7). (A) 5-s injection of salbutamol solution; (B) 5-s injection of salbutamol solution + 5-s injection of salbutamol tmpurlty solution.

Altria % w/w = [MRF x peak area x dllutlon volume x 100 / wt sample (mg)] (9)

This approach has been employed for the determination of the dimeric impurities of salbutamol(8). Fig. 4A shows the separation. The calculated results in % w/w were then directly compared to HPLC and TLC results. 3.2. Percent

Area/Area

Impurity

Procedure

This procedure 1smost commonly employed for ease of operation and simplicity (9). In addition, isolated standards of all impurities may not be available. This procedure infers that all impurities have similar response factors at the detection wavelength to the main component. If the impurity response factors differ significantly, appropriate adjustment of data prior to reporting is necessary 1. Analyze sample solutions and a blank at least m duphcate. 2. Integrate all sample related peaks, ignoring all peaks attributed to the blank. 3. Normalize each peak area to Its correspondmg mlgratlon time: Normalized peak area = (peak area / migration time) (10) 4. Add all the normalized peak areas to give a total normalized area for the electropherogram. 5. Calculate specific impurity levels as: % area = (normalized peak area x 100 / total normahzed areas) (11) 6. If the identity of a peak 1sunknown, it 1soften useful to calculate the relative migration time (RMT) as an identifier. RMT 1scalculated by* RMT = (migration time Impurity / migration time of main component) (12)

Impurity profiles for the duplicate injections of each sample solution should be compared. If the profiles do not differ significantly, impurity results for either injection or an average can be quoted. Typical reporting format may include: No. of lmpurltles Total % area lmpurltles Greatest impurity (% area) Identity of greatest impurity Second greatest impurity (% area)

Identity of secondgreatestimpurity Figure 5 shows the separation of related impurities in salicylamide by MECC (9), which were calculated as % area/areawith a detection limit of
Quantitation

Procedures

57

4 h

Ftg. 5. MECC separation of sahcylamide-related impurities (from ref. 9).

If the response factor for selected impurities differs significantly from the mean component (i.e., typically below 80% or above 120% of the main component response), adjustments should be performed. These corrected data give more representative impurity data. For example, If an impurity gives only a 40% response relative to the main component, it will be considerably underestimated. Conversely, an impurity with a

higher UV response will appear to be overestimated. Corrected % area = (% area x 100 / % relative response factor)

(13)

Data manipulation to generate % area data is usually performed employing validated software programs. 4. Enantiomeric Purity Determination When quantifying enantiomers (1 I), it is common to quote % enantiomerit purity. 1. 2. 3. 4.

Analyze the sample employing the chirally selective CE method. Integrate the peaks attributed to each enantiomer, and ignore all other peaks. Normalize the peak areas to their mtgration times. Calculate % enantiomeric purity by: % enantiomeric purity = (total normalized area- normalized areaen1 / (14) total normalized area) / 100

58

I 00

7 60

8 20

a a0

9.10

10.00

RETENTION

Fig. 6. Determmatton (from ref. 12).

TIME

10.60

11.10

11.80

11 to

4 11 00

(MINUTES)

of 0.1% of the undesrred enantromer

m a choral drug

Table 1 Effect of Peak Area Normalizatton on Peak Area Data for an Enanttomertc Separatrona Enanttomer 1 Peak area, observed Peak area, normahzed Mrgratton time, mm

1,199,781

103,827 11.55

Enantromer 2

Peak area ratio

I ,246,293 103,841 12 00

1.04 1 00

n Normallzatton of the dataconfirmsthe correctarearatlo for a racemlccompound where total area = sum of two enantiomers peak areas and area en1 = normalized area of undestred enantromer (or second migrating enanttomer if racemic).

If the compound is racemic, an enantiomeric purity of 50% would be expected. If 1% of the undesired enantiomer is present, then a percent enantiomeric purity of 99% would be obtained. Figure 6 shows the determination of a chiral drug containing 0.1% of the undesired enantiomer (12). Irrespective of which method is used, it is essential to use normalized peak areas (2). This is exemplified by the data obtained for the repeated analysis of a racemic drug (Table 1). A peak area ratio of 1.OOshould be

Quantitation

Procedures

59

obtained for a racemrc compound. The unnormalized areas indicate a ratio of 1.04, which suggests that more of the second detected enantiomer is present. However, the expected ratio of 1.OOis obtained when calculating using normalized areas (2). The racemic drug, clenbuterol, was chirally resolved by seven mdependent pharmaceutical companies in an crossvalidation exercise (13). The peak area ratio, when employing normalized peak areas, gave a value of 49.8:50.2 with an RSD of 0.6% on the results from the seven companies. 5. Determination of Small Ions by Indirect Detection This detection principle is widely employed in the determination of small ionic species, such as inorganic anions and metal ions (see Chapter 20 for further details). For example (‘I#), metal ions are analyzed using a low-pH electrolyte containing imidazole at 5 rnM. When monitoring at 214 nm, this provides a high UV background signal. The passage of the metal ions through the detector displaces the lmidazole molecules, resulting in a reduction of the background signal giving negatrve peaks. The output signal of the detector is reversed to give apparently positive peaks to simplify integration. Chapter 7 contains a section on the princrples of indirect UV detection. Quantitative analysrs using these methods (IS) generally employs external standards of Analar-grade inorganic materials, such as NaCI, to obtain response factors for the individual ions of interest. Use of appropriate internal standards has been shown to improve peak area precision (I 5). References 1. Altrta, K D (1993) Quantnative apphcattons of capillary electrophorests to the determination of pharmaceuticals and drug related Impunties. J Chromatogr 646, 245-257 2. Altrta, K. D. (1993) Essential peak area normalisation m captllary electrophorests Chromatographra 35, 177-182. 3. Ewmg, A G., Wallingford, R. A., and Olefirowlcz, J. (1989) Capillary electrophoresis. Anal Chem 61,292A-303A 4. Watztg, H and Dette, C (1993) Precise quantitative capillary electrophorests. Methodologtcal and mstrumental aspects. J. Chromatogr 636, 3 l-38 5. Altria, K. D. and Ftlbey, S. D. (1993) Quantrtattve analysts of sumatrrptan by capillary electrophoresis J, Lrquld Chromatogr. 16,228 l-2292. 6. Jackson, P E. and Haddad, P (1993) Optlmtsatton of inJectton techmque m captllary electrophorests for the determmation of trace levels of anions in envnonmental samples J Chromatogr 640,48 l-487

60

Al tria

7. Altria, K. D and Luscombe, D C. M (1993) Apphcatron of captllary electrophoresrs as a quantrtattve identity test for pharmaceuticals employing on-column standard additton. J Pharm Bzomed Analysis 11,415-420 8. Altrta, K D (1993) Quantttattve analysts of salbutamol related impurities by capillary electrophorests. J Chromatogr 634,323-328 9 Swartz, M E. (1991) Method development and selecttvny control for small molecule pharmaceutrcal separations by capillary electrophoresls J Lzquzd Chromatogr 14,923-938. 10. Korman, M., Vindevogel, J., and Sandra, P. (1993) Separation of codeme and its by-products by capillary zone electrophoresls as quahty control tool m the pharmaceuttcal mdustry J Chromatogr 645,3&S-370 11, Altrta, K D., Goodall, D. M , and Rogan, M M (1994) Quantttatrve appltcattons and validation of the resolutton of enantiomers by capillary electrophoresrs Electrophoreszs 15,824-827. 12 Werner, A., Nassauer, T., Krechle, P., and Erm, F (1994) Choral separation by capillary zone electrophorests of an optmally-acttve drug and ammo acids by hostguest complexatton with cyclodextrms J Chromatogr 666,37!&379. 13. Altrta, K D., Harden, R. C , Hart, M , Hevtzi, J , Halley, P A , Makwana, J V , and Portsmouth, M J (1993) An inter-company cross-validation exerctse on capillary electrophorests 1. Chrral analysis of clenbuterol J Chromatogr 641, 147-153. 14. Beck, W and Engelhardt, H. (1992) Capillary electrophoresls of orgamc and inorganic cations with mdu-ect UV detection Chromatographza 33,3 13-3 16 15 Altria, K D., Rogan, M. M , and Goodall, D M. (1994) Quantrtatrve determmatton of drug counter-ton stotchiometry by caprllary electrophoresis Chromatographza 38,637-642.

CHAPTER6

Optimization of Precision in Quantitative Analysis Kevin

D. Altria

1. Introduction Early reports of quantitative analysis by CE on homemade systems indicated that peak area precisions of -5% relative standard deviations (RSD) could be expected. The advent of reliable commercial mstruments and improved methodology can now enable RSD figures of l-2% or better to be routinely obtained (1). The following chapter provides some guidelines toward optimizing precision. The major source of imprecision remaining when using commercial instrumentation is injection volume variability. Reproducible nanoliter sample volumes present a formidable engmeering challenge. Use of an internal standard will improve precision (2). The method should be well controlled in terms of consistent migration times (3), since peak area is related to both sample concentration and migration time. If there is a slight drift in migration times, peak area normalization (4) can be conducted to improve peak area precision (5). Injection precision (1) is also highly related to sample concentration, being worse at low sample concentrations (Fig. 1). This is because of increased variance contributions from such factors as integration errors and solute adsorption onto the capillary surface. It is therefore suggested that whenever possible, a high sample concentration and injection volume should be employed. This has the effect of reducing separation efficiencies, but improving precision. This use of high concentrations also allows the simultaneous determination of related impurities. From

Methods Edlted by

m Molecular Bology, Vol 52 CapHary Electrophoresrs K Altrla Copyrtght Humana Press Inc , Totowa, NJ

61

AZtria

62

* *

* * I

500

”

‘1

b

1000

”

”

mg/l

!

+

1500

Frg. 1. Varlatron of precrsronwith sampleconcentratron(from ref Z). 2. High Sample Concentration This is the most important consideration, since several studies (I, 67) have shown a correlation of improved peak area precision with increased sample concentration. This is attributed to a reduction in integrator errors and minimizes the effects of any solute adsorption that may occur onto the capillary wall. Sample solubility, peak shape, and resolution requirements must all be considered when optimizing the sample concentration. 3. Temperature Control Temperature affects both viscosity and electrophoretic mobility (8). Therefore, it is important to maintain a constant temperature (9) throughout a seriesof injections. This need for temperature control applies to the capillary, sample vials, and electrolyte reservoirs. Viscosity decreases with increased temperature. Therefore, when temperature increases, more sample solution enters the capillary for the same injection time. Electrophoretic mobility increases with temperature, which reduces migration times. It is important to allow the sample solutions to attain a constant temperature on the CE autosampler, since the volume injected is related to the sample solution viscosity (IO). Since in CE the area of peak is related to both sample concentration and migration time, temperature differences

Precision in Quantitative

63

Analysis

during an analytical sequence will cause shifts in migration times and apparent peak areas, leading to poorer precision. 4. Injection

Times

Using hydrodynamic injection, a pressure difference is generated across the capillary for a set period of time (l-20 s). The pressure dlfference is instrument-specific, and the exact pressure level is monitored durmg injection. Most commercial instruments are self-regulating in that, if the pressure difference 1s below the set level, the injectlon time is extended to compensate. Conversely, if the pressure difference 1sabove the set limit, the injection time is reduced accordingly. In all instances, if the monitored pressure difference is too far from the prescribed level, no injection would be made. If short injection times, such as 1 or 2 s, are employed, the ability to self-regulate injection is reduced. Therefore, it is advisable to employ longer injection times, such as 5-10 s. To achieve this, it may be necessary to decrease sample concentration. When using a positive pressure injection system, liquid is displaced from the capillary during injection. This liquid appears as drops at the detector end of the capillary. If the drop falls durmg injection, the pressure difference across the capillary is altered, causing imprecision. To eliminate this possibility, it is advisable to perform the injection step with the detector end of the capillary immersed in a vial containing separation electrolyte. 5. Capillary

Rinsing

It has been shown (I, II) that rinsing the capillary between injections can improve (12) migration time precision to give migration time precisions of
64

Al tria Table 1 Preclslon Data Obtamed for Different InJectIon Times (from ref 13)

Sample K+Na K+Na K+Na

Injection time, s

No of mjectlons

RSD %, K

RSD %, PAR

1 2 2

9 9 45

36 11 2.8

11 03 02

6. Internal Standard Much of the variance in precision is attributable to injection volume fluctuations. Therefore, incorporation of an appropriate internal standard(2) will mnumize this source of error. It is important to match appropriately sample and internal standard concentrations.It is necessaryto ensure that the internal standard does not corn&ate with any sample-related compounds. The purity and stability of the internal standard should be selected, such that no internal standard-related impurities interfere with the sample component peaks. Use of an internal standard also partially compensates for variations in area owing to interrun migration time variation. The advantages of employing an internal standard are highlighted by data from the validation (13) of an assay method for potassium using sodium as an internal standard (Table 1). The precision of injection for the potassium peak alone ranged from l . l-3.6% RSD. However, the precision for peak arearatios (PAR) was improved and ranged from 0.2-l. 1% RSD. 7. Electrolyte Considerations Peak tailing can lead to poor precision because of variable integration. Peak tailing can be reduced by increasing the electrolyte concentration. Excessive electrolyte concentration can, however, lead to a generation of unacceptably high levels of current, which lead to poor precision of migration time. Therefore, rt is necessary to optimize electrolyte in terms of composition and concentration. When performing multiple analysis, electrolysis of the electrolyte in the buffer reservoirs can occur (14,15), resulting in a pH gradient being formed across the capillary (this effect has been termed buffer depletion). These changes may affect both migration times and selectivity. Figure 2 shows the first and fourth injection (14) using a low-capacity buffer. It should be noted that the migration time and selectivity vary, A stable method was obtained by selection of a more appropriate buffer.

Precision in Quantitative

Analysis

65

B I V

0

I

I

5

10

j. 1s 0

I I

I

5

10

15

ThWmin

Fig. 2. First and fourth nqectlons of an atenolol test mixture usmg a lowcapacity buffer.

To minimize buffer depletion, large volumes of electrolyte (5 mL or greater) should be employed. Higher ionic strength reduces the significance of this depletion. It may be appropriate (‘II) to specify use of an electrolyte vial for only a selected number of analyses. After this number of injections, the analysis is performed employmg vials containing fresh electrolyte. It may be necessary, for an extremely long sequence, to utilize several changes of electrolyte. Switches of buffer vials can be preprogrammed into the PC controlling the CE instrument. 8. Preconditioning Preconditioning of the system in certain methods (I, I I, I@, particularly high-pH free solution capillary electrophoresis (FSCE) and micellar electrokinetic capillary chromatography (MECC), can lead to improved precision. It is our experience that the first injection in a sequence can produce significantly different peak area and selectivity to subsequent analyses. This may be the result of an equilibration of the capillary surface and a stabilization of electrolyte composition under electrophoretic conditions. It may therefore be appropriate to include a precondition analysis in the sequence. This precondition may simply

Altria comprise analysis of sample dissolving solvent. Alternatively, the capillary could be filled with the electrolyte, and the appropriate voltage applied for 20-30 min. Incorporation of these preconditioning steps also allows the solutions on the autosampler to reach a constant temperature. Use of temperaturecontrollable autosampler tray may assist in this aspect. 9. Sample Consideration The following points should be considered when performing quantltative analysis: 1. Match lomc strength and vlscoslty of samples and standards.Viscosity influences the volume Injected mto the system (8) Therefore, It 1sfimdamental to match the vlscosltles of both standards and samples Samples, such as bioflmds, will have very dtfferent vlscositles compared to standard solutions. If this 1s lmposslble, standard addition calrbratlon procedures could be performed. 2. Evaporation losses should be mmlmlzed by employmg sealed vials and aqueous-based sample solvents with the mmlmal level of volatile organic solvents possible. Often, small sample volumes of Cl mL are employed using mlcrovial sample contamers. Evaporation losses from these mlcrovials can be very significant (Z 7) over the course of a few hours. The losses may be reduced by using a cooled autosampler tray and ensurmg that the mlcrovlals are stored m humid condltlons (this can be achieved by inserting the microvials into a large vial containing a few drops of water m the bottom). Use of an internal standard alleviates any potential difficulties

owing to evaporation. 10. Capillary Washing Poor precision may result (Z6) if sample solution is transferred onto the outside of the capillary during the injection process. When the separation voltage is applied, some of this transferred material may migrate into the capillary, giving a nonreproducible higher sample loading. To prevent this effect from occurring, an additional step in the method may be incorporated: Step 1: Rinse. Step 2: Rinse electrolyte. Step 3. Inject sample. Step 4: Inject electrolyte or water (1 s). Step 5: Separate.

Precision in Quantitative

Analysis

67

The purpose of step 4 is to wash the capillary exterior to avoid any crosstransfer of sample solution. 11. Capillary Cutting If the capillary end is jagged, sample solution may be irreprodwcibly transferred from the sample solution. It is therefore important to break the capillary using a cutting stone or a diamond-tipped cutter. If this source of imprecision 1s suspected, examine the capillary tip using an appropriate eyepiece. Replace the capillary if necessary. 12. Solution Height Considerations Dipping the capillary into the solution (18,19) causes a small amount of sample solution to enter by capillary action. The amount of this “inadvertent injection” is related to the internal diameter of the capillary and the height of the sample solution in the vial. Siphoning of sample solution into the capillary can occur if the height of the sample solution vial is higher than the level in the detector end electrolyte vial. This would give a higher sample loading than if the levels were identical. Conversely, a lower sample solution level would result in a siphoning flow in the opposite direction, giving a lower on-column sample loading. Unless controlled, this can be a further source of imprecision. This problem will be increased moving to wider-bore capillaries. To reduce this effect, it may be necessary (II) to pipet a set volume of sample solution into each sample vial to ensure a constant solution level. A siphoning flow can occur if a height exists between the solution levels in the high-voltage and low-voltage electrolyte vials. For example, the siphoning flow will be toward the detector if the level is higher in the high-voltage electrolyte vial. In this instance, the migration time will be reduced as the siphoning flow will be superimposed on the mobilities and EOF governing the separation. The magnitude of this flow is such that a 2-mm height difference may have a 2% change in migration time for a 50-pm capillary, whereas a 10% difference may occur with 100-pm capillaries. Repeated rinses from an electrolyte vial will cause a gradual decreasein solution height level, resulting in an increased siphoning flow against the direction of migration. This will be observed as a drift in migration times and corresponding peak areas.This effect can be avoided by performing rinses from a separate vial from those used for separation purposes.

Al tria

68 Table 2 Method Settmgs for Optlmal Preclslona Cycle Set temperature Rinse Injection Injection Separate

Settmg Constant temperature Typically 2 mm Typically 5 s IS

As appropriate

Vlal number, at samphng end

Vlal number, at detector end

1 3 1 2

5 4 4 4

“See Rg 3 for vial numbers From ref 21

13. Voltage

Ramp When the separation voltage is initially applied across the capillary containing the sample solution, much of the load is applied across the sample zone, which usually has a higher electrical resistance. This situation leads to accelerated heating of the sample zone and thermal expansion. This expansion can in turn lead to a small, nonreproducible amount of the sample to be forced out of the capillary at the onset of the separation (20), To minimize this effect, it is recommended to ramp up the voltage gradually to the final level over the course of 20-30 s. This facility is available with many commercial systems. In addition, this effect may be minimized (16) by incorporating a water or electrolyte slug after the sample zone. Therefore, if some material is lost, it will be unimportant, since it will only be water or buffer. 14. Speed of Sampling It has been shown (19) that delays between sample introduction and reinsertion of the capillary tip into the electrolyte vial can cause a loss of precision. Therefore, the timing of this procedure should be well controlled. This feature is built into fully automated CE systems. Use of an internal standard will eliminate this problem. 15. Method Settings for Optimal Precision on Automated Equipment The best settings for a CE method to obtain maximum precision (21) is given in Table 2. The vial numbers refer to those shown in Fig. 3.

Precision in Quantitative

69

Analysis High voltage supply

Buffer vlal

Buffer vial

3 @2 as Sample 0

vial

Fig. 3. Optlmlzed

Empty vial

4

*

0

5

system setup (from ref. 21).

References 1 Watzlg, H and Dette, C (1993) Precise quantltatlve capillary electrophoresis Methodological and instrumental aspects J Chromatogr 636,3 l-38 2. Dose, E. V and Gulochon, G A (1991) Internal standardlsatlon technique for capillary zone electrophoresls. Anal Chem 63, 1154-l 158. 3. Ewing, A G , Walhngford, R. A, and Olefirowicz, T. M (1989) Capillary electrophoresis. Anal Chem 61,292A-303A. 4. Altria, K D. (1993) Essential peak area normalisation in capillary electrophoresrs Chromatographla 35, 177-l 82 5 Altria, K. D., Harden, R. C., Hart, M., Hevizl, J., Hailey, P A., Makwana, J. V., and Portsmouth, M. J. (1993) An inter-company cross-validation exercise on capillary electrophoresls 1 Choral analysis of clenbuterol J Chromatogr 641, 147-153 6. Ryder, S. (1992) Determination of sodium vmyl sulphonate in water-soluble polymers usmg capillary zone electrophoresls. J Chromatogr. 605, 143-147. 7. Altria, K. D , Rogan, M. M., and Goodall, D M. (1994) Quantitative determmatlon of drug counter-ion stoichlometty by capillary electrophoresls Chromatographza 38,637-x542. 8. Rose, D. J and Jorgenson, J. W. (1988) Characterlsation and automation of sample

introduction methods for capillary electrophoresls Anal Chem 60,642--648 9. Goodall, D. M , Wllhams, S. J., and Lloyd, D. K. (1991) Quantttattve aspects of capillary electrophoresis TrAC 103, 272-279. 10. Rush, R. S. and Karger, B. L. (1990) Sample inJection with P/ACE system 2000. Importance of temperature control with respect to quantltation. Beckman Techmcal Bulletzn TIBC 104, I,2

Al tria 11 Thomas, B R., Fang, X G , Chen, X , Tyrell, R J , and Ghodbane, S (1994) Vahdated mlcellar electrokmetrc caprllary chromatography method for the quality control of the drug substances hydrochlorothtazide and chlorothrazrde. J Chromatogr 657,383-394. 12 Coufal, P , Stuhk, K , Claessens, H A., and Cramers, C A (1994) The magnitude and reproductbihty of the electroosmottc flow m sthca captllary tubes. JHRCC 17, 325-334 13 Altrta, K. D , Wood, T , Kttscha, R , and Roberts-McIntosh, A (1995) Valtdatlon of a capillary electrophorests method for the determmatton of potassmm counterton levels in an acrdrc drug salt. J Pharm Bzomed. Analyszs 13, 33-38 14 Shafaatr, A and Clark, B J (1993) Development of a caprllary zone electrophoresis method for atenolol and its related tmpurtttes m a tablet preparatton Anal Proc 30,48 1483. 15 Sun, Y -L , Zhang, C -X , Ling, D -K , and Sun, Z -P (1994) Vartatton of pH of the background electrolyte as a result of electrolysts in CE JHRCC 17,563,564. 16 Altria, K D , Clayton, N G , Harden, R C , Hart, M , Hevtzt, J , Makwana, J V., and Portsmouth, M J (1994) An inter-company cross-vahdatton exercise on captllary electrophorests testing of dose uniformity of paracetamol content m formulations Chromatographza 39, 180-l 84 17 Mormg, S. E., Colbum, J. C , Grossman, P. D , and Lauer, H H (1990) Analyttcal aspects of an automated capillary electrophorests system. LC-GC 8,34-40 18 Dose, E V. and Gurochon, G A (1992) Problems of quantitative inJection in capillary zone electrophoresis Anal Chem 63, 123-128 19. Frshman, H. A., Amudi, N M., Lee, T. T , Scheller, R H , and Zare, R N. (1994) Spontaneous mJectton m microcolumn separattons Anal Chem 66,23 18-2329 20. Knox, J. H and McCormack, K A (1994) Volume expansion and loss of sample due to mittal self-heating in capillary electrophoresis (CES) systems

Chromatogruphza 38,279-282 2 1. Altria, K D. and Fabre, H (1995) Approaches to optimrsation of precision m capillary electrophorests. Chromatographza 40, 3 13-320

CHAPTER7 Optimization Kevin

of Sensitivity D. Altria

1, Introduction The injection volumes involved in CE are very small (in the order of l-10 nL). In addition, the area of capillary employed for on-column detection may be only 50 x 200 pm. Both these factors influence the detector sensitivity to a large extent. CE is less sensitrve when directly compared to HPLC with typical injections of 10-50 pL and l-cm detector cells. The difference may be up to an order of magnitude (2) when comparing at the same UV wavelength. Several strategies may be employed (Table 1) to maximize the sensitivity in CE. These include use of low UV wavelengths, increased capillary bore, and optimized sampling procedure. 2. Detection Wavelength Many solutes have considerably enhanced UV activity at low UV wavelengths (i.e., 190-220 nm). Excessive background UV absorbance from the organic solvents employed in HPLC generally prevents operation at these wavelengths. However, since the electrolytes employed in CE are predominantly aqueous-based,the background absorbance is not excessive and methods can be routinely operated at low wavelengths. Adopting this approach can allow direct detection of solutes that may have required derivatization prior to HPLC. Examples include the direct detection of sugars (2) and amino acids employing detection at 195-200 nm. Equivalent concentration sensitivity for salbutamol and related impurities was obtained (3) for CE and HPLC employing detection wavelengths of 200 and 276 nm, respectively. From

Methods m Molecular Ebology, Vo/ 52 Capillary Electrophoresrs Edtted

by K Altna

Copynght

Humana

71

Press

Inc , Totowa,

NJ

Al tria

72 Table 1 Summary of Approaches Available for Increasmg Sensitivity Action to Improved sensmvtty Employ low-UV

wavelength

Increase capillary bore Increased mlection time Approprtate use of electrokmettc mlection Increased electrolyte strength Optimize electrolyte composmon Decrease operating voltage Decrease temperature Captllary modrficattons Sample derrvattzation Indirect detection Wide-bore capillaries

Increased detector slit wtdth

Drawback(s)

to consider

Increased background notse-detennme wavelength to gave optimum stgnal-to-noise ratio Increased current, reduced EOF givmg possible alteration m selecttvtty Reductton m separatton effictency; excesstve ttme will result m run failure Samphng bias for more mobtle tons, sample matrix effects on qectton amount Increased current and associated notse Effects on selectivity and current Increase m analysis trme Increase m analysis time Reduction m separation efficiency and resolution, Increased cost Addmonal samplmg handhng Extra method development considerations EOF profile disturbed, adjustments to rmse and mjection times, stphonmg effects more pronounced Reduction m separatton effictency and resolution

3. Capillary Bore Increasing the capillary bore improves detector sensitivity considerably. This gain is caused primarily by an increase in the volume of caplllary employed as the on-column detection area and a similar increase in the volume of sample solution introduced into the capillary. Typically, 50- and 75-pm capillaries are routinely employed, since problems of reduced separation efficiency and increasing current occur moving to wider-bore capillaries. Band broadening resulting from nonideal EOF increases with capillary bore. Levels of EOF also decrease significantly with increased capillary bore, which will influence separation selectivity. The ability to dissipate heat is also reduced with increased capillary bore as the effective surface area is reduced. This increased heat can lead to reduced separation efficiency, and if too excessive, can cause outgassing or boiling of the electrolyte. The sample volume introduced hydrodynamically into the capillary is related to the pressure difference across the capillary. Wider-bore capillaries have considerably

73

Sensitivity

Fig. I. Separationof drug-relatedimpurities using a 180~pmcapillary (from ref. 4). reduced back pressures, and rinse regimes require careful adjustment (see Section 12). Capillaries with bores as wide as 180 pm (4) have been successfully used to enhance sensitivity greatly. Figure 1 shows the detection of impurities in a drug substance using a 180~pm capillary. The impurities at the 0.1% level are almost off the full-scale deflection of detector. However, attention should be given to optimizing injection time in conjunction with capillary bore (see Section 12). 4. Injection

Time

The injection time employed in hydrodynamic sampling determines the volume of sample introduced into the capillary. The increase in peak area is linear with injection time. Peak height increases are not necessarily in as linear a fashion. Separation efficiency is reduced with increased injection time, which will have implications on resolution achieved. Excessive sampling times can lead to a breakdown in the conductance along the capillary, causing a run failure. Therefore, injection times can only be increased to a limit that would be determined in method optimization. This breakdown in conductance is most pronounced when

74

Al tria

samples are dissolved m organic solvents, since these are nonconductive. Samples prepared in water, although giving good separation efficiencies for sample components, have low conductivity. Therefore, to extend sampling times, it may be appropriate, for example, to dilute the sample with a low-tome-strength solution (5). Typically, samples may be dissolved in a solution of electrolyte employed for separation, diluted 1 to 10 with distilled water. The sample-dissolving solvent should be optimtzed during method development. 5. Injection Procedure Several means of optimizing sampling to increase sensitivity have been reported. These mclude the use of electrokinetic mjection and multiple electrolyte systems. Chapter 16 discusses some advanced means of Increasing sensitivity. In electrokinetic injection, sample ions enter the capillary by virtue of their mobility and any EOF present. A bias exists with this injection procedure in that a greater number of more mobile ions enter than less mobile ones. When attempting to maximize sensitivity, this effect can be advantageous. For example, to determine trace levels of small inorganic cations, application of a voltage of +5 kV for 10 s will result in a sensitivity improvement of 2-3 orders of magnitude (6) compared to pressure injection. Using this approach, it is possible to determine low-ppb levels of metal cations with indirect detection. The limit of detection for these cations using hydrodynamic injection with similar detection is -1 ppm. To optimize injection of anions, it is necessary to suppress EOF or to alter the direction of EOF. Anions are electrokmetically injected employing a negative voltage, and similar gains in sensitivrty to that described for cations can be achieved. Electrokinetic injection does not give significant sensitivity increases for less mobile tons and is less effective for neutral species, since these enter the capillary only by virtue of the generation of any EOF. 6. Electrolyte Composition The ionic strength and pH of the electrolyte should be optimized to reduce peak tailing. Higher ionic strengths reduce tailing and give improved separation efficiencies. If possible, the srgnal-to-noise ratio should be obtained for electrolytes under consideration, since the background absorbance will vary between electrolytes. For example, when

Sensitivity operating at low UV detection wavelengths, such as 200 nm, borate and phosphate buffers are especially useful, because their background UV absorbances are low compared to electrolytes, such as citrate or acetate. The major source of zone dispersion is molecular diffusion. Diffusion is related to sample molecular weight, electrolyte viscosity, and time. The electrolyte viscostty can be increased to maintain peak sharpnessby addition of agents, such as cellulose, PVA, or glycerol. These additions do, however, suppress EOF, which is also viscosity-dependent 7. Operating Voltage Molecular diffusion is related to analysis time. Therefore, it is useful to operate at higher applied voltages. This can be achieved by either mcreasing the voltage or reducmg the capillary length. Limitations concerning current and electrolyte ionic strength will need to be considered. 8. Temperature Thermal vibration of the capillary can occur at increased temperatures. This vibration will be shown by an increase in detector noise. Operating at high current levels can induce a similar effect owing to internal heatmg within the capillary and associated refractive index changes 9. Capillary

Modifications Several approaches have been described for extending the effective pathlength of the area of capillary employed for detection. These have included use of a Z-shaped portion of capillary (7) and creation of a bubble within the capillary at the detection window. Bubble cells are commercially available from Hewlett Packard (Palo Alto, CA), whereas Z-cells are available from Perkin Elmer (ABI, Foster City, CA) or LC Packings (Zurich, Switzerland). Figure 2 shows the possible sensitivity gain when employing a Z-cell capillary. Rectangular capillaries have also been utilized (8). However, these are not yet commercially available. Capillaries are also available with a small portion of reversed-phase packing material inserted in the injection end of the capillary (9). Extended electrokinetic injection of the sample causes sample ions to migrate into the capillary and become adsorbed onto the packing. After this injection a short, l-s injection of solvent, such as methanol, removes the material from the packing. Using this approach, electrokinetic injection times of several minutes can be employed, and good

76

Al tria

I

2

HS OPTICAL CELL

3 3 f-,

0

;-~~

2

4

4

6 TIME

0.18%

8

10

12

(min)

Fig. 2. Sensitlwty gains possible using Z-cell capillary (from ref. 22).

peak shape and efficiency were still maintained. Detection limits of up to a IOOO-fold over conventional hydrodynamic injection were shown. 10. Derivatization Sensitivity can also be improved by appropriate derivatization of the sample. The derivative may have a considerably higher UV absorbance or may be selected to allow fluorescence detection. The majority of reports (IO) have centered on preseparation denvatlzation. This obviously involves additional sample handling steps. Appropriate derivatization can enable use of laser-induced fluorescence monitoring, which may be up to 1000 times more sensitive than UV absorbance. Some recent reports have indicated use of on-column derivatization (II, 12) in which the sample and derivatizatlon chemicals are injected

Sensitivity

77

FS

0 1 1. Empty capillary

FS A

FS A

0

0 2. Filled caplllary

3. Separation

Fig. 3. Prmclples of mdlrect detection.

onto the capillary and allowed to react prior to separation. This approach is particularly attractive if the derivative has poor stability. On-column chelation is another useful option to improve detection. For example, UV-transparent metal ions are complexed on-column with EDTA to produce strongly UV absorbance anionic chelates, which are well resolved (13) with good detection limits. 11. Indirect Detection This method of detection is widely employed in CE, since simple inorganic or small organic ions are widely analyzed. These species have only limited chromophores and cannot be readily detected by conventional UV absorbance detection. Indirect detection involves the addition of strongly UV active species into the electrolyte to give a constant high UV signal in the detector. This detector signal decreases when solutes have a limited chromophore pass through the signal, resulting in the generation of negative peaks (Fig. 3). Commercial instruments have the ability to reverse detector output such that peaks are easily integrated. The choice and concentration of the UV-absorbing species have a large impact on the performance and sensitivity of the method (14-18). The concentration of the background absorber primarily governs the sensitivity obtainable and the linear dynamic range possible. If the concentration is too high, the UV responsechange for a lower concentration sample will be insufficient to be detectable. Conversely, if the concentration is too low, the linearity will be limited to an unacceptable degree. Therefore, compromise concentrations of l-10 mA4 are typical. The mobility of the absorber in the run buffer should approximately match that of the sample constituents. If the mobility of the solute exceeds that of the UV-

78

Altria 2 69-l

1 2 00

1 3 00

I 2 50

/ 3 50

mlnutas

Fig. 4. Separationof a rangeof metal cations usmg indirect UV detectron (from ref. 18). absorbing ion, then a fronting peak is obtained (K+, Fig. 4). If the mobilities of the solute ion and UV-absorbing ion are exactly matched, then a sharp, Gaussian peak will be obtained @a+, Fig. 4). If the sample is slower, then an apparently tailing peak will be obtained (Li+, Fig. 4). In this separation, the mobility of the background absorber imidazole 1s0.44 cm2/kV/s, which exactly matches the mobility of Ba+. If an exact mobility match IS not immediately available (19), it may be appropriate to alter the mobility of the UV absorber to match the mobility of the sample more closely. For example, P-cyclodextrin was added to match the mobility of 2,6 napthelenedicarboxylate to agree more closely to that of selected aliphatic acids. Indirect detection is extensively employed in the detection of small inorganic and organic ions. Reference to these sections in Chapter 20 will cover the range of possible UV absorbers and applications more comprehensively. Generally, the buffer system should be optimized to give a maxrmum background absorbance of 0.1 aufs (17). The electrolyte composition should be considered to produce a minimal amount of internal heating, smce this 1sexhibited as increased baseline noise owing to thermal vibra-

Sensitivity tion of the capillary. Therefore, methods are typically operated at conditions that produce 5-10 PA. Optimal sensitivity m indirect detection is also obtained at a wavelength where there is a maximum difference between the absorbance of the solute and the UV absorber. Therefore, detection wavelength should be optimized to give the greatest signal-to-noise ratio.

12. Use of Wide-Bore

Capillaries

This is an effective means of increasing sensitivity, smce the detection pathlength is the size of the capillary diameter. Although attractive (Section 3.), several complications must be considered when increasing capillary bore. 1. EOF IS strongly related to capillary bore. A change m capillary bore may, therefore, have sigmficant impact on separation selectlvlty, especially at high pH. 2 A doubling of capillary bore increases the current level obtamed for a set applied voltage by a factor of four. This may result m excessive current or may require operation at a reduced voltage level. 3. Flow rate through the caprllary 1srelated to the fourth power of the dlameter, because the back pressure along the capillary 1s greatly reduced by mcreasing capillary bore. Therefore, sampling and mJectlon times must be adjusted accordingly when capillary bore 1sincreased 4. Moving to capillary bores of 100 pm or greater can cause serious distortions of the separation achieved usmg conventional bore capillaries. The EOF profile becomes more laminar, which causesband broadening. Height differences between electrolyte levels in the buffer vials can result m a siphoning flow. Siphoning flow has a lammar profile, but 1sminimal when employmg conventional bore capillaries. Siphoning can become a slgmficant factor usmg wide-bore capillaries, and careful attention should be paid to electrolyte levels in the vial. 5. When employmg wide-bore caplllanes, it may be beneficial to eliminate EOF by using coated capillaries or by the addition of polymeric additives to the electrolyte. Polymeric additives, such as hydropropyl methyl cellulose or polyvinyl alcohol, are water-soluble, and small additions (< 1% w/w) significantly increase electrolyte vlscoslty (20). Increased viscosity 1sbeneficial for several reasons; EOF flow decreases with viscosity, siphoning flow rate IS reduced with viscosity, and rinse and injection times may be increased as the increased electrolyte viscosity increases the back pressure across the capillary. The disadvantage of these approaches is that only anions or cations may be quantified within a single analysis and that MECC cannot be performed.

80

Altria

Despite these apparent drawbacks, tt is posstble to operate routmely with IOO-pm capillaries (21). 13. Detector

Slit Width

Viewing a longer portton of the capillary will also lead to gains in sensitivity. For example, changing from a 200~pm slit width to 800~pm will generally double the signal-to-noise for a given peak. The expected fourfold increase is not achieved, since the noise is also increased to a lesser extent. Since each peak IS monitored by the detector for a longer time, observed separation efficrenctes will decrease and resolutton of closely migrating peaks will be reduced. References 1 Altrra, K D (1993) Sensmvrty optrmlsation for use of caprllary electrophoresrs m pharmaceutical analysrs LC-GC Znt 11,438-442 2 Hoffstetter-Kuhn, S , Paulus, A , Gassman, E , and Wrdmer, H M (1993) Influence of borate complexatron on the electrophoretrc behavlour of carbohydrates m caprllary electrophoresls Anal Chem 63, 154 l-l 547 3. Altrra, K D. (1993) Quantrtatlve analysis of salbutamol related rmpurrties by capillary electrophoresls J Chromatogr 634, 323-328 4 Altrra, K. D (1993) Capillary electrophoresrs for pharmaceutrcal research and development LC-GC Int 6,6 16-620 5. Chlen, R. L and Burghl, D S (1992) On-column sample concentration using field amplificatton m CZE. Anal Chem 64,489A-496A 6 Jackson, P E and Haddad, P. (1993) Optrmrsatron of mJectlon technique m capillary electrophoresrs for the determmatron of trace levels of anions m envnonmental samples J Chromatogr 640,48 l-487 7. Chervet, J P., Van Soest, R. E J., and Ursem, M (1991) Z-shaped flow cell for UV detectton m caprllary electrophorests. J Chromatogr 543,43!+-449 8 Tsuda, T., Sweedler, J. V , and Zare, R. N (1990) Rectangular caplllarles for capillary zone electrophorests. Anal Chem 62,2 149-2 152 9 Swartz, M. E. and Merron, M (1993) On-lme sample preconcentration on a packedinlet caprllary for lmprovmg the sensrttvrty of caprllary electrophoretlc analysrs of pharmaceutmals J Chromatogr 632,209-2 13 10. Amankwa, L. N , Albmk, M , and Kuhr, W. G (1992) Fluorescence detectron m caprllary electrophoresls TRAC 11, 114-120. 11 Ruyers, H. and Van der Wal, S J. (1994) Fully automated analysts of ammo acid enantlomers by derlvatisatron and chiral separatron on a capillary electrophoresrs instrument J Lzquzd Chromatogr 17, 1883-l 897. 12 Reinhold, N J , TJaden, U. R , and Vand der Greef, J (1994) Automated on-caprllary isotachophoretrc reaction cell for fluorescence derrvatrsatron of small sample volumes at low concentratrons followed by capillary zone electrophoresrs J Chromatogr 673,255-266

Sensitivity

81

13 Motomrzu, S , Oshrma, M., Matsuda, S., Obata, Y , and Tanaka, H (1992) Separatton and determmatron of alkalme-earth metal Ions as UV absorbing chelates wrth EDTA by capillary electrophoresrs Determination of calcmm and magnesium in water and serum samples. Anal Scz. 8,6 19-624. 14 Wang, T. and Hartwtck, R. A (1992) Norse and detectron hmrts of indirect absorption detectton m capillary zone electrophoresrs J Chromatogr 607, 119-125. 15 Foret, F , Fanali, S , Ossrcmr, L , and Bocek, P (1989) Hindu-ectphotometrrc detection m capillary zone electrophorests. J Chromatogr 470,299-308. 16. Vorndan, A G., Oefner, P J , Scherz, H., and Bonn, K (1992) Indirect UV detectton of carbohydrates in caprllary zone electrophoresis. Chromatographla 33, 163-168. 17 Nrelen, M W. F. (1991) Quantttative aspects of mdtrect UV detection m caprllary zone electrophoresrs. J Chromatogr 588, 321-326 18 Beck, W. and Engelhardt, H (1992) Capillary electrophorests of orgamc and morgame cations wrth indirect UV detection Chromatographza 33, 3 13-3 16. 19 Tindall, G. W , Wilder, D R., and Perry, R L (1993) Optimrsmg dynamrc range for the analysis of small ions by captllary zone electrophoresrs J Chromatogr 641,163-167

20 Belder, D and Schomburg, G (1992) Enantromer separatron of tocannde analogues by cyclodextrm modified electrokmettc chromatography JHRCC 15,686-693 2 1 Thomas, B R , Fang, X. G., Chen, X , Tyrell, R. J , and Ghodbane, S. (1994) Vahdated mtcellar electrokmetrc capillary chromatography method for the quahty control of the drug substances hydrochlorothraztde and chlorothrazrde. J Chromatogr 657,383-394. 22 Mormg, S. E , Parraud, C , Albm, M , Locke, S , Thrbault, P , and Tmdall,

G W (1993) Enhancement of UV detectron sensitrvrty for captllary electrophoresis Am Lab July, 22

CHAPTER8

Method Kevin

Validation D. Altria

1. Introduction The criteria for validation applied to CE methods (1-3) are similar to those employed for other quantitative separative techniques, such as HPLC (4). The extent of tests applied will vary according to the nature of the application and the extent of intended usage. Generally, such aspects as accuracy, precision, reproducibility, linearity, robustness, and method of transfer are evaluated during validation. Suggestedmethod validation approacheswill be given for the three key application areas of purity testing, main peak assay, and chiral analysis. All tests do not necessarily need to be performed for each method, and those most appropriate should be conducted and recorded by the analyst. Table 1 gives a suggested range of testing. 2. Selectivity 2.1. Purity Determination Appropriate selectivity of the method should be demonstrated for all known likely synthetic or degradative impurities (1,&d). This would be performed by analyzing test solutions spiked with all available impurities at expected levels. If appropriate, the method may be further challenged by analyzing stressed samples. The stressed samples may be generated by exposure to high temperatures, pH extremes, and both natural and UV light. The repeatability of selectivity should be assessed by repeated injections of a suitable sample or test mixture (.5), since electrolysis (depletion) of the buffer can occur leading to changes in selectivity. Selectivity should be demonstrated at the levFrom

Methods m Mokcular Biology, Vol. 52 Caprllary Electrophoresa Edited by K Altna Copynght Humana Press Inc , Totowa, NJ

83

84

Altria Table

1

Vahdatlon Requwements Test

Identity confirmation

Choral and achlral purity

Main component assay

J J J

J J J J J J J J J

J J J J J J J J J

Accuracy Precwon Linearity Sensitwlty Selectwty LOD

LOQ

Solution stablhty Robustness

J J

domperidoneR33812

I

I

a

0

Fig. 1. Separation

I

I

u

I

I

I

II

IS

4

5

I

I

I

I

17

Time (min)

of all major impurities

I

of dompertdone

in a batch (A) and

a sample solution spiked with 0.1 (B) and 1.O%(C) of each lmpurtty (reproducedfrom ref. 20). els required for quantitation. Figure 1 shows separation of all major impurities of domperidone in a batch and a sample solution spiked with 0.1 and 1.O% of each impurity.

Method Validation

10 00

85

12 00

14 a0

16 00

Mlgratlon

8.00

time (mm )

2000

2200

2400

Fig. 2. Separation of a pure (+) enantiomer of fluparoxan spiked with I % of the (-) enantlomer to confirm migration order (reproduced from ref lo).

2.2. Main Component Assay Selectivity for likely interferents should be demonstrated in addition to the likely sample-related impurities. For example, when determining drug content in a formulation, samples of placebo formulattons should be prepared and analyzed to confirm no interfering peaks. 2.3. Chiral Purity Determination During validation of a chiral determmation method, tt 1snecessary to demonstrate that no synthetic or degradatrve tmpurrties would interfere with the determination of either enantiomer at the required levels. The migration order of the enantiomers should be confirmed by spiking experiments with the pure enantiomer and the racemate if available. Figure 2 shows separation of a pure (+) enantiomer of fluparoxan spiked with 1% of the opposite (-) enantiomer to confirm migration order. 3. Detector Linearity It is necessary (3) to demonstrate detector lineartty over the intended operating range for the method. For example, if the intention of a specific method is to quote impurity levels at the 0.1% level, then tt may be appropriate to prepare standards at 0.1, 1, 10, 50, 100, and 150% of the

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target concentratton. These standards should be injected in duplicate, and the peak area obtatned plotted against percent of nominal concentration. Acceptable correlation coefficients (0.99 or greater) and an intercept close to the origin should be achieved. Many reports (7-9) have shown that this performance is possible with standard CE mstruments. 4. Rectilinearity

When determining impurity levels or trace enantiomer content, tt is also necessary to demonstrate detector rectilinearity. This involves maintaining the main component at a constant concentration and varying the content of the minor component to mimic real samples; where samples containing variable levels of impurities will be analyzed. Acceptable rectilmearities have been reported for lmpurrtres of an quinolone anttbiotic (1). Correlation coefficients of >0.99 have been reported (IO) for fluparoxan enantiomer levels of l-10% of the desired enantiomer content. A further example of this validation aspect has been provided (II) in the determination of levels of the undesired enantiomer of a drug in which the main component was held constant and the trace enantiomer spiked over the range 0. l-l 5% (correlatton coefficient 0.9995). Detector rectilinearity should be demonstrated by analyzing samples spiked with known levels of the appropriate impurities of interest. The main component concentration should be held constant, whereas the spiked impurity levels should be varied over the required range. For example, if the expected content of individual impurities is likely to range from O.l-l.O%, then a sample at the concentration specified in the method should be prepared and aliquots of this sample solution should be spiked with levels of the impurity of interest. For this example, it may be appropriate to spike with 0.1,0.25,0.5,0.75, and 1.O%. An unspiked sample should also be analyzed to quantify the residual level of the selected impurity present in the sample. The peak area of the impurity should be plotted against % w/w spiking. The correlation coefficient of the line should be in excess of 0.9, and the intercept value should be similar to the peak area obtained for the unspiked sample. 5. Response

Factors

The detection wavelength is typically a maximum for the main component. This may not necessarily be the case for the impurities whose chemical structures and UV spectra may differ greatly from the parent

Method Validation

__--

--.____--Impurity

I-

t x a

,

, z E

87

-c

--------.

F

‘8

--..

I I I I ’

, PD w

,

!

Impurity D ‘...--~

-._... .---..._ ---.______----

--..

Impurity I,,

, s 0,

I,

I s z

I

I

(I, s z

--.==.

L

G 1

( z z

Wavelength

Fig. 3. Spectra of ramtldine and three of its major impurities (reproduced from ref. 12).

molecule. If the UV absorbances of both the main component and impurity are different, then the quoted % area/area may not be a true indication of the level of impurity present. If the impurity has a significantly lower UV activity at the detection wavelength, then it will be underestimated. Conversely, if the impurity has a significantly higher UV activity, it will be overestimated. If the impurities have similar UV spectra, then the response factors are similar. Figure 3 shows the spectra of ranitidine and three of its major impurities (12) obtained from a diode array CE detector. To measure response factors, standards of the available impurities and the main component are prepared in duplicate, and analyzed in duplicate, employing the method conditions. The normalized areas are used to calculate response factors (RQ Rf = [normalized peak area / P x wt (mg)]

(1)

Altria where P = purrty of material, which accounts for all impurities, including moisture, solvents, inorganic residue, counterion content (if prepared as a salt), and related Impurities, i.e., 106total impurity content. Typically, rf the Rf of an impurity is 80-120% of that of the main component, it may be considered to be equivalent to the main component and no correction would be necessary. However, rf the response factor difference exceeds these hmits, rt may be necessaryto adjust the reported impurity content, i.e., if the impurity has an Rf of only 40% of the rmpurity, the reported value would be: (normahzed peak area lmpurlty / total normalized area) x (1 / 0.40) x 100 (2)

6. Limit of Detection CLOD) Thus value denotes the mnumum detectable level of impurities. The LOD is often defined (2) as the sample concentration that produces a peak with a height three times the level of the baseline noise. The LOD may alternatively be expressed as the smallest peak that can be detected as a % area/area of the electropherogram when determining purity, i.e., the LOD of 0.1% of one enantiomer in the presence of the main component may be possible. These LOD values will be dependent on the UV activity of the solute, the capillary bore, and the sample loading. Frgure 4 shows the LOD for a quinolone antibiotic analyzed using a low-pH CE method. 7. Limit of Quantitation (LOQ) The LOQ value refers to the lowest level of impurity that can be precisely and accurately measured. The LOQ may be calculated (IO) as ten times the signal-to-noise. Typically, replicate analysis of samples at the LOQ should give an RSD value of 10% or better. 8. Precision Repeated analysis (typically 10 sequential injections) of both a sample and a calibration solution should be conducted. Acceptable precision in terms of migration times and peak areas should be demonstrated to a predetermined level. Precision in CE is typtcally on the order of 0.5-2% RSD for main peak assay (7,&I.?) (with or without internal standard). Repeatable preparation of samples and standards should also be demonstrated. For example, 10 individual standards should be prepared and analyzed in duplicate. The precrsion for the response factors obtained

89

Method Validation

1

I

1

14.20

I

14.40 Retentlon

Fig. 4. LOD for a qumolone (reproduced from ref I).

I

14.60 14.80 tlme In minutes

antlblotx

I

15 00

I

15.20

analyzed using a low-pH CE method

Table 2 Reporting Format for Related Impurity Content Injection 1

Injection 2

Average

Total no. of lmpuritles Total % impurities % Principal impurity RMT of principal impurity % Second greatest impurity RMT of second greatest impurity

should be within acceptable levels (typically l-2% RSD). Similarly, 10 individual samples should be prepared and analyzed in duplicate, and the precision for the pooled assay values should be acceptable. Repeated analysis of the same sample solution should give consistent results (Table 2 shows the reporting format) in terms of number of impurities, levels and relative migration time for each impurity, and total level of impurities. For trace impurities, the precision would be expected to be ~10% RSD (I, 14). Migration time and relative migration times should be about 1% RSD and
90

Altria

method for determining the enantiomeric ratio of clenbuterol (23), peak area precision was l-2% RSD, peak area ratio precision was
Method Validation

91

lished on a case-by-case basis decided by the companies and laboratories concerned. Generally, this activtty will be concerned with the simultaneous testing of identical samples. Comparisons of results generated by both laboratories should not differ stgmficantly. This aspect has been shown in CE by a series of mtercompany crossvalidatton exercises (13,17), in which methods have been repeated by seven independent pharmaceutical companies. 10. Freedom from Interference Samples of the dissolvmg solvent and matrix-related components should be analyzed to confirm absence of interference. For example, if analyzing drug and drug-related impurities in a tablet formulation, a solutron of a placebo formulatron should be prepared and analyzed. 11. Recovery It is necessary to demonstrate full extraction of components from the sample matrix. This is achieved by spiking drug and related rmpurttres into the matrix of concern (18). For example, m the case of a pharmaceutical formulation, it would be appropriate to add known levels of drug and related impurities to a placebo mixture (2,19). Analysis of the spiked sample should confirm acceptable extraction. Demonstration of acceptable recovery is especially important in bioanalysis (20), where sample pretreatments can be extensive. 12. Crossvalidation It is useful to demonstrate the accuracy of the method by performing the same determination using an alternative method of analysis. Typical combinations may be CE and HPLC or CE and TLC. If similar results are obtained by two enttrely different separation techniques, then greater confidence can be placed m the validity of the results achieved by the method under examination. 12.1. Related Impurities CE and HPLC, when used together (21,22), are very complementary techniques for related impurity determinations, since common wavelengths can be selected and results quoted as % area/area.Comparisons with other separative techniques, such as TLC, become more complicated, since response factors need to be calculated and applied for each impu-

92

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rity. Results obtained by CE and HPLC for salbutamol impurities were statistically compared (22) and shown not to be statistrcally different. 12.2. Main Peak Assay Combinations of CE with a variety of analytical techniques have been reported for main component analysis. This crosscorrelation has been predominantly shown using CE and HPLC (7-9). Correlation studies between HPLC and CE results from the testing of a range of drug formulations produced correlatron coefficients of >0.999 between the data sets. 12.3. Chiral Analysis Currently, this testing is predominantly performed by HPLC. Therefore, it is most appropriate to crossvalidate wrth extstmg HPLC methods if possible. Several reports (23) have shown good agreement between the two techniques. 13. Robustness (Ruggedness) Robustness relates to the sensitivity of the method to small deliberate deviations from the method. For instance, if the method states an operating temperature of 3O”C, will acceptable performances be maintained at either 25 or 35”C? Two approaches to method robustness are possible: univariate or multivariate. The univariate approach involves systematically varying each parameter sequentially. Thus “one-by-one” approach has been performed for the determination of enalapril content in tablets by CE (24). The multivariate assessment involves simultaneous evaluation of several parameters using a predefined matrix. Typical experimental designs that may be employed include Placket-Burman (2.5)and Central Composites. See Section 2., Chapter 20 for further details of the applications of experimental designs in CE. CE has been employed to determine levels of drug-related impurities (26). Both fractional factorial and central composite designs were employed in the robustness testing of this method. Typically, each parameter may be varied by 5-10% above and below the value set in the method. Parameters examined may include temperature, pH, electrolyte concentration, rinse times, additive concentration, detector wavelength, and sample loading. The responses measured may include resolutions, peak efficiencies, relative and absolute migration times, peak areas, and migration order. For example, in the determination of impurities by an MECC method employing 20 Wborax, pH 8.5,

Method Validation containing 30 mA4 SDS at 25”C, preseparation rinse with electrolyte for 2 min, and detection at 240 run with a sample concentration of 0.5 mg/mL, it may be appropriate to explore the effects of the following ranges: Rinse time: 1.5,2.0,2.5 min Borax concentration: l&20, 22 mA4 pH: 8.3,8.5, 8.7 SDS concentration: 28,30, 32 mA4 Temperature: 23,25, 27OC Wavelength: 235,240,245 Sample concentration: 0.45,0.5,0.55 mg/mL

These values approximate to slight variations to the method. If one of these extremes is highlighted as having a significant effect on selectivity or on quantitative results, then tight limits for that parameter would be specified in the method. In order to minimize the number of analyses, experimental designs, such as fractional factorials and central composites, may be employed (26,27). The results from robustness testing should

be statistically examined to identify the tolerance limits that should be prescribed on each parameter in the method. 14. Peak Homogeneity Comigration of peaks is possible in CE as in any other separative technique. Therefore, it is useful to investigate the purity of separatedpeaks. Approaches to peak purity determination in CE are similar to those employed in HPLC. The principal methods are fraction collection (28) and spectral characterization. Common approaches would be used m related impurity determinations, main component assay, and enantiomerit separations. 14.1. Fraction Collection Micropreparative fraction collection is possible in CE (see Chapter 9). Fractions can be taken from the CE separation and analyzed using an alternative separative technique, such as HPLC (28,29). If a single peak is indicated by the secondary test method, this gives further confirmation of peak purity. 14.2. Spectral Analysis Many commercial CE instruments are now equipped with diode array facilities, which can be used in assessing peak purity. Standards of the main component and impurity can be analyzed, and their UV spectra

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recorded and compared (30). If good correlation between the spectra is not achieved, this can indicate comigration of peaks. This approach is llmlted in that closely related impurities will have similar UV spectra to each other and the main component. 15. System Suitability Testing The performance of the instrument 1s normally assessed prior to initiation of an analytical test sequence. As in HPLC (3), this testing commonly involves an assessment of parameters, such as selectivity, precision, and separation performance. For example, a test mixture contaming components that represent the critical resolutions may be repeatedly analyzed, for example, five times. In this instance, the system suitability may be specified as: 1. Resolution of C2.0 for all components 2. Precwon of inJectIon for mam component of <2% 3. Peak effhency of MOO0 for mam component. Other system suitability parameters less commonly employed may include linearity and LOD. Linearity may be assessed by employing three calibration solutions, for example, covering 7%125% of the target sample concentration. A measurement of LOD may be of significance when performing trace-level quantitatlon. A maximum permissible peak tailing factor may also be specified in the method. Provldmg that the system meets these mimmum requirements, the analysis may proceed. In some instances, test injections are also positioned at the end and/or throughout the sequence to demonstrate the continued suitability of the instrument and reagents through the analytical sequence. 16. Solution Stability The shelf-life of all reagent, calibration, and sample solutions should be determined. Until this is performed, all solutions should be made up fresh daily. Typically, the stability is established by performing fresh solutions to establish the purity of the sample and calibrations and to generate assay results for the samples. This testing represents d 1 of the stability determmatlon. The solutions should be stored under recorded conditions (i.e., protected from light at room temperature or as appropriate). The analysis is repeated after, for example, 3 d, using freshly prepared sample, calibration, and reagent solutions. Two analytical sequences

Method Validation

95 Table 3 Method Vahdatlon Checkhst

Test

Result

Selectivity Linearity (range -to - nominal cone ) Rectllmeanty (range - to - nommal) Response factors Limit of detection (- mg/L, % nominal cont.) Limit of quantitatlon (- mg/L, - % nominal cone ) Preclslon of migration time (- RSD, n = -) Peak area (- RDS, n = -) Reproduclblhty between analysts between capillaries between mstruments Freedom from Interference Recovery Crossvalidatlon Ruggedness/robustness Peak homogeneity

would be performed in which both stored and fresh calibrations and sample solutions are initially analyzed with fresh reagents and then reanalyzed in a second sequence using the stored reagents. The impurity content for stored and fresh samples should be determined, and the assay results should be calculated using response factors from both the stored and fresh calibrations.

All solutions would be deemed fresh if: 1. Assay results calculated usmg response factors from fresh and stored cahbrations were shown to be identical.

2. Assay results for stored and fresh samples were identical. 3. Selectlvlty should be identical for stored and fresh reagents. 4. Impurity levels m stored sample and cahbratlon solutions should not be significantly increased compared to fresh solutions.

If d 3 results are acceptable, then further testing at 7- and 14-d, and l- and 3-mo time-points may be appropriate. The shelf-life for calibrations/samples would generally be shorter than for reagents. The shelflife and storage conditions for all solutions should be documented in the method. Table 3 gives a checklist for the tests performed in validation protocols.

96

Altria References

1 Altrra, K. D and Chanter, Y L (1993) Vahdatton of a caprllary electrophorests method for the determmatton of a qumoline anttbtottc and Its related tmpurmes J Chromatogr 652,459-463. 2. Thomas, B. R,, Fang, X G., Chen, X , Tyrell, R. J., and Ghodbane, S (1994) Vahdated mtcellar electrokmettc captllary chromatography method for the qualny control of the drug substances hydrochlorothiazide and chlorothrazide J Chromatogr 657,383-394. 3 Clarke, G S (1994) The validatton of analyttcal methods for drug substances and drug products m UK pharmaceuttcal laboratortes J Pharm Boomed Anal 12, 643-652 4 Edwardson, P. A D., Bhaskar, G , and Fanbrother, J E (1990) Method vahdatton m pharmaceuttcal analysrs J Pharm Boomed Anal 8,929-933 5 Shafaatt, A and Clark, B J (I 993) Development of a caprllary zone electrophoreSISmethod for atenolol and Its related impurines m a tablet preparatton Anal Proc 30,48 I-483. 6 Korman, M , Vindevogel, J., and Sandra, P (1993) Separation of codeme and Its by-products by capdlary zone electrophorests as quahty control tool m the pharmaceuttcal mdustry J Chromatogr 645,3&k370 7 Altrta, K. D and Ftlbey, S. D. (1993) Quantttattve pharmaceutrcal analysrs by captllary electrophoresls J Llquld Chromatogr 16, 228 1-2292 8 Ackermans, M. T., Beckers, J. L , Everaerts, F M., and Seelen, I G J A (1992) Compartson of tsotachophorests, captllary zone electrophorests and htgh-performance lrquld chromatography for the determmatton of salbutamol, terbutalme sulphate and fenoterol hydrobromtde m pharmaceuttcal dosage forms. J Chromatogr 590,341-353 9 Tsat, E W , Smgh, M. M , Lu, H H , Ip, D. P., and Brooks, M A (1992) Apphcanon of captllary electrophorests to pharmaceuttcal analysts. Determinatton of alendronate m dosage forms J Chromatogr 626,245-250. 10 Altrra, K. D , Walsh, A R., and Smtth, N. W. (1993) Vahdation of a captllary electrophoresrs method for the enanttomerrc purity testmg of fluparoxan. J Chromatogr 645, 193-l 96. 11 Werner, A , Nassauer, T., Krechle, P., and Erni, F. (1994) Choral separation by capillary zone electrophorests of an optically-acttve drug and ammo acids by host-guest complexatton w&h cyclodextrms J Chromatogr 666,375-379 12. Altria, K. D. and Grace, G (1994) m preparation 13 Altrta, K D , Harden, R C., Hart, M., Hevtzt, J , Harley, P A., Makwana, J V , and Portsmouth, M J (1993) An Inter-company cross-vahdatron exercise on caprllary electrophoresis 1. Choral analysis of clenbuterol. J Chromatogr 641, 147-l 53 14 Swartz, M E. (1991) Method development and selectivrty control for small molecule pharmaceutical separattons by captllary electrophorests J LIqutd Chromatogr 14,923-938. 15 Flurer, C. L and Wolmk, K. A (1993) Quantttatton of gentamrcm sulfate m mJectable soluttons by capillary electrophorests. J Chromatogr. 663, 259-263

Method Validation 16 Madrup, G. (1992) Rugged method for the determination of deaminatron products in msulm solutions by free zone captllary electrophorests usmg an untreated fusedsilica caprllary. J Chromatogr 604,267-28 1. 17. Altrra, K. D., Clayton, N. G., Harden, R. C., Hart, M., Hevtzi, J., Makwana, J. V., and Portsmouth, M. J (1994) An inter-company cross-validation exercise on capillary electrophorests testing of dose uniformity ofparacetamol content m formulations. Chromatographiu 39, 180-l 84. 18. Sun, P., Mariano, G J., Barker, G., and Hartwtck, R A (1994) Compartson of micellar electrokinettc capillary chromatography and high-performance liquid chromatography on the separation and determination of caffeine and tts analogues in pharmaceutical tablets Anal Lett 27,927-937 19. Thomas, B. R. and Ghodbane, S. (1993) Evaluation of a mixed mtcellar electroklnetic capillary electrophorests method for validated pharmaceutical quality control. J Liquid Chromatogr 16, 1983-2006 20. Prunonosa, J., Obach, R , Dtez-Cascon, A., and Gouesclou, L (1992) Compartson of high-performance hqutd chromatography and high-performance capillary electrophoresis for the determmatton of cicletanme in plasma J Chromatogr 581, 219-226 21 Pluym, A., Van Ael, W , and De Smet, M (1992) Capillary electrophoresrs m chemtcal/pharmaceutrcal quality control TRAC 11,27-32 22. Altrta, K. D. (1993) Quantitative analysts of salbutamol related lmpurtttes by capillary electrophorests. J Chromatogr 634, 323-328 23. Rogan, M. M., Altrta, K D , and Goodall, D. M (1994) Enanttoselectrve separations using capillary electrophoresis. Chwality 6,2-O. 24. Thomas, B. R. and Ghodbane, S (1993) Evaluation of a mixed mtcellar electrokrnetrc captllary electrophorests method for validated pharmaceuttcal quality control. J. Lzqurd Chromatogr 16, 1983-2006 25. Vindevogel, J. and Sandra, P (1991) Resolutton optlmtsatton m mlcellar electrokinetic chromatography. use of Plackett-Burman statistical design for the analysis of testerone esters Anal Chem 63, 1.530-l 536. 26 Altria, K D and Filbey, S. D. (1994) The apphcatton of experimental design to the robustness testing of a method for the deterrnmatton of drug related impurities by capillary electrophoresis. Chromatogruphaa 39, 306-3 10 27. Filbey, S. D and Altria, K D (1994) Robustness testing of a capillary electrophorests method for the determmation of potassium content m the potassium salt of an acidic drug. J Cap Electrophoresis 1, 190-195. 28. Camillerr, P., Okafo, G. N , Southan, C., and Brown, R (1991) Analytical and mrcropreparattve capillary electrophoresis of the peptides from calcitomn Anal Bzochem. 198,36-42. 29. Altria, K D. and Dave, K (1993) Peak homogeneity determination and mtcropreparative fraction collection by capillary electrophoresis m pharmaceuttcal analysis. J Chromatogr 633,22 l-225. 30. Beck, W., Van Hoeck, R., and Engelhardt, H. (1993) Application of a diode-array detector m capillary electrophorests Electrophoresls 14, 540-546

CI-IAPTER9

Fraction Kevin

Collection D. Altria

1. Introduction Fraction collection by CE is a potentially important area in which there are several approaches necessary (I-8) to yield sufficient material following fraction collection. Even using optimized conditions, the amounts collected employing preparative CE are extremely small and are on the order of nanogram quantities. However, these levels can be useful to perform operations such as: (1) peak purity verification by reinjection on CE or onto another analytical method, such as HPLC, and (2) sample identity confirmation by characterization of the material by another technique, such as amino acid sequencing or off-line mass spectrometry. The key advantage to preparative CE compared to, for example, preparative HPLC, is that the fraction collection can be readily performed from tiny sample volumes, since typical CE injection volumes are only l-50 nL/injection. The technicalities of performing preparative CE are demanding and require use of a programmable autosampler. In principle, however, the operation of preparative CE is identical to normal CE, and standard equipment is employed. Sample solutions are injected, separated, and detected in a conventional fashion. The separated components are then collected as they emerge from the detector end of the capillary. To allow this collection, it is necessary to stop the separation voltage and position the autosampler tray such that the capillary then dips into a collection vial, containing a few microliters of water or dilute buffer. The voltage is then restarted, and the required sample components migrate out of the capillary into the collection vial. When all the sample zone has emerged from the capillary, the voltage is stopped. From

Methods m Molecular Biology, Vol 52 Caprllary Necfrophoresrs Edited by K Altna CopyrIght Humana Press Inc , Totowa, NJ

99

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100

The times required for calculating the exact time for the swttching between vials can be calculated from the total capillary length, the distance to the detector, and the migration time of the peak of interest (I). Therefore, the switching time to collect a peak detected at 7.8 min on a 57-cm long capillary (50 cm to the detector) is: x 7.8 = 8.9 mm

(1) The exact time of the switch should, however, be programmed to be at least 0.1 min before the expected time to ensure the entire peak is collected. The length of peak collection should also be suffictent to cover collection of the required peak only. Typically, if the required peak is well resolved from interfering peaks, then the collection step may be for 0.5 min. Using this example of a peak that was detected at 7.8 min, the following method may be appropriate: 57150

Step 1: Rinse with generatlonsolution (0.IM NaOH or slmllar). Step 2: Rmsewith electrolyte solution. Step 3: Set temperature. Step 4: Inject sample. Step 5: Apply voltage for 8.8 mm (capillary dipping into electrolyte vial at detectorend). Step 6. Apply voltage for 0.5 mm (capillary dtpping into fraction collection vral at detectorend). There are three main approaches to optimizing the amount collected by preparative CE. These are to use concentrated sample solutions, perform several sequential runs, or employ relatively large-bore capillaries. 2. Use of Concentrated Sample Solutions The limitation 1s that resolution may be lost with increased sample loading, For example, 25 mg/mL fluparoxan solutions (I) and 5 mg/mL calcitonin solutions (2) have been used in micropreparative work. 3. Use of Wide-Bore Capillaries Increasing the bore dramatically increases the volume of sample injected into the capillary. However, the required resolution may be lost at these higher injection volumes, and considerations must be given to joule heating effects. A 100 1-1x 57 cm polyacrylamide-coated capillary has been used (3) to collect peptlde components from a four-component

Fraction Collection

Fig. 1.The preparativeseparationof samplecontaining four peptldes(reproducedfrom ref. 3). text mix of peptides. The fractions were collected into a IO-PL microvial of separation buffer. The separation was operated under stacking conditions (see Chapter 16), which allowed a 20-s injection on this relatively large-bore capillary, which is equivalent to a 500-nL injection volume. Figure 1 shows the preparative separation of the sample. The collected fractions were reexamined by CE to confirm purity levels. Fifty nanograms of the individual peptides were collected (3). This was sufficient material to conduct tirther analyses,such as amino acid sequencing. 4. Repeated Injections Repeated injections in an automated sequence would also increase the total amount collected. This facility IS possible with well-controlled methods where very consistent migration times are obtained. Therefore, particular attention should be paid to the use of appropriate rinse cycles and to operation using a constant temperature. It may be advisable to perform two test Injections prior to starting a preparative sequence to

10.2

Altria

allow the system to equilibrate and to reach a constant temperature (see Chapter 3, Section 10. on good working practices). For example, a fraction sample of a fluparoxan impurity was obtained following 27 injections of a drug substance solution. Injection sequences of up to 30 injections were shown (4) to give collectron yields of 70-100% for injected amounts of various peptides and proteins. The expected amount collected can be calculated for a given number of injections using a particular set of conditions (I). For example, 27 replicate injections of a 25 mg/mL solution containing a 2.5% impurity, using injection settings that give a 11.8-nL injection volume with collection of the fraction mto 20 l..tLof water, yielded a lo-ppm (mg/L) solutton. Each injection is equivalent to a loading of 295 ng. However, the impurity is only present at 2.5%. Therefore, each injection gives a loading of 7.4 ng of the impurity. Twenty-seven replicate injections give a total amount of impurity collected as 198 ng into 20 ltL of water. This gives a final solution concentration of 10 pg/mL (ppm), which is suitable for further mvestigations. 5. Notes 1. The configuration on the ABI mstrument requnes that the detector end reservoir is fixed and cannot be used for fraction collection purposes. Therefore, a scheme has been devised (4) m which sample solution IS vacuum-injected and a rinse cycle is used to suck the sample slug to the detector end of the capillary. A negative voltage is then apphed, causmg the sample components to mtgrate back along the capillary, pass through the detector, and emerge from the capillary at the sample mtroduction end of the capillary. Again the voltage IS stopped immediately prior to

emergence of the sample zone from the capillary, and the capillary is dipped into a collection vial and the voltage restarted. A user bulletin (user bulletm number 4) is available from ABI that gives precise details of this operation. 2. Bundles of capillaries m an array format (5) have been shown to be of use m particular applications. Beckman Instruments are expected soon to launch a clmical CE mstrument (Tradename, Paragon) in which several capillaries are simultaneously employed, This instrument would have obvious application to preparative operations.

References 1 Altrra, IL D. and Dave, Y. K. (1993) Peakhomogeneitydetermination and mrcropreparativefraction collection by capillary electrophoresisin pharmaceuticalanalySW J. Chromatogr

633,221-225.

Fraction

Collection

103

2. Camilleri, P , Okafo, G. N , Southan, C , and Brown, R (1991) Analytical and micropreparative capillary electrophoresrs of the peptides from calcitomn Anal Blochem 198,36-42 3. Schwer, C and Lottspeich, F. (1992) Analytical and mrcropreparatrve separatron of peptrdes by capillary zone electrophoresis using drscontinuous buffer systems J Chromatogr. 623,345-355. 4. Albin, M., Chen, S -M., Loure, A, Pairaud, C., Colburn, J., and Wrktorowrcz, J. (1992) The use of caprllary electrophoresis m a micro-preparattve mode methods and applicatrons. Anal Blochem 206,382-388. 5. Huang, X. C , Quesada, M. A , and Mathres, R A (1992) DNA sequencing usmg capillary array electrophoresis. Anal Chem 64,2 149-2 154 6. Herold, M. and Wu, S -L (1994) Automated peptide fraction collection m CE, LCGC Int 7, 554-558. 7. Lee, H. G. and Desiderro, D. M. (1994) Preparatrve caprllary zone electrophorests of synthetic peptides. Conversion of an autosampler mto a fractron collector J Chromatogr 686,309-3 17. 8 Lee, H. Ci. and Desideno, D. M (1994) Optrmrsation of the loading hmrt for capillary zone electrophoresrs of synthetic oprod and tachykinm peptrdes. a study of the interactions among the amount of peptide, resolutron, saturation, mJectton volume and the capillary diameter. J Chromatogr 662,35-45.

CHAPTER 10

Troubleshooting Kevin

D. Altria

1. Introduction This chapter covers the possible solutions to problems that may be encountered in routine operation. Further details may be available in specific instrument manuals or by contacting appropriate service specialists. The most likely problem areas are: 1. Voltage/currentproblems-Chart 1 2. No peaksobtained-Chart 2. 3 Poor peak shape/height-Chart 3. 4. Poor peak time precision-Chart 4. 5 Poor peakareaprecision--Chart 5. 6. Poor sensitivity--Chart 6. 2. Chart l-Voltage/Current Problem 2.1. Blocked

or Broken

Capillary

Capillaries can block if stored filled with electrolyte, which will crystallize out with time. To test if a blockage has occurred, an empty vial should be placed on the autosampler. A rinse cycle is then performed from this empty vial, which should result in a stream of air bubbles in the “detector end” of the electrolyte reservoir. If no bubbles appear after 1-2 min, then the capillary is blocked. A series of rinse steps may clear the blockage. If this is unsuccessful, then a new capillary should be installed. If the capillary is broken, no current will be generated when a voltage is applied across the capillary when filled with an electrolyte. Visual inspection of the capillary will confirm this problem. A new capillary should be installed. From

Methods I# Molecular Bology, Vol 52 Capllary Electrophoresti Edlted by K Altrta CopyrIght Humana Press Inc , Totowa, NJ

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106

Chart 1. Voltage/current problem.

2.2. Correct VoltagelCurrent The level and polarity of the voltage specified in the method should be checked.Application of too high a voltage will causea rapid increasein the temperaturewithin the capillary, possibly boiling the electrolyte or causing out-gassing of air bubbles. Either of thesetwo occurrenceswill result in breakdown of the electrical circuit. Application of the incorrect voltage polarity will result in sampleions migrating in the oppositedirection along the capillary to that required. 2.3. SamplelElectrolyte Vial Levels If the vials are insufficiently filled, a rinse or injection cycle will cause air to enter the capillary, resulting in an electrical breakdown. This problem should be consideredif a long injection sequenceis performed with rinsing from a single electrolyte reservoir.

Troubleshooting

107

pil +,

m sample?

~~1

Chart 1. continued.

2.4. Voltage Leak If all areasaround the high-voltage connectionsare not kept clean and free from electrolyte, the voltage may escapeto earth rather than pass acrossthe capillary. Principal problem areasinclude electrolyte drops on buffer vial caps.Theseshould be checkedprior to an analysis. Operation at subambienttemperaturecan lead to condensationproblems. Urea is occasionally used as an electrolyte additive, which is known to “creep” acrosssurfaces,leading to voltage leakages.Regular careful cleaning of electrode areas with cotton wool dampenedwith water will minimize voltage leakageproblems. Specific maintenanceadvice will be included in instrument manuals. 2.5. Injection Time Typically, samplesare preparedin water or may contain someorganic solvent. Therefore, the sample zone is relatively nonconductive and, if

108

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the zone length is too long, will causea breakdown tn the conductivity along the capillary This effect is reduced by shortening the sample injection time. 2.6. Buffer If the buffer is too concentrated, then excess current will be drawn, which will result m breakdown in the circuit owing to electrolyte boiling or out-gassing. The electrolyte should be diluted accordingly or a lower voltage applied. 2.7. Correct Vials The positioning and contents of the vials on the autosampler should be verified and replaced if incorrect. 2.8. SampZe Solvent If the sample 1s dissolved in an inappropriate solvent (such as pure organic solvent or of high ionic strength), this may cause a breakdown owing to localized heating in the sample region. This effect can be reduced by reducing the sampling time or by dilution of the sample with water. 2.3. Air Bubble in Sample If only small sample volumes are available, microvials are used to hold the samples. When using these microvials, it is possible to trap air in the bottom of the vial. This will result in injection of air mto the capillary, resulting in electrical breakdown. The sample microvials should be refilled with sample solution to remove the trapped air. 3. Chart ~-NO Peaks Obtained Blocked or broken capillary--see Chart 1. Correct voltage/current set--see Chart 1. Sample vial level--see Chart 1. Voltage leak--see Chart 1. 3.1. Analysis Time Too Short The analysis time should be extended (or voltage increased) and the sample reanalyzed. 3.2. Sample SoZu bili ty If the sample is insoluble in the electrolyte, it may drop out of solution within the capillary. If this is suspected, an alternative electrolyte should be selected in which the sample is sufficiently soluble.

Troubleshooting

109

Chart 2. No peaks obtained.

110

Altria

1311 Chart 2. continued. 3.3. Correct

Buffer

and Vials

The positioning and contentsof the vials on the autosamplershould be verified and replaced if incorrect. 3.4, Air Bubble

in Sample-See

3.5. Capillary

Chart

1.

Not AZigned

If the capillary detection window area is not correctly aligned within the detector, no peaks will be observed, since the UV light will be adsorbedby the polyimide coating on the capillary. This can be checked following guidance from specific instrument manuals and the capillary realigned if necessary. 3.6. Lamp

Burned

Out

This can be checked following guidance from specific instrument manualsand the lamp replaced if necessary.

111

Troubleshooting

0

Change and reanalyze

Chart 3 Poor peak shape/height. 3.7. Data

Collection

The connections to the data system should be checked. The correct operation of the data system should be checked, and the system replaced if appropriate. 4. Chart 3-Poor Peak Shape/Height 4.1. Correct

EZectroZyte

Used

If the electrolyte concentration is too high, excessive current will be drawn, possibly leading to temperature-related peak distortion. Conversely, if the electrolyte concentration is too low, the conductivity of the sample will lead to peak distortion or tailing. The electrolyte concentration should be optimized during method development. 4.2. Correct

VoltagelCurrent

Excessive voltages will result m temperature-related peak distortion. However, too low a voltage will result in extended migration times and diffusion-related peak broadening.

Altria

Chart3. continued. 4.3. Injection

Time

Excessivesamplevolumes will causemixing of the samplewithin the capillary, resulting in loss of resolution and separation efficiency. The injection time should be optimized during method development. 4.4. Sample

Matrix

Extreme peak distortion can occur if the sample is present in a highionic-strength solution or high organic solvent levels (I). Reduction in sampleinjection time or dilution of the samplewill reducepeak distortion. 4.5. Temperature

Too high a temperaturecan causepeak splitting and distortion, and lower temperatures increase analysis time, therefore the temperature should be optimized during method development.

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113

Chart 4. Poor peak time precision. 4,6. Sample

Adsorption

Peak tailing owing to sample adsorption may be reduced by several approaches,such as use of pH extremes,high-ionic-strength buffers, or coatedcapillaries.Appropriateuseof rinsecyclesbetweeninjectionscanbe usedto removeunquantifiedmaterial from the capillary, which may cause adsorptionproblems.An increasein electrolytestrengthor decreasein pH can reducesample/surfaceinteractions,which may be causingpeak tailing. 4.7. Sample

Concentration

Sampleoverloading will dramatically reducepeak efficiency and resolution. The sampleshould be diluted or a shorter injection time selected. 5. Chart 4-Poor Peak Time Precision Chapter 6 discussesfactors affecting precision in greater detail.

114

Altria

5.1. Temperature Electrophoretlc mobihty 1sdirectly related to temperature, and therefore, fluctuations in temperature during a sequence will cause variations in mlgration times. The operating temperature should be specified in the method. 5.2. Capillary Surface Variations m the capillary surface will lead to changes m the EOF, resultmg in mlgratlon time vanablhty. Appropriate capillary conditionmg and use of rinse cycles of base, acid, water, or electrolyte should ensure a consistent EOF during a sequence. These cycles should be establashed durmg method development. 5.3. Electrolyte Depletion Extensive apphcatlon of a voltage across an electrolyte solution can cause electrolysis of the buffer to occur (2,.?). This may result in drifts m mlgratlon time and/or changes in selectivity. Therefore, it may be approprlate to program use of fresh vials of electrolyte during an extended series of injectlons (4) 5.4. Standard/Sample Composition The ionic strength and organic solvent content of the sample may alter EOF, leading to changes in migration time. Therefore, it is necessary to match the composition of samples and standards.

6. Chart

B-Poor

Peak Area Precision

Chapter 6 discusses factors affecting precision in greater detail. 6.1. Temperature Sample viscosity decreases with increased temperature. The sample volume introduced into the capillary is also related to viscosity. Therefore, an increase m temperature will result in a higher sample loading onto the capillary, giving a higher peak area. A constant temperature should be specified in the method. 6.2. Migration Time In CE, the area of a peak is proportional to both the solute concentration and its migration time (5) Therefore, variations in migration time will produce apparent variations m peak area. The factors controlling migration time precision are covered in Chart 4.

115

Troubleshooting

Chart 5. Poor peak area precision.

6.3. Peak Size Peak areaprecision is improved with peak size. Use of higher sample concentrations or longer injection times produces larger peaks. Errors relating to peak integration and solute adsorption are reduced with increasedsample loading. 6.4. Evaporation Evaporation during a sequencewill lead to a gradual increasein areas during a sequence.Samplevials should be sealedif possible or an internal standardemployed. 7. Chart 6-Poor Sensitivity Chapter 7 discussesfactors affecting sensitivity in greater detail.

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Chart 6. Poor sensitivity. 7.1. Sample Load Sample injection time should be increased as much as possible while still retaining required resolutions. 7.2. Capillary Alignment If the detection window area of the capillary is not correctly aligned, a loss in sensitivity will result. The alignment should be checked according to the instrument manual. 7.3. Capillary Bore An increase in capillary bore will improve sensitivity, but may have implications relating to resolution and selectivity (see Section 2., Chapter 7). 7.4. Noise Spikes Particulates in the sample or electrolyte may migrate along the capillary and pass through the detector. Theseparticulates will appear as needle-sharp

Troubleshooting

117

Chart 6. continued peaks. The electrolyte should be filtered through an appropriate filter. A sample filtration step would require further additional validation. 7.5. Lamp Poor lamp intensity or intermittent lamp output will be observed as extreme baseline noise. Instrument-specific lamp performance diagnostic tests should be performed and the lamp replaced if necessary. 7.6. Voltage Increased voltage causes higher capillary vibration, and both shortand long-term baseline noise. A reduction in voltage, although extending analysis time, will reduce this source of noise. 7.7. Temperature Capillary vibration noise is also related to temperature, being more pronounced at higher temperatures. A reduction in temperature will reduce this effect, but may unduly alter selectivity and resolution and extend analysis time.

Altria

118 References

1 Ackermans, M T , Everaerts, F M , and Beckers, J L. (1991) Determmatton of some drugs by mrcellar electrokmetrc captllary chromatography The pseudoeffecttve mob&y as parameter for screening. .I Chromatogr. 585, 123-13 1 2 Shafaatr, A and Clark, B J (1993) Development of a caprllary zone electrophoreSISmethod for atenolol and rts related rmpurmes m a tablet preparatron. Anal Proc 30,48 1-483 3 Zhu, T , Sun, Y -L., Zhang, C -X , Lmg, D -K , and Sun, Z -P (1994) Varratron of pH of the background electrolyte as a result of electrolysis in CE JHRCC 17,563,564. 4 Thomas, B R., Fang, X G , Chen, X, Tyrell, R J , and Ghodbane, S (1994) Valrdated micellar electrokmetrc captllary chromatography method for the quahty control of the drug substances hydrochlorothtaztde and chlorothrazrde. J Chromatogr 657,383-394 5 Altrta, K D. (1993) Essentral peak area normahsatron m caprllary electrophorests Chromatographta 35, 177-182

CHAPTER11

to Running

Quick Guide a Successful Separation Kevin

D. Altria

1. Introduction This chapter is intended to act as a brief guide to the operations required to perform a series of CE analyses and subsequent care of the capillary. 2. Preanalysis Procedures 1. Specify all parameters to Include sample concentration and dissolving solvent, electrolyte type and concentration, capillary length and bore, injection volume, rinse settings, wavelength, applied voltage, and analysis time. All these wrll have been optimized during method development (Chapter 4). 2. Check all connections and settings. The majority of mstruments perform rinses and sample injection by means of an external gas supply. In addition, most instruments involve use of a cartridge-type device to house the capillary. The appropriate capillary, or cartridge, designated for use for the specific separation should be inserted mto the Instrument. 3. Self-diagnostic tests are performed when the power IS turned on. Accuracy of wavelength, gas pressure, temperature, and voltage are generally tested. The lamp should be independently switched on if necessary. It is recommended to allow 15 mm for the mstrument and lamp to warm up. If the instrument employs a filter-based detector, the appropriate filter should be selected to allow it to warm up. Failure to preselect the appropriate filter wtll result in a driftmg baseline for the first 15 mm of operation 4. All of the operatmg parameters (as listed m step 1) should be programmed into a method file on the controlling personal computer. From

Methods m Molecular Biology, Vol 52 Caplllary Electrophoresds Edited

by K Altrra

CopyrIght

Humana

119

Press Inc , Totowa,

NJ

120

Altria

5. The electrolyte and/or regeneration solution should be filtered through a 0.45+m filter to remove any parttculates Failure to do this can result in notse spikes as the parttculates migrate through the detector. 6 Fill a sufficient number of autosampler vials with regeneration solutton and electrolyte. Fill the vials equally to the prescribed level m the specific mstrument manual. If the vials are unevenly filled, a siphoning flow can occur, which causesmtgration ttme variability If a large number of analysesare performed, levels m the vials being rinsed from would be constderably lowered. Therefore, if a long sequence 1s to be performed, rmses should be conducted from different vials to those used during separation. If the vials are not filled enough, the electrode and capillary will not reach the solutton, resulting m a run failure. Ensure that the caps of the vials are clean and free from electrolyte. Contamination can cause electrical arcing and would result m a run failure. Repeated analyses from a single electrolyte reservoir can alter the nature of the electrolyte owing to electrolysts. This occurrence ts termed “buffer depletion,” and may cause changes m electrolyte concentratton and pH Buffer depletion may result m changes in selectivity and drifting mtgration times. To reduce or prevent this depletion, frequent changes m electrolyte vials should be programmed mto the sequence. Place a sufficient number of vials onto the autosampler m the posittons designated m the method and/or sequence. Ensure sufficient levels of solution are in rinse vials, since repeated rmses lower the level and eventually the level may be reduced such that the capillary cannot reach the liquid. A rinse would then fill the capillary with air and would result in a run failure. 7. Fill the sample vials with sufficient sample solution to allow the captllaty to reach the ltqutd. Filter the sample solutton as necessary (this would require addttional valtdatton). If sample volumes are limrted, plastic mtcrovials may be employed with mnumum sample volumes of -10 pL. If these mtcrovtals, or similar, are employed, ensure that no au bubbles are present. These bubbles are often caused by trapped an at the bottom of the vial. If au bubbles are present, the vials should be refilled or the vial should be gently tapped until the au-bubble 1sshifted. Place the sample vials m the autosampler positions designated m the method/sequence. 8. Condttton the capillary as necessary, The first ever use of a capillary wtll involve a 20-mm rinse with 0.M NaOH to regenerate surface stlanols Coated captllaries may have specific regenerating requirements and reference should be made to the mstructions supplied with the capillary Followmg the conditioning, rinse and fill the capillary wtth the required electrolyte.

Successful Separation

121

9. Close the autosampler door/cover to activate the safety interlocks The mterlocks ensure that it is not possible to accessthe capillary/electrodes while the voltage is being applied. 10. An appropriate test mixture should be analyzed to ensure that the system is suitable for operation. This injection also allows the system to settle. 11. The integrator should be assigned with the correct analysis time and number of data files corresponding to the number of injections m the sequence.The detector output should be reversed if indirect detection is to be employed.

3. Postanalysis

Procedures

1. The capillary should be rinsed for 2-5 min with water to remove electrolyte. A vial of water should be placed on the instrument to allow this operation. 2. The capillary should be flushed with an prior to storage. This is to prevent any residual electrolyte crystallizing out Inside the capillary to form a blockage. An empty capped vial is placed on the autosampler and a 2-5 min rinse performed from the empty vial. 3. Carefully remove the capillary for storage. The capillary, or cartridge, should be labeled with an identifier indicating the history and dimensions of the capillary. If the capillary 1shoused in a cartridge, it should be stored in the box provided. Loose capillary can be coiled and stored m plastic boxes available from the instrument suppliers. The fragile capillary wmdow section may be protected using a plastic tubing sheath. A 2-in. section of narrow-bore plastic tubing, as used in HPLC, could be cut and the capillary threaded through until the capillary window section is covered. 4. If the instrument is to remain unused for an extended period, it may be appropriate to turn the mstrument off. However, if further imminent use is intended, the lamp alone may be switched off to preserve lamp life. 3.1. Capillary It is recommended that:

Care

The capillary be rinsed for 20 min with 0. IM NaOH on its initial use. The capillary be flushed with 0.M NaOH (or acid) and equilibrated with the running buffer prior to each injection as appropriate. The capillary be rinsed with water and air blown through it at the end of each day. This prevents buffer precipitation and subsequent capillary blockage. Individual capillaries be dedicated to specific methods to avoid “memory effects” that may lead to nonreproducible separations. An initial injection of a blank, or sample solutton, be performed prior to commencing an analytical sequence to allow the capillary wall to equilibrate to the run buffer.

122

Altria Table 1 Preanalvsls ChecklIst Actlon

Comphes (J)

Date Analyst System Correct capillary size/length Correct analysis time on CE instrument Correct analysis time on Integrator Correct voltage/current selected Correct mjection parameters Buffer and rinse vials sufficiently full Correct number of sample vials All sample vials full with no air bubbles Vials correctly posltloned on autosampler Safety door closed Blank solution included in sequence Correct wavelength selected Correct temperature specified Integrator parameters correct Test mjectlon satisfactory

Table 1 provides a preanalysis checklist that could be completed in order to ensure that all the correct procedures have been performed prior to starting an injection or series of injections.

CHAPTER

Micellar

Electrokinetic Koji

Otsuka

and

12 Chromatography

Shigeru

Terabe

1. Introduction Recently, capillary electrophoresis (CE) or high-performance capillary electrophoresis (HPCE) has become popular as a high-resolution separation method. The technique was first Introduced by Mikkers et al. (I), Jorgenson and Lukacs (2), and Hjerten (3), as an instrumental version of electrophoresis. Although CE can generally give a higher resolution within a shorter time compared with conventional high-performance liquid chromatography (HPLC), only ionic or charged solutes can be separated by CE, in principle. This was a serious limitation of CE, but the development of electrokinetic chromatography (EKC) (4) has been able to solve such problems. EKC is based on chromatographic principles using homogeneous solutions containing an ionic “carrier” and the same apparatus as CE. The unique characteristic of EKC is that both neutral and charged analytes can be separated electrophoretically. Among various modes of EKC, micellar EKC (MEKC) (5-7), which uses micellar solutions of ionic surfactants, has become the most popular technique for the separation of small neutral molecules. Many papers on fundamental characteristics and applications of MEKC have been published (8), and some reviews of MEKC are available (9-12). In this chapter, the separation principle and chromatographic consideration of MEKC will be described tirst as basic properties of MEKC. Then, strategies for selectivity manipulation will be discussed briefly, followed by the description of some applications. From

Methods m Molecular Biology, Vol 52 Cap/llary Electrophoresm Edlted by K Altria CopyrIght Humana Press Inc , Totowa, NJ

125

Otsuka and Terabe

126 --_-_------___--___---

-m----------m-

O-J = Surfactant (negatrve charge) I= I = Solute

w

Electroosmotlc = Electrophoresls

Flow

Fig. 1 Schematic lllustratlon of the separation prmclple of MEKC (ZI) Reprinted with permIssIon from Terabe (1992), Mxellar Electrokmetlc Chromatography; Beckman, Cahfomla.

2. Principle

of Separation

The separation principle of MEKC is schematically shown in Fig. 1 (I 1). A capillary or fused silica 1sfilled with an ionic surfactant solution, in which the concentration of the surfactant 1s higher than its crltical mlcelle concentration (CMC), so that mlcelles are formed. When an anionic surfactant, such as sodium dodecyl sulfate (SDS), is employed, the micelle is forced toward the positive electrode by electrophoresls. The electroosmotic flow migrates toward the negative electrode owing to the negative charge of the capillary surface. The electro-osmotic flow 1s larger than the electrophoretic migration of the micelle under neutral or basic conditions, and therefore, the anionic SDS micelle also migrates toward the negative electrode at a retarded velocity. When a neutral analyte 1s injected into the micellar solution, it will be distributed between the micelle and bulk solution. The analyte will migrate at the same velocity of the micelle when it is incorporated mto the micelle, and at the electro-osmotic velocity, since it is free from the micelle and exists m the bulk solution. Thus, the migration velocity of the analyte depends on the distribution coefficient of the mlcellar solubllizatlon. As long as the analyte is neutral, It must migrate at a velocity between the two extremes, i.e., the electro-osmotic velocity, v,,, and the velocity of the micelle, v,,, as shown m Fig. 2A (6). In other words, the migration time of the analyte, tR, 1slimited between the migration time of the bulk solution, to, and of the micelle, tmc(Fig. 2B) (6).

MEKC

127

A

Micelle E;I

L inj.

Water

Solute lpil column

Water w det.

Solute

Micelle

I

1 tlllC

Bi I to

I 0

f Fl

N Time

Fig. 2. Schematicof the zone separationin MEKC (A) and chromatogram(B) (6). Reprintedwith permissionfrom Terabeet al. (1985)Anal. Chem.57,834. When an acidic solution or pH below 5.0 is employed, the electroosmotic flow becomes smaller than the electrophoretic velocity of the SDS micelle, and then the micelle migrates toward the positive electrode (13). When a catiohic surfactant, e.g., dodecyltrimethylammonium bromide, is employed instead of SDS, the direction of the electro-osmotic flow will be reversed or toward the positive electrode through the adsorption of the surfactant molecule to the inside wall of the capillary (14). 3. Chromatographic

3.1. Chromatographic

Properties

Parameters

3.1.1. Capacity Factor Capacity factor, K, can be defined in the case of conventional chromatography (6): k’ = (%lc / %J (1) where y1,, and naq are the amount of the analyte incorporated into the micelle and in the aqueous solution, respectively. Then we can obtain the relationship between the capacity factor and the migration time as: k’ = (tR- to) / [to (1 - ttJtmc)] It can be rewritten as:

tR= [(l + k’) / 1 + (t,,/t,,)k’] to

(2)

128

Otsuka and Terabe 2

0004AU

6 AL I 0

I

i. 1

12 Capacity

I

I

/‘I

5 10 2050~ Factor

1'0 Time (mln)

Fig. 3. An example of MEKC separation of the test solutes (6) (1) methanol, (2) resorcmol, (3) phenol, (4) p-mtroamlme, (5) nltrobenzene, (6) toluene, (7) 2-naphthol, (8) Sudan III. Mlcellar solution, 50 mM SDS m 100 mM borate-50 mM phosphate buffer, pH 7 0, capillary, 50 pm id x 650 mm (effective length, 500 mm); applied voltage, 15 kV; current, 33 PA; detectlon wavelength, 2 10 nm; temperature, 35°C. Reprinted with permission from Terabe et al (1985) Anal Chem 57,834. Here, the reciprocal of to/t,, or t,,ltO is a parameter representing the migration time window. If the analyte is not incorporated into the micelle or does not interact with the micelle at all, the migration time of such a solute 1s equal to t0 and hence k’ = 0. On the other hand, when the analyte 1s totally incorporated into the micelle, the migration time becomes tR, and k’ becomes infinity. Thus, the migration time window is limited between to and t,,. A typical example of MEKC separation of neutral compounds 1s shown in Fig. 3 (6). In this figure, the scale of the capacity factor is inserted to show the relationship between the migration time and capacity factor.

MEKC

When tmcis infinity or the micelle never comes out from the capillary, that condition is attained only when the absolute value of the electroosmotic velocity is identical to that of the electrophoretic velocity of the micelle in opposite directions. Equation (3) becomes: (4) This is the same situation as in conventional chromatography, that is, the parameter of the migration time window is equal to infinity. When to is infinity or the electro-osmotic flow is completely suppressed, Eq. (3) becomes: tR = (1 + l/k’)t,, (5) tR=(l

+/cyto

In this case, the aqueous phase never comes out from the capillary, and only the micelle migrates toward the positive electrode through the aqueous phase. Thus, electro-osmotic flow is not essential m MEKC. According to Eq. (2), to, tR, and t,, are required to obtain the capacity factor. As a marker of the electro-osmotic flow or to, methanol is usually used becausethe distribution coefficient of methanol between the micelle and aqueous phase is negligibly small. Although methanol is transparent to UV, it can be detected as a baseline deflection with a UV detector owing to a refractive mdex change. Sudan III or IV is often employed as a tracer of the SDS micelle (6), which is completely incorporated into the micelle. Timepidium bromide is also useful as a tracer for an anionic micelle (15). It should be noted, however, finding good tracers for to and tmc applicable to every condition is difficult. 3.1.2. Resolution

Resolution, R,, in MEKC is given as: R, = (N’“/4)

[(a-

1)/a] [Ii241 +I?;)]

{[(l -to)/&,,,]

/ [l + (to/t&‘,]}

(6)

Here, N is the theoretical plate number, a the separation factor equal to k;lk’,, and k’i and k; are the capacity factors of analytes 1 and 2, respectively. Effects of these parameters on resolution are briefly discussed below. 3.1.2.1. PLATE NUMBER Resolution increases proportionally with an increase in square root of the plate number. Usually average plate numbers for most analytes are lOO,OOO-200,000.Normally, the higher the voltage applied, the higher

Otsuka and Terabe

130

the plate number that can be attained, unless excessive Joule heating 1s generated at the higher apphed voltage. Since the diffusion coefficrent of the micelle 1s small, solutes having larger capacity factors can yield higher plate numbers. 3.1.2.2. SEPARATIONFACTOR The separation factor reflects the relative drfference of the distrrbutron coefficrents between two analytes and is a unique variable to a given separation conditron. Thus, we can manipulate the value by changing the type of micelles or the bulk solution using modifiers. 3.1.2.3. CAPACITYFACTOR The optimum value of the capacity factor IS represented as (t,,,lt0)1’2. Under neutral conditions, the optimum value is close to 2 for most long alkyl chain surfactants. The capacity factor can be related to the distribution coefficient, K, between the micelle and aqueous phase as: (7) Here, V,,,, and Vaq are the volume of the micelle and aqueous phase, respectively. The phase ratio, V,,,,/V,,, can be wrrtten by using the concentration of the surfactant, C,, and specific volume of the mtcelle, V as: (V,, / V,,) = [ v (C,,- CMC) / 1 -V (CT,,- CMC)] (f-9 Then, at low mrcellar concentratrons, Eq. (7) can be rewritten as: k’ = Kt; (C,,- CMC)

(91 This reveals that the capacity factor increases linearly wrth an increase in the surfactant concentratton. The capacity factor can be easily adjusted by manipulating the surfactant concentratron through Eq. (9) If the CMC is known. 3.1.2.4. ELECTROOSMOTICFLOW The effect of the electroosmotic flow can be discussed m terms of the migration time ratio, to/&,,., or the migration time window, t,,ltO. The velocity of the mrcelle is given as: V

mc= [ k. + cL&Nl E

(10)

where E is the electrical field strength. Then:

t0~4tlc= [ 1+ rep

/ pealE

(11)

MEKC

131

16

Fig. 4. Dependenceof the capacityfactor (k’) on the SDS concentration(csus) (6). Reprintedwith permission from Terabeet al. (1985)Anal. Chem. 57, 834. The mobilities peaand I.t&rnc) usually have different signs, and the ratio ,uep(mc)/~eois between 0 and -1. Therefore, to/t,, is smaller than 1. The smaller the value of toltmc becomes, the larger the resolution will be. If a negative sign for to/t,, is assumed when peP(mc)/ peais smaller than -1, an extremely high resolution is expected for an analyte having the capacity factor close to -(t,,ltJ according to Eq. (6), although a quite long migration time is required (13). 3.2. Thermodynamic Parameters As mentioned above, the capacity factor increases linearly with an increase in the concentration of the surfactant. The dependence of the capacity factor on the SDS concentration is shown in Fig. 4 (6). This reveals that the distribution coefficient is almost constant regardless of the SDS concentration. The distribution coefficient measured at different temperatures should follow the van’t Hoff equation: InK = - (AHOIRT) + (ASOIR) (12)

132

Otsuka and Terabe Table 1 CMC of SDS and the Partial Specific Volume (V) of the SDS Mlcelle Buffela CMCImA4

i/mLlg

TempYC

B-P

PIPES

BES

Urea

B-P

20 22 25 30 35 40 45 50

28 29 25 26 3.0

38 42 43 42 44 48

44 45 53 5.9 59 64

0 8562

31 33 3.3 35 36 38

-

0 0 0 0

8610 8686 8710 8758

Urea 0 8126 0 8160 0 8242 0.8248 0 8290

-

OB-P, 100 mMborat+50 mMphosphate buffer, pH 7 0, PIPES, 20 mMPIPES-20 mMNaOH, pH 7 0, BES, 100 mA4 BES-100 mM NaOH, pH 7 0, urea, 5M urea m 100 mA4 boratc50 mA4 phosphate buffer, pH 7 0

where AHo is the enthalpy change associated with micellar solubilization or the transfer of the solute from the aqueous phase to the micelle, Aso the corresponding entropy change, R the gas constant, and T the absolute temperature. Thus, Eq. (12) allows the calculation of the enthalpy and entropy changes in micellar solubilization from the temperature dependence of K. In various buffer systems, such as borate-phosphate (B-P), piperazine-N,N’-bis(2-ethanesulfonic acid) mono sodium salt (PIPES)sodium hydroxide, N,N’-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BESksodium hydroxide, and B-P with urea, CMC and the partial specrtic volume were measured for the SDS mrcelle (16) as shown in Table 1. Table 2 lists distribution coefficients of test solutes at different temperatures between the SDS micelle and a B-P buffer. Corresponding enthalpy, entropy, and Gibbs free energy changes were calculated as shown m Table 3. Obviously, the effect of the buffer solution is not significant on these thermodynamic quantities. The entropy changes for resorcinol, phenol, p-nitroanilme, and 2-naphthol were negative, and the contribution of A.S” to AGO was significant except for phenol. These solutes favored the aqueous phase from the viewpoint of entropy.

MEKC

133 Table 2 Dlstrlbutlon Coefficients of Alkylphenols Between the SDS Mlcelle and a 100 mM Borat+SO mM Phosphate Buffer, pH 7.0” Temp I “C

Solute o-Cresol m-Cresol p-Cresol 2,6-Xylenol 2,3-Xylenol 3,4-Xylenol 2,4-Xylenol p-Propylphenol p-Butylphenol p-Amylphenol

30

35

40

45

100 104 112 203 233 250 269 788 2320 7120

93.0 96.5 104 187 214 230 248 729 2140 6660

86.4 89.9 96 8 173 197 212 229 668 1940 5870

81.1 84 5 90.7 161 183 197 213 622 1800 5580

‘Separation solution, 50 mM SDS m 100 mM boratc50 tiphosphate

50 76.3 79 5 85 5 150 170 183 198 571 1620 4900

buffer, pH 7 0

Table 3 Enthalpy, Entropy, and Gibbs Free Energy Changes m Mlcellar Solublhzatlon of Alkylphenols by the SDS Mlcelle” Solute o-Cresol m-Cresol p-Cresol 2,6-Xylenol 2,3-Xylenol 3,4-Xylenol 2,4-Xylenol p-Propylphenol p-Butylphenol p-Amylphenol

AH”lkJlmol -11.1 -10 8 -109 -12 4 -12.7 -12 7 -12 4 -13 1 -14.4 -150

AS”/J/mollK 16 29 33 3.2 3.3 39 55 12 3 16 9 24.3

AGO/kJ/mol, 35’C -11 6 -117 -119 -13.4 -13.7 -13 9 -14.1 -169 -19 6 -22 5

OTheseparation solution was the same as m Table 2.

4. Selectivity Manipulation Selectivity in chromatography can be discussed by using the separation factor, a. The separation factor can be manipulated with chemical considerations. The micelle in MEKC corresponds to the stationary phase in reversed-phasehigh-performance liquid chromatography (RP-HPLC),

Otsuka and Terabe whereas the bulk solution or the surrounding aqueousphase correspondsto the mobile phase, from the viewpoint of selectivity manipulation. Mainly, the following four factors can be controlled to manipulate selectivity: 1 2. 3. 4.

The mlcellar structure; Temperature, pH, and Additives to the aqueousphase. 4.1. Effect of the Micellar Structure 4.1.1. Surfactants A surfactant molecule has a hydrophobic and hydrophilic group, and both groups affect selectivity in MEKC. Since most analytes interact with the micelle on its surface, the hydrophilic or ionic group is generally more important than the hydrophobic one in determining selectivity. For example, SDS and cetyltrlmethylammomum bromide (CTAB) show considerably different selectivity as shown m Fig. 5 (17). The structural difference between SDS and CTAB is mainly ionic groups: sulfate in SDS and quaternary ammonium in CTAB. The hydrophobic groups are slmilar, only different in the length of alkyl chains. Similar results have also been published (18). In Table 4, differences in distribution coefficients of the test solutes among three surfactants, such as SDS, sodium tetradecyl sulfate (STS), and sodium dodecanesulfonate (SDDS), are shown. The distribution coefficients for resorcinol, phenol, p-nitroaniline, and nitrobenzene are virtually identical between SDS and STS, which have the identical ionic groups, but different alkyl chain lengths. On the other hand, significantly different distribution coefficients are observed between SDS and SDDS, which have the ldentlcal alkyl chains, but different ionic groups. These results suggest that many compounds are adsorbed on or at least strongly interact with the surface of the micelle. Bile salts, such as sodium cholate, sodium deoxycholate, and sodmm taurodeoxycholate, which form helical mlcelles (19), can have slgnificantly different selectivity compared with the long alkyl chain surfactants (20). Bile salts are naturally occurring chiral surfactants, and hence, they can also be used for enantiomeric separation (19,21-23). 4.1.2. Mixed Micelles In MEKC, ionic micelles are usually used, and they can be easily modlfied by adding ionic or nonionic surfactants to form mixed micelles. The

MEKC

135 A

I

1

OOOZAU

J 0

I

6

B

I

I

4

I, 0

0OWaAU

I,,

12

,

,

20

16

I 0

I

I 4

8

Time I mln

I I I 0 12 Time / min

I

I

16

Fig. 5. Effect of the surfactant structures on selectivity (17). (1) water, (2) aniline, (3) nitrobenzene, (4) m-nitroaniline, (5) p-nltroamlme, (6) o-nitroamline. Micellar solution, (A) 100 rmI4 SDS in 100 mM borate50 mM phosphate buffer, pH 7.0, (B) 50 mM CTAB in 100 mM Tns-HCl, pH 7 0; capillary, 50 pm id x 650 mm (effective length, 500 mm); applied voltage, 15 kV; detection wavelength, 230 run; temperature, ambient. Table 4 Coeffictents at 3Y’C

Distribution

Dlstrlbution Solute Resorcinol Phenol p-Nitroamline Nitrobenzene Toluene 2-Naphthol

coefficient

SDS0

STSb

SDDSC

21.6

20.8 52.3

27.7 56.1 84.3

52.1 103

135 318 656

%odlum dodecyl sulfate. “Sodium tetradecyl sulfate cSodium dodecanesulfonate

100 138

111

345 789

288 698

136

Otsuka and Terabe

mixed micelle consisting of ionic and noniomc surfactants 1s usually larger than the original iomc micelle, and has a lower charge density, and hence, a lower electrophoretic mobility. Consequently, a narrower migration time window is obtained (24), and also different selectivity is expected (24) since the surface of the mixed micelle is different from that of the original one.

4.2. Effect

of Temperature

The distribution coefficient is dependent on temperature: It decreases with an increase in temperature, which causes a reduced migration time. The increase in temperature also causes increases m v,, and vep(mc) to the same extent because of reduced viscostty. Dependencies of the distribution coefficients on temperature are different among solutes. Therefore, temperature affects selectivity as shown in Fig. 6 (26). It should be noted that temperature seriously affects the migration time, although its effect on selectivity is not remarkable, and hence, it is important to maintain temperature precisely to obtain reproducible results.

4.3. Effect 0fpH Effect of the constituents of the buffer is not significant, whereas the pH is a critical factor for ionizable analytes. If the ionized form of the solute has the same charge as the micelle, it will be incorporated into the micelle less than its neutral form. Figure 7 demonstrates the dependence of the apparent capacity factor on the buffer pH for some chlorinated phenols (2.5). Here, the apparent capacity factor was calculated by Eq. (2) regardless of whether the solutes were ionized or not. For acids, the increase in pH will promote ionization. Then the dtstribution coefficient to the anionic micelle or SDS will be reduced. It should be noted that the change of the buffer pH, especially in the lower pH region, causes a significant change in the electro-osmotic velocity as mentioned in the previous section (13).

4.4. Effect of Additives

to the Aqueous

Phase

The most versatile and effective methods to manipulate selectivity in MEKC are the use of additives to the aqueous phase as well as the choice of surfactants. In conventional HPLC, modifications with additives to the mobile phase is well established, and the knowledge can be applied in a similar manner to MEKC. Of course, we must understand the difference between the micellar phase in MEKC and the stationary phase in

MEKC

137

6

4i /-/-y-q7 l-4 31

33

T-1 , ,03-L

Fig. 6. Van’t Hoff plots of alkylphenols (16). (I) o-cresol, (2) m-cresol, (3)p-cresol, (4) 2,6-xylenol, (5) 2,3-xylenol, (6) 3,4-xylenol, (7) 2,4-xylenol, (8) p-propylphenol, (9) p-butylphenol, (10) p-amylphenol. Micellar solution, 50 mMSDS in 100 mMborate50 Wphosphate buffer, pH 7.0; apphed voltage, 10kV. Reprinted with permission from Terabe et al. (1993) J. Microcol. Sep. 5,23.

HPLC to use such additives. There are four main categories of additives that are useful in MEKC: 1. Cyclodextrins (CDs); Ion-pair reagents;

2. 3. 4.

Urea; and Organic modtfiers. 4.4.1. Cyclodextrins

Recently, CD has become a popular additive or stationary phase in chromatography. In most cases, CD’s capability of recognizing specific molecules that fit its hydrophobic cavity is used for chromatographic separations. The use of CDs is especially effective for the separation of

138

Otsuka and Terabe

1-

l

l I 6

I 6

I 7

I 9

PH

Fig. 7. Dependence of apparent capacity factors (Pap& of chlorinated phenols on pH (25). (1) phenol, (2) 2-chlorophenol, (7) 2,5dtchlorophenol, (14) 2,4,Wrtchlorophenol, (17) 2,3,4,5-tetrachlorophenol, (20) pentachlorophenol. Micellar solutton, 100 mM SDS in 50 mA4 phosphate-borate buffer; capillary, 50 ym id x 650 mm (effective length, 500 mm); applied voltage, 15 kV; detection wavelength, 220 nm; temperature, 35OC Reprinted with permission from Otsuka et al. (1985) J. Cizromatogr. 348, 39. aromatic isomers and aromatic enantiomers that have a chiral center close

to the aromatic ring. Originally, CD is electrtcally neutral and not affected by the electrophorests. This means that CD itself cannot be used as a carrier m EKC, unless an iomc group IS introduced into CD. The surface of CD is hydrophilic, and hence, we can assume that CD is not mcorporated into the mtcelle. A surfactant molecule, however, may be included into the CD cavity. The separation principle of CD-modified MEKC (CD-MEKC) is schematically shown in Fig. 8 (26). In this system, CD migrates at the same velocity as the electro-osmotic flow. The analyte molecule, which is assumed to be neutral, both included by CD and in the aqueous phase,

139

MEKC -0------_--

------------

G--

Anionic Surfactant

(

Electroosmotrc

-

Analyte

,-

Electrophoretvz Mrgratron

Flow

Cyclociextrrn

Fig. 8. Schematicof the separationprinciple of CD-MEKC (26) Reprinted wrth permission from Terabeet al (1990)J Chromatogr 516,23 migrates at the same velocity as the electro-osmotic flow. On the other hand, the analyte migrates at a different velocity from the electro-osmotrc flow when it is incorporated into the ionic micelle. In the case of highly hydrophobic analytes, they seem to be totally incorporated into the micelle in the absenceof CD. This means that the addition of CD reduces the apparent distribution coefficient of the analytes between the bulk phase and the micelle and makes rt possible to separatesuch solutes. The higher the concentration of CD becomes, the smaller the distribution coefficient will be observed. In CD-MEKC, therefore, the capacity factor can be manipulated by varying both the concentrations of CD and the micelle. An example of CD-MEKC separation of hydrophobic compounds is shown m Fig. 9 (26). CD-MEKC is also effective for enantiomerit separation, which will be discussed later. It should be noted that CD-MEKC is a different technique from CDEKC, although these two terms are somewhat confusing. In CD-MEKC, CD is added to micellar solutrons, and while in CD-EKC, an ionic CD derivative is used as a carrier of EKC in a solution without micelles. 4.4.2. Urea Urea is usually used to increase the solubility of hydrophobic compounds in water. In MEKC, a successful separation of highly lipophilic

Otsuka and Terabe

140

;o Time (mln)

Fig. 9. Separation of 11 trichlorobiphenyl isomers by CD-MEKC (26): BIPH = blphenyl. Separation solution, 60 rmI4 y-CD, 100 mM SDS, and 2M urea m 100 &borate50 &phosphate buffer, pH 8.0; capillary, 50 pm id x 650 mm (effecttve length, 500 mm); applied voltage, 15 4 kV; current 50 PA. Reprinted with permission from Terabe et al. (1990) J. Chromatogr. 516,23.

compounds was achieved with an SDS solution containing high concentration of urea, as shown m Fig. 10 (27). By adding urea to the micellar solution, the electro-osmotic velocity is slightly reduced, whereas the migration velocity of the micelle is considerably reduced, which causes the reduced capacity factors. Urea is also effective to improve peak shapes, especially m the separation of ammo acids (28). Although a remarkable change in the selectivity is not attained by the urea addition, a slight change in the selectivity can be recognized, especially for the separation of closely related compounds. 4.4.3. Organic Modifiers Similar to the case of HPLC, an organic solvent miscible with water can be used as an additive to the micellar solution to manipulate the capacity factors or selectivity. In HPLC, highly hydrophobic compounds can be analyzed by using a high concentration of the organic solvent, whereas in MEKC, a high concentration of the organic solvent cannot be employed because of the breakdown of the micellar structure. In general, the usable maximum content of the organic solvent is approx 20%. The use of the organic solvent usually provides an improved resolution and/or a change in the selectivity. In MEKC, methanol (2932),2-propanol (33), and acetonitrile (30) are used as the organic modifiers, and they contribute to reduce the electro-osmotic velocity and

MEKC

141

A

6

Time (mln)

Fig 10. The effect of urea addition to the SDS solution (27) (1) hydrocortlsone, (2) hydrocortisone acetate, (3) betamethasone, (4) cortisone acetate, (5) triamcmolone acetonlde, (6) fluocmolone, (7) dexamethasone acetate, (8) fluocinonide. Separation solution, 50 mA4 SDS In 20 mA4 borate-phosphate buffer, pH 9.0. (A) Without urea and (B) with 6M urea; capillary, 50 pm Id x 650 mm (effective length, 500 mm); applied voltage, 20 kV, detection wavelength, 210 nm. Reprmted with permission from Terabe et al. (1991) J Chromatogr 545,359. expand the migration time wmdow. An example of the use of methanol for the separation of aromatic sulfides is shown in Fig. 11 (29). Recently, Tanaka (34) has reported that the use of a fairly high concentration of methanol, e.g., 80 to almost 100% (v/v), in an SDS solution is effective for the MEKC separation of some hydrophobic compounds. In such circumstances, it is unclear whether the SDS micelle still exists in the solution, but some interactions between the solutes and the SDS molecule or micelle might occur. Imasaka and coworkers (3.5) have reported that the addition of N,Ndimethylformamide (DMF) to a bile salt micellar solution is effective for

142

Otsuka and Terabe l-

1

6

L 0 c I

I 10

1

I

30

50

Fig. 11 Separation of 11 aromatic sulfides (29) (1) benzyl methyl sulfide, (2) benzyl ethyl sulfide, (3) benzyl propyl sulfide, (4) benzyl tsopropyl sulfide, (5) methyl phenyl sulfide, (6) ethyl phenyl sulfide, (7) phenyl propyl sulfide, (8) isopropyl phenyl sulfide, (9) butyl phenyl sulfide, (10) tsobutyl phenyl sulfide, (11) s-butyl phenyl sulfide, (12) Sudan III. Separation solutton, 30 mM SDS, pH 7.0, contammg 20% (v/v) methanol, captllary, 50 urn td x 900 mm (effecttve length, 750 mm); applied voltage, 22 kV, current, 20 PA; detection wavelength, 210 nm, temperature, ambient Reprinted with permtsston from Otsuka et al. (1986) Ncppon Kagaku Kauh, 950 the separation of polyaromatrc hydrocarbons (PAHs) as shown in Fig. 12. Similarly, the use of a high concentratton of dimethyl sulfoxide (DMSO)

~IIthe SDS-MEKC systemISfound to be useful for the analysis of PAHs (36). 4.4.4.

Ion-Pau-

Reagents

In MEKC, the use of an ion-pair reagent causes a remarkable change m separation characteristics, which is mainly because of the charge of the micelle. When a tetra-alkylammonium salt is added to the SDS micellar solution, anionic analytes form paired ions with the ammonium

ion, and hence, the electrostatic repulsion between the anionic SDS micelle and the anionic analyte is reduced. That formation of the paired ion IS promoted with an increase of the concentration of the ammonium salt, that is, the higher the concentration of the ammonium salt, the larger

143

MEKC I2

I

I

I

0

4

8

Time

I

12

16

/ min

Fig. 12. Separation of PAHs by MEKC with DMF (3.5) (1) fluoranthene, (2) pyrene, (3) perylene, (4) benzo[a]pyrene, (5) 2,3-benztriphenylene, (6) dlbenz[a,h]anthracene. Separation solution, 70 mM sodium deoxycholate in Trls buffer containing 20% DMF; capillary, 50 pm rd x 600 mm (effective length, 500 mm); detection, He-Cd laser-Induced fluorescence. Reprinted wrth pernnssion from Kaneta et al.

the capacity factor of the anionic analyte. On the other hand, a cationic analyte competes with the ammonium ion in pairmg to the anionic micelle, so the migration time of the cation decreases with an increase in the concentration of the ammonium salt. The effect of the addition of tetra-alkylammonium salts to SDS micellar solutions on the selectivity is shown m Fig. 13 (1.5). The normal CZE separation, without SDS, using the buffer containing the salt is also shown. The effect strongly depends on the structure of the ion-paring reagent, e.g., the length of the alkyl chain. 4.4.5. Metal Salts Cohen et al. (37) reported the effect of the addition of metal salts to the SDS micellar solution. By adding magnesium, zinc, or copper(I1) to the

144

A

3

8 B

:I 4

5

I

Otsuka and Terabe

a

7 +

Time (mln)

,

I

r

I

0

5

10

15

Time (mm)

Time (mm)

Fig. 13. Separation of cephalosporin anttbtotlcs by (A) CZE, (B) MEKC with SDS, and (C) MEKC with SDS and tetramethylammomum salt (15). (1) C-TA, (2) ceftaztdtme, (3) cefotaxime, (4) cefmenoxrme, (5) cefoperazone, (6) cefpiramide, (7) cefptmlzole, (8) cefmmox, (9) ceftrtaxone. Separation solution, (A) 20 mM borate-phosphate buffer, pH 9.0, (B) 50 mM SDS added to (A), (C) 40 mM tetramethylammomum bromide added to (B); caprllary, 50 pm rd x 650 mm (effective length, 500 mm); applied voltage, 20 kV; detection wavelength, 2 10 nm Reprinted with permtssion from Ntshr et al. (1989) Anal. Chem 61,2434.

SDS micellar solution, the separation of oligonucleotides

was successfully achieved, and good selectivity could be obtained, as shown in Fig. 14.

5. Applications 5.1. General

Scope

Almost 10 years have passed since the first paper on MEKC (5) was published, and a number of applications of MEKC have appeared. Although separation characteristics of MEKC are similar to those of RPHPLC, the range of analytes applied to MEKC is limited compared to

MEKC

145

Fig. 14. Separation of 18 oligonucleotides, each with 18 bases, by MEKC with a metal ion (37). Separation solution, 50 mA4 SDS, 3 mM Zn(II), and 7M urea m 20 mM Tris-5 mM sodium phosphate buffer; capillary, 50 urn id x 850 mm; applied voltage, 22 kV; current, 10 PA; detection wavelength, 260 m-n;temperature, 25’C. Reprmted with permission from Cohen et al. (1987) Anal. Chem 59, 1021.

RP-HPLC: MEKC is mainly employed for the separation of small molecules, because the size of the micelle is relatively small and not able to incorporate big molecules, such as proteins, whereas RP-HPLC can treat such molecules as analytes. Regardless of such a limitation, MEKC has been recognized as a useful and powerful technique in various analytical fields, because of its advantages over RP-HPLC. The main advantage of MEKC is the higher separation efficiency. Nishi and Terabe (38) have shown some such examples, especially in the pharmaceutical analyses. Other advantages of MEKC over RP-HPLC are as follows: 1. MEKC analysis can be carried out with smaller amounts of sample and separation solutions. 2. Separation usually can be completed within a shorter time.

146

! 0

Otsuka and Terabe

/ 10

20

Time

/

mln

10

20 Time

30 / mln

Fig. 15. Separation of PTH-ammo acids by SDS-MEKC (A) wtthout urea (14) and (B) wtth urea (27) The peaks are labeled with one-letter abbrevtattons for amino acid. Separation solutton, (A) 50 mM SDS, pH 7.0, (B) 100 mM SDS contammg 4 3M urea, capillary, (A) 50 pm td x 650 mm (effecttve length, 500 mm), (B) 52 urn td x 500 mm (effecttve length, 300 mm); applred voltage, (A) 10 kV, (B) 10.5 kV; detectton wavelength, (A) 260 nm, (B) 220 nm, temperature, (A) 35’C, (B) ambient Reprinted with permtssions from (A) Otsuka et al. (1985) J Chromatogr 312,219 and (B) Terabe et al (1991) J Chromatogr

545,359

3. Maintenance of the separation capillary, e.g., cleaning or replacmg, can be easily operated. A typical application of MEKC is the separation of closely related compounds. A mixture of phenylthrohydantoin amino acids (PTH-AAs) was successfully separated by using an SDS, as shown m Fig. 15A, and a dodecyltrimethylamtnonium bromide (DTAB) solution (24). By adding urea to the SDS mrcellar solution, better resolution and selectivity could be obtained, as shown in Ftg. 15B (27). Separation of all isomers of chlorinated phenols, including phenol, could also be achieved with an SDS solution as shown in Fig. 16 (25). These separations cannot be carried

out by a simple Isocratic HPLC, i.e., a gradient method is required. The overall discussions on the MEKC applications are available in the review by Janmi and Issaq (9).

147

MEKC

14 11 7 I

h.li I

5

10 Ttme

4

I

15 lmm

Fig. 16. Separation of chlormated phenols by MEKC (25) Phenols, (1) phenol, (2) 2-chloro, (3) 3-chloro-, (4) 4-chloro-, (5) 2,3-dtchloro-, (6) 2,4-dtchloro-, (7) 2,5-drchloro-, (8) 2,6-drchloro- (9) 3,4-dichloro-, (10) 3,5-dtchloro-, (11) 2,3,4-trichloro-, (12) 2,3,5-trichloro-, (13) 2,3,6-trtchloro-, (14) 2,4,5-trrchloro-, (15) 2,4,6-trichloro-, (16) 3,4,5-trrchloro-, (17) 2,3,4,5-tetrachloro-, (18) 2,3,4,6-tetrachloro-, (19) 2,3,5,6-tetrachloro-, (20) pentachloro-, separation solutton, 70 mJ4 SDS, pH 7.0; capillary, 50 pm td x 650 mm (effecttve length, 500 mm); applied voltage, 10 kV, current, 17 PA; detection wavelength, 220 nm; temperature, 35°C. Reprinted with permlssron from Otsuka et al. (1985) J Chromatogr. 348,39.

5.2. Optical Resolution Recently, optical resolution has become one of the major objectives m chromatographic separation, and many papers on optical resolution by HPLC have appeared. A number of reports on chiral separations by MEKC have also been published at the present stage. In MEKC, the following two methods are usually employed to achieve optical resolution: (1) MEKC with chiral surfactants and (2) cyclodextrin-modified MEKC (CD-MEKC). Brief reviews on chiral separations by MEKC and also by CE have been pubhshed previously (39-41). A more extensive treatment is given in Chapter 14. 52.1. MEKC with Chiral

Surfactants

Chiral surfactants mainly used in MEKC for optical resolution are as follows: Amino acid derivatives, such as sodium Wdodecanoyl-L-

148

Otsuka and Terabe

I

,

0

20 Time

I 40 / mln

Frg 17. Chual separatton of six PTH-m-ammo actds by MEKC wrth SDVal (45): Corresponding AAs: (1) Ser, (2) Aba, (3) Nva, (4) Val, (5) Trp, (6) Nle. (0) Acetomtrile. Micellar solution, 50 nuI4 SDVal-30 mM SD%-O.SM urea, pH 9.0, containing 10% (v/v) methanol; capillary, 50 ym td x 650 mm (effective length, 500 mm); applied voltage, 20 kV; current, 17 PA; detection wavelength, 260 nm, temperature, ambient. Reprmted with perrmsston from Otsuka et al. (1991) J Chromatogr. 559,209.

(Z&42-45) and sodium N-dodecanoyl-L-glutamate (SDGlu) (46), which were effective in resolution of PTH-DL-AAS, as shown in Fig. 17 (45). Digitonin, which is a glycoside of digitogenin, could achieve the optical resolution of some dansylated m-amino acids (Dns-m-AAs), used as the mixed micelle with SDS or bile salts (4446). Bile salts are useful to chnal separations, as mentioned previously. By using sodium taurocholate (STC) and sodium taurodeoxycholate (STDC), some Dns-DL-AAs were optically resolved (21). Some chiral drugs, e.g., diltiazem hydrochloride and trimetoquinol hydrochloride, have also been valinate

(SDVal)

149

MEKC

resolved (22,23,4 7). Enantiomeric separation of binaphthyl analogs by MEKC with bile salts was reported by Cole et al. (48). As other chiral surfactants, saponins, such as glycyrrhizic acid and p-escin, have been used for optical resolution of Dns- or PTHDL-AAs (49). 5.2.2. Cyclodextrin-Modified

MEKC (CD-MEKC)

As mentioned previously, CD-MEKC is capable of optical resolution, especially of aromatic and related enantiomers. Some Dns-oL-AAs were optically resolved by CD-MEKC using SDS solutions containing p- or ?/-CD (50). Not only the underivatized CDs but also some CD derivatives, e.g., 2,6-di-O-methyl-P-CD, in SDS solutions can be used for the resolution of the optical isomers (51). Recently, the CD-MEKC system has become one of the most popular techniques for chiral separations in HPCE: Optical resolution of some labeled amino acid enantiomers (52) and RS-chlorpheniramine (53) has been reported. It should be noted that the CD-added CE system without micelles is usually more effective than CD-MEKC for chiral separation of ionic compounds, especially for the analyte having a high electrophoretic mobility, and CD-MEKC and CD-added CE are complementary techniques to each other. 6. Conclusion As mentioned previously, many papers on MEKC, which include fundamental characteristics and applications, are available at this present stage. Since only brief discussion on some aspects of MEKC are described in this chapter, it is necessary to refer to some of that literature where more detailed information is available. Especially for optimization of MEKC, which was not discussed in this chapter, theoretical discussions by Foley (54), Vindevogel and Sandra (55), and Khaledi and coworkers (56,57) should be cited, along with the review article (II). There are some EKC techniques other than MEKC: Cyclodextrin EKC (CDEKC) (58), ion-exchange EKC (IXEKC) (59), and microemulsion EKC (MEEKC) (60,61). In CDEKC, a cyclodextrin derivative having an ionic function is used instead of the micelles in MEKC. Similarly, polymer ions and microemulsions are used in IXEKC and MEEKC, respectively.

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Electrokinetic chromatography, which is indeed a branch of HPCE, has become the normal technique for high-resolution separation of neutral species by electrophoresis, and will be used in wider fields in the future. Notes Added in Proof Recently, mass spectrometry (MS) has become one of the powerful detection schemes in CE. The development of the MEKC-MS system, however, did not successfully progress, since most surfactants normally used in MEKC often deteriorate ionization efficiency and cause high background signals in an electrospray ionization-MS (ESI-MS). One solution to these limitations is to use high-mol-wt surfactants, instead of a conventional one, such as SDS, as pseudostationary phases in MEKC Terabe and coworkers (62,63) used butyl acrylate-butyl methacrylatemethacryhc acid copolymer sodium salts (BBMA), and investigated their use in MEKC-MS systems. In an ESI-MS system, BBMA was successfully used for the separation and detection of some quaternary ammomum salts, alkaloids, and sulfamids (64). In an atmospheric pressure chemical ionization-MS (APCI-MS) system, some xanthine derrvatives were able to be separated and detected (65). The use of high-mol-wt surfactants in MEKC is also useful for the alteration of the selectivity and theoretical treatments on separation characteristics becausethe high-molwt surfactant forms the molecular micelle or the micelle with a single molecule. As mentioned m Section 4.4.3., separation of hydrophobic compounds, such as polycyclic aromatic hydrocarbons (PAHs), by MEKC is still in progress. Some PAHs were successfully separated with a CD-MEKC mode, i.e., using and y-CD-SDS solution (66), and also by MEKC with an SDS-acetone solution (67). As new pseudostationary phases in EKC other than micelles, starburst dendrimers were introduced by Tanaka et al. (34,68,69). They showed remarkably different selectivity from that m the SDS-MEKC system and could be used for the separation of various PAHs. Other techniques for the separation of hydrophobic compounds, nonaqueous CE (70), and hydrophobic interaction electrokinetic chromatography (HI-EKC) (71) were demonstrated. References 1. Mikkers, F E. P , Everaerts, F M , and Verheggen, Th. P. E. M. (1979) Highperformance zone electrophorem J Chromatogr 169, 1 l-20.

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2 Jorgenson, J W and Lukacs, K D. (198 1) Zone electrophoresis in open-tubular glass capillaries. Anal. Chem 53, 1298-l 302 3 HJerten, S. (1983) High-performance electrophoresis: the electrophorettc counterpart of high-performance liquid chromatography J Chromazogr 270, l-6. 4 Terabe, S (1989) Electrokmetic chromatography: an interface between electrophoresis and chromatography. Trends Anal. Chem. 8, 129-134 5. Terabe, S , Otsuka, K , Ichikawa, K., Tsuchiya, A., and Ando, T. (1984) Electrokinetic separations with mtcellar soluttons and open-tubular capillaries. Anal. Chem 56, 111-113 6 Terabe, S , Otsuka, K., and Ando, T. (1985) Electrokmetic chromatography with mxellar solution and open-tubular capillary. Anal Chem 57,834841 7 Terabe, S , Otsuka, K., and Ando, T. (1989) Band broadenmg in electrokmetic chromatography wtth micellar solutions and open-tubular captllaries. Anal Chem 61,25 l-260 8 Kuhr, W G. and Monnig, C A. (1992) Capillary electrophoresis Anal Chem. 64, 389R-407R 9. Janini, G. M. and Issaq, H J (1992) Micellar electrokinettc capillary chromatography: basic considerations and current trends. J Lzquzd Chromatogr 15,927-960 10. Vindevogel, J and Sandra, P (1992) Introduction to Mzcellar Electrobnetcc Chromatography Huthrg, Heidelberg 11 Terabe, S (1992) Mzceilar Electrokinetzc Chromatography Beckman, Cahforma 12 Terabe, S (1993) Micellar electrokmetic chromatography, m Capillary Eiectrophoreszs. Theory, Methodology, and Appbcatlons (Guzman, N A , ed ), Marcel Dekker, New York, pp. 65-87 13 Otsuka, K and Terabe, S. (1989) Effects of pH on electrokinetic velocities in micellar electrokinettc chromatography J Mzcrocol Sep 1, 150-154 14. Otsuka, K , Terabe, S , and Ando, T. (1985) Electrokmettc chromatography with micellar solutions* separation of phenylthiohydantoin-ammo acids J Chromatogr 312,219-226 15 Nlshi, H., Tsumagari, N , and Terabe, S (1989) Effect of tetraalkylammonmm salts on mtcellar electrokinettc chromatography of ionic substances Anal Chem 61,2434-2439. 16 Terabe, S., Katsura, T., Okada, Y., Ishihama, Y., and Otsuka, K. (1993) Measurement of thermodynamic quantities of micellar solubihzation by micellar electrokinetic chromatography wnh sodium dodecyl sulfate. J Mcrocol Sep 5,23-33. 17 Ishthama, Y. and Terabe, S , unpublished data 18. Nrshi, H., Fukuyama, T., Matsuo, M., and Terabe, S. (1990) Effect of surfactant structures on the separatton of cold medtcme Ingredients by micellar electrokinettc chromatography J Pharmaceut Sci 79,519523 19. Cole, R 0 , Sepamak, M J., Hinze, W. C., Gorse, J., and Oldiges, K. (1991) Bile salt surfactants m micellar electrokmetx capillary chromatography. apphcation to hydrophobic molecule separatrons. J. Chromatogr 557, 113-I 23. 20. Ntshr, H., Fukuyama, T., Matsuo, M., and Terabe, S. (1990) Separation and determination of lipophilic cortxosteroids and benzothtazepm analogues by mxellar electrokinetic chromatography using bile salts. J. Chromatogr. 513,279295.

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21 Terabe, S., Shlbata, M , and Mlyashlta, Y. (1989) Chiral separation by electroklnetlc chromatography with bile salt mlcelles J Chromatogr 480,403-4 11 22 Nlshl, H , Fukuyama, T., Matsuo, M., and Terabe, S. (1990) Chrral separation of dlltlazem, trlmetoqumol and related compounds by mlcellar electrokmetlc chromatography J Chromatogr 515,233-243 23 Nlshi, H , Fukuyama, T , Matsuo, M., and Terabe, S (1990) Chiral separation of trlmetoqumol hydrochloride and related compounds by mlcellar electrokmetlc chromatography using sodium taurodeoxycholate solutions and application to optical purity determination Anal Chum Acta 236, 281-286. 24 Rasmussen, H. T., Goebel, L K., and McNalr, H. M (1991) Optimization of resolution m mlcellar electrokmetlc chromatography J Hzgh Resolut Chromatogr 14,25-28

25. Otsuka, K , Terabe, S., and Ando, T (1985) Electrokmetlc chromatography with mlcellar solutions retention behavlour and separation of chlorinated phenols J Chromatogr 348,39--47. 26 Terabe, S , Miyashita, Y , Shlbata, 0 , Barnhart, E R., Alexander, L. R , Patterson, D G , Karger, B. L , Hosoya, K., and Tanaka, N (1990) Separation of highly hydrophobic compounds by cyclodextrm-modified mlcellar electrokinetlc chromatography. J. Chromatogr 516,23-3 1 27 Terabe, S , Ishlhama, Y., Nlshi, H , Fukuyama, T , and Otsuka, K (1991) Effect of urea addition m mlcellar electrokmetlc chromatography. J Chromatogr 545,359-368 28 Otsuka, K and Terabe, S. (1990) Effects of methanol and urea on optical resolution of phenylthiohydantom-m-ammo acids by micellar electrokinetlc chromatography with sodium N-dodecanoyl+valinate Electrophoresis 11,982-984 29 Otsuka, K., Terabe, S., and Ando, T. (1986) Separation of aromatic sulfides by electrokinetic chromatography with micellar solution (m Japanese). Nippon Kagaku Kalshl, 950-955 30 Gorse, J., Balchunas, A. T., Swaile, D F , and Sepamak, M J (1988) Effects of organic mobile phase modifiers in mlcellar electrokmetlc capillary chromatography. J Hugh Resolut Chromatogr Chromatogr. Commun. 11,554-559. 3 1 Bushey, M M and Jorgenson, J. W. (1989) Separation of dansylated methylamme and dansylated methyl-d3-amme by mlcellar electrokmetlc capillary chromatography with methanol-modified phase. Anal Chem 61,491-493 32 Bushey, M. M. and Jorgenson, J W (1989) Effects of methanol-modified mobile phase on the separation of isotoplcally substituted compounds by micellar electrokinetic capillary chromatography. J. Mlcrocol. Sep 1, 125-130. 33. Balchunas, A. T and Sepamak, M J. (1988) Gradient elutlon for mlcellar electrokinetic capillary chromatography. Anal. Chem 60,6 17-62 1. 34. Tanaka, N., Fukutome, T., Tanigawa, T., Hosoya, K., Klmata, K., Araki, T , and Unger, K. K. (1995) Structural selectivity provided by starburst dendrlmers as pseudostationary phase in EKC J Chromatogr A. 699,33 l-341 35 Kaneta, T., Yamashita, T., and Imasaka, T. (1992) Separation of polyaromatlc hydrocarbons by laser-induced fluorescence detection--mlcellar electrokmetlc chromatography, Abstracts ofPapers, 12th Symposmm on Capillary Electrophoresis, Himeji, Hyogo, Japan, Abstract 8.

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36 Otsuka, K , Koike, R., and Terabe, S. (1993) Effect of addmon of organic modlfiers in mrcellar electrokinetic chromatography, Abstracts of Papers, 65th Meetmg of the Chemrcal Socrety of Japan, Tokyo, Japan; Abstract 3A252. 37. Cohen, A. S , Terabe, S., Smith, J. A., and Karger, B. L. (1987) High-performance capillary electrophoretrc separatron of bases, nucleosides, and olrgonucleotrdes retentron manipulation vta mtcellar solutions and metal additives. Anal Chem 59, 1021-1027. 38 Nishi, H and Terabe, S. (1990) Applications of micellar electrokmetic chromatography to pharmaceutrcal analysis Electrophoreszs 11, 69 l-70 1 39 Otsuka, K. and Terabe, S (1993) Enantromerrc separation by mrcellar electrokrnetrc chromatography. Trends Anal, Chem. 12, 125-l 30 40 Otsuka, K. and Terabe, S (1993) Choral separation by capillary electrophoresis and electrokmetic chromatography, in Capillary Electrophoresis Theory, Methodology, and Appllcatlons (Guzman, N. A., ed.), Marcel Dekker, New York, pp 6 17-629 41. Terabe, S , Otsuka, K., and Ntshi, H. (1994) Separation of enanttomers by caprllary electrophoretrc techniques J Chromatogr A 666,295-3 19 42. Dobashi, A , Ono, T , Hara, S , and Yamaguchr, J. (1989) Optical resolution of enantiomers with chiral mixed micelles by electrokmetic chromatography Anal Chem 61,1984-1986. 43. Dobashi, A., Ono, T., Ham, S., and Yamaguchr, J (1989) Enantroselectrve hydrophobic entanglement of enantiomertc solutes wtth chnal functionahzed micelles by electrokinetic chromatography J Chromatogr. 480,413420 44. Otsuka, K and Terabe, S. (1990) Enantromeric resolution by mrcellar electroklnetrc chromatography with chual surfactants J Chromatogr 515,221-226. 45 Otsuka, K., Kawahara, J , Tatekawa, K , and Terabe, S. (1991) Chual separatrons by mrcellar electrokmetrc chromatography with sodium N-dodecanoyl+-vahnate. J. Chromatogr 559,209-214 46. Otsuka, K., Kashihara, M., Kawaguchr, Y., Koike, R., Huamttsu, T., and Terabe, S. (1993) Optical resolution by high performance capillary electrophoresrs. mrcellar electrokinetic chromatography with sodium N-dodecanoyl+-glutamate and drgitonin. J. Chromatogr A 652,253-257. 47. Nishr, H., Fukuyama, T , Matsuo, M., and Terabe, S. (1989) Chnal separatron of optical isomeric drugs usmg mrcellar electrokmetic chromatography and bile salts. J Microcol Sep 1,234-241 48. Cole, R. O., Sepamak, M. J , and Hinze, W. L. (1990) Optimizatron of bmaphtyl enantiomer separations by CZE using mobile phases contammg bile salts and organic solvent. J. Hugh Resolut. Chromatogr. 13,579-582 49. Ishihama, Y. and Terabe, S. (1993) Enantiomerrc separation by micellar electrokrnetrc chromatography using saponins J. Llquld Chromatogr. 16,933-944. 50. Mryashita, Y. and Terabe, S. (1990) Separation of dansyl ot.-amino acids by micellar electrokmetrc caprllary chromatography wrth and without cyclodextrms using P/ACE system 2000. Appkcatlon Data, Hugh Performance Capillary ElectrophoreSIS, Beckman, DS-767 5 1. Nrshr, H., Fukuyama, T., and Terabe, S (1991) Chiral separation by cyclodextrmmodified micellar electrokmetrc chromatography. J Chromatogr 553, 503-5 16

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52 Ueda, T , Krtamura, F , Mttchell, R , Metcalf, T., Kuwana, T , and Nakamoto, A (1991) Choral separation of naphthalene-2,3-dtcarboxaldehyde-labeled ammo acid enanttomers by cyclodextrm-modified mtcellar electrokmettc chromatography with laser-Induced fluorescence detection. Anal Chem 63,2979-298 1 53 Otsuka, K and Terabe, S (1993) Optical resolution of chlorphemramme by cyclodextrm added captllary zone electrophorests and cyclodextrm modttied mrcellar electrokmettc chromatography. J Lzquzd Chromatogr 16, 945-953 54 Foley, J P (1990) Optimization of mtcellar electrokmetic chromatography Anal Chem 62, 1302-1308 55 Vmdevogel, J. and Sandra, P (199 1) Resolutton opttmrzatton m mtcellar electrokinetic chromatography use of Plackett-Burman stattsttcal design for the analysis of testosterone ester Anal Chem 63, 1530-1536 56 Strasters, J K and Khaledi, M G (1991) Mtgration behavior of catiomc solutes in micellar electroktnetic caprllary chromatography Anal Chem 63, 2503-2508 57 Khaledt, M. G , Smith, S C., and Strasters, J. K (1991) Micellar electrokmetic capillary chromatography of acidic solutes: migration behavior and opttmtzation strategies Anal Chem 63, 1820-1830 58 Terabe, S , Ozakt, H , Otsuka, K , and Ando, T (1985) Electrokmetic chromatography with 2-O-carboxymethyl-P-cyclodextrm as a movmg “statronary” phase J Chromatogr 332,2 11-217 59 Terabe, S. and Isemura, T. (1990) Ion-exchange electrokmettc chromatography with polymer tons for the separation of tsomerrc ions having identical electrophoretic mobthttes Anal Chem 62,65&652 60 Watarar, H. (199 1) Mtcroemulston capillary electrophorests Chem Lett 39 1-394. 61. Terabe, S , Matsubara, N., Ishthama, Y , and Okada, Y (1992) Mtcroemulston electrokmettc chromatography* comparison with micellar electrokmettc chromatography J Chromatogr 608,23-29 62. Terabe, S., Ozakt, H., and Tanaka, Y. (1994) New pseudo-stationary phases for electrokmehc chromatography* a htgh-molecular surfactant and proteins J Chw Chem Sot 41,251-257. 63 Ozaki, H , Ichthara, A , and Terabe, S (1994) Mrcellar electrokinetic chromatography using high-molecular surfactants. use of butyl acrylate/butyl methacrylate/ methacrylic acid copolymers sodium salts as pseudo-stationary phases J Chromatogr A 680, 117-123. 64 Ozaki, H , Ito, N , Terabe, S , Takada, Y., Sakatrt, M , and Koizuml, H (1995) Micellar electrokmetic chromatography-mass spectrometry (MEKC-MS) using a high-molecular surfactant. on-line couplmg with an electrospray tomzatton mterface J Chromatogr A , in press 65. Ozaki, H., Ito, N., Terabe, S., Takada, Y ., Sakatrt, M., and Koizuml, H. (1995) MEKC-MS using high-molecular surfactants. Abstracts ofPapers, 56th Forum of Analytical Chemtstry, Osaka, Japan, Abstract 1DO 1. 66. Terabe, S., Mtyashita, Y., Ishthama, Y., and Shtbata, 0 (1993) Cyclodextrin-moditied mtcellar electrokmetlc chromatography. separation of hydrophobic and enantiomertc compounds J Chromatogr 636,47-55.

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67 Otsuka, K , Hrgashimori, M , Korke, R., Karuhaka, K , Okada, Y , and Terabe, S. (1994) Separation of hpophthc compounds by mtcellar electrokmetrc chromatography wrth orgamc modttiers Electrophoreszs 15, 128CLl283 68. Tanaka, N , Tanigawa, T , Hosoya, K , Krmata, K , Arakr, T , and Terabe, S ( 1992) Starburst dendrimers as carriers in electrokinetrc chromatography Chem Lett 959-962 69 Tanaka, N., Fukutome, T , Hosoya, K., Ktmata, K., and Arakr, T (1995) Polymersupported pseudostatronary phase for EKC. EKC m a full range of methanol-water mixtures with alkylated starburst dendrimers. J Chromatogr , m press 70. Sahota, R. S and Khaledi, M G (1994) Nonaqueous capillary electrophorests AnaE Chem 66, 1141-l 146 7 1 Ahuja, E S and Foley, J P (1994) Separatron of very hydrophobrc compounds by hydrophobic mteractron electrokmetic chromatography J Chromatogr A 680, 73-83.

CHAPTER13 Capillary

Gel Electrophoresis And&s

Guttman

1. Introduction There is a great deal of interest in analytical biochemistry m the separation and identification of biologically important polymers, such as DNA protein and complex carbohydrate molecules (1,2). For relatively short single-stranded DNA units (i.e., oligonucleotides) and carbohydrate molecules, there is a need to separate by a single base difference (for DNA sequencing) (3) or even for identical chain length with a different sequence (identification of primers, probes, and antisense DNA molecules) (3,4). For the double-stranded DNA molecules, there is an interest to analyze and identify DNA molecules in the form of restriction fragments or PCR products. Using various types of sieving media allows us to do these kinds of separations. In capillary gel electrophoresis, crosslinked or noncrosslinked sieving matrices can be employed (5-7). The crosslinked gels, i.e., chemical gels, have a well-defined pore size. Noncrosslinked, or so-called physical gels, have a dynamic pore structure. This major difference provides the noncrosslinked linear polymer networks with much higher flexibility when compared to the crosslinked gels. One can operate at high temperatures (up to 5&7O”C) while applying extremely high field strengths (up to lo3 V/cm range) without any damage to the linear polymer network formulations (8’. It is important to note that the crosslinked gels are not usable under such extreme conditions (9). The other main advantage of the linear polymer network system is that it can be easily replaced in the capillary column by simply From

Methods Edrted by

m Molecular Biology, Vol 52 Caprllary Electrophoresrs K Altna Copynght Humana Press Inc , Totowa, NJ

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rinsing the gel matrix through the capillary by pressure or vacuum. Therefore, if the column becomes contaminated, the gel is easily replaced extending the lifetime of the system. Employing the replaceable concept, there is a possibility of the use of pressure injection compared to the crosslinked gels where electrokinetic injection mode is the only possibility (10). It is important to note that in addition to convenience, pressure injection permits quantitative analysis. 2. Materials 2.1. Apparatus

In all these studies, the power supply of the capillary electrophoresis apparatus (homemade or commercial) was used m reversed polarity mode, with the cathode on the injection side and the anode on the detection side. The separations were monitored on-column at 214 nm for the protein and carbohydrate, and at 254 nm for the DNA and the dansylated amino acid samples. The temperature of the gel-filled capillary columns was maintained in all experiments at 20°C * 0.5”C even at high field strengths by the Peltier device controlled cooling system (II). The electropherograms were acquired and stored on an Everex 386/33 computer using the System GoldTM software package (Beckman Instruments, Inc., Fullerton, CA). 2.2. Chemicals

The crude 70-mer and the slab-gel-purified 99-mer oligonucleotides were the gift of N. Bischoffer (Genentech, South San Francisco, CA). The human K-ras oncogenes (dGTTGGAGCT-C-GTGGCGTAG, dGTTGGAGCT-G-GTGGCGTAG, dGTTGGAGCT-T-GTGGCGTAG) were purchased from Pharmacia (Piscataway, NJ). The DNA restriction fragment mixture, @Xl 74 DNA-HaeIII digest, was purchased from New England Biolabs (Beverly, MA). The 102-mer was synthetized in-house. All the DNA samples were diluted to 50 pg/mL with water before injection. Ultrapure electrophoresis grade acrylamide, Tris, boric acid, EDTA, urea, ammonium persulfate, and TEMED were employed in the experiments (Schwartz/Mann Biotech, Cambridge, MA). Orange G (Sigma, St. Louis, MO) was used in the electrophoretic separations as an internal standard at 0.0 1% concentration. The dansylated n,L-amino acids (Dns-or,-AA) and the SDS protein test mixture (14,400-97,400 Dalton) were purchased from Sigma. ANTS-

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labeled maltooligosaccharioles were from Glyko (Novato, CA). Before injection, the samples were diluted to 0.2-2 mg/mL with the eCAP SDS-200 sample buffer and were boiled m a water bath for 5 min after adding 2.5% P-mercaptoethanol as reducing agent and 0.0 1% Orange-G as the internal standard. All the DNA, amino acid, protein and carbohydrate samples were stored at -2OOC or freshly used. The buffer and gel solutions were filtered through a 1.2~pm pore size filter (Schleicher and Schuell, Keene, NH) and carefully vacuum degassed. 3. Methods 3.1. Denaturing Gels for Single-Stranded OZigonucleotide Separations and DNA Sequencing For the separation of single- and double-stranded DNA molecules, the polymerization of the different concentration (high- and low-viscosity) linear noncrosslinked polyacrylamides was accomplished in fused silica capillary tubing (Polymicro Technologies, Inc., Phoenix, AZ) m 100 mM Tris-boric acid, 2 m44 EDTA, pH 8.5, buffer (7). For stabilization, the high-viscosity linear polyacrylamide gel (6-l 2% acrylamide) was covalently bound to the wall of the column by means of a bifunctronal agent, (methacryloxypropyl)-trimethoxysilane (Petrarch Systems, Bristol, PA) (12). The polymerization was initiated by the amount of ammonium persulfate (2-4 pL, 10%) and catalyzed by tetramethylethylenediamine (TEMED) (2-4 FL), which caused full polymerization of the given percentage acrylamide solution within 20-40 min at room temperature. 3.2. Nondenaturing Gels for Single- and Double-Stranded DNA Separations In a second column type, the use of low-viscosity linear polyacrylamide (3-6% acrylamide) without binding to the capillary wall permits replacement of the gel-buffer system in the capillary column by means of the rinse operation mode of the P/ACE apparatus (i.e., replaceable gel). The samples were injected either electrokinetically (typically: 0.0 15-O.15 W) into the prepacked column or by pressure (typically: 5 s, 0.5 psi) into the replaceable polyacrylamide gel-filled capillary column. The denaturing polyacrylamide gel column contained 7M urea.

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Gels for Capillary

EZectrophoresis

For the enantiomeric separation of Dansylated m-amino acids, the polyacrylamide gel contained specific chiral selectors, such as p-cyclodextrin. The 5% acrylamide, 0.17% N,l\r-methylene-bisacrylamide (BIS) gel was polymerized in the presence of the chiral agent. The buffer composition of that was: 0. IA4 Tris, 0.25M boric acid, pH 8.3, 7M urea, and 75 rnM P-cyclodextrin. 3.4. SDS-Protein

and Carbohydrate

Columns

In all the SDS-protein capillary electrophoresis experiments, the eCAPTM SDS-200 (Beckman Instruments, Inc., Fullerton, CA) capillary electrophoresrs size separation kit was used. For the carbohydrate separations, the eCAP Neutral coated capillary was used with 25 Macetate, pH 4.75, buffer containing appropriate amount of sieving polymer. In both instances, the 47-cm long (40 cm to the detector) and 0.1 -mm id coated capillary columns were washed with 1NHCl before each run. The sieving matrices in both cases were low-viscosity gel formulations that were not bound to the capillary wall. This permits replacement of the gel-buffer system in this coated capillary column by means of the pressure rinse operation mode of the P/ACE apparatus (i.e., replaceable gel). The samples were injected by pressure (typically 30-60 s, 0.5 psi) into the replaceable gel-filled capillary column. 3.5. Specific Applications 3.5.1. Denaturing Capillary Electrophoresis Separation of Single-Stranded Oligonucleotides

Denaturing polyacrylamide gel-filled capillary columns are utilized mainly in size separations of relatively short (up to several hundreds of bases) single-stranded DNA molecules. The most commonly used denaturing agents are urea and formamide. Figure 1 shows an example of using a denaturing polyacrylamide gel-filled capillary column containing 7M urea for the purity check of a synthesized single-stranded oligodeoxyribonucleotide (70- and 99-mer). The electropherogram shows full separation of all the failure sequences in addition to the peak of interest, the 70-mer, and the effectiveness of slab-gel purification of the 99-mer. This figure illustrates the very high resolving power of this technique, and the usefulness of the ability of fast separation and identification of single-stranded DNA molecules in molecular biology. These gels also

Capillary

161

Gel Electrophoresis 70-

B

mer

99-mer

crude

purlfled

Fig. 1. Denaturing capillary gel electrophoresis separation of synthettzed oligomers. (A) Crude 70-mer. (B) Slab-gel-purtfied 99-mer on a 5% acrylamide (crosslinked with 0.17% BIS) gel columns (see ref. 5). have micropreparative capability (13). It is possible to collect any of the peaks in Fig. 1A in sufficient concentration and purity to perform mlcrosequencing for identification of possible failures of the oligonucleotide synthesis. Several research groups are currently exploring the use of CE as an alternative to slab-gel electrophoresis for automated DNA sequence

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determination (14-Z 6). The large surface-area-to-volume ratio of the capillary permits higher electric fields than are used typically with slab gels (because of more efficient heat dissipation), resulting in very rapid and efficient separation of sequencing reaction products. The capillary format is readily adaptable to automated sample loading and on-line data collection. With CE, detection of separated DNA sequencing fragments is performed by laser-induced fluorescence (LIF) detection The sensitivity of the LIF method allows sequencing reactions to be performed on the same template and reagent scale as that of manual DNA sequencing with autoradiographic detection. The identity of the terminal base of each DNA sequencing fragment is encoded in the wavelength and/or the intensity of the fluorescent emission. 3.5.2. Nondenaturzng Capillary Gel Electrophoresis Separatbons of Single- and Double-Stranded DNA Molecules Nondenaturmg polyacrylamide gels are utilized when separation is based on the size and charge of the biopolymer. It is important to note that secondary structure differences can also be recogmzed utilizmg this kind of gel matrix. Figure 2 shows a nondenaturing polyacrylamide capillary gel electrophoretic separation of three 19-mers (human K-ras oncogene probes). Orange-G was used as internal standard in the separation in order to increase precision of migration time measurement (17). The three 19-mers in Fig. 2 differ only in a single change in sequence in the middle (position 10) of the molecules. This difference causes some secondary structure difference in the molecules. It is believed that these columns are able to separate these species based on that change. The same type of nondenaturing gels can be used in lower concentration for the separation of double-stranded DNA molecules, such as restriction fragments. Figure 3A shows a separation of a $X174 DNA HaeIII digest restriction fragment mixture by using replaceable polyacrylamide gel matrix. It is important to point out the baseline separation of the two closest size fragments, the 27 1 and 281 bp. This pair had proven difficult to separate by other types of sieving buffer systems, such as cellulose derivatives (IS). The separation of double-stranded DNA molecules can be improved by using special additives. The effect of an intercalator additrve, ethrdium bromide, on the separation of the previous test mixture can be seen in Fig. 3B. Ethidium bromide intercalates

Capillary

Gel Electrophoresis

163

)G

Fig. 2. Nondenaturmg capillary polyacrylamlde gel electrophoresls separation of a human K-ras oncogene probe mixture (19-mew). Peaks: 1 = dGTTGGAGCT-G-GTGGCGTAG, 2 = dGTTGGAGCTC-GTGGCGTAG, 3 = dGTTGGAGCT-T-GTGGCGTAG (see ref 14)

between the two strands of the DNA double helix. Since it is oppositely charged, it reduces the migration times of all the fragments. Because of this particular complexation phenomenon (one ethidium bromide molecule/5 bp) and the increasing rigidity of the complex, the larger DNA molecules move more slowly. Therefore, the separation time window opens up so the peak capacity is increased. Ethidium bromide is quite useful to manipulate the migration time and separation, making it possible to separate even the identical chain lengths of different sequences for double-stranded DNA molecules (IO). This enhanced separation is again based on the secondary structure differences caused by different primary sequences. Separation of DNA restriction fragments can be nnproved by using different temperatures during the electrophoresis (8), as well as using gradient methods, such asthe field strengthgradient separationtechnique (I 3). These

Guttman

164

g% CR

z 2

z 2

x Ei

=: ;:

x it

Fig. 3. Effect of the mtercalator additive, ethldmm bromide, on the separation of the $X 174 DNA-HaeIII digest restrlctton fragment mixture. Separatlon without (A) and with (B) 1 pg/mL ethldmm bromide m the gel-buffer system (see ref 10).

methods are useful in optimizatron of the time requirement of the separations, as well as the actual selectivity and resolution that can be achieved. 3.5.3. Chiral

Capillary

Gel Electrophoresis

Capillary polyacrylamlde gel columns can also be used with complexing agents to achieve special selectivittes. As m affinity electrophoresis (IO), the additive can either be covalently bound to the gel matrix or just incorporated into the polymeric fiber gel matrix. The latter method is always preferable, since it requires no special chemistry. Under an applied electric field, the complex will migrate according to its overall charge in the gel when the complexmg agent IS not bound to or entrapped in the matrix. As an example of selectivity manipulation, cyclodextrins (CD) can be used as complexing agents in polyacrylamide capillary gel electrophoresis (20). CDs are nonionic cyclic polysaccharides of glucose with the shape of a toroid or hollow truncated cone. The

Capillary

165

Gel Electrophoresis

‘: I

34

1

2

I 0

”

”

”

1”

! ” 10

”

”

’

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I 20

”

“1

min

Fig. 4. High-effrclency capillary gel electrophoresls separation of Dns-m-amino acids usmg P-cyclodextrin m the gel as chnal selector. Peaks: 1 = Dns-L-Glu, 2 = Dns-n-Glu, 3 = Dns-L-Ser,4 = Dns-D-Ser,5 = Dns-L-Leu, 6 = Dns-D-Leu(seeref. 17). cavity IS relatively hydrophobic, whereas the external faces are hydrophilic. The torus of the larger circumference contains chiral secondary hydroxyl groups. By incorporation of j3-cyclodextrin into the gel matrix, the complexing agent is practically immobilized (since the CD has no charge) particularly when the pore size of the gel is smaller than the size of the P-CD (cl .4 nm). Cyclodextrins can be used for chiral separations, since chiral selectivity can arise from the entrance of the cavity with the chiral glucose moiety. Figure 4 shows a high-resolution chiral separation of a mixture of dansylated amino acids based on the chiral recognition of the P-cyclodextrin incorporated into the polyacrylamide gel matrix. 3.5.4. SDS-Protein

Capillary

Gel Electrophoresis

Figure 5 shows a separation of a protein test mixture in the mol-wt range of 14,00&98,000 Dalton in 16 min by means of a polymer network filled capillary column (eCAP SDS-200). An internal standard (Orange G) was used in order to increase migration time precision, and thereby improve the accuracy of the mol-wt determination of an unknown protein. The actual peak shape in the case of capillary gel SDS-protein elec-

Guttman

166

I

;

L

.ge cm

8 m

x N

:: m

x w

Fig. 5. Capillary SDS gel electrophoresls of SIX proteins on an eCAP SDS-200 capillary. Peaks: (1) lysozyme (14,400), (2) soybean trypsm mhlbltor (21,500); (3) carbomc anhydrase (mol wt 29,000); (4) ovalbumm (mol wt 45,000); (5) bovine serum albumin (mol wt 66,000); (6) phosphorylase B (97,400). A trackmg dye, Orange G (OG), was added to the sample. Condltlons: Injected amount, 100 ng protein. Detection 2 14 nm. Run temperature, 2OT; applied electric field, 300 V/cm; current 25-30 PA (courtesy of Beckman Instruments).

trophoresis IS a function of the complexation phenomena between the unfolded protein chain and the SDS micelles. Thus, depending on the actual protein molecule, peak efficiencies can be different. As an example m Fig. 5, the slower migrating phosphorylase B peak (#6) is sharper than the preceding ovalbumin (#4). The same column and sieving matrix can be used for even higher mol-wt proteins up to several hundred thousand Dalton (21). Figure 6 shows the mol-wt calibration curve of the standard protein mixture of Fig. 5. This particular gel can be used in fast screening methods for product purity checks, as well as mol-wt determination. The same replaceable concept applies here as previously described for the replaceable polyacrylamide gels. Depending on the purity of the sample, the polymer network can be replaced after each run

Capillary MOBILITY

167

Gel Electrophoresis (lOE-5)

1.1 i

I 100

07 10

30 LOG MW llOE3)

Fig. 6. Relationshrp between the logarithmic mol wt and the mobility. Data points from the electropherogram in Fig. 5. Correlatton coefficient: 0.995 (courtesy of Beckman Instruments) if necessary. This mmimtzes column contamination column lifetime.

3.5.5. Carbohydrate Separation by Capillary

while increasmg

Gel Electrophoresis

In recent years there has been considerable activity in the development of simple, rapid, and reliable separation methods for the analysis of complex carbohydrates released from a variety of glycoconjugates, including glycoproteins, glycolipids, proteoglycans, and glycosammoglycans (4). The high resolving power of CE for the separation of complex carbohydrates has been demonstrated earlier in capillary zone electrophoresis (22) and in capillary gel electrophoresis (23) modes. Since most carbohydrate molecules are not charged and have no significant absorbance of UV light at the common wavelength range of CE-detection systems (200600 nm), a derivatization procedure is required before analysis. This derivatization usually involves the stoichiometric labeling of the reducing end of the oligosaccharide with a labeling reagent (#,22). In most instances, the UVMuorescence dye disodium 8-amino- 1,3,6-naphtalene trisulfonate (ANTS) was employed as a labeling tag followed by reduction using NaCNBH,. The fluorescently labeled oligosaccharides are then separated and quantified by CE using UV (214 nm) or laser-induced fluorescent (LIF) detection (360 nm He-Cd laser).

Guttman

168 3

8 fii 2 P 2s LL 1 0 I

0

6

u I

I

I

I

5

IO

15

20

Migration

Time (min)

Fig. 7. Capillary electrophoretlc-LIF separation of the ANTS-labeled wheat starch digest Numbers correspond to the degree of polymenzatlon (see ref. 23). Figure 7 shows a fast (Cl 5 mm), high resolution separation of wheat starch digest at 20°C on a 40-cm long capillary column. The average plate count for these separations was higher than one million plate/meter, which corresponds to 3500 plate/m/s for the maltose (Fig. 7, peak/# 2). References 1 Landers, J. P (ed.) (1994) Handbook of Capzllary Electrophoreszs CRC, Boca Raton, FL 2 LI, S F. Y. (1993) Caprllary Electrophoresu. Elsevier, Amsterdam, The Netherlands. 3 Schwartz, H and Guttman, A (1995) Separation of DNA by Capillary Electrophoreszs Beckman Primer 607397, Fullerton, CA 4 El Rassi, Z (ed ) (1995) Carbohydrate Analyszs Elsevler, Amsterdam, The Netherlands 5 Cohen, A S , Najanan, D R , Paulus, A., Guttman, A , Smith, J. A., and Karger, B L. (1988) Rapld separation and purlficatlon of oligonucleotldes by high performance capillary electrophoresls Proc Nat1 Acad Ser. USA 85,9660-9663 6. Lux, J. A., Ym, H F , and Schomburg, G (1990) A simple method for the production of gel-filled caplllarles for capillary gel electrophoresis J Hzgh Resolut. Chromatogr

13,436,437

7 Helger, D. N , Cohen, A. S., and Karger, B. L. (1990) Separation of DNA restriction fragments by high performance capillary electrophoresls with low and zero

Capillary

Gel Electrophoresis

169

crosslmked polyacrylamide using continuous and pulsed electric fields J Chromatogr. 516,3348. 8. Guttman, A. and Cooke, N (1991) Effect of the temperature on the separation of DNA restriction fragments in capillary gel electrophorests. J Chromatogr 559, 285-294 9. Tanaka, T. (1981) Gels. Scl Am 244, 124-138 10 Guttman, A. and Cooke, N (1991) Capillary gel affinity electrophoresis of DNA fragments. Anal Chem 63,2038-2042. 11 Nelson, R. J., Paulus, A , Cohen, A S , Guttman, A., and Karger, B. L. (1989) Use of peltier thermoelectric device to control column temperature in high performance capillary electrophoresis. J Chromatogr 480, 111-127. 12 Cohen, A. S. and Karger, B L. (1987) High performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins J Chromatogr 397,409-4 17 13. Guttman, A. and Mazsaroff, I (1992) Economical performance analysis m preparative capillary gel electrophoresis, m New Approaches In Lzquzd Chromatography, Intercongress, Budapest, Hungary, 63-75 14 Pentoney, S L., Konrad, K. D , and Kaye, W. (1992) A single-fluor approach to DNA sequence determmation using high performance capillary electrophoresis. Electrophoresrs 13,467-474. 15 Rutz-Martinez, M. C , Berka, J., Belenki, A., Foret, F , Miller, A W., and Karger, B L (1993) DNA sequencing by capillary electrophoresis with replaceable linear polyacrylamide and laser induced fluorescence detection. Anal Chem 65,285 l-2858 16. Dovichi, N. (1994) Handbook of Capzllary Electrophoresls (Landers, J P., ed ) CRC, Boca Raton, FL 17 Guttman, A , Nelson, R. J., and Cooke, N (1992) Prediction of migration behavior of ohgonucleottdes in capillary gel electrophorests J Chromatogr 593,297-303 18. Schwartz, H E , Ulfelder, K. J , Sunzeri, F J , Busch, F J., and Brownlee, R. G (199 1) Analysis of DNA restriction fragments and polymerase cham reaction products towards detection of the AIDS (HIV-l) virus m blood. J Chromatogr 559, 267-283 19. Guttman, A , Wanders, B., and Cooke, N (1992) Enhanced separation of DNA restriction fragments by capillary gel electrophoresis using field strength gradients. Anal Chem 64,2348-235 1. 20. Guttman, A , Paulus, A., Cohen, A. S., Grinberg, N., and Karger, B L. (1988) Use of complexing agents for selective separation m high performance capillary electrophoresis, chwal resolution via cyclodextrms incorporated with polyacrylamide gels. J Chromutogr 448,41-53. 21. Guttman, A , Shich, P , and Cooke, N. (1992) P/ACE SDS capillary gel electrophoresis of protcrns Beckman Technzcal Information Bulletrn, #DS-827. 22. Chiesa, C and Horvath, C. S. (1993) Capillary zone electrophoresis of maltoohgosaccharides derivatized with 8-ammonaphtalene-1,3,6-trisulfomc acid J, Chromutogr 645,337-352 23 Guttman, A , Cooke, N , and Starr, C. (1994) Capdlaty electrophoresis separation of ohgosacchandes I. effect of operational variables Electrophoresls 15, 15 18-l 522

CHAPTER 14

Chiral by Capillary Manus

Separations Electrophoresis

M. Rogan

and Kevin

D. Altria

1. Introduction Many papers have appeared concerning the application of capillary electrophoresis (CE) to the resolution of chiral compounds. Predominantly, separations have been achieved in Free Solution Capillary Electrophoresis (FSCE) employing cyclodextrins (CDs) as chiral selectors. This is because of their ready availability and broad applicability. Chiral resolutions have also been achieved m micellar electrokinetic chromatography (MEKC) through the use of chirally selective micelles. However, the variety of approaches has increased significantly. In this chapter, the use and applicability of these methods are considered. Chirally selective CE methods can have significant advantages over HPLC counterparts in terms of increased ruggedness and reductions in both method development time and cost. Acceptable performance levels are achievable, and validated methods are routinely used m many laboratories. In excess of 100 papers have appeared on this subject, and several reviews have been published (I-6). This chapter is divided into sections describing the various selectivity options, method development and optimization, and the application of chiral CE methods. 2. FSCE In chiral FSCE, optically active reagents are added to the electrolyte in order to achieve enantioselective interactions with the solute molecules. Choral recognition agents used in FSCE have included CDs, proteins, carbohydrates, and crown ethers. From

Methods

Edtted

m Molecular

by K Altna

Biology,

Copyright

Vol 52 Caprtlary E/8CtrOphOf8s/s Humana Press Inc. Totowa, NJ

171

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Fig. 1. Principles of chiral separation for basic compounds using CD additives. A,+ and Ad+ are “I” and “d” enantiomers,respectively. 2.1. CE Employing CDs The most widely reported chiral separation mode is operation at low pH utilizing CDs as chiral recognition agents for the resolution of basic compounds. There are currently wide ranges of both native and derivatized CDs commercially available. The native CDs, a, p, y, possess different numbers of glucose subunits, being six, seven, and eight, respectively. Enantioselective recognition is often explained by interactions between the CD (which possessesmany chiral centers) and the guest enantiomers. By derivatization of the surface hydroxyl groups on the cyclodextrin, the solubility and enantioselective properties can be significantly altered. Hydroxypropyl- and dimethyl-cyclodextrins are two such derivatives, which when compared to the native forms, differ significantly. In addition, CDs having acidic or basic ionizable groups are available for separation of uncharged solutes. These derivatived CDs are available from a number of chemical suppliers. The basis for the separation is the stereospecific interaction of the analyte with the CD cavity and surface (see Fig. 1). At low pH, EOF is minimal, and the uncharged CD does not migrate. Basic analytes are protonated at this pH and migrate toward the detector by virtue of their charge. When the solute interacts with the CD, its mobility is greatly reduced. If the two enantiomers have differing stability constants, then one enantiomer will migrate more slowly than the other, and chiral resolution will be achieved. Figure 1 shows the process diagrammatically. A number of groups have exploited CDs in the free solution mode. For example, Fanali (7,8) has reported the enantiomeric resolution of

Chiral Separations

6

173

.

i

i

mm

Fig. 2. Chiral resolutton of dtmethidine and a range of related compounds (reproduced with permission from ref. 9) Peak 1 = N-demethyl dimethldme, 2 = dlmethidme, 3 = N-demethyl-6-methoxy-dlmethtdme, 4 = 6-methoxy-dtmethyl-dlmethidme, 5 = dlmethldme-N-oxide, pH 3, electrolyte contammg hydroxypropyl+CD, ephedrine, norephedrine, epinephrine, norepinephrme, and tsoproterenol using dimethyl-P-CD. Dimethidine and a range of related compounds (9) have been simultaneously chirally resolved (Fig. 2). Free solution CE employmg CDs can also be performed at high pH

(10, II). Under these conditions, a different separation mechanism exists. The acidic solute is negatively charged and opposes the EOF. The strong EOF sweeps the neutral CD to the detector, and when the anionic solute interacts with the migrating neutral CD, its migration velocity is reduced. Therefore, the enantiomer having the most interactions migrates before the other stereorsomer, a result that is the reverse of that obtained at low PH (W. Alternatively, ionizable CDs can be employed for the separation of neutral solutes or for the improved separation of charged analytes (12). For example, sulfated CDs can be used to affect separation of neutral species at high-pH values. Carboxymethyl-P-cyclodextrin (C-Den) was used to resolve six enantiomeric dansylated amino acids (13).

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R

Fig 3. Complex of primary amine with crown etherwhere R ISa substituent group or groups

2.2. FSCE Employing Crown Ethers Crown ethers are macromolecules that can be employed to achieve chiral separation of primary amines. A typical structure of a crown ether/ primary amine complex is given in Fig. 3. The separation process is similar to CD-based FSCE in that the enantromers of prrmary ammes form complexes with the crown ether and their migration is retarded. The NH3+ group of the solute binds with the crown ether via hydrogen bonding, and enantrorecogmtion depends on the secondary interactions of the crown ether substituents with the functional groups on the analyte. Kuhn et al. (14) were the first to report the use of [18] crown-6-tetracarboxylic acid (18C6) as a selector in CE for separation of tryptophan and DOPA enanttomers. No buffer substituents were used in this work becausethe cations were found to interact compettttvely with the additive. The enantiomers of phenylalanine, phenylglycine methoxamme, and octopamine have been resolved using 18C6 (15). Crown ethers have also been employed for the resolution of a number of optrcally active amines of pharmaceutical interest (6,16, I 7). 2.3. FSCE Employing Enantioselective Metal Chelation In this separation mechanism, a transition metal/ammo acid (R or S) complex is added to the electrolyte. The coordination number of the complex is not fully satisfied, and therefore, solutes of an appropriate structure can form ternary diastereomeric complexes. If there are differences

Chiral Separations

175

in complex stability for the two stereoisomers, enantioselective recognition can be achieved. The more complexed enantiomers migrate faster than their lesser complexed counterparts. This approach has been used to resolve enantiomerically seven pairs of dansylated amino acids (18) using a Cu2+-aspartame complex as a background electrolyte additive. 2.4. FSCE Employing

Carbohydrates

It has been shown that additions of 2-10% of maltodextrins (mixtures of linear linked D-glucose polymers) can be used for chiral resolution of acidic drugs (‘19), such as nonsteroidal anti-inflammatories (NSAIDs), and anticoagulants, such as warfarin. In a second report, dextrins were employed for the chiral separation of a range of drug molecules and showed different enantioselectivities to CDs (‘20). 2.5. FSCE Employing

Proteins

Proteins are widely utilized in HPLC as chiral phases, since they possess multiple chiral centers. Similarly, proteins, such as bovine serum albumin (BSA) (21-23), and ovomucoid (OVM) (24) have been successfully employed in choral FSCE. Typical protem concentrations are on the order of a few milhgrams per milliliter, which makes them a realistic possibility from a cost perspective. The separations are typically performed at pH 7, where most proteins are negatively charged and migrate against the EOF. Favorable solute interactions with the proteins retard migration and lead to the possibility of chiral resolution. BSA has been successful in chiral separation of a range of drugs, including warfarin, benzoin and tryptophan (24), and leucovorin (21,22). Valtcheva et al. (25) successfully employed the protein cellobiohydrolase I (CBH I) as an additive in FSCE to resolve a series of P-blockers (propranolol, alprenolol, metoprolol, and pindolol). The addition of organic solvents, such as 2-propanol, was shown to be of importance for obtaining chiral separation. 2.6. FSCE Employing

GZycopeptides

In this separation mode, analytes selectively bind to chiral glycopeptides, such as vancomycin. The enantioselective binding of vancomycin to various chiral dipeptides has been extensively studied (26). Armstrong et al. (27) have recently reported the use of vancomycin to resolve over 100 different racemic drugs and amino acids, often with very high enantioselectivities.

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3. Capillary Gel Electrophoresis Chapter 13 deals with the general use of capillary gel electrophoresis. For chiral separations, capillaries can be filled with gels containing a chiral selector, such as a CD (28) or a protein. Incorporation of the additive into a suitable gel immobilizes the analyte-selector complex, thus maximizing the separation potential. A range of amino acids were resolved employmg a capillary tilled with a polyacrylamide gel containing P-CD (28). Resolution was improved by increasing the CD concentration, addition of methanol, and a reduction m operating temperature. Another recent report (29) has shown copolymerization of ally1 carbamoylated-P-CD with acrylamide to form gels. Separation of seven choral molecules, mcludmg ammo acids, were shown. Electrolytes containing P-CD polymers have been used in the efficient separation of a range of basic drugs (30). Gels have also been produced by the crosslinking of BSA with glutaraldehyde (31). This gel was employed to separate the enantiomers of tryptophan. BSA has also been employed in FSCE as an electrolyte (21), additive for chiral separations. 4. MEKC Terabe (32) mtroduced MEKC as a means of separating neutral species. Chapter 12 discusses in detail the prmciples of MEKC. It was reasoned that selectivity is achieved owmg to differences m the analyte partitioning mto the hydrophobic micelle core and/or ion-pair mteractions with the hydrophilic head groups. Chn-al separations can be achieved by using a choral surfactant or by adding a choral selector, such as a CD, to the MEKC electrolyte.

4.1. MEKC

Employing

Cyclodextrins

It is believed that CDs are solubilized by SDS micelles by effectively lining the interior of the micelle. This gives the CD a transient net negative charge, which therefore migrates against the EOF. When enantiomers are solubilized by the micelle, the stereoisomer forming the more stable complex with the CD (A, in Fig. 4) elutes later Barbital, thiobarbital, pentobarbttal, and phenobarbital have been resolved using this CD-based MEKC (33). y-CD was the most useful selector for enantiomerically resolving these relatively large molecules. Derivatized amino acids have also been separated using y-CD (34).

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177

Fig. 4. CD-modified SDS MEKC diagram where A, and Ad are “1” and “d” enantiomers,respectively.

4.2. MEKC Employing Mixed Micelles It is possible to employ combinations of different chiral and achiral surfactants to alter separation selectivity in MEKC. Several reports have described chiral separations, which were achieved using SDS or a similar achiral surfactant in conjunction with a chirally selective surfactant. A nonionic chiral surf’actant (Digitonin) was mixed with anionic SDS to resolve optically sixderivatized amino acids (35), aswell asbenzoin and warfarin (36). 4.3. MEKC Employing Chiral Surfactants Bile acids are naturally occurring, steroid-based chiral surfactant compounds, which are available as sodium cholate, deoxycholate, or taurocholate salts. Bile salt micelles have a planar sheet-type structure and have been used in MEKC for the chiral analysis of some derivatized amino acids (37,38) and optically active drugs (39,40), such as diltiazem and trimetoquinol. Addition of organic solvents, such as 2-propanol and methanol (411, has been shown to enhance separations. Modification of the ubiquitious SDS-type surfactant to form monomers, such as dodecoxycarbonyl-valine (42) and dodecanoyl-1-serine (43),,has been shown to be a viable option for achieving chiral separations. The migration order of the solutes can be reversed by changing from the (1)~surfactant to its (d)-form.

5. Electrochromatography This separation method involves use of CE capillaries filled (or coated) with HPLC-type packing material. Chapter 15 describes the background to electrochromatography.

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Fused silica CE capillaries have been coated with a 0.2~mm layer of Chnasil-Dex (permethylated P-CD) and used to resolve chnally 1, l’binaphyl-2,2’-diyl-hydrogen phosphate and 1-phenyl ethanol (44,451. Capillaries filled with HPLC packing material coated with or-acid glycoprotein (AGP) have been used to resolve a range of compounds (46), including benzoin, hexobarbital, pentobarbital, alprenolol, and propranolol. Addition of organic solvents, such as acetonitrile, was shown to improve chiral resolution. 6. Preseparation Derivatization As with chromatographic techniques, preseparation derivatizatton of analytes with a chiral reagent to form a diastereoisomer can prove to be a useful means for enantiomertc resolution of chiral compounds. Diastereoisomers can be resolved by achnal separation methods. For example, amino acids were derivatized using Marfey’s reagent to form the corresponding diastereomertc pairs (47). These pairs were then resolved using FSCE and MEKC with no chiral additives. Enantiomeric amphetamines and related compounds were derivatized with 2,3,4,6-tetra-0acetyl-P-u-glucopyranosyl isothiocynate (GITC) and resolved by SDS-based MEKC (481, and forensic samples were analyzed. 7. Method Development The followmg options are suggestedas starting points in method development and, as such, may produce some chiral recognition. Methods should then be optimized using suggestions given in Section 8. Method development and optimization can be relatively swift in chiral CE, since the high degree of automation available with CE instruments allows a range of electrolyte systems and operating parameters to be assessed overnight. The most appropriate electrolyte composition can then be selected for future optimization. Samples are best prepared in water or a 1:10 dilution of the run electrolyte to maximize stacking effects (see Chapter 16 for a fuller discussion of stacking). Alternatively, if samples are only soluble in organic solvents, then the minimum amount of organic solvent should be used to solubilize the sample. 7.1. Basic Compounds The majority of pharmaceuticals contain amine functions, which can be protonated at low pH. Typically, 50 mMNaH2P04 buffer pH adjusted

Chiral

Separations

179

Fig. 5. Method development guidelines for a basic compound.

to 2.5 with cont. H3P04may be a suitable starting electrolyte. The solute should be run using this electrolyte to ensurean adequateresponseand appropriateinitial analysis time. A useful initial experimental setupmay be to usea 40 cm x 50 urn capillary with a +20 kV appliedvoltage; 15mA4 may be selectedas the initial CD concentration.As a rough guide, if the compound contains no aromatic rings, then a- or derivatized a-CD should be considered.It may be more appropriate if the compound contains a single aromaticring to considerp- or derivatized P-CDs.Analytes containing multiple rings would suggestthe use of y-CDs. Addition of 20 mM crown ether can be explored for analytes containing primary amine groups. Figure 5 shows a flowchart indicating the possible progression of method development. 7.2. Acidic

Compounds

The sampleshould be run using 20 mM borax to ensurean appropriate initial analysis time and detector response.CD should be added to this electrolyte; 15 mM hydroxypropyl+CD may be a useful starting point (Fig. 6). As for basic compounds,the CD type and concentration should be optimized. It may be useful to consider use of anionic derivatized CDs, such asthose having acidic groups. If the use of CDs is unsuccess-

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Fig. 6. Method development guidelines for an acidic compound. ful, then use of a chiral MEKC electrolyte or an FSCE electrolyte containing a protein or oligosaccharide should be attempted. 7.3. Small

Neutral

Compounds

It may be appropriate for small compounds containing less than two aromatic rings to employ 20 mM borax, 50 mM SDS, and 15 rnA4 P-CD. The CD type and concentration should be optimized (Fig. 7). If no resolution is achieved, then use of mixed micelles or alternative chiral surfactants should be considered. 7.4. Large

Neutral

Compounds

Bile salts are able to solubilize large neutral compounds and are used at mid-high-pH values; 20 mA4 borax containing 50 mM sodium cholate is a useful starting point. Alternative bile salts include taurocholate, dexoxycholate, and taurodeoxycholate. Again, the concentration of the bile salt should be optimized (Fig. 8). Chiral resolutions can often be improved, or achieved, using addition of organic solvents, such as isopropanol. Combinations of bile salts and CDs have also been successful.

Chiral Separations

181

Fig. 7. Method development guidelines for small neutral compounds. 8. Method Optimization The following factors are known to influence chiral resolution: PH. Cyclodextrin concentration and type. Electrolyte composition. Organic solvents. Operating temperature. Polymeric additives. Capillary coating. Sample loading. Table 1 indicates the possible effects of increasing a number of operating variables on resolution and analysis time. 8.1. Effect of Operating pH The ionization of both analyte and CDs is also controlled by choice of operating pH. MEKC is generally performed at pH 7.0 or above. Thus, the optimization of pH is essential (49) to achieve selectand and selector ionic states, which best favors the interactions governing enantioselectivity. Another consequence of pH changes is the alteration in solute

Rogan and Aitria

182

Fig. 8. Method development guidelines for large neutral compounds. Table 1 Table of the Effects of OperatingParameters Variable

Range

Voltage Current Capillary length Capillary bore PH

5-30 kV 5-250 pA 20-l 00 cm 25400 pm 1.5-11.5

Organic solvents Urea

l-30% v/v l-7A4

Ion-pair reagent CD type and size Cyclodextrins

l-20 mM Various l-100 mA4

Polymer

0.1-0.5%

Electrolyte cont.

S-200 mM

Injection time Bilt salt

l-20 s lo-50 mA4

Effect of increasingvariable Reducedanalysistime, somelossin resolution Reducedanalysistime, somelossin resolution Increasedanalysistime, gain in resolution Increasedcurrent, someloss in resolution IncreasedEOF, increasedionization of acids,reducedionization of bases Gain or loss of resolution Increasedsolubilization of hydrophobic solutes(and CDs) Can reduceor increaseresolution Large impact on chiral selectivity Increasedviscosity, reducedEOF, increased solute migration if complexation occurs ReduceEOF, longer migration times, increasedefficiency Increasedresolution,increasedcurrent,lower EOF and solute ionization, reducedtailing Improved signal, someloss of resolution Choice has large impact on chiral selectivity

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183

electrophoretic mobility and, hence, migration time. The pH of the electrolyte should be optimized m terms of resolution and speed of analysis. 8.2. Cyclodextrin Type and Concentration The hydroxyl groups on the native CDs can be chemically modified to form various derivatives. A range of different CDs should be explored to establish which gives the best chiral resolution (.50,51). The extent of chemical derivatization, known as the degree of substitution, varies between CD manufacturers. The magnitude of the chiral separation has been shown to be dependent on this degree of substitution (11,.51). Since the chiral recognition mechanism is based on complexation, increasing the concentration of CD present improves the probability of interactions. However, from equilbrium studies, an optimum CD concentration exists for each chiral resolution (52). Increasing CD concentration above this level leads to a decrease in resolution. Optimum concentrations ranging from 5 (53) to 112 mM (54) have been reported and are dependent on the extent of complexation. 8.3. Electrolyte Composition Resolution is improved by increasing electrolyte concentration (50,51) owing to a decrease in peak tailing. However, the concentration should not be raised excessively, because this will lead to poorer resolution owing to an increase m Joule heating within the capillary. The composition of the electrolyte is also critical to the selectivity achieved in CDbased separations (55). For example, baseline resolution of quinagolide enantiomers was achieved employing a phosphate electrolyte at pH 1.5 (55). However, at the samepH, poorer resolution was obtained for a glycme/ HCl electrolyte, and no chiral recognition was observed in a citric acid/ HCl system. This is thought to be the result of competitive binding of the electrolyte counterions with the selector. 8.4. Organic Solvents The use of organic solvents, such as methanol, has been shown to improve chiral resolution in certain instances (53,56). Chiral separation of propranolol was only achieved using FSCE with 30% methanol (4A4 urea, 40 mA4 CD) (53). Therefore, during method development, a range of different types and concentrations of organic solvents should be considered if resolution is insufficient.

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8.5. Operating Temperature At lower temperatures, the mass-transfer kinetics of the analyte-CD complex is affected. A lowering of temperature has been shown to improve resolution (SO). Therefore, it is recommended that the lowest feasible temperature should be employed to maximize resolution. However, a correspondmg increase in migration time is observed. 8.6. Polymeric Additives Enantioselectivity and separation efficiency can be dramatically improved by addition of a polymeric additive to the electrolyte. Solution viscosity increases, which increases peak efficiencies, and peak tailmg is reduced. For example, 0.1% methyl hydroxyethyl cellulose was found to improve separation of chloramphenicol enantiomers greatly (4). An alternative additive is poly vinyl alcohol (PVA), which has been used to separate tocainide and related compounds (5 7). 8.7. Capillary Coatings It has been demonstrated that coating of the capillary with polyacrylamide can enhance chiral selectivity (53). Reasons for this include the elimination of EOF and prevention of reversible adsorption of solute onto the walls, thereby reducing peak tailing. It is also possible to improve peak shape and chiral resolution by dynamically coating the capillary with cellulose or polyvinyl alcohol (57). 8.8. Sample Loading Increases in either sample concentration or injection time lead to a decrease in peak resolution (.50,51). Improved peak area precision is obtained at higher loadings. Therefore, a compromise must be reached. High sample loadings are neccessary when attempting to quantify trace levels of an undesired enantiomer in a single enantiomeric compound. 8.9. Statistical Method Optimization A Plackett-Burman design has been used to screen the effects of various separation parameters in a chiral CE separation of the bronchodilator clenbuterol (58). The results allowed conclusions to be drawn that compared favorably with those resulting from a univariate approach, but within much fewer experiments. 9. Method Performance Validation of chiral CE methods includes (51,59,60) assessments of limits of detection and quantitation for the undesired enantiomer, linear-

185

Chiral Separations 5

BCH (+) enantlomer

I BCH (-) enantlomer

400

0.00

Retentlon

14.00

tlme In mlnutea

19.00

24.00

Fig. 9. Analysis of a single enantiomer containmg 0 3% of the undesu-ed enantlomer (reprinted with permlsslon from ref. 66). Separation conditions: electrolyte, 50 mMdlmethyl-P-cyclodextrin m 50 Mborax adjusted to pH 2.5 with cont. H,P04; detection, 2 14 nm; voltage, 13 kV; capillary, 47 cm x 50 pm.

ity of detector response, robustness, recovery, precision, freedom from interference, and method robustness. 9.1. Detection Limits Single enantiomeric chiral compounds may often be produced to high enantiomeric purities, with ~1% of the undesired enantiomers present. Detection levels of
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enantiomeric ratios. Adequate and acceptable detector lmearities for both main peak concentration and for varying levels of the undesired enantiomer have been reported with typical linearity data of >0.998 described (X,55,59,62). 9.3. Precision Essentially, each enantiomer acts as an internal standard for the other, and therefore, good precision for peak area ratios for enanttomeric mixtures can be obtained. For example, in an intercompany crossvalidation exercise (67) for the separation of racemic clenbuterol, all seven participating companies obtained RSD values of
Evaluation

Successful method transfer (67) between seven independent laboratories of a method for the resolution of clenbuterol enantiomers demonstrated method robustness. Ruggedness testmg of a chnal CE method (51) involved use of CD from different suppliers, different instruments, different capillaries, and different days. 9.6. Crossvalidation Good agreement between enantiomeric purity results generated by both CE and HPLC methods has been reported (65) for the testing of a single enantiomer drug. Similar agreement between HPLC and CE has also been reported in the enantromeric purity testing of phenoxy acid herbicides (68).

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I 12 16

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’

’

3

26

1

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Fig. 10. Separation of a 60:40 mixture of fluparaoxan enantiomers (reprinted with permission from ref. 59). Separation conditions: electrolyte (150 mA4 P-cyclodextrin, 10 mA4borax, 10 mA4 Tris, 6M urea), lsopropanol (80:20 v/v) adjusted to pH 2.5 with cont. H3P04; detection, 214 nm, voltage, 16 kV; capillary, 57 cm x 50 pm.

10. Application Areas The quantitative uses of chirally selective CE methods has centered on enantiomeric purity testing of drug products, analysis of formulations, reaction rate monitoring, and forensic and clinical applications. 10.1. Enantiomeric Purity Testing Enantiomeric purity testing has been reported for norephedrine (62), ephedrine (621, fluparoxan (59), Sandoz drug EN 792 (5.5), and Lilly

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Fig. 11. Separation of a 60:40 mrxture of fluparoxan enantiomers (reprinted with pernnssion from ref. 62). Separation conditions: electrolyte, 40 g/L drmethyl+-cyclodextrin m 30 mM Trrs-phosphate, pH 3; detectton, 200 nm; voltage, 25 kV; capillary, 70 cm x 50 pm drug LY248686 (51). A chirally selective MEKC method has been used to assess the optical purity testing of batches of trimequinol hydrochloride (40). The optical purity of production batches of herbrctdes has been tested by CE (68). Figure 11 shows analysis of two optically pure drugs

containing 1% of their stereoisomers. 10.2. Reaction

Rate Monitoring

The robust nature of chiral CE separations makes the monitoring of chirally selective processespossible. To date, only one report has appeared concerning this area. A chirally selective CE method was employed to monitor the enzymatic transformation of a racemate to a single enantiomer (64). The reaction was monitored over a 5 l-h period. The final product was shown to contain only 0.5% of the undesired stereoisomer. 10.3. Formulation

Stability

Testing

Interconversion of enantiomers can occur with time. Therefore, it is often necessary to incorporate a chirally specific assay in stability studies when analyzing chiral compounds. In addition, it is important that the assay be both quick and simple, since many samples may be analyzed. The enantiomerlc purity of I-epinephrine m a pharmaceutical formula-

Chiral Separations

189

tion has been monitored (63), employing Z-pseudoepinephrme as an internal standard. Peak area ratio precisions of 1.8% RSD with 99% recoveries and detector linearities of >0.998 were reported. Stored samples were tested, and enantiomeric purity results were within specification. Samples stored past product shelf-life were shown to fail the specification set regarding undesired enantiomer level. The chiral analysis of phenylalanine in a pharmaceutical dosage form was achieved (10) within 70 s using a short capillary. The enantiomeric composttion of epinephrine m commercial samples was assessedby chn-al CE (81. 10.4. CLinical and Forensic Analysis Applications of CE in therapeutic drug level monitormg in bioflulds have been reviewed (69) and are described in Chapter 20. Often extensive sample work-up is required in HPLC owing to the presence of matrix components. These components may mask the peaks of interest or impair separation performance. However, in CE, it has been shown that biosamples may often be directly analyzed with no sample pretreatment. Clenbuterol enantiomers spiked in urine at a level of 10 mg/L have been resolved (60) following direct injection of the spiked urine sample. Hexobarbital at 50 ppm has been chnally resolved (70) from direct injection of rat plasma. The preferential metabolism of the (-) enantiomer of warfarin in patients undergoing warfarin therapy has been confirmed (71) by CE. Figure 12 shows an electropheropherogram of a patient undergoing warfarin treatment. Chlorowarfarin (ClWf) was used as an internal standard. Separation was achieved using an electrolyte containing CD at pH 8.5. A validated assay for the chiral separation of cicletanine in both plasma and urine has been reported (9). Chiral resolution of leucovorin and its major metabolite 5-methyl-tetrahydrofolate spiked into plasma has been shown (72) at therapeutic levels (low micromolar). Enantiomeric ratios of various amphetamines in several forensic samples have been determined by MEKC (48). The results obtained compared favorably with those generated by GC-MS. Separation of ephedrine enantiomers in urine by MEKC has been shown with simple filtration of the urine sample prior to injection (42). 10.5. Analysis of Related Substances If the compound under investigation is chiral, there is a strong likelihood that its related impurities are also chiral. It is often possible to

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Fig. 12. Electropheropherogram of a patlent undergomg warfarm treatment (reproduced with per-ml&on from ref. 72). Separation condltrons* electrolyte, 8 mM methyl-P-CD, 100 mA4 sodium phosphate buffer, pH 8.35, methanol (98:2 v/v); detection, 3 10; voltage, 20 kV; capillary, 72 cm x 50 pm. resolve simultaneously both the main compound and its related compounds using a single set of operating conditions. This is not normally possible in HPLC, where operating conditions are very specrtic to the resolution of the main compound. For example (9), dimethidine and several chiral-related compounds were resolved (Fig. 2). Use of a high concentration of dimethyl-P-CD allowed resolution (Fig. 13) of the bronchodilator salbutamol and several of its chiral and achiral-related impurities (54). A number of dilttazem-related substances were srmultaneously resolved using bile-salt-based MEKC conditions (40). 11. Conclusions CE is a useful addition to the techniques available for the resolution

and quantitation of enantiomers. Methods have been validated and can give similar performance levels to those obtained by HPLC. Successful method transfer has also been demonstrated. Particular features of chirally selective CE methods include simplicity, ruggedness, and low cost of reagents and capillaries. Application areas of quantitative CE analysis reported to date include enantiomeric purity testing of drugs and herbicides, reaction rate monitoring, stability testing, and the analysis of

191

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Mtgration hme (mms)

Fig. 13. Resolution of salbutamol and several of its choral and achu-al lmpuritles (reproduced with permission from ref. 54). Salbutamol peaks III.1 and 111.2.Separation conditions: electrolyte, 112 mM dimethyl+CD, phosphate/ citrate buffer, pH 2.5, detection, 214; voltage, 15kV; capillary, 72 cm x 75 pm.

clinical and forensic samples. Undoubtedly, the number of quantitative applications areas will greatly expand within the near future as CE becomes more established across a variety of industries. Notes Added in Proof A recent extensive review paper has been published by Ntshi and Terabe (73) that gives details of 146 relevant papers covering applications and separation principles. The broad applicability of cyclodextrins has continued to be demonstrated; for example, 22 racemic basic compounds were resolved using CDs and ion-pair reagent (74), 42 different racemates were separated using modified+cyclodextrins (75), and 22 chiral drugs were resolved (76) using mixtures of CDs and bile salts. The selectivity options have been expanded to include the antibiotic ristocetin A, which was used to chirally resolve (77) over 120 racemates. The ability to reverse enantiomeric migration order by selecting appropriate derivatized cyclodextrins has been shown (78). Validation reports continue to expand; for instance, an optimized method for separation of

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naproxen enantiomers has been shown (79) to give good validation data, including a 0.1% LOD. A similar detection of 0.06% was reported (80) for the unwanted enantiomer of a cholesterol-lowering drug using a CDmodified MEKC method. Validatron also included robustness studies, linearity, recoveries at low enantiomer spiking levels, and correlation with HPLC data. References 1 Rogan, M M , Goodall, D. M , and Altria, K D (1994) Enantioselecttve separations using capillary electrophorests Chzrallty 6,25-40 2 Kuhn, R and Hofstetter-Kuhn, S (1992) Chnal separation by caprllary electrophorests Chromatographla 34,505-5 12 3 Snopek, J , Jelmek, I , and Smolkova-Keulemansova, E (1988) Mrcellar, mclusion and metal-complex enantioselective pseudophases m high-performance electromlgratron methods. J Chromatogr 452, 57 l-590 4. Snopek, J , Souu, H , Novotony, M., Smolkova-Keulemansova, E , and Jelmek, I (199 1) Selected apphcations of cyclodextrm selectors m capillary electrophoresis J Chromatogr 559,2 15-222 5 Otsuka, K and Terabe, S. (1993) Enanttomeric separation by micellar electrokinetlc chromatography TrAC 12, 125-130 6 Bereuter, T L (1994) Enantioseparation by capillary electrophoresis LC-GC Int 7, 78-93. 7. Fanah, S. (1989) Separation of optical tsomers by capillary zone electrophorests based on host-guest complexation wtth cyclodextrms J Chromatogr 474,441446 8. Fanalt, S. and Bocek, P (1990) Enantlomertc resolutton by using capillary zone electrophorests: resolution of racemic trytophan and determmatton of the enantiomer composition of commercial pharmaceutrcal epmephrme Electrophoreszs 11, 757-760. 9. Heuremann, M and Blascke, G. (1993) Chiral separation of basic drugs using cyclodextrms as chiral pseudo-stationary phases m capillary electrophoresis J Chromatogr 648,267-274 10. Sepamak, M. J , Cole, R O., and Clark, B. K (1992) Use of native and chemically modified cyclodextrms for the captllary electrophorettc separation of racemates. J Liquid Chromatogr. 15,1023-l 040 11. Valko, I. E., B&et, H A. H., Frank, J., and Luyben, K. C A. M. (1994) Effect of the degree of substttution of (2-hydroxy)propyl+cyclodextrm on the enanttoseparation of organic acids by captllary electrophorests J Chromatogr 678, 139-144. 12 Nardt, A., Eltseev, A , Bocek, P , and Fanah, S. (1993) Use of charged and neutral cyclodextrms in captllary zone electrophoresis enantiomertc separation of some 2-hydroxy acids. J Chromatogr. 638,247-253 13. Terabe, S , Ozakt, H , Otsuka, K., and Ando, T (1985) Electrokmetic chromatography with 2-0-carboxymethyl-P-cyclodextrm as a moving “stationary phase ” J Chromatogr 332,2 1 l-2 17

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14. Kuhn, R., Stoecklm, F., and Erm, F. (1992) Choral separations by host-guest complexation with cyclodextrms and crown ethers by capillary zone electrophoresis Chromatographla 33,32-36 15 Hohne, E , Krauss, G J , and Gubitz, G (1992) Capillary zone electrophoresis of the enantiomers of aminoalcohols based on host-guest complexation with chiral crown ethers. J Hugh Res Chromatogr. Comm 15,698-700 16. Kuhn, R., Erni, F., Bereuter, T., and Hauser, J. (1992) Choral recognmon and enantiomeric resolution based on host-guest complexation with crown ethers m capillary zone electrophoresis. Anal Chem. 64,28 15-2820 17. Walbroehl, Y. and Wagner, J (1994) Enantiomeric resolution of primary ammes by capillary electrophoresis and high-performance liquid chromatography using choral crown ethers. J Chromatogr 680,253-26 1 18 Gozel, P., Gassman, E., Michelsen, H., and Zare, R. N. (1987) Electrokmetic resolution of amino acid enanttomers with copper (II)-aspartame support electrolyte Anal Chem 59,44-49 19. D’Hulst, A. and Verbeke, N. (1992) Choral separation by capillary electrophoresis with carbohydrates J Chromatgr 608,275-287 20 Quang, C. and Khaledi, M G. (1994) Direct separation of the enantiomers of pblockers by cyclodextrm-medtated capillaryzoneelectrophoresisJHRCC 17,9!LlOl 2 1 Barker, G. E , Russo, P , and Hartwick, R A (1992) Chiral separation of leucovorm with bovine serum albumin using affinity capillary electrophoresis. Anal Chem 64,3024-3028 22 Sun, P , Barker, G. E , Hartwick, R. A., Grinberg, N , and Kaliszan, R. (1993)

Chrral separations using an immobilised protein-dextran polymer network in affinity capillary electrophoresis J Chromatogr 652,247-252. 23. Sun, P., Wu, N., Barker, G , and Hartwick, R. A (1993) Chiral separations usmg dextran and bovine serum albumin as run buffer additives m affmty capillary electrophoresis. J. Chromatogr 648,475-480. 24. Busch, S , Kraak, J. C , and Poppe, H. (1993) Chiral separations by complexation with proteins in capillary zone electrophoresis. J Chromatogr. 635, 119-125 25 Valtcheva, L., Mohammad, J., Pettersson, G., and HJerten, S. (1993) Choral separation of P-blockers by high-performance capillary electrophoresis based on nonimmobilised cellulase as enantioselective protein. J Chromatogr. 638,263-267 26 Carpenter, J. L., Camilleri, P , Dhanak, D , and Goodall, D M. (1992) A study of the binding of vancomycm to drpeptides using capillary electrophoresis. J Chem Sot Chem Commun. 804,806-8 10. 27. Armstrong, D. W., Rundlett, K. L., and Chen, J.-R. (1994) Evaluation of the macrocyclic antibiotic vancomycin as a chiral selector m capillary electrophoresis Chzrakty 6,496-509. 28. Guttman, A , Paulus, A., Cohen, A. S., Grinberg, N , and Karger, B. L (1988) Use of complexing agents for selective separation m high performance capillary electrophoresis. Choral resolution via cyclodextrms incorporated within polyacrylamide gels. J Chromatogr. 448,41-53. 29. Cruzado, I. and Vigh, G (1992) Choral separations by capillary electrophoresis using cyclodextrin-containing gels. J. Chromatogr. 608,42 l-425

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30 Aturkt, Z and Fanah, S. (1994) Use of P-cyclodextrm polymer as a choral selector m capillary electrophoresrs J Chromatogr 680, 137-146 3 1 Bnnbaum S and Nrllson S (1992) Protem based capillary affinity gel electrophoresis for the separation of optical Isomers. Anal Chem 64, 2872-2874 32 Terabe, S. (1989) Electrokmetrc chromatography, an interface between electrophoresrs and chromatography. TrAC 8, 129-l 34 33 Nrshr, H , Fukuyama, T , and Terabe, S (199 1) Chrral separation by cyclodextrmmodified mrcellar electrokmettc chromatography J Chromatogr 553, 503-5 16. 34 Ueda, T., Kitamura, F , Mrtchell, R., and Metcalf, T (1991) Choral separatron of napthalene-2,3-drcarboxyladehyde-labeled ammo acrd enantromers by cyclodextrm-modified mtcellar electrokmettc chromatography wtth laser-Induced fluorescence detectron Anal Chem 63,2979-298 1 35 Otsuka, K and Terabe, T (1990) Optical resolution by mrcellar electrokmetrc capillary chromatography with choral surfactants J Chromatogr 515,221-226 36 Otsuka, K and Terabe, S (1990) Effects of methanol and urea on optrcal resolutron of phenylthiohydantoin-DL-ammo acrds by mrcellar electrokmettc capillary chromatography with sodrum N-dodecanoyl+vahnate. Electrophoreszs 11,982-984 37 Terabe, S , Shtbata, M , and Mryashtta, Y (1989) Choral separation by electrokrnetrc chromatography with bile salt mtcelles J Chromatogr 480,404-411 38 Terabe, S , Shtbata, M , and Mryashtta, Y (1989) Choral separation by electrokinettc chromatography with bile salt mrcelles. J Chromatogr 480,404-4 11 39. Nishr, H., Fukuyama, T , Matsuo, M., and Terabe, S (1989) Choral separations of optical rsomeric drugs using mrcellar electrokmetrc capillary chromatography and bile salts J Mzcrocolumn Sep 1,234-24 1. 40 Nisht, H , Fukuyama, T , Matsuo, M., and Terabe, T (1990) Choral separations of dtltrazem, trrmetoqumol and related compounds by mrcellar electrokinetrc caprllary chromatography wtth brie salts J Chromatogr 515,233-243 4 1. Cole, R. 0 , Sepamak, M. J , and Hinze, W. L (1990) Opttmrzatron of bmaphthyl enantiomer separations by captllary zone electrophoresrs usmg mobile phases containing bile salts and organic solvents J Hzgh Resolut Chromatogr 13,579-582 42. Mazzeo, J R., Grover, E R., Swartz, M. E , and Petersen, J. S (1994) Novel choral surfactant for the separation of enantromers by mrcellar electrokmetrc capillary chromatography. J Chromatogr. 680, 125-135. 43. Otsuka, K., Karrchaka, K., Hrgashrmorr, M., and Terabe, S. (1994) Optrcal resolution of ammo acid derivatives by mrcellar electrokmetic chromatography with Ndodecanoyl+serine. J Chromatogr 680, 3 17-320 44 Mayer, S. and Schurig, V. (1992) Enantiomer separatron by electrochromatography on caprllanes coated with chin&-dex J Hzgh Res Chromatogr Comm 15,129-13 1 45 Mayer, S. and Schurtg, V (1993) Enantromer separatton by electrochromatography m open tubular columns coated with chuasrl-dex. J LzquzdChromatogr. 16,9 15-93 1 46 LI, S and Lloyd, D. K (1993) Drrect chiral separations by capillary electrophoreSISusmg capillarres packed with oracid glycoprotem choral stationary phase. Anal,

Chem 65,3684-3690. 47. Tran, A. D., Blanc, T., and Leopold, E. J (1990) Free solutron capillary electrophoresis and mrcellar electrokmetrc resolutron of amino acid enantiomers and peptides isomers with L- and D-Marfeys reagent. J Chromatogr 516,241-249

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48 Lurte, I. S (1992) Mtcellar electrokmettc captllary chromatography of the enantiomers of amphetamine, methamphetamme and their hydroxyphenethylamme precursors J Chromatogr 605,269-275. 49. St Pierre, L. A. and Sentell, K B (1994) Cyclodextrms as enantioselecttve mobile phase modifiers for choral capillary electrophoresis. Effect of pH and cyclodextrm concentration. J Chromatogr 657,291-300 50. Altria, K. D , Goodall, D M , and Rogan, M. M (1992) Chual separation of CLammo alcohols by capillary electrophoresls using cyclodextrms as buffer additives I Effect of varying operating parameter Chromatographza 34, 19-24 5 1 Rickard, E. C. and Bopp, R. J. (1994) Optimtsatton of a capillary electrophorests method to determine chnal purity of a drug J Chromatogr 680, 60%62 1 52 Wren, S A C and Rowe, R. C. (1992) Theoretical aspects of choral separations m capillary electrophoresis. I. Inmal evaluation of a model J. Chromatogr 603, 234-241. 53 Fanah, S (1991) Use of cyclodextrms m capillary electrophorests. resolutton of terbutalme and propranolol enantiomers. J Chromatogr 545,437444 54 Rogan, M. M , Goodall, D M., and Altria, K. D (1994) Enanttomeric separation of salbutamol and related impurities using capillary electrophorests. Electrophoreszs 15,808-S

17

55 Kuhn, R , Stoecklm, F , and Erm, F (1992) Chual separations by host-guest complexation with cyclodextrin and crown ether m capillary zone electrophorests Chromatographza 33,32-36

56 Penn, S. G , Lm, G , Bergstrom, E T , Goodall, D M., and Loran, J S (1994) Systematic approach to treatment of enantiomeric separations m capillary electrophorests and ltqutd chromatography 1 Initial evaluation using propanolol and dansylated amino acids. J Chromatogr. 680, 147-155 57. Belder, D and Schomburg, G. (1992) Enantiomer separation of tocamtde analogues by cyclodextrm modified electrokinettc chromatography. JHRCC 15,686-693 58. Rogan, M M , Altria, K D , and Goodall, D. M (1994) Plackett-But-man experimental design m chual capillary electrophorests Chromatogruphza 38,723-729. 59 Altria, K. D , Walsh, A R., and Smith, N W (1993) Vahdatton of a capillary electrophoresls method for the enantiomeric purity testing of fluparoxan J Chromatogr 645,193-l 96 60. Altrta, K D., Goodall, D. M., and Rogan, M M (1994) Quantitative applications and validation of the resolution of enantiomers by capillary electrophoresls Electrophoreszs 15, 824-827.

61. Pluym, A., Van Ael, S., and De Smet, M. (1992) Capillary electrophorests in chemtcal/pharmaceutical quality control. TRAC 11, 27-32. 62. Ntelen, M. W. F (1993) Choral separation of basic drugs using cyclodextrin-modifled capillary zone electrophoresis. Anal Chem 65, 885-893 63. Peterson, T. E. and Trowbrtdge, D. (1992) Quantttatton of I-epmephrme and the determmatton of the d-/l- epinephrme enanttomer ratto m a pharmaceutical formulation by capillary electrophoresis J Chromatogr 603,298-301 64 Rogan, M. M , Drake, C., Goodall, D. M., and Altria, K. D (1993) Enanttoselecttve enzymatic biotransformation of 2’-deoxy-3’thtacytidme (BCH 189) monitored by capillary electrophorests Anal. Bzochem 208, 343-347

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65. Rogan, M M., Goodall, D M., and Altria, K. D (1994) Enantioselective separations using capillary electrophoresis. Chwalq 6,254O 66 Altria, K. D and Rogan, M M (1994) Comparison of capillary electrophoresis and HPLC m the pharmaceutical industry Chromatogr Anafysls April/May, 3S8 67 Altrta, K. D , Harden, R. C , Hart, M , Hevtzt, J., Halley, P. A., Makwana, J V., and Portsmouth, M. J. (1993) Inter-company cross validation exerctse on capillary electrophoresis J Chromatogr 641, 147-153 68 Ntelen, M W F (1993) (Enantio-) separation of phenoxy acid herbictdes usmg capillary zone electrophoresis J Chromatogr. 637, 8 l-90. 69. Thormann, W , Ltenhard, S., and Wemly, P. (1993) Strategies for the momtormg of drugs m bioflmds by mtcellar electrokmetic capillary chromatography. J Chromatogr 636, 137-148. 70. Francotte, E., Cherkaoul, S., and Faupel, M (1993) Separatton of the enanttomers of some racemic aromatase inhibitors and barbiturates by capillary electrophoreSIS. Chrralqv 5,5 16526 71 Garetl, P , Gramond, J P , and Guyon, F (1993) Separatton and determmation of warfarm enantiomers in human plasma samples by capillary zone electrophoresis using a methylated P-cyclodextrm-containing electrolyte. J Chromatogr 615, 3 17-325 72 Shibukawa, A., Lloyd, D. K , and Wamer, I W. (1993) Simultaneous chnal separation of leucovorm and Its maJor metabolite 5-methyl-tetrahydrofolate by capillary electrophoresis using cyclodextrms as choral selectors* estimation of the formation constant and mobility of the solute-cyclodextrm complexes Chromatographra 35,4 19-429 73 Nisht, H. and Terabe, S. (1995) Review. Optical resolutton of drugs by capillary electrophorests techmques J Chromatogr 694,245276 74 Quang, C and Khaladi, M (1995) Extendmg the scope of choral separation of basic compounds by cyclodextrm-mediated capillary zone electrophorests J Chromatogr 692,253-265. 75 Aumatell, A., Wells, R. J., and Wong, D. K. Y. (1994) Enanttometic dtfferentiatton of a wide range of pharmacologically active substances by capillary electrophoresis using modified P-cyclodextrins. J Chromatogr 686,293-307 76. Aumatell, A. and Wells, R. J (1995) Enanttomettc dtfferenttatton of a wade range of pharmacologically active substances by cyclodextrm-modified mtcellar electrokinetic capillary chromatography usmg a brie salt. J Chromatogr. 688, 329-337 77 Armstrong, D W , Gasper, M. P , and Rundlett, K. L. (1995) Highly enantio-selective capillary electrophorettc separations with dilute soluttons of the macrocychc antibiotic ristocetm A J, Chromatogr. 689, 285-304. 78 Schmitt, T. and Engelhardt, H. (1995) Optimlsatton of enanttometic separations m captllary electrophorests by reversal of the migration order and using different derivatized cyclodextrms. J Chromatogr 697,56 l-570. 79 Guttman, A and Cooke, N. (1994) Practtcal aspects m choral separation of pharmaceuticals by capillary electrophoresis. II. Quantttative separation of naproxen enanttomers. J. Chromatogr. 685, 155-159. 80 Noroskl, J. E., Mayo, D J., and Moran, M. D (1995) Determmation of the enantiomer of a cholesterol-lowermg drug by cyclodextrin-modified mtcellar electrokinetic chromatography J Pharm Blamed Anal 13,52-54

CHAPTER15

Capillary

Electrochromatography Iain

H. Grant

1. Introduction In conventional liquid chromatography, the flow of eluent is generated by the application of a pressure gradient, which is usually provided by means of a high-pressure mechanical pump. However, under certain conditions, the mobile phase can be forced to migrate through the chromatographic medium by the application of an electric field. This phenomenon of electrically induced fluid flow is commonly referred to as electroosmosis or electro-osmotic flow. Liquid chromatography making use of this effect, where the pump is substituted by a high-voltage power supply, has been given a number of descriptions, including chromatoendosmosis and electroendosmosis. However, the term electrochromatography has been adopted by authors of the most recent publications and accordingly will be the one used here. The use of an electrical field in liquid chromatography has been discussed by several workers (I, 2). However, the first discussion since the advent of modern HPLC, proposing the use of electro-osmotic flow as an alternative to pressure driven flow was given by Pretorious and coworkers in 1974 (3). Electra-osmotic flow is a familiar concept to anyone with experience in capillary electrophoresis (CE) in any of its various forms. This phenomenon, however, is not restricted to open capillaries, such as those used in conventional CE, micellar CE, and so forth, but may also occur in tubes packed with small particles of silica gel, either as unmodified silica or chemically derivatized reverse-phase materials, From

Methods m Molecular B/ology, Vol 52 Caprllary Electrophoresm Edkxi by K Altria CopyrIght Humana Press Inc , Totowa, NJ

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The introduction of a chromatographic stationary phase into an electrophoresis environment, either in the form of a packed bed or a thm surface film on the walls of a capillary, mtroduces the possibility of chromatographic partrtion as an alternative selectron mechanism that, m the case of ionic species, will be superimposed on the electrophoretrc separation, or in the case of neutrals, will provide the sole means of differentiating between the various components. Like open tubular CE, the use of a high voltage dictates that electrochromatography should be car-r-ted out in capillaries having a very narrow diameter and is therefore subject to the same mstrumental requirements. Thus, despite the fundamental differences in the separation mode, electrochromatography bears a close resemblance to CE and its associated techniques. Accordmgly, the devel-

opment of electrochromatography has, to a large extent, been made possible by the advances m CE in recent years. This IS illustrated by the fact that one of the pioneering papers on CE also reported separations of neutral hydrocarbons in packed capillaries filled with IO-pm diameter

reversed-phase silica gel (4). Despite its obvious potential as a hrgh-performance separation method exhibiting both selectivity and efficiency, electrochromatography

is still by far the least exploited of all captllary

electroseparation techniques. 2. Limitations of Conventional CE Despite the remarkable efficiency of open tubular CE and its ease of

use, there are still a number of shortcomings that do not apply to conventional HPLC. These can be summarized as follows. l

l

l

The analytes must carry an electrical charge unless a micellar additive is used in the carrier electrolyte. The relative differences in the electrophoretic mobilities of structurally similar species, such as a pharmaceutical substance and its degradation products, are often far smaller than the relative differences m capacity factors (k’) found m HPLC. Consequently, there are many situations where a mixture can easily be resolved using HPLC with only a few thousand theoretlcal plates, whereas CE, despite a considerably greater efficiency, lacks sufficient selectivity for complete resolution. In contrast to LC, there are, in CE, relatively few parameters available for

optimrzation of the selectivrty.With the exceptionof the carrierelectrolyte pH, most other variables have a comparatively minor Influence on the selectivity.

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Fig. 1. Separation of polycychc aromatic hydrocarbons by capillary electrochromatography.Column: 50 cm x 40 pm id, packing material: ODSHypersi15 pm, mobile phase:70:30acetomtrile:water6 mMNaH,P04, analytes (in order of elutlon) 1. naphthalene,2. 2-methylnaphthalene,3. fluorene, 4 phenanthrene,5. anthracence,6. pyrene, 7. 9-methylanthracene,8. unknown, 9. unknown. Applied field: 64 kV/m (5). By carrying out the analysis in a packed capillary containing an LC packing medium, the limitations outlined above can largely be overcome owing to the introduction of chromatographic partition as the separation mechanism. This has the great advantage of introducing, to the array of electroseparation methods, a degree of predictability, since neutral species will be separated in exact accordance with the rules governing LC analysis. An example showing the separation of several polycyclic aromatic hydrocarbon species by electrochromatography in a capillary packed with S-pm diameter ODS derivatized silica gel is shown in Fig. 1 (51. As would be expected for electrically neutral species, the elution order and k’ values are identical to those found for the equivalent analysis by pressure-driven HPLC.

200

Grant 3. Theoretical

Considerations

3.1. Limitations of Conventional HPLC In order to appreciate the potential advantages of using an electrical field as opposed to a pressure gradient to generate the flow of eluent m LC, it is necessary to consider the factors that limit the performance of conventional pressure-driven LC. When compared to CE, conventional HPLC, with comparable analysis times, exhibits a relatively low efficiency in terms of plate number. If a higher efficiency is required, the price is generally a longer analysis time. The required analysis time for a given plate number (N) in HPLC dependsprimarily on the available pressure drop. This can be summarized by the following expression from Knox and Saleem (6) * t mm= N2

L,n2 cbrl 1AP (1) where t,,, represents the minimum analysis time for an unretained species, h,,, the minimum reduced plate height, $ the dimensionless flow resistance parameter, q the mobile phase viscosity, and AP the available pressure drop. In conventional HPLC, operatmg at approx 200 bar, it is usually possible to generate plate numbers of ca. 10,000 in a time of 2 min or less for an unretained component. However, if HPLC were to be operated at the same plate numbers as are typical in CE, i.e., ca. 500,000, the above expression suggeststhat m order to maintam the same analysis times, the pressure drop would have to be increased 250-fold, i.e., to 50,000 bar, assuming all other factors were to remain unchanged. The particle size of the packing medium must also be chosen to ensure that the system is operated at h,,, when using the full available pressure drop. The particle diameter (d,) satisfying these conditions is given by the following expression, also from Knox and Saleem (6).

dp= W hmnvm,,rl $D, /AP)“2

(2)

where v mlnrepresents the reduced velocity at which h,,, occurs and D, the diffusion coefficient of the analyte in the mobile phase. If one applies the same conditions of plate height and pressure drop as in the previous example, Eq. (2) suggests that particles with a diameter of 1 ~1or less should be used for the packing medium. Although it does not appear too unrealistic to produce particles with such dimensions, it could not be

Capillary

201

Electrochromatography

Fig. 2. Origin of electro-osmoticflow. Eachparttcle is surroundedby its own electrical double layer owing to the self-iomzatlon of residualsllanol groupsor adsorptionof chargedadditives. considered practical to operate HPLC with pressure drops significantly higher than those used at present. In principle, it would be possible to find acceptable pressure drops and analysis times for very high values of N by carrying out HPLC in open tubular capillaries with retentative walls, as in capillary gas chromatography. However, unlike in gas chromatography, where the diffusion coefficients of the analytes in the carrier gas are typically lo4 times greater than those in liquids, the capillary diameters for open tubular HPLC have to be very much smaller than those typically employed in CE. 3.2. Electroosmotic

Flow

in Packed

Columns

In the case of a column packed with a conventional HPLC packing material, such as silica gel, the electro-osmosis arises becauseof the electrical double layer at the surface of the particles, as illustrated m Fig. 2, and not from the double layer at the wall of the capillary. The bed structure of a packed capillary column can be modeled as a network of interconnecting capillaries runnmg in random directions between the particles of the packing medium. It can be shown that the mean diameter of these interparticle channels is approximately one-fifth to one-quarter of the mean particle diameter. Provided there is a negligible degree of overlap between the electrical double layers on the opposing walls of a given interparticle channel, the effective eluent linear velocity resulting from electro-osmosis is given by the following expression:

202

Grant

where y is a dimensionless factor, with a value estimated to be in the range 0.4-0.7, which accounts for the tortuosity and porosity of the packed bed, E, and E, represent the relative permitivity and the permitivity of free space, respectively, < the zeta potential at the particle/electrolyte interface, E the applied field, and q the viscosity of the electrolyte. If the product ( y E, E, c/q ) is denoted neo,then Eq. (3) reduces to: ueo = lleoE (4) which closely resembles the familiar expression for the ion migration velocity in CE. The main point to note from the above is the implication that the diameter of the packing medium does not influence the flow velocity of electrically driven flow. The situation contrasts markedly with pressureinduced flow, where the linear velocity for a given pressure gradient is proportional to the square of the particle diameter. The fact that the flow rate m electro-osmotic flow is essentially mdependent of particle diameter implies that no penalty is incurred by reducing the particle diameter in an attempt to increase efficiency and suggests that the rapid generation of very high plate numbers should be possible using very small particles. It should, however, be emphasized that Eq. (3) is only valid provided the channel diameter is significantly greater than the electrical double-layer thickness in order that double-layer overlap does not occur. If overlap does occur, the flow velocity is dependent on the mean interparticle channel diameter and therefore becomes a function of the particle diameter as in pressure-driven flow. Based on an earlier theoretical treatment by Rice and Whitehead (71, it has been suggested by Knox (8) that double-layer overlap begins to have a significant effect on the electro-osmotic flow velocity when the particle diameter is reduced to less than about 40 times the “thickness” of the electrical double layer. This, of course, places limits on how small the particles can be before a reduction of the flow velocity is observed. The effective “thickness” (6) of the electrical double layer for a 1: 1 electrolyte can be expressed as: 6 = (E, E, R Ti 2cF2 )I’*

(5)

where R is the universal gas constant, T the absolute temperature, F the Faraday constant, and c the electrolyte concentration. Typically, CE is operated with a background electrolyte concentration in the range 10-50 mM. At an electrolyte concentration of 10 mM, 6 would have an

Capillary

Electrochromatography

203

approximate value of only 3 nm. Using the condition that the particle diameter should not be reduced to below 40 6, the smallest particle diameter that could be used at this ionic strength would be as small as 0.12 pm. If particles with a diameter of 1 pm were to be used, the electrolyte concentration would only have to be 0.1 mM m order to avoid sigmficant electrical double-layer overlap. Given that the smallest particles used routinely in conventional HPLC have a mean diameter of ca. 3 pm, it is clear that there is considerable scope for reducing the particle diameter before one runs into problems of double-layer overlap. However, the use of a packed column does not remove the requirement for a high background electrolyte conductivity in the analysis of charged species, which must be taken into account when selecting the ionic strength of the mobile phase. For the analysis of electrically neutral species, it is only necessary to have a minimal electrolyte concentration that is just sufficient to ensure a small enough value of 6 and, consequently, minimal self-heating. For this reason, m situations where only electrically neutral species are present, it should be possible to use capillaries that have significantly wider bores than those used in conventional CE, which would lead to improved detection limits. 3.3. Band

Broadening

in Electrochromatography

The introduction of a chromatographic stationary phase brings with it the same band broadening processesthat occur in conventional LC. Thus, the plate height (H) in packed column electrochromatography is represented by the van Deemter equation: H=BD,/u+Cud,*/D,+Ad,

(6)

where A, B, and C represent the familiar van Deemter constants. Thanks to the plug-like velocity profile of electro-osmotic flow, the value of A is often found to be significantly smaller in electrochromatography than in HPLC. In the above expression, all terms except the axial diffusion (BD,/u) decrease with decreasing particle diameter. Thus, if 4 is small enough for the A and C terms to have a negligible contribution in comparison to the B term, the above expression reduces to a simple limiting form that is exactly analogous to the expression commonly used for plate height in CE (cf. H = 2D, / u), i.e: H=BD,/u

(7)

204

Grant

u

1 I 0

I

I

I

10

I

I

20

Time / minutes Fig. 3. Electrochromatography on 1S-urn diameter particles Column: 50 cm x 50 pm Id, packing material: C18-Exs11 1 5 pm, mobile phase. 70.30 acetonitrile:water, analytes (1n order of elutton) 1. acetone, 2. benzyl alcohol, 3. acetophenone, 4. dimethylphthalate, 5. amsole, 6. methyl benzoate, 7. toluene, 8. phenyl benzoate, 9. naphthalene. Applied field: 50 kV/m. By pernuss1on of Capital HPLC Limtted (1993).

If, for example, one were to use a particle diameter of 0.5 pm, this would lead to an A term contribution of ca. 0.5 pm and a C term contribution of only 0.025 pm, whereas the diffusion-only term would contribute 2 pm to the overall plate height, i.e., axial diffusion dominates. Thus, provided the particles are small enough, the presence of the packing material should not lead to any loss of efficiency when compared with the diffusion-only case. In this way, it should be possible to operate electrochromatography with efficiencies, m terms of plate number (N), similar to those found in open tubular CE. Figure 3 shows the separation of a simple test mixture of electrically neutral species by electrochroma-

Capillary

Electrochromatography

205

tography using a 50 cm x 50 p.m id column packed with particles as small as 1.5 pm in diameter. In this case, an efficiency of ca. 140,000 theoretical plates (280,000/m) is achieved with an unretained time of only 8 min. 3.4. Electrochromatography in Open Tubular Columns The use of open tubular capillaries in pressure-driven LC has never become popular because of the requirement for the use of very narrow capillary bores. In order to operate under near-optimal conditions, the capillary diameters must be very much smaller than those used in CE. However, there are several reports of electrochromatography being carried out in open capillaries with wall-coated stationary phases, and in many cases, remarkably high plate numbers have been achieved, typically in columns with diameters of 10 pm or less (9,10). If electro-osmotic flow is used in place of pressure-driven flow m open capillaries, the band broadening for analytes that are either unretained or only very slightly retained is drastically reduced, leading to high plate numbers. However, the advantage of electro-osmotic flow over pressuredriven flow becomes less significant as the capacity factor (k’) increases. In order to achieve a degree equilibration between the two phases, one relies on random diffusion transporting the analyte molecules across the axis of the capillary. Thus, for significantly retained compounds, the efficiency of open tubular electrochromatography is strongly dependent on the capillary diameter and represents only a modest improvement over the pressure-driven case. Thus, changing from pressure driven to electrically driven, open tubular LC does not get around the requirement for extremely narrow-bore capillaries, if large plate numbers for retained compounds are to be achieved. By contrast, the choice of column bore for packed capillaries is governed only by the same thermal considerations that must be taken into account in conventional CE. 4. Practical Aspects of Electrochromatography Despite its close resemblance to CE, there are a number of practical considerations that must be taken into account over and above those for open tubular CE. 4.1. Detection In packed column electrochromatography, the presence of the particles slightly complicates detection by UV absorption. The conventional CE

Grant

206 I llgh

VOkdgC

I

Fig. 4. Schematic diagram of a packed capillary for use in electrochromatography method using a focused beam passing through the separation capillary itself is not particularly effective in packed columns owing to the limited UV transmission characteristics of silica particles, especially at the short wavelengths used in CE. It is therefore more effective to carry out the detection in an unpacked portion of capillary tubing after the components have been eluted from the packed zone. This can be accomplished either by forming a butt connection between the packed capillary and the open detection capillary, or by forming porous frit a few centimeters from the outlet side of the capillary prior to packing. This ensures that during the packing process a small length of the tubing remains free of packing material, which then functions as the detection zone. A typical arrangement is shown in Fig. 4. One minor disadvantage with this arrangement is that the separated zones will be diluted by a factor of (1 + k’) prior to detection. In the case of fluorescence detection, it is not necessary for the excitation beam to penetrate the packed bed fully, and therefore, the packing material may occupy the full length of the capillary. In this way, the dilution of the eluting bands by the (1 + k’) factor would be avoided. 4.2. Mounting of Capillaries Conventional CE instruments are able to generate a pressure drop of up to 1 atm across the length of the separation capillary. Although this 1s normally sufficient to flush open capillaries with conditioning electrolytes and the running buffer, it is nowhere near the pressure required to fill packed capillaries with the desired chromatographic mobile phase.

Capillary

Electrochromatography

207

Thus, with the current commercially available CE instrumentation, it is necessary to use an alternative pumping system, such as an HPLC pump, in order to fill the packed capillary with the desired mobile phase. It is then necessary to mount the capillary in the CE instrument as quickly as possible to minimize the possibility of dry areas owing to evaporation from the capillary extremities. However, once filled with liquid, the mobile phase can easily be changed electro-osmotically by stmply changing the electrolyte in both the inlet and outlet buffer reservoirs. 4.3. Bubble Formation A major practical difficulty with packed column EC is the avoidance of gas bubbles m the packed bed, which have a tendency to form at the elevated temperatures resulting from Joule heating. In free solution or micellar CE, the electrolyte is generally filtered through a 0.2-urn membrane in order to remove any particulate matter. Thus, in conventional CE, the carrier electrolyte doesnot contain particles that may act as nucleation sites for bubble formation. Even if bubbles do form in CE, despite rigorous degassing, they are simply carried out of the capillary by the electro-osmotic flow and, at worst, give rise to a sharp spike on the detector signal. However, in a packed capillary, there is no shortage of available sites that may facilitate bubble formation, Moreover, once a bubble has formed in a packed bed, it is immobilized by the particles. Consequently, the rate of heat generation at this point is increased owing to the increased electrical resistance. Thus, once initiated, bubble formation is self-accelerating and the end result is a rapid drying out of the column. Once dry areas have formed in the column, the operator 1sleft with no alternative but to remove the column from the instrument and fill it once more with the electrolyte from a high-pressure pump. This problem of bubble formation puts limits on the concentration of the background electrolyte and the electric field that can be applied. The future exploitation of electrochromatography requires a solution to the problem of bubble formation. This would require a very efficient cooling system, which would have to be active over the entire length of the packed bed, or the system might be operated with the full length of the packed region maintained at an elevated pressure. 5. Applications Despite the fact that the technique is still very much in its infancy, and at the time of writmg there still exists no dedicated instrumentation, it is

Grant

208

4

I I’ 0

‘1

’

1 10

‘1

”

Time

I 20

4

““I”” 30

I 40

/ mmutes

Fig. 5. The separation of Flutrcasone Propronate from related rmpurttes. Column* 60 cm x 50 mm id, packmg material: Spherrsorb ODS 1 3 mm, mobile phase: water:acetonitrrle 20:80 (1 mA4borate) Applied field: ca 40 kV/m By permission of ref. 5 now beginning to find applications in analytical research laboratories, particularly in the pharrnaceutrcal industry (21-13). The best examples

of this so far have come from the work of Smith and Evans (I I) in which electrochromatographic

separations have been obtained for various

classes of pharmaceutical substancesand their related products. Figure 5 shows the separation of a synthetic steroid, fluticasone propionate, from a number of related impurities. Using a column packed with 3 pm diameter particles, chromatographic efficiencies in excess of 300,000 plates/m were obtained for several components. In this case, presumably because of the higher efficiency, electrochromatography was able to resolve an impurity that had previously never been observed. Smith and Evans (11) has also demonstrated high-efficiency EC separations of prostaglandins and cephalosporin antibiotics. Other pharmaceutical applications mclude the work of Yamamoto and coworkers (I.?), who have demonstrated very fast electrochromatographrc separations of the calcmm channel blocker isradipme and its byproducts. The general use of electrochromatography as a routine analytical technique awaits the development of suitable instrumentation together with

Capillary

Electrochromatography

209

packing materials having mean particles diameters of 1 pm or less. The fact that only very small quantities of the packing material are required may well open up new possibilities in terms of derivatization chemistries, which would otherwise be prohibitively expensive, e.g., certain optically active reagents. It can therefore be confidently predicted that when the instrumentation and materials become readily available, there will be a massive growth in the number of real applications. References 1 Lecoq, H. (1944) Bull de Sot. Roy des Ser. (Lkge) 13,20 2. Strain, H H. (1939) J. Amer Chem Sot 61, 1292. 3. Pretorius, V., Hopkins, B J , and Schieke, J. D. (1974) J Chromatog 99, 23 4. Jorgenson, J. W and Lukacs, K de A. (1981) J Chromatog 218,209 5. Knox, J. H and Grant, I H (1991) Chromatographla 32, 3 17 6. Knox, J. H. and Saleem, M (1969) J. Chromatogr. Scl 7,614 7. Rice, C. L and Whitehead, R. (1965) J Phys Chem 69,4017. 8 Knox, J H. (1988) Chromatographla 26,329. 9. Tsuda, T., Nomura, K., and Nakagawa, G. (1981) J Chromatogr 248,241 10. Bruin, G. J M , Tack, P. P H , Kraak, J. C , and Poppe, H. (1990) J Chromatogr 517,557

11. Smith, N W. and Evans, M. B (1994) Chromatogruphza 38,649 12. Boughtflower, R. J., Chaberlam, M. C., Paterson, C. J., and Underwood, T. (1994) Poster presentatton and the 20th Int. Symp. on Chromatogr. (Bournemouth, UK). 13. Yamamoto, H., Baumann, J , and Erm, F. (1992) J Chromatogr 593,3 13

CHAPTER16 Application and Limits of Sample Stacking in Capillary Electrophoresis Dean S. Burgi

and Ring-Ling

Chien

1. Introduction A blessing and curse of capillary electrophoresis (CE) is the small amount of material that can be injected into the column (l-4). If one has a tiny quantity of substance to analyze, volumes on the order of 2-l 0 nL can be injected into a capillary column with good reproducibility; thus, precious material 1snot wasted. Analyses of material from a single cell have been reported (5). However, such a small volume generates a host of difficulties for conventional chromatographic detection technologies. For example, the detection of small volumes using on-column UV detection is hindered by the short optical path defined by the diameter of the column. Although the mass limit in CE can be very low because of the small volume, the concentration limit for UV detection is usually on the order of lOAM, which is several orders higher than detection hmits for high-performance liquid chromatography (HPLC). Laser-mduced fluorescence allows low concentration detection (on the order of 1WgM), but most analytes do not fluoresce. Also, most fluorescent labels are not excited by the available laser wavelengths, and laser technology 1s still complicated and expensive. Thus, one would like a way to concentrate the sample on-column to improve detection without a loss in resolution and analysis time. Several techniques have been reported to perform on-column concentration to enhance the detection in CE: isotachophoresis (ITP), From

Methods m Molecular Biology, Vol 52 CapNary Electrophoresls Ed&d by K Altna Copynght Humana Press Inc , Totowa, NJ

211

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Burgi and Chien

field-amplified CE, switching valves, solid-state extraction, and pH differences, to name a few. For example, Everaerts et al. and other groups applied ITP to achieve a high sample concentration within a pre-CE column (612). In ITP, the sample is inserted between two different buffers containing leading and trailing electrolytes that have sufficiently higher and lower electrophoretic mobilities, respectively, than the sample. All sample bands ultimately migrate between the two electrolytes and move through the column at the same velocity, and the concentrations of samples in their migrating zones will be adjusted according to the concentration of the leading electrolyte. Although ITP can produce very sharp bands, the major disadvantage is the necessity of using a discontinuous buffer system. Also, information about the sample’s mobilittes have to be known to achieve good separations (11,12). Debets et al. have developed a switching valve using a six-port injection valve and report a 2-3 orders of magnitude improvement over direct injection (13). Guzman et al. used a small C- 18 packed region inside the front end of the column to extract and enrich the sample before injecting the material onto the column (14). Chien and Burgi have investigated the use of a continuous buffer system to increase the concentration on-column (15-19). On-column sample concentration using a continuous buffer system, also known as the sample stacking technique, was introduced to CE first by Mikkers et al. (10). In the continuous buffer, sample stacking is achieved as a result of the movement of sample ions across a stationary boundary, that is generated by a difference in the buffer concentration of the sample region and the rest of the column. Because of the concentration difference, the ions experience a lower electric field in the support buffer region than in the sample region; thus the ions’ velocity decreases as they cross the stationary boundary. The slower-moving ions will stack up into a smaller sample zone volume, thereby increasing the concentration. The concentration differences that generate the different electric fields can be a change in pH and/or concentration of support buffer (20-23) in a continuous buffer system. In the simplest form of sample stacking, a large plug of sample dissolved in water is introduced hydrostatically into the capillary. The sample ions form into a narrow band when they migrate into the region with more concentrated background electrolyte. Moring et al. have reported an increase of a factor of 10 m detectability in CE with sample stacking (21).

Sample Stacking

213

An alternative continuous buffer method for enhancement of signals is to use electroinjection with samples prepared in a highly diluted buffer or water. In conventional electroinjection for CE, samples are prepared in a buffer solution that has the same concentration as that used in the separation. The amount of ions injected into the column under this condition is rather limited. However, if the sample is prepared in a diluted buffer that has the same composition as the background buffer inside the column, an enhanced electric field strength at the injection point exists when the high voltage is applied. This field-amplified sample injection can yield a large enhancement in the amount of ions injected into the column. The concentration in the sample zone can be increased from lo-fold to ZOOO-foldwith sample stacking (19). All concentrating methods have limits in their application. If the concentration of the sample zone becomes within two orders of magnitude of the support buffer concentration, the peak shapes might be distorted. Everaerts has reported in depth on the changing electric field generated by the sample zone (6-10). The zone itself can set up ITP conditions according to Kolausch regulating function and will either front or tail depending on whether the sample has a slower or faster mobility that the support buffer. For field-amplified CE, there exists a point in which the sample stacking process perturbs the separation process. Laminar flow, heating of the sample zone, and loss of separation voltage all combine to force one to balance between resolution and the concentrating process, 2, On-Column

Transient Isotachophoretic Preconcentration There are many options that exist for isotachophoretic preconcentration (I&12,24). Two simple methods demonstrated by Foret et al. will be addressedhere (12). Method A uses a leading electrolyte (LE) in the support buffer or background buffer. The sample is dissolved in the leading electrolyte and a large volume is injected into the column. The injection end of the column is then placed into a reservoir containing a terminating electrolyte (TE). The voltage is applied for a fixed time to focus the sample into sharp ITP bands (Fig. 1). After the sample is focused, the column is removed from the TE buffer reservoir and placed in the support buffer reservoir. The voltage is then applied to separate the sample by the normal CE process.

214

Burgi

and Chien Ground

LE

B

- 30

kv

Ground &KC I

LE

C

-30 kV

lEl

AB

lTEl

Ground

C

LE

Ial

LE

Fig. 1. Method A: The terminating electrolyte is replaced after focusing. (A) In the injection step, the sample is injected into a column filled with leading electrolyte. (B) In the focusing step, the termmating electrolyte IS placed into the reservoir and the voltage is applied. (C) In the separation step, the reservoir is replaced with leading electrolyte and the voltage is reapplied. The focusing step assumes the LE has a higher mobility than the sample and the TE. The sample concentration will then adjust itself according to the concentration of the leading electrolyte. Kohlraush regulating function (25) states that the sample concentration can be written as: cs= CdPshe+ PqYPle(Ps+ l-q (1) where C, is the concentration of the sample zone, Ct, is the concentration of the leading electrolyte, and ps, l+, l.tqare the mobilities of the sample, leading electrolyte, and counter ion, respectively. It takes a finite time period for the sample zones to move and concentrate themselves between the LE and TE. Stacking is complete when all the co-ions in the original sample zone migrate past the front of the sample ions. t = Ye~[u4e

-

l-d&4

(2)

where iVi, is the number of moles of the leading ion in the support buffer, El, is the electric field strength m the boundary between LE and the

Sample Stacking

215

sample zone, and A is the cross-sectional area of the column. In general, one performs ITP in the constant current mode, thus: I = he&J (3) = Wehe + P&%A (4) where k,, is the specific conductivity of LE and F is the Faraday constant. Combining Eqs. 2-4, we obtain:

(5) A similar expression, replacing pie by pte and changing the sign properly, can be derived for the case when the sample is dissolved in the terminating electrolyte. After the focusing step, the reservoir with the TE is replaced by a reservoir containing LE. The co-ions in the LE will then move rapidly in the column through the zone of the TE and destack all the sample bands. The amount of time for this destacking period can be estimated from the difference in velocities of the co-ions of leading electrolyte in the zones of the LE and TE (Fig. 1D). (6)

whereXis the distance from the focused sample zones from the injection end, and E,, -El, is the difference in electric field strengths in the terminating and leading zones, respectively. In general, depending on various parameters, the focusing time periods are on the order of 10 min and the time for the separation step to begin is on the order of 5 min. Since the times of focusing and separation are dependent on the volume of the injected sample and the concentration of the sample buffer, care must be used in the injection process to obtain reproducible migration times. Also, the concentrations of the major components in the sample itself can perturb the migration times if they vary greatly from sample to sample. Desalting may be needed to control the concentration of co-ions in the sample zone. To maintain a constant velocity from the detection of the zones, constant current instead of constant voltage must be used. Method B uses only one electrolyte in both the ITP focusing step and the CE separation step, and the sample buffer contains a high concentration of an ionic component with high mobility and like charge. The requirement of this method is that the co-ion must be a low mobility ion

216

Burgi and Chien A

-30 kV

Ground I

BGE

B

BGE

- 30 kV

Ground 1

BGE

C

BGE

-30 kV BGE

Ground

ABC LE

BGE Ill

-

Fig. 2. Method B: The sample contams the leading electrolyte. (A) In the injection step, the sample is injected into a column filled with the background electrolyte. (B) In the focusmg step, the leadmg electrolyte m the sample focuses the sample. (C) In the separation step, the leading electrolyte moves away from the sample and the sample separates based on its ion mobility

that acts as the TE during the transient ITP step. After the current is applied, the high mobility co-ions in the sample zone will move to the

front because the LE and the sample components are stacked at the rear of the zone by an isotachophoretic process. With time, the LE will dissipate into the support buffer and the sample zones will destack and continue to migrate and separateby the normal CE process (Fig. 2). A simple theoretical model of the type of system is described m detail by Gebauer et al. (26). Typically, the time necessary for stacking will be similar to that described in Method A.

The on-column transient ITP, as described in Method A or B, is a powerful tool for preconcentration of samples. One disadvantage of these methods is that the migration length in the separation step will vary, although it can be calculated, and is shorter than the normal CE process. In other words, migration time will vary from injection to injection so an internal standard is needed. In addition, the resolution between sample zones is diminished because of the shorter amount of time spent m the column. Several methods have been devised to solve this problem.

217

Sample Stacking

One is the use of counterflow to keep the focusing zones at the mjection end of the column (24,. In this method, the correlation between the current and the sample zone velocity during the focusing step is used to calculate the pressure needed to counterbalance the sample velocity in ITP and the timing to switch from ITP to CE. By keeping the ITP zones near the injection end, one can obtam reproducible CE migration times. Also, differing sample matrixes can cause migration times to change. Sodium, potassium, or ammonium ions can vary from sample to sample. Thus, a desalting step is needed to dilute this effect. 3. Field Amplification and Sample Stacking in Continuous Buffer System In the simplest form of field-amplified capillary electrophoresis, a long plug of sample containing sample ions prepared in a lower concentration buffer is injected into a column of higher concentration buffer. High voltage is then applied to the column, causing electrophoresisto occur. There are two phenomena of interest; one is the electric fields generatedacross each region and the other is the local electroosmotic flow in each region. Let us consider the electric field distribution first. If a capillary column of length L is filled with two different concentration buffers of the same electrolyte, assuming a length XL of the column is filled with concentration 1 and a length (1 - x)L of the column is filled with concentration 2, where 0 I x I 1, then the local field strengths Er and E2 m the two regions could be approximated by: E, = yE()l[‘yx + (1 -x)]

(7)

and

+ (1 - 41

(8) where y = p1/p2is the ratio of resistivities of buffer 1 and buffer 2, respectively, and E0 = V/L is the field strength of a uniform system, whether it is buffer 1 or buffer 2. We have also assumed that the concentration of analytes is very small and its contribution to the resistivities is negligible. Equation 1 shows that whereas the field strength inside the lower resistance region is less than the original uniform field strength, the field strength inside the higher resistance region will be amplified (increased) by a field enhancement factor rl[rx + (I --x)1. This enhancement factor can be easily several hundred to a thousand if the concentration of the two buffers is chosen properly. E2Eo4v

218

Burgi

and Chien

Although the absolute value of the electric field strength in regions 1 and 2 will depend on X, the ratio between them will remain a constant and is equal to y, the ratio of the resistivities or conductivities. In general, the resistivities are inversely proportional to the concentration of the electrolytes that make up the buffers. Hence, the ions inside the lower concentration region will experience a higher electric-field strength and move faster than the ions inside the higher concentration region. Thus, the velocity of the ions will change once they cross the concentration boundary. For a large y, the ions inside the injected sample plug will experience very high electric-field strength and move very rapidly toward the high-concentration support-buffer region. Once they pass the concentration boundary, they will experience a lower electric-field strength and slow down. Since the flux of the ions across the concentration boundary has to be conserved, we have: GIVqllI = G2vep,2 (9) where C, 1,G2 and vepl1l vep12 are the concentrations of analyte ions and their electrophoretic velocities inside the injected sample plug and the region of higher concentration buffer, respectively. Since the electrophoretic velocity of the ions is simply proportional to the electric field strength, Eqs. 1 and 2 yield = YG (10) Equation 10 shows that the concentration of sample ions that migrated into the high concentration buffer region will be enhanced by the field ratio y. Since the total number of sample ions has to be conserved, the length of the sample zone in the high concentration buffer region also has to be reduced by the same factor y. Neglecting the diffusion effect, the effective plug length or the length of the sample zone after stacking is G2

x, = x,tl,h (11) where J&, is the inmal plug length of the injected sample. If there is no electroosmotic flow, the flux of ions out of the concentration boundary will be exactly equal to the flux into it. Consequently, the concentration boundary will be stationary, neglecting the effect of diffusion. In other words, the apparent movement of the concentration boundary in fieldamplified capillary electrophoresis comes solely from the electro-

Sample Staclzing

219

osmotic flow of the whole bulk solution. This pseudostatronary concentration boundary is the major difference between field-amplified capillary electrophoresis and isotachophoresis. In isotachophoresis, where one has different electrolyte solutions in different regions of a single column, the electric fields are inversely proportional to the effective mobilitres of the solution inside each region, The boundary is then moving at a constant velocity. Although the concentratton boundary is pseudostationary, the zone of injected analyte ions, on the other hand, migrate through it because of the electrophoretic motion. This thin zone of ions then moves through the support buffer and separates into individual zones by conventronal free-zone electrophoresis. The stacking mechanism occurs for both positively and negatively charged species. The positive species stack up in front of the sample plug and the negative species stack up in back of the sample plug. The neutral compounds are left in the sample plug and coelute out. Theoretrcally, the amount of stacking is simply proportional to the field enhancement factor; the larger the difference m concentrations, the narrower the peak; i.e., there will be more stacking. An extrapolation can be made that a rather long sample plug prepared m water or very low concentration buffer should be stacked into a very thin zone in the high concentration support buffer. However, a laminar flow (back pressure effect) is generated inside the column because of the mismatch between local electroosmotic velocities and bulk velocity. The laminar flow will broaden the sharp zone generated by the stacking process. The larger difference in concentration will result in a bigger laminar flow. These two effects, stacking and broadening, work against each other, so there is an optimal point as to the length of water plug one can introduce into the column and still achieve the same high resolution. Another source of band broadening 1s that the temperature in the sample plug can increase as the result of the high field strength. The degree of heating is a function of the plug length (22). The heated region will radiate out into the support buffer region and generate a thermal gradient along the length of the column. This thermal gradient can cause band broadening and introduce thermal decomposition of thermally labile samples. Care must be taken when high concentrations of inorganic buffers are used as support buffers.

220

Burgi

and Chien

4. Laminar Flow and Peak Broadening in Field-Amplified Capillary Electrophoresis In field-amplified capillary electrophoresis, the electric field strength will be distributed according to Eq. 7. Also, because the zeta potential is a function of the ionic strength of the solution, the electroosmotic mobility is greater in the lower concentration region (27). The combmation of these two factors results in a large difference in the local electroosmotic velocities between the two regions Nevertheless, the amount of compound flow across any plane perpendicular to the column has to be the same according to the continuity principle in a noncompressible fluid. Thus, the bulk solution and the concentration boundary are moving at an average velocity vb. The difference in the local electroosmotic velocities and the bulk velocity will generate a hydrostatic pressure across the local regions. The pressure difference generates laminar flows in both the high and low concentration regions. The resulting average laminar velocities in each region are exactly equal to the difference of the bulk electroosmotic velocity, vb, and the local electroosmotic velocities, vel and ve2. val = veI - vb

(12)

vz.2 = vb - ve2

(13)

where v,~ and va2are the average laminar velocities in the low and high concentration regions, respectively. In a first order approximation, a simple relationship exists between the bulk velocity vb and the local electroosmotic velocities in the two buffer regions: vb = xv,1 + (1 -4v,2 (14) where v,J = 1 or 2, are the local electroosmotic velocities. These local electroosmotic velocities, v,i and ve2,are simply proportional to the local electric field given in Eq. 1, so that: "ej = veoj EjIEO (15) where veOJis the electroosmotic velocity in a column filled with only a single bufferj of a fixed concentration, Substituting Eq. 14 into Eq. 15, we obtain the equation for the bulk velocity of the solution inside the capillary column: vb=

{?~ve,ll[yx+(l

-x)1)

+ i(l

-~)ve,24~x+(1

-x>l)

(16)

Sample Stacking

221 Table 1 Relevant Parameters for Two Special Cases yx
1

yx>>

1

El

YEO

~52 V el

Eo

(l/x) Eo 0

Peal

W)

V e2

Veo2 V eo2

"b

Veal

0 V eel

Equation 16 shows that the average electroosmotic velocity of the system is not only weighted over the filled lengths of their components, but also weighted over their partial resistance. Thus, a small plug of low concentration buffer can greatly influence the average bulk electroosmotic velocity of the system. As the sample plug length increases the influence it has on the bulk, electroosmotic velocity becomes greater. In turn, the localized electroosmotic velocity changes and starts to manifest itself as a perturbation of the plug profile, i.e., a laminar flow is generated in the column. Table 1 lists some relevant parameters for two special cases. For the case where the sample length, X, is short and Y.X<< 1, the bulk velocity of the buffer inside the column is equal to the local electroosmotic velocity in the high concentration region. The electroosmotic pressure is small; thus, there is little laminar flow and peak broadening is minimized. In the case where x is short but YX >> 1, the local electroosmotic velocity m the high concentration region approaches zero. The bulk velocity of the buffer equals the homogeneous electroosmotic velocity of the low concentration buffer. The large mismatch between the velocities means the peak broadening is at its maximum and the sample plug is pushing the high concentration buffer region through the column like a piston. If the plug length is very long, the column behaves as if the low concentration region is absent and the ions stack up against the concentration boundary and move through the column with the sample plug. 5. Thermal Heating in Sample Stacking If the support buffer pulls a large current under the applied voltage, heating can occur in the sample plug. Vinter et al. investigated the thermal properties of the sample using a thermally labile nuclease as his

222

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and Chien

probe molecule. The total power of the system is additive; the power pulled by the sample plug and the power pulled by the rest of the column: P,= P, + Pb= R,12 + Rb12

(17)

Since the resistance of each region is dependent on the length of the region and the resistivity (p): P,=m(x/p,+

(1 -x)Ipb}

(18)

As seen from Eq. 18, the power in the sample plug can be several times larger than the power in the rest of the column if the support buffer has a much larger concentration than the sample plug. If the support buffer pulls a large current, the temperature rise in the sample plug can approach the boiling period of water. Also, a thermal gradient is formed across the concentration boundary and will perturb the narrow zone formed. 6. Improvement of Resolution in Sample Stacking To increase the amount of sample injected beyond the optimal conditions while retaining high resolution, one has to be able to remove the sample buffer after the stacking process is completed. One method is to use a switching valve and move the stacked sample zone physically away from the sample buffer into the support buffer. Another method is to remove the sample buffer by pumping it out of the column using the electroosmotic flow. This pumping technique will work only on ions that have negative mobility with respect to the bulk electroosmotic flow (28). With a negatively charged silica wall, the negative sample ions are stacked at the back end of the plug of sample buffer and will follow the plug as it moves through the column under the applied electric field. Thus, if one reverses the polarity of the electrodes from the normal separation configuration immediately after sample injection, the sample buffer will be pushed out of the column ahead of the negative sample ions. When the sample buffer is almost completely out of the column, which can be monitored by the electric current, the polarity is reversed again to the normal configuration and separation of the negative species occurs. To separatepositive species, the charge on the silica wall is made positive by adding cetyltrimethylammonium bromide (CTAB) to the buffer, thus changing the direction of the electroosmotic flow (29). There is some diffusion broadening caused by the amount of time the sample traverses the column, but a hundred-fold improvement in sample injection can be accomplished (28).

Sample Stacking

223

7. Sample Stacking of Extremely Large Sample Volume As the sample buffer is pushed out of the column by the electroosmotic flow, the negative ions overcome it and stack inside the column (28,30). This is possible if the field enhancement inside the sample buffer region is such that -vepl

’

vb

(1%

where vePlis the local electrophoretic velocity of the analyte species i. The negative sign indicates the electrophoretic velocity has to be in a direction opposite to the electroosmotic flow. The electrophoretic velocity of the analytes inside the sample buffer region is simply proportional to the local field strength and can be expressed as: V epl=

PeplYEOi[YX

+ Cl

-X)1

w-0

For yx >> 1, Eq. 20 can be simplified as: V epj = CLep1~0/~

(21)

The bulk electroosmotic velocity of an extremely long sample buffer can be calculated from Eq. 2 1. As the length of the sample buffer region decreases,the bulk velocity simply changes from the homogeneous electroosmotic velocity of the sample buffer to that of high concentration support buffer. In general, the difference in the homogeneous electroosmotic velocity between two different concentrations is not very large, especially if compared with the large enhanced electrophoretic velocrty. As a result, it can be approximated by: vb = PeoEO

c-w

where peais the electroosmotic mobility of the sample buffer. Substituting Eq. 2 1 and 22 into Eq. 19 yields: -ClepllX

’

Pea

(23)

Thus, the maximum filled length without loss of any analytes is: (24) For example, if the electrophoretic mobility of the analytes is half of the electroosmotic mobility of the buffer, we can then fill up to 50% of the column with the sample solution and stack the analyte into a sharp zone. xmax

=-Pept/Peo

224

Burgi and Chien

8. Diethylenetriamine (DETA) Pump To concentrate a large volume of sample the sample buffer must be removed. As seen in Section 7., the electric configuration of the electrode can be used to pump the sample buffer out of the column. However, precise switching of the electrode IS needed for reproducible migration times. The DETA pump method allows the electrode’s configuration to be maintained (31). This method uses the fact that the sample buffer sets up a localized region in the column that has a different electroosmotic flow than the rest of the column, and the separation voltage is greatly reduced across the sample zone for large volume Injections. The DETA in the support buffer suppressesthe electroosmotic flow by reducing the zeta potential on the column wall. Once the sample is introduced into the column, the DETA on the wall dissolves into the water of the sample plug, which m turn increases the zeta potential m the sample region (Fig. 3). After the voltage IS applied, the local electroosmotic flow m the sample region will move the bulk solution toward the negative electrode of the system. At the same time, the ions in the sample region will stack themselves up against the boundary between the sample region and the support buffer region. The ions will stay at the boundary until the water plug is pumped out of the column because most of the applied electric field is dropped across the water plug. As the support buffer region ISpulled back mto the column from the reservoirs, the DETA m the solution will again suppress the zeta potential on the column wall. After the sample buffer leaves the column, the full applied field is dropped across the whole column and the stacked sample zones start to separate by the normal CE separation process. This method has been shown to work for high mobility anionic species, but investigation into other charged species is ongoing. 9. Conclusion We have described how to use transient ITP and field amplification to perform on-column concentration in CE. Several different techniques are available depending on the desired resolution, the injection method, and desired species of ions. Each technique has its advantage but all have at least a loo-fold improvement m detection limits of the analytes. This sensitivity is comparable to the best results obtained from HPLC and opens CE into the uses as an trace level analytical tool.

225

Sample Stacking

A-

H20/ @ sample 0

Buffer

0 hydrodynamic

0

ground

-3,0kV

‘QQ. I

’

htgh

I

I

0

Buffer

C

qectlon

Buffer voltage

IS applred

-3OkV

I

I

ground I

D

Fig 3. A schemattc dtagram of how the water pumps rtself out of the column. (A) Once the sample ts injected into the column, the DETA on the caprllary wall dissolves mto the water of the sample plug, increasing the < potential m the sample region. (B, C) After the voltage IS applied, the tons stay at the boundary until the water plug IS pumped out of the column. (D) The water is completely pumped out of the column and the anions separate under normal CE condttions.

References 1. Hjerten, S. (1967) Chromatogr Rev 9, 122-219. 2 Mikkers, F. E. P , Everaerts, F M , and Verheggen, Th. P E M (1979) J Chromatogr 169, 1l-20 3 Jorgenson,J W and Lukacs, K D. (1981) Anal Chem 53, 1298-1302 4 Virtanen, R (1974) Acta Polytech Stand. 123, l-67 5 Ewing, A G., Wallmgford, R A., and Olefirowlcz, T M (1989) Anal Chem 61, 292R-303R 6 Everaerts, F. M., Verheggen, Th. P E M., and Mikkers, F E P. (1979) J Chromatogr 169,2 1-38

Burgi and Chien 7 8 9. 10 11. 12 13 14 15 16 17 18 19

20. 21 22 23 24 25

Foret, F , Sustacek, V , and Bocek, P (1990) J Mzcrocol Sep 2,229-233 Dolmk, V , Cobb, K A , and Novotny, M (1990) J Mzcrocol Sep 2, 127-13 1 Jandik, P. and Jones, W R (1991) J Chromatogr 546,43 l-443 Mlkkers, F E. P , Everaerts, F M., and Verheggen, Th P E M (1979) J Chromatogr 169, l-10. Foret, F., Szoko, E , and Karger, B. L (1992) J Chromatogr 608,3-12. Foret, F., Szoko, E , and Karger, B L (1993) Electrophoreszs 14,417-428 Debets, A. J. J., Mazereeuw, M., Voogt, W. H , Van iperen, D. J , Lmgman, H , Hupe, K. P , and Brmkman, U A T. (1992) J Chromatogr 608, 15 1. Guzman, N. A, Trebrlcock, M. A , and Advrs, J P (1991) J Lzq Chromatogr 14,997 Chren, R -L and Burg], D S. (1991) J Chromatogr 559, 141-152 Chren, R -L and Burgr, D. S (1991) J Chromatogr 559, 153-161 Chren, R-L. and Helmer, J L (1991) Anal Chem 63, 13541361 Burgr, D S. and Chren, R -L (1991) Anal Chem 63,2042-2047 Chren, R.-L and Burgr, D. S (1992) Anal Chem 64,489A Mrchov, B M. (1989) Electrophoreszs 10,686-689 Mormg, S. E , Colbum, J C , Grossman, P D , and Lauer, H H (1989) LC-GC 8, 34-46. Vmter, A , Everaerts, F M , and Soeberg, H (1990) J Hugh Resolutzon Chromatogr 13,63%642 Burgr, D S and Chren, R.-L (1991) J Mzcrocol Sep 3, 199202. Remhound, N J , TJaden, U R , and van der Greef, J (1993) J Chromatogr 641, 155-162 Everaerts, F M , Beckers, J L , and Verheggen, Th P E M (1976) Isotacho-

phoresu-Theory, 26 27 28. 29 30 31

Instrumentation

and Appllcatlons,

J Chromatogr

Library,

vol 6, Elsevrer, Amsterdam Gebauer, P , Thormann, W , and Bocek, P (1992) J Chromatogr 608,47-57 Chren, R.-L and Helmer, J C. (1991) Anal Chem 63, 1354-1361 Burgr, D. S and Chten, R -L. (1992) Anal Chem 64, 1046 Tsuda, T (1989) J Liq Chrom 12,250l Burgr, D. S. and Chren, R -L. (1992) Anal Blochem 202,306 Burgr, D S (1993) Anal Chem 65,3726.

CHAPTER17

Analysis of Bases, Nucleosides, and (0ligo)nucleotides by Capillary Electrophoresis Herbert

E. Schwartz

and

Kathi

J. Ulfelder

1. Introduction This chapter reviews the CE analysis of bases,nucleosides, and singlestranded (oligo)nucleotides. The analysis of double-stranded DNA and the technology associated with capillary gel electrophoresis (CGE) is covered elsewhere in this book (Chapter 13) and in recent reviews (I-3). A number of nucleosides and nucleotides have been used in chemotherapy as antiviral agents, e.g., 3’-azido-3’-deoxythymidine (azT), and 2’,3’-dideoxyinosine (ddI), and 2’,3’-dideoxycytidine (ddC) in the treatment of AIDS. Quantitative methods for nucleotides within cells are of interest in clinical studies dealing with therapeutic drug monitoring or cell metabolic studies. Detecting base damage within DNA is increasingly used with a variety of sensitive, high-resolution analytical methods (4). Oligonucleotides are used in a variety of applications, e.g., hybridization probes, gene cloning, primers for DNA sequencing and PCR, DNA fingerprinting, and antisense therapeutic reagents. Traditionally, chromatographic methods (ion-exchange-, ion-pairing, and reversedphase LC) or enzymatic methods have been used for purification and purity analysis of synthetic oligonucleotides (5); classical electrophoretic methods are also used for this purpose, as well as for the analysis of large (kilo to megabase-sized) DNA and RNA (6). In 1983, Tsuda et al. (7) From

Methods m Molecular Brology, Vol 52 Caprllafy Electrophoresrs Ed&d by K Altria Copyright Humana Press Inc , Totowa, NJ

227

228

Schwartz

and Ulfelder

were the first to report a capillary zone electrophoresis(CZE) separation of nucleotides. Studies involving micellar electrokinetic chromatography (MEKC) followed m the mid-80s (see Section 3.). Recent progress in capillary gel electrophoresis (CGE--see Section 5.) now routinely permits high-resolution separations of ollgonucleotides, for example, for purity control of synthetic products. In this chapter, first CZE and MEKC examples of bases, nucleosides, and nucleotides are discussed in which UV absorbance is used as the detection method. Work in which methods other than UV absorbance are used (e.g., laser-induced fluorescenceLIF) is reviewed next, followed by a discussion of the separation of relatively small (Cl 50 bases) oligonucleotides and large (150-800 bases) polydeoxyoligonucleotides. To the best of our knowledge, CE has not been routinely applied yet to cellular RNA analysis (e.g., ribosomal RNA) and will therefore not be discussed in this chapter. 2. CE Techniques

Used for Bases, Nucleosides, and Nucleotides 2.1. CZE In this mode of CE, the electrophoretic separation takes place in free solution, without a gel m the capillary. The separation is based on the charge-to-mass ratio of the analytes, and selectivity is governed by the difference in the effective mobilmes of the analytes. Resolution may be optimized by exploiting acid-base equilibria. CZE is applicable to the separation of bases, nucleosides, and nucleotides (separation primarily based on charge differences), but is not well suited for medium to larger oligonucleotides. In the latter case, the charge-to-mass ratio of the analytes is nearly the same and a different mechanism (i.e., sievingsee Section 5.) must be operational to achieve separation. Selection of the appropriate buffer, pH, and capillary column is of key importance m the separation of nucleotides. The buffer pH determines the degree of ionization of the analytes, and consequently, their effective mobilities and resolution. Various buffer and capillary systems have been used for nucleotide separations by CZE. Untreated as well as coated capillaries (see Section 2.1 1.) may be used. In the first CE publication on the separation of nucleotides, Tsuda et al. (7) used a pH 7, 20-m phosphate buffer in conjunction with 80 pm id glass capillaries. In this work, the electro-osmotic flow (EOF) was in the opposite direction of the electrophoretic flow of the nucleotides, driving the analytes toward

Analysis

by CE

the detection point. In most CE work with untreated fused silica capillaries, the EOF is toward the cathode (CZE in the “normal” polarity mode). Nucleotide concentrations were determined in blood, liver, and kidney samples. In a later paper (81, nucleotides in organs of guinea pig were determined. Ethylene glycol (0.5%) was used as a buffer additive to reduce peak asymmetry and to moderate the EOF. Relatively modest theoretical plate numbers of 30,000-40,000 were achieved. As will be discussed later (Section 4.1.), with stacking techniques, lo-fold higher plate numbers may be achieved. It is interesting to note that, m Tsuda et al.‘s paper (7), the possibility of moderating or reversing the EOF with cationic surfactants already was mentioned, a prmciple that has found great utility later on in CZE and MEKC of biomolecules (see Section 3. on MEKC). Reversed EOF was also utilized by Huang et al. (9) for the separation of several ribonucleotides. To the 12-tiformate, pH 3.8, buffer, 0.1 mA4 cetyltrimethylammonium bromide (CTAB) was added while the capillary was preconditioned (“dynamically coated”) with CTAB. One-tenth of a millimolar is below the critical micelle concentration of CTAB (0.92 mM), and consequently, CZE rather than MEKC conditions prevail. The reversed EOF method proved faster and more reproducible than the one with normal EOF. CMP, GMP, AMP, and UMP were determined in calf liver, rabbit liver, and baker’s yeast, and found to agree reasonably well with published values. With the proposed method, the absolute RNA content in tissue extract can be determined. As the authors pointed out, the greatest advantage of CZE over other methods 1sm the required sample quantity. In HPLC, typically a 1OO-foldhigher sample load is required. In addition, the CZE method is fast (ca. 15-mm run time) and offers high resolution. In a later study from the same laboratory, Ng et al. (10) determined intracellular nucleotlde profiles from two different cell lines. Nine nucleotide species were identified, all eluting within 25 min. A 140-a borate, pH 9.4, buffer was used. Figure 1 shows the CZE profiles in human blood lymphocytes and leukemic cells. It can be seen that the triphosphate nucleotides elute before the mono- and diphosphate nucleotides. The runs were performed on an automated instrument (Beckman [Fullerton, CA] P/ACETM), and were very reproducible in terms of migration time and peak area. However, at present, more peaks may be Identified with HPLC, since larger injection volumes can be used to detect trace compounds.

230

Schwartz

and Ulfelder

‘- A 4-

2m‘0

;; B 5 a

o-

5 lo9

B

Fig. 1. CZE profiles of nucleotlde pools m (A) human peripheral blood lymphocytes and (B) leukemic cells. A 140~mM borate, pH 9.4, buffer was used. Reproduced with permlsslon from ref. 10.

A similar CE system, involving untreated, 75-urn td caprllarres and a sodium borate, pH 8.3, run buffer, was used by Hernandez et al. (II) to determine cyclic nucleotides. These compounds are purinic base derivatives, which mediate many of the intracellular biochemical events triggered by neurotransmitters and hormones. The authors combined CE (as the detection step) with brain perfusion techniques, such as mrcrodialysis, to study the biochemrcal changes underlying brain functions. Prelrmrnary results show good linearity and reproducibilrty for the method, whereas the cyclic nucleotides can be resolved and quantitated in the subpicomole range. A therapeutic drug-monitoring application was described by Lloyd et al. (12). The antileukemic agent, cytosine-P-n-arabinoside (Ara-C), was determined in human serum. During the course of the CE methodology development, the authors found that determining very low levels

Analysis

by CE

231 NH:

OH

0000’

2

H

Time (mm)

I 9

Fig. 2. CZE separation of ara-C extracted from serum. A 20-mM citrate, pH 2.5, buffer was used. Reproduced with permlssion from Beckman Application Note DS-792.

(i.e., submicromolar) of Ara-C was problematic. With UV detection, inadequate sensitivity does not allow a direct determination (see also Section 4.). However, by using solid-phase extraction for concentration and sample clean-up, it was possible to determine Ara-C in the l-l 0 uA4 range. This procedure removes most of the protein and allows doubling of the Ara-C concentration. The assay was validated over a concentration range of l-l 0 ).tM. Responsewas linear in this range, with a correlation coefficient of 0.996 for the calibration plot. An electropherogram is shown in Fig. 2. Compared to HPLC, the proposed assay has a rapid

232

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and Ulfelder

Fig. 3. Time-course of an enzymatic reaction monitored by CZE. Racemic BCH-I89 was transformed into the less toxic (-) enantiomer. A sodium phosphate, pH 2.3, buffer with 27 mM dlmethyl+cyclodextrm added as chn-al selector was used as the run buffer. Reproduced with permission from Rogan and Altria, “Introductron to the Theory and Applications of Chnal CE,” Beckman Instruments, part nr. 726388. analysis time (no need to run a gradient to remove late-eluting compounds) and is free from endogenous substances. Rogan et al. (13) showed that CE is well suited to monitor enzymatic biotransformation reactions of nucleoside analogs. The racemic, antiviral drug 2’-deoxy-3’-thiacytidme (BCH 189, Glaxo, Ware, Hertfordshire, UK) underwent an enantiospecific deamidation to yield the (-) enantiomer, which is less toxic and therefore preferred for clinical use over the racemic drug. The time-course of the enzymatic reaction was followed for 51 h, and the CE electropherograms are shown in Fig. 3. After 40 h,
Analysis

233

by CE

typically involve expensive chiral stationary phases, the CE method IS simpler, reliable, and precise. In a study by Nguyen et al. (14), purine nucleotides in fish tissue were determined. In most fish, ATP degrades quickly to IMP, which, in turn, degrades to inosine and hypoxanthine. A “freshness” index was derived from the concentrations of certain nucleotides, and the results by CE were compared to an enzymatic assay in which the analytes were converted to uric acid (15). A 100~mM CAPS, pH 11, buffer was used in conjunction with a SO-pm id capillary. The analytes of interest, consequently, were driven to the detector by the strong EOF present. Good correlation between peak area and nucleotide concentration was observed. To obtain reproducible results, the authors regenerated the capillary with 1NNaOH after each analysis. Figure 4 shows the comparison of the concentrations of IMP, inosine, and hypoxanthine obtained by CE and enzymatic assays. As mentioned earlier, the selection of the pH is important with respect to the resolution of the analytes. When a sample contains substances with very similar mobilities and also spans a wide pK, range, it is practically impossible to achieve separation of all the analytes within a reasonable analysis time. Sustacek and coworkers (16) described an elegant solution to this problem by forming a “dynamic” pH gradient within the capillary. The composition and pH of the electrolyte at the injection end of the capillary were modified using an external pump supplying the modifying electrolyte. Hence, CZE selectivity can be controlled in a similar way to gradient elution in HPLC. Chosing a model mixture of 11 purine and pyrimidine bases varying in pK, from 1.9 to 6.0, baseline resolution was obtained by initiating the pH at 3.5, but decreasing the pH to 2.2 during the separation. Attempts to resolve the mixture at either pH 3.5 or 2.2 were not succesful. It should be mentioned that the instrumentation for generating the dynamic pH gradient is not commercially available. 2.1.1. Coated Capillaries

A few recent publications mention the use of coated capillaries for nucleotide separations by CZE. In the work discussed in the previous section, untreated fused silica capillaries were used, resulting in moderate to strong EOF conditions. Generally, the strategy with coating the capillaries is to moderate or eliminate the EOF, which would result in more efficient (and possibly more reproducible) separations. Takigiku

234

Schwartz

5

0

100

200

Concantratlon

300

400

by

500

and Ulfelder

600

CE ( P M)

Fig. 4. Correlation of enzymatic and CE assaysm tissue extracts for hypoxanthme (top trace), mosme (middle trace), and IMP (bottom trace). Reproduced wrth permlsslon from ref. 14.

and Schneider (I 7) devised a method with a polyacrylamide-coated column and reversed polarity, i.e., the anionic nucleotides migrated toward the positive electrode. The capillary was coated according to the procedure of HJerten (28) and yielded zero EOF. The method was able to resolve easily the common eight ribonucleoside triphosphates (NTPs) and deoxyribonucleoside triphosphates (dNTPs). Reproducibility and quantitation

were assesssed with two commercial

instruments,

the

Bio-Rad HPE 100 and Beckman’s P/ACE System 2000. Figure 5 shows the electropherogram of the NTPs and dNTPs with an analysis time of x20 min (HPLC separations generally take much longer, i.e., ca. 60 min). The overall net charge of the analytes determines their migration

Analysis

235

by CE

0

5

10

1.5

20

TIME (MINUTES)

Fig. 5. Electropherogram of an NTP and dNTP mixture. A 50-mM phosphate, pH 2.7,2 mA4EDTA run buffer was used. Peak id: 1, UTP; 2, dTTP; 3, ITP; 4, GTP; 5, dGTP; 6, dCTP; 7, CTP; 8, dATP; 9, ATP. Reproduced with permission from ref. I7 rates. Under the low-pH conditions used (phosphate buffer, pH 2.7), the

uracil, thymine, and inosine moieties are neutral, whereas guanine, cytosine, and adenine moieties are positively charged. As expected, the compounds with the largest negative charge showed the fastest elution time. Size may also play a role, as can be concluded from the slow migration rates of the relatively large adenine nucleotides compared to the cytidine nucleotides. Linearity (peak area vs sample concentration) was found to be nearly three orders of magnitude for both instruments. Good linearity was also obtained when plotting the injection time vs the nucleotide concentration; the migration time precision varied from 0.04 to 0.6% RSD for the P/ACE instrument. The minimal detectable concentrations (3:l signal-to-noise ratio) were determined for a number of ribonucleosides (120-7500 pmol/mL range). It should be noted that injection under stacking conditions (see also Section 4.) would considerably improve the detection limits. A similar crosslinked polyacrylamide-coated capillary was employed by Huang et al. (19). With this capillary, EOF was negligible in the

Schwartz

236

and Ulfelder

3

0

lb

;0

40

i0

50

min

Fig. 6. Electropherogram of rlbonucleotldes extracted from HeLa cells A 50-W Tns-HCl, 30-d phosphate, pH 5 3, run buffer was used. Peak id: 1, UTP; 2, CTP; 3, ATP; 4, UDP; 6, CDP, 7, ADP; 8, GDP; 9, XMP; 10, UMP; 11, CMP; 12, IMP; 13, AMP; 14, GMP. Reproduced with permlsslon from ref. 19.

pH 3-9 range, and the coating appeared mert to the injected biological samples. Minimum detectable levels were m the l-10 p.A4 range with UV detection at 254 nm. Figure 6 shows the electropherogram of a HeLa

cell extract. Migration time precision was in the O&1.2% RSD range. Quantitative results, i.e., the amount of ribonucleotide per 1.5 x lo7 cells, were determined for several ribonucleotides (42.4 nmol for ATP, 27.4 nmol for ADP, 6.5 nmol for AMP). It can be seen that RSDs (migration time) varied from 0.6 to 1.2%. The sharp peaks in Fig. 6 are partly the result of a sample stacking effect during the inJection step (see also Section 4.1.). The cell extract contained a lower salt concentration than the run buffer and, on injection, the analyte zone was sharpened. A capillary coating, consisting of four polymeric layers, was used by Smith and El Rassi (20) for the separation of nucleotides. This capillary featured a fuzzy polar network of polyether chains, which reduced electrostatic analyt+capillary-wall interactions and allowed for controlled (anodic) EOF. A separation with an average theoretical plate count of

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12 27 12 95 14 28 14 53 15 32 15 76 16 00 17 07 17 84 20 18 21 74 21 79 22 74 23 82 24 28 25 13 27 58

Urldme (Urd) Cyttdme ICyd) Cytostne ICYI) Thymfdtne(Thd) Thymme (Thy) 2’.Aztdo-2 deoxycylldme (AZC) Adenosme (Ado) 2 -0eowyguanosme (dGuo) 2’ Deoxyadenosme (dAdo) 2’Guanasme Deoxyguanoslne 5’ monophosphate rGMP) 2’ Deoxyadenosme-5’ 5 mOnophoSphate IdGMP) 2 Deoxycytfdlne monophosphale(dAMP) Cyttdme 2 5 monophosphate(dCMP) monophosphate (2 CMP) Cyildlne 3’.monophosphate (3’CMP) 3’ Aztdo-2’.deoxythymtdme (AZT) Unknown

Fig. 7. MEKC electropherogram of 16 nucleic acid derivatives and analogs A 25-mA4 sodium phosphate, pH 6.9, lOO-mM SDS run buffer was used Reproduced with permlsslon from ref. 24.

240,000 plates/m (in IO-min run time) was reported using a pH 7.0 phosphate buffer. 3. MEKC MEKC takes advantage of differential partmonmg of analytes into a pseudostationary phase consisting of micelles. Anionic as well as cattome surfactants have been used for this purpose. In this section, several examples will be discussed employing these surfactants as buffer additives for the separation of nucleic acids. Generally, untreated fused silica capillaries are used with MEKC, although coated capillaries also have been used. Burton et al. (21), Cohen et al. (22), and Row et al. (23) were the first to use MEKC for the separation of purine bases, nucleosides, and (oligo)nucleotides. When operating at neutral pH (e.g., with a sodium phosphate, pH 7, buffer), the bases and nucleosides are uncharged, and their separation resulted from differential partitioning within the micelles; hydrophobic analytes typically have a large partition coefficient and are more retained than hydrophilic ones. Elaborating on the earlier MEKC work, Ohms (24) used a 2%mA4phosphate buffer that contained 100 mA4 SDS. Figure 7 shows the separation of 17 nucleic acid derivatives or analogs. The analyte-SDS partitioning effect resulted in the following

238

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relative migration times for the basesand nucleosides: U < C < T < G < A; nucleosides < bases; and ribose < deoxyribose derivatives. The migration time of nucleotides appeared to be a function of the phosphorylation site, i.e., 5’ < 2’ < 3’. Substituents (methyl, acetyl, or azido for hydroxyl) greatly enhanced the migration time for certain nucleic acids. Recently, it was found by Kaneta et al. (25) that addition of glucose to micellar run buffers enhanced selectivity and extended the “elution window” (a measure of the separation range possible with MEKC). Glucose appearedto decreasethe distribution coefficient of certain analytes (those with hydrophilic functional groups) into the micellar phase. The effect of methanol and glucose as buffer additives on the separation of nucleosides IS demonstrated in Fig. 8. Methanol is frequently added in MEKC to increase the elution range. It can be seen m Fig. 8A and B that adenosine and 2’-deoxyadenosine (peaks 7 and 8) are well separated. This was not the case without methanol. Using l.OM glucose (Fig. SC) greatly improved the resolution of the cytidine/deoxyuridine (peaks 2,3) and adenosine/dexyadenosine (peaks 6,7) pairs. MEKC was also effective in the separation and detection of DNA adducts in DNA exposed to alkylating agents. Optimum conditions (using SDS as micellar phase) were reported by Lecoq et al. (26) for normal and modified nucleic acid bases,deoxy- and ribonucleosides, and 3’- and 5’-monophosphate nucleotides. Although HPLC can also be used for these types of separations, MEKC permits much faster analysis times (ca. 10 min). Migration time precision of selected nucleotides was in the 0.3-1.1% RSD range. The limit of detection with an acceptable signalto-noise level was determined as 100 fmol. Comparisons of HPLC and CE methods were also made by Lahey and St. Claire (27) and Singhal et al. (28). The latter group investigated MEKC as a possible alternative to reversed-phase HPLC for the separation of dideoxynucleosides. These compounds are currently used for the treatment of HIV-l-positive individuals (azT, dd1, and ddC) or are in human clinical trials (d4T and ddA). A preliminary MEKC separation of seven dideoxynucleosides in the 0.5-0.9 pmol range is shown in Fig. 9. A 40-w SDS, 50-M, pH 6.5, phosphate buffer was employed. Whereas in the majority of MEKC publications untreated fused silica capillaries are used, Lux et al. (29) mvestigated the effect of the coating and the EOF on the separation of nucleobases. A capillary with a nonpolar (polymethylsiloxane, OV-1) and a polar coating (polyethylene glycol,

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239

A

Fig, 8. Effect of additives on the MEKC separation of nine nucleotldes. (A) 10% methanol, 13.5 kV; (B) 20% methanol, 15.8 kV; (C) 1M glucose, 17.8 kV. A 50-Wphosphate, pH 7.0, 150~mM SDS run buffer was used. Peak id: 1, urrdine; 2, cytidine; 3, deoxyuridine; 4, deoxycytidine; 5, thymidine; 6, guanosme; 7, adenosine; 8, deoxyadenosine; 9, deoxyguanosme. Reproduced with permrssion from ref. 25.

Carbowax 20M) were compared to an untreated fused silica capillary. The coated capillaries are commercially available (J & W Scientific) and are typical GC-type capillary columns. Figure 10 shows that the nonpolar capillary yields a higher EOF and poorer resolution than that obtained with the untreated fused silica capillary. This is because SDS binds to the polysiloxane wall coating, thereby increasing its negative charge density. The relatively polar polyethylene glycol coating shows a decrease in EOF (compared to the untreated capiillary) and also gives the best resolution, Another way to manipulate (and change the direction of) the EOF is through the use of cationic surfactants, as will be discussed next.

240

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I

6

I

8

10

and Ulfelder

I I2

Time, mm

Fig 9. MEKC of seven dideoxynucleosides. A 50-U phosphate, pH 6 5, 40 mA4 SDS run buffer was used. Peak id: 1, contaminant, 2, d4T, 3, ddC; 4, dd1; 5, azT; 6, ddA; 7, Glo-azT. Reproduced with permission from ref. 28. As mentioned earlier, Tsuda et al. (7) were the first to recognize in CE that with certain cationic surfactants, such as cetyltrimethylammonium bromide (CTAB) and dodecyltrimethylammonium bromide (DTAB), the EOF changes direction. The mechanism of this principle is illustrated in Fig. 11. A bilayer is formed at the capillary wall surface, and at a high enough surfactant concentration, the EOF is reversed (reversed polarity conditions of the power supply must be used). The concentration at which this reversal takes place depends on the surfactant, e.g., for CTAB, this concentration is 0.35 wand for DTAB, it is 0.1 mM. When the concentration of the surfactant exceeds its critical micelle concentration, a pseudostationary (micellar) phase is created in the capillary that allows for analyte partitionmg, i.e., the conditions for MEKC. Liu et al. (30) studied the effect of the type of surfactant and its concentration on the resolution of phosphorylated nucleosides. It was found that MEKC with

Analysis

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I

A

4 5

10

115

Fig. 10. MEKC of nucleobases with coated captllarres. (A) Polymethylsiloxane OV-1 coating; (B) uncoated; (C) polyethyleneglycol coating. A 20-n-H phosphate, 50-W SDS run buffer was used. Peak id: 1, water; 2, uridine; 3, cytidine; 4, guanosme; 5, adenosme. Reproduced wtth permission from ref. 29.

0+

-----------_~--___ +.*---*_* + + + + + ~~~~~ +*r---I**!sJ + r +*+ #fc SF-

0-

Fig. 11. Separation mechanism wtth EOF reversal. A bilayer is formed at the capillary wall with the EOF directed toward the anode.

Schwartz

and Ulfelder

the cationic surfactant DTAB gave superior results compared to CZE separations and to MEKC with the anionic surfactant SDS. Twelve common phosphorylated nucleosides could be resolved in only 4 mm Detection limits for selected nucleotides were in the 5-14 pmol range. 4. Detection 4.1. Sample

of Low Amounts of Nucleotides in Biological Samples

Stacking

with

W Absorbance

Detection

Although the mass sensitivity with CE and the on-capillary UV absorbance detection is high, the small detection volumes limit the concentration sensitivity. Typically, with CE, concentration detectability is lo- to loo-fold less than with HPLC. However, various approaches have been used to overcome this general problem associated with CE-UV absorbance detection and were recently reviewed by Albin et al. (31). Essentially, these approaches to improve detectability are on-column sample concentration techniques. One of these techniques, sample stacking, was mentioned in Section 3. (30). The MEKC conditions of Liu et al. (30) were also employed by Perrett and Ross (32,33) for the determination of nucleotides m cell extracts. In addition, they used 1 mMEDTA m the run buffer as a metal chelating agent to prevent metal-nucleotide interaction, which was thought to result in peak tailing. Figure 12 shows that this method can be applied to acid extracts of cells. The upper trace shows the separation of a standard mixture of 15 nucleotides with a 50-Wphosphate, pH 7,100~mMDTAB, 1mA4EDTA run buffer. A neutralized perchloric acid extract of rat tumor is shown in the bottom trace of Fig. 12. The method yielded a linear response up to 200 CLM;migration time and peak area precision ranged from 2.2 to 5.5% and 3.3 to 6.1%, respectively. Chapman (34) used the method of Perrett and Ross (32) in conjunction with electrokinetic injection. This sample-introduction mode yielded much lower detection limits than previously reported with CZE, since sample components are effectively stacked during the injection process. In addition, depending on their charge, undesirable sample components are not introduced into the capillary using the electrokinetic injection. Figure 13 shows the electropherogram of a 50-nA4 test mixture of AMP, ADP, and ATP with an average plate count of 550,000. This number is exceptionally high, considering we are dealing with small molecules. Detection limits with the method of Chapman (34) were estimated to be in the low-nanomolar range.

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A e

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Fig. 12. MEKC separation of nucleottdes in (A) a standard mtxture and (B) a neutralized perchloric extract of rat tumor. The sohd trace shows detection at 255 nm, and the dashed trace, detection at 280 nm. A 50-rn44 sodium phosphate, pH 7.0, 1-m&I EDTA, 100-rmI4 DTAB run buffer was used. Reproduced with permtssion from ref. 33.

4.2. ITP Preconcentration with W Absorbance Detection The concentration detection limits can dramatically be improved by applying the principles of isotachophoresis (ITP) to CE. In ITP, the concentration of sample components is controlled by the leading and terminating electrolytes. The ITP mechanism has been applied as a means of

244

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650

600

1200

1000 Tme

and Ulfelder

14001460

,mm,

Fig. 13. High-efficiency (ca. 0.5 mullion theoretical plates) separatron of a standard mrxture of AMP, ADP, and ATP. Electrokrnetrc inJectron was used with a 50-w sodium phosphate, pH 7, lOO-mM DTAB, 1-d EDTA run buffer. Reproduced with permission from ref. 34. concentrating the analytes prior to their separation by CZE. Thus, with ITP-CE, sample components m very dilute samples-which otherwise would go undetected-can be analyzed. In earlier work, this was achieved by performing the ITP preconcentration step on a separate precolumn and then transferring the concentrated sample plug to the separation capillary. For example, Foret et al. (35) used a homemade ITP-CE system for the separation of nucleotides. Whereas in CZE a 45-nL, 3 x lO-“M sample solution was injected, with ITP-CE, a 10.6~pL, 10”M solution

yielded approximately the same signal-to-noise ratio in the electropherogram. Thus, a ca. 300-fold increase m detectability was achieved with the ITP-CE system. More recently it has been shown by Foret and coworkers (36,3 7) as well as others (38, that with a judicious choice of leading and terminating electrolytes, a commercial CE system (P/ACE) can be used for protein separations with sensitivity gains approaching three orders of magnitude. Figure 14 shows an ITP preconcentrated nucleotide separation; 100 mMMES-Tris, pH 5.7, were used as the background electrolyte, and the separation was performed on a 107 cm x 75 pm polyacryl-

amide-coated capillary (39). The 900-nL vol sample (10-5M of each nucleotide) was dissolved in 20 mM histidme hydrochloride.

Analysis

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30

5

time (mm)

Fig. 14 CZE with ITP preconcentration on a polyacrylamide-coated capillary (100 cm x 75 pm). The background electrolyte was 100 mMMES-Tris, pH 5.7. Sample (1t5M of each nucleotide) was m 20 mM histidme HCl. A 900-nL injection volume was mtroduced into the capillary. Constant current, 13 PA, max. voltage, 26 kV. The first elutmg group contained UTP, TTP, CTP, ATP, and GTP; the second group contained UDP, TDP, CDP, ADP, and GDP; the last group contained UMP, TMP, CMP, AMP, and GMP. Reproduced with permission from ref. 39.

4.3. Detection

Methods

Other than

4.3.1. Radiochemical

W Absorbance

Detectors

Methods based on fluorescence and radiochemistry have been reported permitting high-sensivity detection of nucleosides and nucleotides. Pentoney et al. (40) described two on-line radioactivity detectors for CE. The systems were evaluated with P-32-labeled nucleotides. Although the detection limits were impressive (ca. 1O-‘Ok!),handling and disposing of radioactive materials are well-known disadvantages that may have halted commercial development of radiochemical detectors for CE. 4.3.2. LIF

In principle, LIF approaches allow extremely sensitive detection of fluorescently labeled compounds with up to six orders of magnitude higher sensitivity than UV absorbance.Yeung’s group (41-44) published

Schwartz

and Ulfelder

several approaches involving LIF of nucleotides. Direct as well as indirect fluorescence detection can be utilized, as will be discussed next. 4.3.2.1. INDIRECT LIF DETECTION In the indirect fluorescence approach, a fluorophore, such as salicylate, is used as the run buffer (#1,42). The nonfluorescing analytes can be detected through charge displacement of the fluorophore (generally an anion). An increase in fluorescence is observed when cation analyte ions pass the detector region because more fluorophore has to be present to preserve charge neutrality. On the other hand, when anions are detected, a decreased fluorescence signal is obtained. Results on very small id capillaries (14 pm) were reported with typical limits of detection m the 50-100 attomol range (for comparison, UV detection with 50-pm id capillaries permits ca. 10 fmol to be detected). A 325-nm HeCd laser (41) or a UV Ar ion laser (42) was used for excitation. 4.3.2.2. DIRECT LIF DETECTION Because of the lower background and higher signal-to-noise ratio, even better sensitivity (compared to the indirect method) was achieved with direct LIF of labeled nucleotides (43). Figure 15 shows the MEKC-LIF analysis of dansylated calf thymus DNA digest. A 20-pm id capillary with a phosphate/borate, pH 9.0,45-mMSDS buffer was used with an Ar ion laser operating at 350 nm for the on-capillary fluorescence. MEKC is preferred over CZE for this application, since the dansylated impurities (because of their hydrophobic nature) are slowed down in MEKC and better resolved from the nucleotides. Combined with trace enrichment procedures (HPLC), the MEKC-LIF technique has potential to assess DNA damage in carcinogenesis studies. The mass detection limit was estimated as 6 attomol, corresponding to ca. 4 million molecules. The concentration detection limit with MEKC-LIF approaches that of CE radiochemical detection (40) and is four to five orders of magnitude lower than UV absorbance detection. Measurement of intercellular levels of the 6-thioguanine nucleotide metabolites of the anticancer drug 6-mercaptopurine is under study by Rabel et al. (45). A homemade CE instrument with a 325-nm HeCd laser was used for separation and detection of attomole amounts of 6-thioguanine mono-, di-, and triphosphate nucleotides. The concentration sensivity for the thionucleotides was ca. 2 x lO-‘OM. However, the lowest concentration that could be reproducibly quantitated was a factor 10 higher.

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247

9

L 1,

Time (mm)

II

JI

7

13

16

17

Fig. 15. MEKC-LIF electropherogram of dansylated (dns) calf thymus DNA digest. A 10-Wsodium phosphate, 7-d sodium borate, 45-W SDS, pH 9.0, run buffer was used. Peak id: (A) dns-hydroxide; (B) dns-dTMP; (C) dnsdCMP; (D) dns 5-MedCMP; (E) dns-dAMP; (F) dns-dGMP. Reproduced with permission from ref. 43.

Future work of this group focuses on the applicability of the method to real biological samples, such as lymphocyte and erythrocyte extracts. There is increasing interest in measuring DNA base damage. A recent review by Cadet and Weinfeld (4) describes strategies involving various chromatographic and electrophoretic methods to quantitate photoinduced and oxidative DNA basemodifications. A general problem with these types of assays is that the sensivity often is not adequate to allow the measurement of at least one modification in 104-lo6 normal bases within a few micrograms of DNA. CE with LIF detection is, in principle, sensitive enough to overcome these limitations. Preliminary results of a CZE-LIF method for S-deoxynucleotides using a derivatization procedure with fluorescein and ethylenediamine were presented by Li et al. (46,4 7). A homemade instrument with a 488-nm Ar ion laser was used in this work. After purifying the DNA from biological samples, the DNA adducts are isolated as damaged deoxynucleotides, labeled with fluorescein, and finally detected with CZE-LIF. Figure 16 shows electropherograms of samples exposed to 6oCoradiation (A), hydrogen peroxide (B), and a combined sample (C). It can be seen that the highlighted peak at

A

8

14

20

8

14

20

8

14

20

TIME (min) Ftg. 16. CE-LIF of fluorescein-ethylenediamine-dAMP samples. (A) Sample exposed to 6oCo radtation, (B) sample exposed to hydrogen peroxide; (C) combmed injection of (A) and (B) A 10-n&! Trts-borate, 10% acetonitrtle, pH 10.4, run buffer was used. Reproduced with permission from ref. 47. The peak marked by an arrow is absent in Trace (B), and represents exposure to (j°Co radtation.

Analysis

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16.02 min is absent in trace B and C. The authors speculate that their CE-LIF method may therefore discriminate between DNA damage from ionizing radiation and that of other sources.CE-LIF appearsto be a promising alternative to HPLC studies of this kind because of its speed, resolution, easeof column cleaning, and compatibility with small sample sizes. A detection limit of 20 ZM (1 zmol = 1O-21mol) for a fluorescemlabeled deoxynucleotide triphosphate was reported by Dovichi’s group (48,49) using CE-LIF instrument with a postcapillary sheath-flow cuvet. In this LIF detector, the sample components, while exiting the capillary, are introduced in the center of a flowing sheath stream of run buffer. The cuvet, commonly used in flow cytometry, provides excellent optical quality for high-sensitivity detection. The CE-LIF instrument was used for the fluorescence detection of amino acids and DNA sequencing (see also Section 5.3.). The authors hint that the ultimate sensivity of single-molecule detection soon may be a reality. Native fluorescence of nucleotides can also be exploited in LIF detection schemes, as recently demonstrated by Milofsky and Yeung (44). The 275.4-nm line from an Ar ion laser or the 24%nm line from a waveguide KrF laser can be used to excite native fluorescence. Although not as sensitive as the above mentioned direct LIF method involvmg labeled analytes (43), the proposed method is three orders of magnitude more sensitive than that obtainable with UV absorbance. Detection limits for GMP and AMP were 1.5 x 1o-8 and 5 x 1@M, respectively. 5. CE Techniques

Used for Oligonucleotides

Small oligonucleotides (4-8 bases) can easily be resolved by opentube, free-solution CE methods, i.e., with CZE and MEKC (22). However, as the number of nucleotide units increase, the separation becomes more difficult in free solution. CGE is eminently suitable for the separation of the larger oligonucleotides. Medium-size (10-l 50 bases), synthetic oligonucleotides are used in a variety of applications, e.g., as probes for gene isolation and diagnostics, as primers in PCR reactions and DNA sequencing, and in work dealing with cloning and gene alteration, DNA fingerprinting, and antisense therapeutics. In molecular biology and biochemistry, ohgonucleotides are traditionally analyzed by slab-gel electrophoresis, either with polyacrylamide or agarose gels (6). Generally, polyacrylamide gels are used with DNA sequencing. For the analysis of smaller oligos (<30 bases), HPLC in the anion-exchange or

250

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reveresed-phase modes can also be used. Recent papers compare the performance of HPLC and CE for oligonucleotide separations (5&52). Important applrcations involving classical electrophoresis are the purity control of synthetic oligonucleotides (including antisense DNA) and DNA sequencing. In DNA sequencing gels, resolution of nucleotides from ca. l&800 bases can be achieved. However, in spite of the high resolving power achievable with slab gels (especially at the high end, i.e., 100-l 000 bases), there are some important limitattons with classtcal electrophoresis: 1 Vlsuahzation of the bands requires the use of dyes or radlolabelmg followed by scanning or autoradiography; 2. The gel-handling procedure 1stime-consuming, labor-intensive, and not readily automatable; and 3. Quantitation of impurities is only semiquantitative using off-line scanning devices. CE, in the CGE mode, may overcome these disadvantages. The capillary technique permits on-line detection for real-time monitoring, and staining is not required. Compared to slab gels, CGE separations can be run at considerably higher field strengths (ca. 10x higher), resultmg in faster and often more efficient separations. Typically, nanogram to picogram quantities of oligonucleotides are separated. Basically, CE can be viewed as an instrumental approach to electrophoresis. Therefore, it is reasonable to assume that, in time, almost all applications developed for slab-gel electrophoresis can be transferred to a capillary format. For example, Southern hybridization (53) and protein-DNA interactions by mobility shift assays (54) have already been attempted with the CE approach. It should also be noted that, although somewhat limited, CE can be used in semipreparative mode. For example, Cohen et al. (55) purified a 20-mer oligonucleotide by CGE from a crude preparation. The collected fraction was subsequently used in standard dot-blot assay. In another paper from Guttman’s group (56), other aspects of micropreparative CE of oligonucleotides were discussed. Shieh et al. (57) also presented a procedure for collecting synthetic oligonucleotides from a commercially available gel column. In this section, selected examples of oligonucleotide separations are presented, reflecting the present state-of-the-art m CE, with emphasis on applications dealing with quality control of synthetic preparations and DNA sequencing. More information on the types of gels used with CE, their preparation, limitations, and so forth, can be found m Chapter 13 of

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251

this book, and in recent reviews by Cohen et al. (21, Baba and Tsuhako (52), and Schomburg (3). 5.1. Purity Control of Synthetic Oligonucleotides (lo-150 Bases) Whereas the purity requirements play a relatively minor role in applications where the oligonucleotide is used as a hybridization probe, in other applications, it is essential that, after synthesis, the level of failure sequences is determined. For the CE of oligonucleotides with l&150 bases, crosslinked or linear polyacrylamide gels covalently bonded to coated fused silica capillaries are mostly used under denaturing conditions (urea, formamide, heat). The capillary wall is derivatized with a suitable coating, because the EOF must be eliminated (otherwise the gel would come out of the capillary during use); the coating also provides a means for attachment of the polyacrylamide gel. The gel matrix, whose pore size (as expressed m the % T and% C) can be controlled by judicious choice of the polymerization reagents, separates the analytes by a molecular sieving effect, i.e., small molecules migrate faster than large ones. Gel capillaries of the polyacrylamide type are now commercially available from Beckman Instruments and J & W Scientific. PerkinElmer, Applied Biosystems Division (Foster City, CA) manufactures a proprietary gel capillary (Micro-Gel 100) that is nonpolyacrylamide and does not contain urea. The operating factors affecting resolution and quantification of oligonucleotides separated on this gel-filled capillary were reported by Demorest and Dubrow (58’. Sample detectability was improved by imecting the sample in a low-conductivity solution, increasing the injection time or voltage. Overloading the column resulted in decreasedresolution and obscured the presenceof trace components. The prepacked, lOO+m id capillary available from Beckman (eCAP ssDNA 100) contains 7Murea to prevent the formation of secondary structure of oligonucleotides (59). The capillary is designed for oligonucleotides in the 10-l 00 base range and can be used in the 20-50°C temperature range. An example from work of Egan and Holzman (60) is shown in Fig. 17. Two synthetic oligonucleotide preparations, each with a 67-mer sequence complementary to the other, were analyzed by CGE and slab-gel (PAGE) electrophoresis.The synthetic ssDNA must be sufficiently pure so that, after annealing of the two ss oligonucleotides, the resulting ds DNA product could be used for production of a protein coded by the 67-mers. It can

252

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Fig. 17. Separation of synthetic oligonucleotides by CGE and slab-gel electrophoresis(7% acrylamide). Capillary, eCAP ssDNA 100 (Beckman). (A) 67-mer; (B) complementary67-mer. Reproducedwith permission from ref. 60.

be seenthat the failure sequencesare much better resolved by CGE. In addition, run times are shorter(40 min for CGE; 180min/run for PAGE). In most CE work, the long section of the capillary (from the injection endto the detectorwindow) is usedasthe separationmedium.With CGE, fast purity checks are possible by utilizing the short end of the capillary (59). This approach-useful

when high resolution is not required-is

Analysis

by CE

i

253 HIGH SPEED Short end injection I=7cm)

E

: HIGH RESOLUTION

8.

62 z8

long end injection (I = 40 cm)

Fig. 18. CGE separation of polyrlboadenylic acid (12-18-mers). Capillary, eCAP ssDNA 100 (Beckman). Reproduced with permission from Guttman and Cooke (74).

illustrated in Fig. 18 for a polyriboadenylic acid mixture (12-18 bases). The total capillary length is 47 cm, and the length to the detection window is 7 cm. The left panel shows a Smin, fast separation in the 7-cm side

Schwartz

I/+?

and Ulfelder

Purlfird

Fig. 19. Separation of a crude and purified 119-mer by CGE. Capillary, eCAP ssDNA 100 (Beckman).

of the capillary, whereas the right panel shows a high-resolution, relatively slow (35-min) separation in the 40-cm effective length capillary. Whereas the formulation of the polyacrylamide gel of Figs. 17 and 18 primarily is designed for oligos in the
Analysis

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255

mentary nature of the antisense molecule allows it to hydrogen bond to the above biomolecules, thereby inactivating the genetic message and inhibiting gene expression, However, oligonucleotides with a phosphodiester backbone are very suceptible to cellular nuclease degradation. Therefore, much interest has been directed toward DNA analogs with phosphorus-modified backbones, such as phosphorothioates and methylphosphonates, because of their increased resistance to these nucleases. In fact, a type of antisense, termed peptide nucleic acid (PNA), in which the entire deoxyribose-phosphate backbone is exchanged for a peptide backbone composed of (2-aminoethyl)glycine units, shows promise as a potent antisense molecule (62). Because of the therapeutic nature of these antisenseDNA molecules, purity requirements are stringent. CE of these relatively short (IO-25 bases) oligonucleotides using crosslinked or linear polyacrylamide gel-filled capillaries gives excellent single-base resolution of phosphodiester antisense from its failure sequences,but rather unsatisfactory results (broad peaks resulting m poor resolution) with backbone-modified antisense. Phosphorothtoate ohgonucleotides separations were shown by Warren and Vella (50) using crosslinked polyacrylamide gels, but with less resolution than that seen by anion-exchange HPLC. DeDionisio (63) used a modified procedure with a commercially available gel-filled capillary (Micro-Gel 100, Perkin-Elmer, Applied Biosystems Division) and achieved single-base resolution of a standard phosphorothioate mixture by Increasing buffer pH. However, for typical phosphorothioate DNA commonly synthesized for biological studies, the N- 1 and N-mer could not be baseline-resolved. When using a commercially available linear polyacrylamide gel capillary (eCAP’” ssDNA 100, Beckman, Fullerton, CA), a relatively long capillary (40-cm effective length instead of the standard 30 cm suggested by the manufacturer) was effective in achieving 99% baseline resolution between N-l and N-mer. Figure 20 shows the separation of a 20-mer phosphorothioate antisense using a linear polyacrylamide capillary. Resolution was increased using the longer capillary at the expense of run time. Cohen et al. (2) also analyzed phosphorothioates. A formamidemodified polyacrylamide gel capillary yielded improved resolution of failure sequencesup to 50 bases. Finally, Rose (64) studied the binding kinetics of specific PNAs with oligonucleotides using CGE to separate and quantitate both free PNA and oligonucleotide, as well as bound heteroduplexes. In this case, both

Schwartz

and Ulfelder

Fig. 20. CGE separatronof a 20-mer phosphorothloateantisenseDNA from the 19-mer.Capillary, eCAP ssDNA 100(Beckman). single- and double-stranded molecules were analyzed on the same capillary simultaneously, with high resolution. 5.2. LIF Detection DNA applications involving the use of fluorescence detection are well established in the fields of flow cytometry and electrophoresis. Work in CE combined with fluorescence has been limited owing to a lack of su:lable, sensitive instrumentation. However, an LIF detector (488-nm Ar ion laser) specifically designed for CE now is commercially available (Beckman Instruments). It can be anticipated that CE applications using fluorescence detection of ss- and ds-DNA will dramatically increase in the years to come. For example, various groups involved in capillary-based DNA sequencing utilize CE-LIF systems(see Section 5.3.). Schwartz and Ulfelder (65) reported a sensitive LIF assay for ds-DNA restriction fragments and PCR products. The method involved the use of a fluores-

Analysis

by CE

257

cent, DNA-intercalating dye and a replaceable gel (eCAP dsDNA 1000, Beckman). Currently, there is considerable interest in alternative labeling techniques for fluorescent-based analysis of PCR products. Labeled primers allow direct incorporation into the PCR (66). CE separations of fluorescently labeled primers and probes have been reported by Toulas and Hernandez (67) and Ulfelder (68). The speed and sensitivity (ca. lo-‘*A4 of primer can be detected) of CE-LIF open the way for various applications in molecular biology involving ss- and ds-DNA, such as the analysis of variable number tandem repeats, nuclease mapping, and so on. A study of protein-DNA interaction by means of a mobility shift assay was reported by Maschke et al. (54). Synthetic oligonucleotides were labeled with Joe-dye and detected by LIF. With slab-gel methods, detection typically takes place by autoradiography of P-32-labeled oligonucleotides. The binding to a model protein, restriction endonucleaseEcoRI, was investigated using an open-tube CE system with a TBE/S% acetonitrile run buffer. The authors also presented preliminary data on DNA-RNA hybridization with CE-LIF detection. In this case, 0.5% hydroxypropylmethylcellulose was used as the sieving agent in the TBE run buffer. Chen et al. (53) used polyacrylamide-filled capillaries for the identification of DNA by precolumn hybridization. Parameters that affect the hybridization of DNA in solution were studied. Essentially, the feasibility of performing Southern hybridization m a capillary format (with on-line LIF detection) was investigated. Potential applications include the screening for genetic mutations and infectious diseases in conjunction with PCR. A Joe-tagged oligonucleotide was selected as the probe for the hybridization with complementary DNA. Whereas encouraging results were demonstrated for ss DNA, the ds DNA molecules gave inconsistent results. 5.3. Separation

of Large Oligonucleotides; DNA Sequencing The design of gel columns with increased pore size (i.e., ca. 3% T, 5% C with polyacrylamide) for the separation of larger oligonucleotides (100400 bases) was discussed by Guttman’s group (56), Baba and Tsuhako (52), and Schomburg (3). From a pure separations perspective, the resolving power (i.e., the number of achievable theoretical plates) of these columns is quite astonishing. To the best of our knowledge,

258

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Guttman et al, (56) hold the unofficial world record for plate count (using any separation mode, CE, LC , GC, or SFC) with 30 milhon plates/m calculated for a 160-mer oligonucleotide! Schomburg (3) and Baba and Tsuhako (52) have reported similar large plate numbers, as exemplified by the separation of 430 peaks of a polyuridine-5’-phosphate sample (3) and 470 peaks of a sample of polyadenyhc acid (52). The actual plate count depends on the size of the nucleotide, the type of sample, and the sample load (56). The technology for developing these columns is still evolving and shows real potential for use m capillary-based DNA sequencers. Several groups (see refs. 3, 69-72) are currently involved m research exploring the use of CE with different LIF schemes for this purpose. Recently, replaceable, linear polyacrylamide gels have also been applied to the separation of oligonucleotides up to 450 bases (72,73). Whereas the resolution achievable with these gels is shghtly less than that of the crosslmked type, the replaceable gels appear preferable in terms of ease of use, stability, and reproducibility for routine DNA sequencing applications. An example from Pentoney et al. (72) showing the excellent resolution of fluorescently labeled oligonucleotides feasible with a replaceable gel (polyacrylamide, 6% T, 0% C) is shown in Fig. 21. The authors used a single-fluor approach (“Tabor and Richardson” method) for enzymatic chain termination sequencing of DNA. The CELIF system was employed for capillaries with a single power supply for the analysis of the complementary sequencing reactions. The CE-LIF instrumentation described by Pentoney et al. (72) is primarily designed for relatively inexpensive, routine, automated DNA sequencing.

Notes Added in Proof Several recent advances in nucleotide separations have been reported since this chapter was written, Using CZE, O’Neill et al. (75), described the use of Ucon (Alltech, Deerfield, IL) coated capillaries for the analysis of intracellular nucleotide pools. This neutral, hydrophilic coating reduced macromolecular adsorption and EOF under desirable buffer conFig. 2 1. (opposite page) CE-LIF electropherogram showing the separation of fluorescently labeled DNA fragments generated enzymatically (M 13mp 18 template) using three dldeoxy cham terminators in the concentration ratio 4A:2G: 1C. The separation was performed using a replaceable polymer network solution. Courtesy of S L. Pentoney, Jr.

0 w

0 m

259

-=zr

-

-

r

0 W

0 In

c’

z 2 a C .d

CJ E *i-

260

Analysis

261

by CE

ditions (pH 5-6). Esaka et al. (76), added polyethylene glycol (PEG) to a phosphate buffer to separatenucleotides. In this hydrogen-bonding mode, improved resolution results from the electrostatic interaction between the ether section of PEG and the amide and amino groups in the nucleotides. Tadey and Purdy (77) demonstrated separation of phosphorylated nucleotide isomers using P-cyclodextrin and borate complexation. Tseng et al. (78’ showed LIF detection of adenine analogs at the single-cell level by selective derivatization using chloroacetaldehyde as the fluorogenic reagent. In antisense therapeutics research, Cohen et al. (79) found that chromatographic separation methods worked well for small phosphorothioates, but CGE using linear PA at high density (13-l 8 %T) yielded the optimal resolution. These gels are most often used for DNA sequencing. Using a linear PA gel system, Srivatsa et al. (80) demonstrated the validity of CGE for routine analysis of drug product formulations. The linearity, accuracy, selectivity, precision, and ruggedness of the method were evaluated, with excellent migration time and peak area reproducibility demonstrated by using an internal standard. Bourque and Cohen (81) likewise applied CGE for quantitative analysis of antisense ohgonucleotides in biological matrices. Vilenchik et al. (82) used CGE as an on-column quantitative Southern hybridization tool to monitor antisense DNA binding. Detection by laser-induced fluorescence (LIF) monitored the hybridization of a phosphorothioate target with a complementary fluorescein-labeled DNA probe. Finally, Effenhauser et al. (83) described very fast (~1 min) size separations of fluorescent phosphorothioates using a PA gel in a micromachined glass chip as a CE device. References 1. Landers, J. P., Oda, R. P , Spelsberg, T. C., Nolan, J. A., and Ulfelder, K. J. (1993) Blotechniques 14, 98. 2. Cohen, A. S., Smisek, D. L., and Keohavong, P. (1993) Trends ln Anal Chem. 12, 195. 3. Schomburg, G (1993) Oltgonucleotides, m Capillary Electrophoresis. Theory and Practice (Camtllert, P , ed.), CRC, Boca Raton, Ch. 7, p. 255 4 Cadet, J. and Wemfeld, M. (1993) Anal Chem. 65,675A. 5. Lloyd, L. L., Warner, F. P., and Kennedy, J. F. (1991) Bloseparatlons 2,207. 6. Efcavrtch, W. J. (1990) The electrophoresis of synthettc oligonucleottdes m Gel Electrophoresls ofNuclelc Acids* A Practical Approach (Rickwood, D and Hames, B. B., eds.), IRL Press at Oxford University, New York, p 125 7. Tsuda,T., Nakagawa,G., Sato,M., andYagi,K. (1983) J. Appl Biochem. 5,330.

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8. Tsuda, T , Takagt, K , Watanabe, T , and Satake, T (1988) J Hugh Resolut Chromatogr HRC 11,72 1. 9 Huang, X , Shear, J B., and Zare, R N (1990) Anal Chem. 62,2049 10 Ng, M., Blaschke, T F, Anas, A A, andZare, R N (1992) Anal Chem 64, 1682 11 Hernandez, L , Hoebel, B G , and Guzman, N A (1990) Analysis of cychc nucleottdes by captllary electrophorests using ultravrolet detection, m Analytzcal Brotechnology, Capillary Electrophoreszs and Chromatography (Horvath, C and Nikelly, J G., eds ), American Chemical Society, Washmgton, DC, Ch. 3, p 50 12 Lloyd, D K , Cypess, A M , and Wamer, I W (1991) J Chromatogr 568, 117 13 Rogan, M M , Drake, C , Goodall, D M , and Altrta, K D (1993) Anal Bzochem 208,343 14 Nguyen, A.-L,, Luong, J H T., and Masson, C (1990) Anal Chem. 62,249O 15. Mulchandani, A., Male, K. B., and Luong, J. H T. (1990) Bzotechnol. Bzoeng. 35,739 16 Sustacek, V , Foret, F , and Bocek, P (1990) J Chromatogr 480,27 1 17 Takigtku, R. and Schneider, R E. (1991) J Chromatogr 559,247 18 HJerten, S (1985) J Chromatogr 347, 191 19 Huang, M , Lm, S , Murray, B. K , and Lee, M L (1992) Anal Bzochem 207,23 1 20. Smith, J T and El Rasst, Z (1993) Electrophoreszs 14,396 21 Burton, D E., Sepamak, M J , and Maskarmec, M P (1986) Chromatographza 21,583 22. Cohen, A. S , Terabe, S., Smith, J. A , and Karger, B L (1987) Anaf Chem 59,102 1 23 Row, K H , Griest, W H , and Maskarmec, M P (1987) J Chromatogr 409, 193 24 Ohms, J I. (1989) Applrcatrons Data, DS-738A, Beckman Instruments, Fullerton 25 Kaneta, T , Tanaka, S , Taga, M , and Yoshtda, H (1992) J Chromatogr 609,369 26. Lecoq, A -F., Leuratti, C., Marafante, E , and DiBiase, S (1991) J Hugh Resolut Chromatogr HRC 14,667. 27 Lahey, A and St Claire, R L (1990) Am Lab., November, pp. 68-70. 28 Smghal, R P , Hughbanks, D , and Xtan, J. (1992) J Chromatogr 609, 147 29 Lux, J. A., Ym, H , and Schomburg, G (1990) J Hugh Resolut Chromatogr HRC 13, 145 30 Lm, J , Banks, F., Jr , and Novotny, M (1989) J. Mzcrocolumn Sep 1, 136 3 1 Albm, M , Grossman, P D , and Mormg, S E (1993) Anal Chem 65,489A 32 Perret, D and Ross, G R (1991) m Human Purzne and Pyrzmzdme Metabolism WI Man, Vol 7, Part B, (Harkness, R A , ed.), Plenum, New York, pp l-5 33 Perret, D. (1993) Capillary electrophoresrs for the analysts of cellular nucleotides, in Capillary Electrophoreszs Theory and Practzce (Camtlleri, P., ed.), CRC, Boca Raton, p. 37 1. 34 Chapman, J (1993) AppZzcatlon Znformatzon, A-1726, Beckman Instruments, Fullerton 35 Foret, F., Sustacek, V., and Bocek, P. (1990) J, Mzcrocolumn Sep. 2,299. 36 Foret, F., Szoko, E., and Karger, B L. (1993) Applzcatzon Znformatron, A-1740, Beckman Instruments, Fullerton 37 Foret, F., Szoko, E , and Karger, B. L (1993) Electrophoresrs 14,417. 38 Schwer, C. and Lottspeich, F. (1992) J Chromatogr 623,345. 39 Foret, F , Szoko, E., Vine, W , and Karger, B. L , unpublished results

Analysis 40. 41. 42. 43. 44. 45.

by CE

Pentoney, S L , Jr., Zare, R N , and Quint, J F (1989) Anal Chem. 61, 1642 Kuhr, W. G and Yeung, E S. (1988) Anal Chem. 60,2642 Gross, L and Yeung, E S (1989) J Chromatogr. 480, 169 Lee, T , Yeung, E S , and Sharma, M. (1991) J Chromatogr 565, 197. Mrlofsky, R. E and Yeung, E. S. (1993) Anal. Chem. 65, 153 Rabel, S. R , Trueworthy, R , and Stobaugh, J F. (1993) J Hugh Resolut Chromatogr HRC 16,326 46. LI, W , Moussa, A., and Giese, R W (1992) J Chromatogr. 608, 17 1. 47. Lr, W , Moussa, A., and Geese, R. W (1993) J Chromatogr. 633,3 15 48. Zhang, J. Z , Chen, D Y , Wu, S., Harke, H. R , and Dovrcht, N J. (1991) Clan. Chem. 37,1492 49. Cheng, Y.-F and Dovxhr, N J (1988) Sczence 242,562 50 Warren, W. J and Vella, G. (1993) Btotechmques 14,598 51. Oefner, P J , Bonn, G K , Huber, C G., and Nathakarnkrtkool, S (1992) J Chromatogr. 625,33 1 52. Baba, Y and Tsuhako, M (1992) Trends zn Anal Chem 11,280 53. Chen, J. W , Cohen, A S , and Karger, B. L. (1991) J. Chromatogr. 559,295 54 Maschke, H. E , Frenz, J , Willlams, P. M., and Hancock, W. S (1993) 5th Znternatronal Sympostum on High Performance Caprllary Electrophoresu, Orlando, January, Poster T- 12 1. 55 Cohen, A. S , Najarran, D R , Paulus, A , Guttman, A., Smith, J A , and Karger, B. L. (1988) Proc. Mat1 Acad Scz USA 85,966O 56. Guttman, A., Cohen, A. S , Hetger, D N., and Karger, B L (1990) Anal Chem. 62,137 57. Shteh, P. C H., O’Brten, B , Salmas, T., Guo, M., and Cooke, N. (1991) llth Internattonal Sympostum of Proteins, Pepttdes, and Polynucleottdes, Washmgton, DC, October 58 Demorest, D and Dubrow, R (1991) J Chromatogr. 559,43 59. Guttman, A. and Cooke, N (1991) Am Btotechnol Lab 9, 10 60 Egan, D and Holzman, T (199 1) Applicattons Brief, DS-8 16, Beckman Instruments, Fullerton 61. Marshall, W. S. and Caruthers, M. H. (1993) Sctence 259, 1564 62. Nielsen, P. E., Engholm, M , Berg, R. H., and Buchardt, 0 (1991) Science 254, 1497 63 DeDionisro, L. J Chromatogr 652, 101. 64. Rose, D J. (1993) Anal Chem. 65,3545. 65 Schwartz, H E. and Ulfelder, K. J (1992) Anal. Chem 64, 1737. 66. Mansfield, E. S and Kromck, M N (1993) Bzotechnrques 15,274. 67. Toulas, C. and Hernandez, L (1992) LC GC lo,47 1 68. Ulfelder, K. J (1993) Sczence Innovatton, Conference on New Research Technzques, Boston, August. 69. Swerdlow, H., Zhang, J. Z., Chen, D. Y., Harke, H. R., Grey, R , Wu, S , Dovtchr, N. J , and Fuller, C (1991) Anal Chem 63,2835 70. Smith, L M. (1991) Nature 349,812 71. Mathies, R. A. and Huang, X C. (1992) Nature 359, 167. 72. Pentoney, S. L., Jr., Konrad, K. D., and Kaye, W (1992) Electrophorests 13,467.

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73 Rmz-Martinez, M. C , Berka, J , Belenkn, A., Foret, F , Miller, A. W., and Karger, B L. (1993) 5th international Symposium on High Performance Caprllary Electrophoresu, Orlando, January, Poster T-206 74 Guttman, A. and Cooke, N (1991) Am Bzotech Lab 9, 10 75 O’Netll, K , Shao, X., Zhao, Z., Mahk, A , and Lee, M L. (1994) Captllary electrophorests of nucleottdes on Ucon-coated fused silica columns Anal Blochem. 222, 185-189. 76 Esaka, Y., Yamagucht, Y., Kano, K , Goto, M , Haragucht, H , and Takahasht, J (1994) Separation of hydrogen-bonding donors m capillary electrophorests using polyethers as matrix Anal Chem 66,244 l-2445 77 Tadey, T. and Purdy, W C (1994) Capillary electrophoretic separation of nucleottde isomers via complexatton with cyclodextrm and borate J Chromatogr B 657,365-372. 78. Tseng, H C., Dadoo, R., and Zare, R N. (1994) Selective determmatlon of adenmecontammg compounds by capillary electrophoresis wtth laser-induced fluorescence detection Anal Blochem 222, 55-58. 79 Cohen, A. S., Vrlenchtk, M., Dudley, J L , Gembotys, M. W , and Bourque, A. J (1993) High-performance liquid chromatography and capillary gel electrophorests as applied to anttsense DNA. J Chromatogr 638,293-301 80 Srtvatsa, G S , Batt, M., Schuette, J , Carlson, R H , Fttchett, J., Lee, C., and Cole, D. L (1994) Quantitative capillary gel electrophorests assay of phosphorothtoate ohgonucleottdes m pharmaceuttcal formulattons. J Chromatogr A 680,469-477 8 1. Bourque, A. J and Cohen, A S. (1994) Quantttattve analysts of phosphorothtoate oltgonucleottdes m biologtcal fluids using direct inJection fast anion-exchange chromatography and capillary gel electrophorests J Chromatogr B 662,343-349 82. Vtlenchtk, M , Belenky, A , and Cohen, A. S. (1994) Momtormg and analysts of antisense DNA by high-performance capillary gel electrophorests. J Chromatogr 663, 105-l 13, 83. Effenhauser, C. S , Paulus, A, Manz, A, and Wtdmer, H. M (1994) High-speed separation of anttsense ollgonucleottdes on a mtcromachined capillary electrophorests device. Anal Chem 66,2949-2953

CHAPTER18

Application of Capillary to Pharmaceutical

Electrophoresis Analysis

Kevin D. Altria 1. Introduction The majority of drugs are either acidic or basic water-soluble compounds, and many drugs may be adequately separated by FSCE. For example, an acidic drug may be analyzed in its anionic form at high pH, and basic drugs may be tested at low pH in their cationic form. Figure 1 shows the efficient separation of a test mixture containing 11 basic drugs using a simple low-pH electrolyte. Zwitterionic drugs (those contaming both acidic and basic groups) may be analyzed at either end of the pH range. However, a mixture of neutral and charged drugs would be unresolved by FSCE, and these would require separation by MECC (see Chapter 12 for further details and discussion of MECC, also called MEKC). The determination of drug-related impurities is generally performed by HPLC, which is an established technique with highly automated instrumentation. Impurity profile information is often supplemented by crosscorrelation of results obtained with those from TLC or an alternative HPLC method. In all cases, the selectivity relies on a chromatographic interaction where coelution or irreversible adsorption may occur. CE offers a completely different selectivity process to chromatography and is therefore truly a complementary technique to HPLC. Another key application areaof CE is in the determination of drug enantiomer ratios, since the CE methods developed may be more superior than HPLC in terms of resolution, sensitivity, ruggedness, and simplicity (see Chapter 14 for further details and discussion of chiral separations by CE). From

Methods m Molecular Biology, Vol 52 Cap/llary Electrophoresm Edlted by K Altna Copyright Humana Press Inc , Totowa, NJ

265

266

Al tria

20.00

-I

ci In

15.00

-l-J--J I

0 00

I

5.00 Retention

I

I

10.00 15.00 time In minutes

I

20.00

Fig. 1. Electropherogram showing the separation of 11 basic drugs. Separation conditions electrolyte, 50 mM NaH,PO, (pH 2.4 with cont. H,PO,), sample concentration 0.1 .mg/mL m water; detection, 200 nm; injection, 5-s pressure; voltage, 25 kV; capillary, 57 x 50 pm; Instrument, Beckman P/ACE 2200 (reprinted with permlsslon from ref. 28).

The application of CE to drug analysis was first demonstrated in 1987 (1,2). Subsequentdevelopment of commercially available instruments has led many investigators to apply CE to the analysis of drugs. CE has been applied to drugs from all the major drug classes in a variety of matrices. 2. Determination of Drug-Related Impurities The standard requirements of a drug-related impurities method is that all likely synthetic and degradative impurities are resolved from each other and the main drug, and impurities can be monitored at the 0.1% area/area level or below. The main component and structurally related impurities possess similar chemical properties, which makes resolution difficult. The high separation efficiencres often achieved in CE mean that a small degree of selectivity can provide acceptable resolution. Sultable detection limits for related impurities have been reported (3,4) with determinations at the 0.1% area/arealevel and below. Quality control of

Application

of CE

9.00

267

1100

13 00

15.00

Mlgrauon

ttme (mmutes)

17 00

19 00

2100

Fig. 2. Separation of ranmdme impurities at the 0.1% level. Separation condrtrons* electrolyte, 100 rnMsodmm citrate (pH 2.5 with 1Mcrtrrc acid), sample concentratton 0.5 mg/mL m water; detectron, 230 nm; mJectron, 5-s pressure; voltage, 5 kV; capillary, 37 x 75 pm; mstrument.

codeine and byproducts at the 0.05% level has been reported (5). Figure 2 shows the separationof selectedranitidine impurities at the 0.1% level (6). Data handling in CE IS slightly different when calculating impurity levels, since it is necessary to divide the observed area of each peak by its migration time (7). (See Chapter 5). This normalization is necessary since faster migrating peaks move through the detector at a greater speed than their slower counterparts. Therefore, faster-moving peaks have smaller peak widths and correspondingly smaller peak areas. The sum of these “normalized peak areas” is used to calculate impurities as % area/area.When impurity determinations are to be expressed as % w/w through the use of external standards (providing the precision of migration time is acceptable) this normalization process IS not required. A number of applications have been reported (8,9), many at the 0.1% area/area detection level. A stability-indicating method for enalapril has been reported (IO), which gives detection limits of 0.2% for the momtored impurities. Use of high sample loadings can significantly extend detection limits below 0.1% (I I). However, the linear dynamic range of many cormner-

268

Al tria

coal detectors is often msufficient, and the higher sample loadings result in off-scale peaks. This difficulty can be overcome by use of a “highlow” approach. In this procedure, a short injection time (i.e., 1 s) is employed to produce a separation with the main peak on-scale. A larger injection time (i.e., 10 s) is then used to produce a separation with the main peak off-scale, but with considerably enhanced sensitivity for the minor components. The separation of a basic drug and related impurities at low pH can be achieved by exploitmg fundamental differences m the charge and size of the components. If this selectivity is insufficient, incorporation of an additive, such as cyclodextrin (12,13), or an ion-pair reagent (‘14) into the electrolyte may achieve the desired effect. Components will selectively interact with these additives, resulting in overall changes in separation selectivity. Levels of dimeric salbutamol impurities were determined by HPLC and CE (3). Drug substance batches were tested by CE and HPLC using external standards of the impurities for quantitation. Table 1 shows the good correlation between the two techniques. The time-dependent degradation of an impurity of ranitidme was monitored by CE (15). Sample solution was reinjected several times over the course of 9.25 h. After this period, ~2% of the original substance remained. CE can be extended to allow impurity determinations in relatively water-insoluble drugs. For example, impurities have been determined in a water-insoluble quinolone antibiotic (16). The sample was dissolved in dilute NaOH solution and analyzed with a pH 1.5 electrolyte. Detection limits of 0.1% were demonstrated during validation. A single sample was injected 10 times, and precision values of 0.4 and 0.6% RSD were obtained for migration time and peak area, respectively. MECC can be employed to analyze neutral compounds, such salicylamide impurities, at levels below 0.1% area/area (17) using an SDSbased MECC separation. Nishi and Terabe (18) showed use of MECC to determine 0.1% impurity levels in dilitazem drug substance. Identification of impurities during method development can be obtained by appropriate use of diode array technology or by spiking experiments (19) with samples of the impurities of interest. Using commercial instruments, it is possible to record the spectra of an impurity (20). Figure 3 shows determination of the spectra of an impurity at the 0.1% level.

Application

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of CE

Table 1 Levels of Salbutamol Impurltles m Drug Substance Determined by CE and HPLCa Batch 1 2 3 4 5 6

Bis-ether CE

HPLC, O/ow/w

Dlmer CE

0.14 0.14 0.10 0.10

0 16 0 16

0 08 0 08 0 06 0.07 0 13 0 14 0 07 0 08 0 08 0 07 0 18 0 19 0.05 0 06 0.20 0.22 0 19 0.19 0 39 040

0.20 0 20 0 12 0.15 0 13 0.12 0.3 1

031 7 8 9

10

0.07 0.08 0.38 0.44 0.37 0.37 0 75 0.77

0.11 0.11 0 19 0 19 0 13 0 14 0 14 0.13 0.28 0.26 0.09 0 10 0.38 0.38 0 38 0.35 0 66 0.67

HPLC, O/ow/w 0 0 0 0

08 08 07 06

0 11 0 10 0 06 0 05 0.06 0 05 0 17 0 15 0.04 0.03 0.18 0.19 0 18 0.19 0 33 0 35

“Good crosscorrelatlon was obtamed between the two techmques

3. Main Component Assay Several reports have appeared concernmg the successful correlation of CE and HPLC assay results (4,21-24), indicating that CE is capable of producing accurate results. Existing HPLC instrumentation can routinely achieve
Altria

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Fig. 3. Spectra of a 0.1% area/area impurity in a drug substance. Separation conditions: electrolyte, 50 mM NaH2P04 (pH 2.4 with cont. H,PO,), sample concentration 0.1 mg/mL in water; detectlon, 200 nm; injection, 5-s pressure; voltage, 25 kV; capillary, 57 x 50 pm; instrument, Beckman P/ACE 2200 (reprinted with permtsston from ref. 20). era1 aminoglycoside antibiotics, including neomycin, streptomycm, and sisomycin, were separated (26) and detected using indirect detection. Often combination products are marketed that contain several active ingredients requiring separation and quantitation. Frequently a single set of CE conditions can be applied to a wide variety of drugs. For example, a simple low-pH electrolyte has been employed for the separation and quantitation of 17 basic drugs (27). An additional 11 basic drugs (28) were also resolved using a similar electrolyte. A pH 7.0 electrolyte (Fig. 4) was shown to resolve 16 common sulfonamides (29).

Application

of CE

0

271

6

16

time

24

32

(mtnl

Fig. 4. Separation of 16 sulfonamides. Sixteen sulfonamides (0.1 mg/mL), 57 x 50 pm capillary, pressure mjectron time 2-s, 10 kV, electrolyte 0.02M imidazole-acetate at pH 7.0 (reprinted with permission from ref. 29).

CE has been applied to the testing of a wide variety of formulations, including tablets, solutions for injection, infusion solutions, syrups, eardrops, creams, and rotacaps. Some noteworthy examples include the determination of three bronchodilators, fenoterol, salbutamol, and terbutaline, in six different dosage forms (21). Comparisons were made among the CE data, HPLC, and label claim with correlation coefficients of better than 0.999 reported between HPLC and CE results. In general, CE is not the first technique of choice when faced with the need to separate nonwater-soluble compounds owing to problems with on-capillary precipitation. In some cases, MECC conditions can be appropriate for the separation of benzothiazepines and corticosteroids (30). Methanolic solutions of the samples (e.g., as a cream formulation) were injected directly on capillary. Levels of water-insoluble dapsone in tablets have been determined (31) using methanol as sample dissolving solvent and fenbendazole as an internal standard. Average assays of 105.2 mg/tablet were obtained for a 100 mg/tablet (no indication was available of possible tablet content overage values). Correlation coefficients of 0.999 were obtained for detector linearity in the range lO-100% of target concentration. Levels of the antimigraine agent sumatriptan in injection solutions were determined by CE and HPLC (22). An internal standard was employed to provide improved precision. Results generated by the two

272

Al tria Table 2 Sumatrrptan Contents in InJectIon Solutions as Determmed by CE and HPLC” Sumatrlptan content CE, mg/mL

Sample Batch Batch Batch Batch Batch Batch

2, 2, 3, 3, 4, 4,

“Good

sample sample sample sample sample sample agreement

1 2 1 2 1 2

11 5, 11 6 11 6,11 6 11 7, 11 8 11.6, 11.6 11 7, 11 8 11.7, 11.6 was obtamed

between

HPLC, mg/mL 11 6, 11 6 117,117 11.8, 11 8 11 7, 11.7 11 8, 11 8 11 7, 11.7

the two techmques

techniques were in good agreement (Table 2). Detector linearity in the range 5-150% of target concentration was 0.9993. Peak area ratio data were 0.1-0.8% and 0.5X).7% RSD for sample and standard solutions, respectively. Interday repeatability gave assay data repeatability within 1%. A range of synthetic and degradative impurities were simultaneously separated, and detection hmits of CO.1% were possible. The transfer of an MECC method for the analysis of the paracetamol content in a commercial capsule formulation among seven independent pharmaceutical companies has been shown (32). Figure 5 gives three representative electropherograms showing the repeatability of the separation. All companies reported CE assay values equivalent to the HPLC data and the label claim (298.3, 302.5, and 300 mg/capsule, respectively). Use of an internal standard enabled the average precision data for the calibration response factor to be 1.5% RSD, whereas the assay results gave an RSD of 1.0% for 56 individual determinations. A fully validated quality-control MECC method for hydrochlorothiazide and chlorothiazide drug substance has been reported (33). The Fig. 5. (opposzte page) Separation of (1) paracetamol, (2) caffeine, and (3) 4-hydroxyacetophenone (int. std.) by MECC. Instrument specific settings (1) ABI HT, (2) Beckman P/ACE, and (3) Spectra Physics. Conditions: electrolyte, 40 n-N dlsodmm tetraborate, 125 mM sodium lauryl sulfate; detectlon, 210 or 214 run; capillary, fused slhca 72 cm x 50 pm (1,3) 57 x 50 pm (2); temperature, 40°C (reprinted with permission from ref 32)

0 8 c r

-------

Fig. 5

274

Al tria

method also allowed the quantitation of a selection of synthetic impurities at the 0.1% level. Injection precision of
Application

275

of CE

En 1

En2

Fig. 6. Specimen chiral separations of clenbuterol achieved in three companies. Electrolyte: 30 mMhydroxylpropyl-P-cyclodextrin (typically 0.83 g/20 mL) in 50 mA4 disodium tetraborate, pH adjusted to 2.2 with concentrated orthophosphoric acid (reproduced with permission from ref. 35).

limits of detection (0.3%) and quantitation (1.O%). The method was capable of determining 0.3% of either enantiomer in the presence of the other. Chiral separationsof drugs in biofluids have also been reported (44-46). The simplicity and robustness of CE make it particularly attractive in

Altria

276

Migrauon

“me

(minutes)

Fig, 6. continued this area.Examples include the quantitation of dimethindene enantiomers in urine, following a single 4-mg dose (4.5), and the separations of leucovorm and its major metabolite at therapeutic levels m plasma (46). 5. Stoichiometric

Determinations

Typically, acidic drugs are prepared as their sodium or potassium salts, whereas basic drugs may be produced as chloride, sulfate, succinate, or maleate salts. Chapter 20 details the typical separation conditions employed in these determinations. Generally, indirect UV detection is employed with electrolytes containing a UV-absorbing species, such as imidazole or chromate. This is a new CE application area, and to date, only two reports of the CIA analysis of pharmaceuticals have been reported (4 7,48).

Application

of CE

277 -i E: c

w-3 P 1

Ir

I?. I

I

4.00

9.00

I

14.00

I

19.00

1 24 00

Fig. 7. Analysis of a cholesterol-lowering drug substance batch containing 0.1% of the (-) enantiomer and 0.1% w/w of a cis-diastereolsomenc Impurity. Separation conditions: Electrolyte, 10 mA4 hydroxypropyl+cyclodextrm m 100 mA4borax, 30 mM SDS, detection, 200 nm; voltage, 20 kV; capillary, 457 cm x 50 pm (reproduced with permlsslon from ref. 42). Stoichiometrlc testmg of several drug substances has been conducted (47). Figure 8 shows separations of a batch of a drug prepared as a chloride salt. Clearly, this batch largely contains chloride, but also contains low levels of other inorganic and organic anions as contaminants.

Table 3 shows results for chloride and sulfate content in three drug substances (denoted GRD l-3). Good agreement among CE, theoretical anion content, and microanalysis results was obtained (47). The method performance in terms of linearity (R2 >0.999) and RSD values of l-2% for peak area and migration time was also acceptable. Levels of potassium and sodium content have been determined in various acidic drug substance (47). Results obtained by CE for three drug substance materials agreed well with the theoretical and HPLC results. Good method performance in terms of linearity (0.9999 over the range 10-500 mg/L), precision (0.3-l .5% RSD), and sensitivity (LOD of 1 ppm) was also obtained. CE has been employed to determine a range of inorganic and organic ionic impurities present in drug substance batches (48). Limits of detec-

tion of CO.1% w/w were reported. The method allowed the determination to be conducted on both water-soluble and water-insoluble drug

278

Altria

Fig. 8 Electropherogram of chloride assayof GRD 1drug substance. Separation conditions: electrolyte, 5 mA4 chromate with 0.5 mM tetradecyl trimethyl ammonium bromide; capillary, 75 pm x 57 cm; voltage, -15 kV; detectlon, indirect at 254 nm (reproduced with permission from ref. 47).

Levels of Amomc Counterions Sample Chloride (% w/w) GRD 1 batch A GRD 2 batch A GRD 2 batch B GRD 2 batch C GRD 2 batch D GRD 2 batch E Sulfate (% w/w) GRD 3 batch A GRD 3 batch A

Table 3 m Drug Substances Determined by CE”

Theoretical content 80 96 96 96 96 96 16.6 16.6

Mlcroanalysls

CE results

9.5 9.6 9.5 9.4 9.5

8 0,7.9 9.3,9 3 9.4,9.4 9.2,9.7 9.9, 9 6 93.95

-

167 16.8

OGood agreement was obtamed between CE, mIcroanalysIs, and theoretlcal content

substance materials. Insoluble drugs were dissolved in acetonitriIe:water mixtures. Figure 9 shows the separation of a test mixture of inorganic ions dissolved in acetonitrile:water and analysis of a 1 mg/mL solution

Application

of CE

279

~ugh-id

s 1oi .o d t

po.o-

m- .o

1 mytni

ANION

SCREEN STANDARD

DEUTERATEO PRAVA

l~~~~l~..,l,*..l,~.~l,,,,,~,,,,,,,,,,,,~ 2.6

3.0

3.5

4.0 T1r

4.6

6.0

5.6

6.0

6

t1nutd

Ftg. 9. Electropherogram of (A) 1 pg/mL anion screen standard. The migration order of the anions IS bromtde, chlortde, sulfate, nitrite, citrate, fluorrde, phosphate, carbonate, and acetate. (B) I mg/mL deuterated pravachol. Separation conditions: electrolyte, chromate, dilute sulfuric acid and Waters AnionBT OFM; voltage, 20 kV, inJectton, 20 s hydrostatrc; detection, Indirect UV at 254 nm (reproduced with permisston from ref. 48).

of deuterated pravachol. Various validation criteria were applied to the method, including linearity (typically 0.999 in the range l-100 ppm), precision (1.3-6% RSD for anions spiked at the limit of quantitation, 0.1% w/w), and sensitivity (LOD of 0.05% w/w impurity m drug substance). The robustness of CE for stoichiometric counterion assays has been demonstrated (49) by a method transfer exercise between seven pharmaceutical companies. A formic acid electrolyte containing imidazole was used to quantify sodium levels in sodium cephalothin. Potassium was used as an internal standard. All companies obtained a similar separation and confirmed the theoretical sodium content of 5.5% w/w. Given the simplicity and robustness of the CE methods used for the determination of drug stoichiometry, it is expected that there will be a significant increase in application and perhaps even replacement of existing methods of testing.

280

Al tria

6. Conclusions Capillary electrophoresis is now firmly established as a viable option for the analysis of pharmaceuticals. A recent survey (50) on the application and status of CE within leading US and UK pharmaceutical companies confirmed that CE has become established in many laboratories as a routine technique. The survey also mdicated that CE data have been successfully submitted to regulatory authorities. Specific application areas include the determination of drug-related impurities, drug potency, chiral analysis, and determination of drug counterion content. CE is often viewed as an alternative or complementary to HPLC. Validated CE methods are in routine use in many industrial pharmaceutical analysis laboratones. Validation criteria for CE methods are similar (50,51) to those employed in evaluation of HPLC methods, Use of CE for specific analysis, such as choral analysis, can have benefits in terms of method robustness and ruggedness, cost, and time. Current disadvantages are largely poorer sensitivity when directly compared to HPLC and the limited preparative options. Undoubtedly, technological developments and advances in methodology will strengthen and endorse the position of CE within pharmaceutical analysis, Of considerable note are the possibilities that the further development of new detector options, coated capillaries, and electrochromatography may bring. Notes Added in Proof Increasing reports are now concentratmg on the performance of CE methods during validation (52-56). For example, a CE method has been validated for determination of specific in-process impurity levels (52), to determine the potassium content in an acidic drug salt (.53), determine a range of cephalosporins in various samples (54), assay of xanthine derivatives for quality control of pharmaceutical formulations (55), and for assay of multiple components in an analgesic tablet formulation (56). Invarrabrlity accuracy was demonstrated by comparison with HPLC data. The use of MECC at low pH for separation of alkaloids (57) and drugs with very similar mobilities (58) highlights the increasing selectivity mechanisms being developed. Recent pharmaceutical analysis applications reported include the use of CE (59) to measure levels of residual drug on processing equipment following manufacture of pharmaceutical products, and the application of CE to assay levels of ethanol preservative in syrup formulations (60).

Application

281

of CE References

1 Altria, K. D and Simpson, C. F (1988) Analysis of some pharmaceuticals by capillary zone electrophorests J. Boomed. Pharm Anal 6,801-805. 2 Fujiwara, S. and Honda, S (1987) Determmation of mgredients of antipyretic analgesic preparations by micellar electrokmetic capillary chromatography. Anal Chem 59,2773-2776 3 Altria, K D. (1993) Quantitative analysis of salbutamol related impurities by capillary electrophoresis J Chromatogr. 634,323-328. 4. Pluym, A, Van Ael, W , and De Smet, M (1992) Capillary electrophoresrs in chemical/pharmaceutical quality control. TRAC l&27-32. 5 Korman, M., Vmdevogel, J , and Sandra, P. (1993) Separation of codeme and its by-products by capillary zone electrophoresis as quality control tool m the pharmaceutrcal industry. J Cht-omatogr. 645,366-370. 6 Altria, K. D. and Grace, C., m preparation 7. Altrta, K. D (1993) Essential peak area normalisation m capillary electrophoresis. Chromatographra 35, 177-l 82 8. Zhang, C. X., Sun, Z. P., Lmg, D. K , and Zhang, Y J (1992) Separation of tetracyclme and its degradation products by capillary zone electrophoresis J Chromatogr. 627,28 1-286. 9. Flurer, C. L and Wolmk, K. A. (1993) Quantitation of gentamicm sulfate m mJectable soluttons by capillary electrophoresis J Chromatogr 663,259--263 10. Qin, X. Z., Ip, D P., and Tsar, E W (1992) Determination and rotamer separation of enalapril maleate by capillary electrophoresis J Chromatogr. 626, 25 l-258.

11. Altria, K. D (1993) Application of high-low CE for improved quantitative determination of drug related tmpurmes Chromatographza 35,493496 12 Ng, C. L , Lee, H. K., and Li, S. F Y (1992) Systematic optimisation of capillary electrophoretic separation of sulphonamides. J Chromatogr 598, 133-138 13 Altria, K D (1993) Application of high speed capillary electrophoresis to the analysis of pharmaceutrcals employing commercial instrumentation. J. Chromatogr 636,125-132. 14, Nishr, H., Fukuywara, T., Matsuo, M., and Terabe, S (1990) Chn-al separation of dihtiazem, trimetoqumol and related compounds by micellar electrokmetic chromatography with bile salts J Chromatogr 515,233-243. 15. Altria, K D and Connolly, P. (1993) On-line solution stability determrnation of pharmaceuticals by capillary electrophoresis Chromatographla 37, 176-178 16. Altria, K. D and Chanter, Y L (1993) Validation of a capillary electrophoresis method for the determinatton of a quinolme antibiotic and its related impurities. J. Chromatogr. 652,459463 17. Swartz, M. E (1991) Method development and selectivity control for small molecule pharmaceutical separations by capillary electrophoresrs. J Llqurd Chromatogr 14,923-938. 18. Nishi, H. and Terabe, S (1990) Application of micellar electrokinetic chromatography to pharmaceutical analysts. Electrophoresls 11,691-701.

Altria 19. Altna, K D and Luscombe, D C. M (1993) Application of capillary electrophoresis as a quantitative identity test for pharmaceuticals employmg on-column standard addition J Pharm Blamed Analysis 11,415-420 20 Altna, K D and Rogan, M M (1994) Comparison of capillary electrophoresls and HPLC in the pharmaceutical industry Chromatography and Anaiysu, April/ May, 3-8

2 1 Ackermans, M T., Beckers, J L , Everaerts, F. M., and Seelen, I. G J A (1992) Comparison of Isotachoporesls, capillary zone electrophoresls and hlgh-performance liquid chromatography for the determmabon of salbutamol, terbutalme sulphate and fenoterol hydrobromide m pharmaceutical dosage forms J Chromatogr 590,341-353 22. Altna, K. D. and Filbey, S D (1993) Quantitative pharmaceutical analysis by capillary electrophoresls J Lzquzd Chromatography 16,228 l-2292. 23. Tsar, E. W , Smgh, M M , Lu, H H , Ip, D P , and Brooks, M A (1992) Apphcatlon of capillary electrophoresls to pharmaceutical analysis Determination of alendronate m dosage forms J Chromatogr 626,245-250 24 Sun, P , Mariano, G J , Barker, G , and Hartwlck, R A (1994) Comparison of mlcellar electrokinetic capillary chromatography and high-performance liquid chromatography on the separation and determmatlon of caffeine and Its analogues m pharmaceutical tablets. Anal Lett 27,927-937 25. Watzig, H. and Dette, C (1993) Precise quantitative capillary electrophoresls Methodological and instrumental aspects. J Chromatogr 636,3 l-38 26. Ackermans, M. T , Everaerts, F M , and Beckers, J L (1992) Determmatlon of ammoglycoslde antlblotlcs m pharmaceuticals by capillary zone electrophoresls with indirect UV detection coupled with mlcellar electrokmetlc capillary chromatography J. Chromatogr. 606,229-235 27 Chee, G I. and Wan, T. S M (1993) Reproducible and high-speed separation of basic drugs by capillary zone electrophoresls. J Chromatogr 612, 172-l 77 28 Altria, K D (1993) Capillary electrophoresls for pharmaceutical research and development LC-GC Int. 6,616-620. 29. Ackermans, M T., Beckers, J L., Everaerts, F. M., Hoogland, H , and Tomassen, M. J H (1992) Determination of sulphonamldes in pork meat extracts by capillary zone electrophoresls. J Chromatogr 596, 10 l-l 09. 30. Nlshl, H., Fukuyama, T., Matsuo, M , and Terabe, S. (1990) Separation and determmatlon of lipophlhc cortlcosterolds and benzothiazepin analogues by mlcellar electrokmetlc chromatography usmg bile salts J Chromatogr 513,279-295 31 Ackermans, M. T., Everaerts, F M., and Beckers, J. L. (1991) Determmatlon of some drugs by micellar electrokmetlc capillary chromatography The pseudoeffective mob&y as parameter for screening J Chromatogr 585, 123-l 3 1 32. Altna, K. D., Clayton, N G., Harden, R. C., Hart, M., Hevizi, J., Makwana, J V , and Portsmouth, M. J. (1994) An inter-company cross-vahdatlon exercise on capillary electrophoresls testing of dose umformlty of paracetamol content m formulations. Chromatographla 39, 180-I 84. 33. Thomas, B. R., Fang, X G., Chen, X., Tyrell, R. J , and Ghodbane, S. (1994) Vahdated mlcellar electrokmetlc capillary chromatography method for the quality con-

Application

of CE

trol of the drug substances hydrochlorothiazide

283 and chlorothiazlde

J Chromatogr

657,383-394. 34. Rogan, M. M., Goodall, D M , and Altria, K D (1994) Enantiomeric

separatton of salbutamol and related impurities using captllary electrophoresis. ElectrophoreSES15, 808-8 17. 35. Altrta, K D , Harden, R C , Hart, M , Hevtzt, J , Hailey, P. A, Makwana, J V., and Portsmouth, M J (1993) An inter-company cross-vahdatton exercise on captllary electrophorests 1. Choral analysis of clenbuterol. J Chromatogr 641, 147-153. 36. Altna, K D., Goodall, D. M., and Rogan, M M (1994) Quantitative applications and validatton of the resolution of enanttomers by captllary electrophorests. Electrophorests 15,824-827. 37. Peterson, T. E. and Trowbrtdge, D. (1992) Quantttatton of I-epmephrme and determination of the d-/l- epmephrme enanttomer ratto in a pharmaceuttcal formulatton by capillary electrophorests. J Chromatogr. 603,298-301 38. Kuhn, R., Stoecklin, F , and Erm, F (1992) Chiral separattons by host-guest complexation with cyclodextrm and crown ether in capillary zone electrophorests Chromatographla 33,32-38. 39 Ntelen, M W. F (1993) Chtral separation of basic drugs using cyclodextrm-modtfied capillary zone electrophoresis. Anal Chem 65, 885493 40 Rogan, M. M., Drake, C , Goodall, D. M., and Altrta, K. D (1993) Enanttoselecttve enzymatic biotransformatron of 2’deoxy-3’-thtacytidme (BCH 189) momtored by capillary electrophorests. Anal. Biochem 208, 343-347 41. Nishi, H., Fukuywara, T., Matsuo, M., and Terabe, S. (1990) Choral separation of dtltiazem, trimetoqumol and related compounds by micellar electrokinettc chromatography wrth btle salts J Chromatogr 515, 233-243 42. Noroskt, J E , Mayo, D J., and Moran, M (1995) Determmatton of the enantiomer of a cholesterol-lowering drug by cyclodextrin-modified mtcellar electrokmettc chromatography. J. Pharm Blamed Analysis 13,52-54. 43. Altria, K. D., Walsh, A. R., and Smtth, N. W (1993) Vahdation of a caprllary electrophoresis method for the enanttomertc purity testmg of fluparoxan J Chromatogr 645, 193-l 96. 44. Gared, P., Gramond, J P., and Guyon, F. (1993) Separation and determmation of warhuin enantiomers in human plasma samples by capillary zone electrophoresis usmg a methylated P-cyclodextrin-containing electrolyte. J. Chromatogr 615,3 17-325 45. Heuermann, M. and Blaschke, G. (1993) Chiral separation of basic drugs usmg cyclodextrms as chiral pseudo-stationary phases m capillary electrophoresis J Chromatogr. 648,267-274 46. Shtbukawa, A., Lloyd, D K , and Wainer, I. W (1993) Simultaneous chiral separation of leucovorm and Its maJor metabolite 5-methyl-tetradyrofolate by capillary electrophoresrs usmg cyclodextrms as choral selectors: esttmatton of the formatton constant and mob&y of the solute-cyclodextrin complexes. Chromatographza 35, 419-428. 47. Altria, K. D., Rogan, M. M., and Goodall, D. M (1994) Quantitattve determmation of drug counter-ion stoichiometry by capillary electrophorests. Chromatographla 38,637-642.

Al tria 48. Nan, J B and Izzo, C G (1993) Anion screening for drugs and intermediates by capillary ion electrophoresrs. J Chromatogr 640,445-46 1 49 Altrra, K D , Clayton, N. G., Harden, R. C., Hart, M., Hevtzi, J., Makwana, J V , and Portsmouth, M. J. (1995) Inter-company cross-valtdatron exercise on caprllary electrophoresrs Quantitative determinatton of drug counter-Ion level Chromatographta 40,47-50 50. Altrra, K D and Kersey, M (1995) Caprllary electrophoresrs and pharmaceuttcal analysts: a survey of the mdustrral application and then status of m the United States and Umted Kmgdom LC-GC Jan, 4&46. 5 1 Clarke, G S. (1994) The validanon of analytical methods for drug substances and drug products m UK pharmaceuttcal laboratortes J Pharm Bzomed Analysis 12, 6433652. 52. Shah, P A and Qumones, L (1995) Validation of a mrcellar electrokmetrc caprllary chromatography (MECC) method for the determmatton of p-toluenesulfomc acid impurity m a pharmaceutical intermediate. J Lzq Chromatogr 18, 1349-l 362 53 Altrta, K. D., Wood, T , Kttscha, R , and Roberts-McIntosh, A. (1995) Valrdatron of a capillary electrophorests method for the determinatron of potassium counterion levels m an acidtc drug salt J Pharm Blamed Anal 13,33-38 54 Boonkerd, S , Lauwers, M , Detaevermer, M R , and Mrchotte, Y. ( 1995) Separatron and srmultaneous determmatron of the components m an analgesic tablet formulation by mrcellar electrokmetrc chromatography J Chromatogr 695,97-l 02 55 Korman, M , Vmdevogel, J , and Sandra, P (1994) Application of mrcellar electrokmetrc chromatography to the quahty control of pharmaceuttcal formulattons the analysts of xanthme derivatives Electrophoresls 15, 1304-I 309. 56 Scracchrtano, C J , Mopper, B , and Specchro, J. J (1994) Identrficatton and separation of five cephalosporms by mtcellar electrokmetrc capillary chromatography J Chromatogr 657,395-399 57 BJornsdottir, I and Hansen, S. H. (1995) Determmatron of opium alkaloids m opium by capillary electrophoresrs. J. Pharm. Blamed Anal 13,687-693. 58. Hansen, S H , BJornsdottir, I , and TJornelund, J (1995) Separation of basic drug substances of very stmtlar structure using mrcellar electrokmettc chromatography J Pharm Blamed Anal 13,48%495 59. Altrta, K D. and Hadgett, T. (1995) An evaluation of the use of capillary electrophorests to monitor trace drug restdue levels following the manufacture of pharmaceuticals Chromatographla 40,23-27. 60 Altria, K. D and Howells, J S (1995) Quantitative orgamc solvent determmatron by capillary electrophoresis using indirect UV detection. J Chromatogr 696, 34 l-348

CHAPTER19

Separation of Peptides and Protein Digests by Capillary Electrophoresis Marxell

Herold, and

Gordon David

A. Ross, Rudolf N. Heiger

Grimm,

1. Introduction Peptide analysis is routinely performed using reversed-phase liquid chromatography (RP-HPLC), which achieves separation based on hydrophobicity differences between peptides (1,2). Recently, however, capillary electrophoresis (CE) has increasingly been used for peptide analysis. In capillary zone electrophoresis (CZE), the separation mechanism 1s based mainly on differences in charge-to-mass ratios, and since peptldes are amphoteric, they are ideally suited to electrophoretic analysis. The different separation mechanism of CZE has provided workers with an excellent complementary tool to HPLC. The provision of such a complementary separation mechanism is extremely useful in characterization of complex biological samples and, in particular, where purification of peptides and proteins is often done by preparative HPLC, such an orthogonal technique is essential for purity control (3,4). Method development in CZE is primarily focused on optimization of buffer composition, i.e., pH, ionic strength, the physical properties of the buffering ions, and use of additives (5). In CZE, separation of neutral species is not possible. Therefore, it is important that buffer conditions be selected to maintain a charge on the peptides. The inert nature of the tised silica material enablesthe running buffer to be varied from pH 2 to 12, From

Methods m Molecular B/o/ogy, Vol 52 Caprllary Electrophoresls Edited by K Altna Copyright Humana Press Inc , Totowa, NJ

285

286

Herold et al.

and optimal separation is generally obtained using buffers with a pH 1 or 2 units above or below the p1 value of the peptides. Complex peptide mixtures are commonly separated at low pH (between 2.0 and 3.5). A further benefit of using such extreme pH values is that this reduces peptide adsorption to the capillary walls. At low pH, peptide adsorption via coulombic interaction is minimized by protonation of the wall (rendermg the wall essentially neutral), whereas at high pH, where both the peptides and surface are anionic, coulombic repulsion is induced. Alternative CE separation modes applicable to peptides include micellar electrokinetic chromatography (MEKC) (6-8), where the peptides are separated by virtue of their hydrophobic interaction with a micellar “pseudostattonary” phase, capillary isoelectric focusing (CIEF), and capillary rsotachophoresis (CITP). With CIEF (9) separation of peptides, detectlon is limited to those peptides containing tryptophan or tyrosine residues since the ampholytes used to establish the pH gradient absorb at wavelengths <280 nm. CITP (IO) has also been used for peptide analysis, although it should be stressed that neither of these alternatrve methods is as widely applied as the CZE mode. 2. Materials and Methods All CE experiments were performed usmg an HP3DCE system from Hewlett-Packard (Waldbronn, Germany). The system comprises a CE unit with built-in diode array detector and an HP ChemStation for system control, data collection, and data analysis. The analysis of peptides derived from tryptic digestion of hemoglobin (Hb) was performed using either modular CE instrumentation (II) or an SP 1000 CE instrument (ThermoSeparations Ltd., UK). 3. General Buffer Effects Electrolyte buffers in CZE should fulfill at least two prerequisites, First, they should generate low Joule heating (low conductivity), and second, they should be transparent at UV wavelengths down to 200 nm. Furthermore, peptides and proteins should be well solubilized, and the chosen buffers should provide good pH control and have adequate buffering capacity. In most cases, phosphate and borate salts, or a combination of both are used for peptide separations. Both buffer types are transparent at low UV wavelengths, and phosphate, m particular, can be used over a relatively broad pH range. However, the high conductivrty of these salts,

CE of Peptides

287

mAU

10 I=9

e 4

14

L.-------------4

6

8

ID

mM uA

.._. -____.-_---_----_-.-12 14 ,a 100

1

phosphate.

*e mM

20 phosphate,

pH

7

PH

7

PH

7

_ Inin

Fig. 1. Effect of phosphate buffer concentration on the separation of a tryptlc digest of P-lactalbumin-A. Capillary 80 (72) cm x 50 pm, temperature 3O”C, electric field 3 10 V/cm, inJection 200 mbar x s.

particularly when used in relatively high concentrations (>80 mM), gives rise to increased Joule heating in the capillary. Since higher phosphate concentrations are advantageous for peptide and protein separations (see Section 3.1.), smaller inner diameter fused silica capillaries, which have improved heat dissipation properties, are often used to counteract the effects of excessive Joule heating. Alternatively, so-called Good’s buffers can be used (e.g., Tris, CAPS, HEPES, and so forth), but thesebuffers are often opaque in the low UV range. 3.1. Usage

of Phosphate

BufferslEfiects on Reproducibility In our hands, the use of low-pH sodium phosphate buffer for peptide analysis has proven very successful. In Fig. 1, the influence of the buffer concentration on a peptide map separation is demonstrated (12).

288

Herold et al. RSD 0.60% RSD 0.33%

1

:’

10

“:,

.

15

‘,

:“”

20

:

25

”

mln

Fig. 2. Reproduclbllity of peptidemapof recombmanthGH (Herold, M. and Wu, S.-L. [1994] LC-GC July, with penssion). Capillary 80 (72) cm x 50 pm, temperature35”C, run buffer 105mMsodlum phosphate,pH 2.0, electric field 3 10 V/cm, mJectron150 mbar x s. At low pH, silanol ionization IS reduced and phosphate buffers dynamically modify the capillary wall, creating an adsorbed phosphate layer that is more easily protonated (13). This combination, especially at higher phosphate concentrations, reduces the residual negative wall charge, which avoids peptide adsorption, resulting in increased peak efficiency. Therefore, intensive washing between runs with phosphate buffer and/or phosphoric acid improves the reproducibility of the peptide analysts at low pH (Fig. 2). In this case, the capillary was equilibrated between each run as follows: Flush with 0. 1Mphosphorrc acid for 3 min and then flush with prefiltered buffer for 10 min (14). The buffer was automatically replenished between each run in both the inlet and outlet vial. If possible, sodium hydroxide washing should be avoided to maintain the dynamic

CE of Peptides phosphate coating. Sometimes, however, “sticky” biological samples demand a thorough washing with sodium hydroxide or other solvents. Reproducibility of automated CZE peptide separations can be further improved by using the previously mentioned buffer replenishment. During electrophoresis, the composition and volume of the inlet and outlet vial may change. Therefore, buffer replenishment ensures identical starting conditions for every run. The HP 3DCE instrument has this function built in and enables the instrument to replace the buffer solvent completely in the outlet and inlet vial (0.6-0.8 rnL each) automatically between each run. Since the reservoir bottle for electrolyte buffer has a volume of 500 n-L, this allows automated replenishment for more than 300 analyses. It should be stressed that the use of capillary preconditioning, which is appropriate to the analytical conditions, is essential. It is often not possible to transfer one preconditioning method to another separation method or to different kinds of biological “real-life” samples. Therefore, when developing a new separation method, the preconditioning method should also be redefined. There are a number of other parameters that can be optimized in peptide separation. The sensitivity of peptides’ electrophoretic behavior to pH is shown in Fig. 3, which indicates that even small pH changes can result in distinct changes in the separation selectivity. The capillary temperature can also influence the separation (Fig. 4). Increasing temperature decreases the buffer viscosity, which will not only increase the electro-osmotic flow, but will also increase the peptide’s mobility by ca. 2%/“C. This will speed up the separation, but may also reduce peak resolution. 4. Sample Handling/Matrix Effects Peptides are often stored or solved in salt- or additive-containing buffers as a consequence of the purification process. Peptide samples derived from enzymatic digests of proteins may contain several components in the solvent. To facilitate enzymatic digestion, proteins are typically denatured by unfolding, and reduction and alkylation of disulfide bridges. Therefore, resulting matrices typically include up to 50 mA4 buffer, and up to 1M GuCl or urea, together with other additives. Figure 5 shows some of the effects such matrices can have on a peptide separation (12).

j

pH 3.0

pH 2.5

200

pH 2.0 i

6

Time (min)

‘O

14

Fig. 3. Effect ofpH change m the phosphate running buffer. Capillary 64 (56) cm x 50 pm, temperature 5O”C, run buffer 50 mM sodium phosphate, pH 2, 2.5, 3, electric field 390 V/cm, injection 300 mbar x s Sample: standard peptldes m elutlon sequence, anglotensin 1, angiotensm II, xenopsln, somatostatm, leucme enkephalin.

CE

of Peptides

200

45 ‘C

/

--L-b---L 10

20

_T--~ 30

Time

--_-

_ 40

(min)

Fig. 4. Effect ofcapillary temperature change. Capillary 80 (72) cm x 75 pm, temperature 25,35,45”C, run buffer 105 Msodium phosphate, pH 2.0, electric field 250 V/cm, injection 250 mbar x s.

High sample buffer concentrations may lead to large nonpeptide peaks (Fig. 5B,C), which can interfere with interpretation of the peptide map. High sample salt concentration is more problematic, since this leads to peak anomalies and recovery problems. However, desalting of the sample overcomes the problem completely. Figure 6A shows a CZE peptide map of aspartate aminotransferase in typical salt conditions consisting of 50 mA4 HEPES buffer and about 100 rnM GuCl. After desalting the sample on a reversed-phase C- 18 HPLC column, the CZE peptide map shown in Fig. 6B was obtained. In our experience, removing the salt from the sample is easier than adjusting the electrophoretic conditions to accommodate the original sample. Alternatively, it is often possible to avoid high sample buffer concentrations by using acetate or ammonium bicarbonate buffers, which can

Herold et al.

292 10 mM

3TA”

Phosphate

C II

8

B

Tlms lmln)

10 mM

Phosphate

20

+ 4 M Urea

50 mM HEPES

ml” 20

10 mM Phosphate

+ 2 M GuCl

10

Fig. 5. Effect of sample buffer additives on separation of a tryptlc digest of bovine serum albumin. Capillary 80 (72) cm x 50 pm, temperature 25”C, run buffer 30 mA4 sodium phosphate, pH 7.0, electric field 3 10 V/cm, injection 200 mbar x s. be evaporated from the sample prior to CE analysis. If samples contain high organic solvent concentratrons, the conductivity of the sample plug

will be very low. On applymg high voltage, the temperature in the plug will increase, which may lead to bubble formation, disrupting the CE analysis. Loss of resolution has been observed with CE analysis of peptide samples that contain SDS (15). Heating in the capillary can be reduced in a number of ways. Smaller internal diameter capillaries may be used to dissipate heat more effectively. Alternatively, if this is not possible (i.e., for micropreparative applications), active capillary cooling to 115°C will help. Another possibility is to initiate the separation using a smooth voltage gradient to avoid the fast increase of Joule heating in the sample plug of concentrated peptides or proteins (16).

293

CE of Peptides

In 50 mMHEPES and 100 MMGuCl

After Desalting on reversed phase Ci8 mAU IO

mAU 3.0

8 2.5 6 2.0 4 1.5

2 15

20

25

30

i

35

Time (min> Fig. 6. Influence of sample matrix on separation of a tryptlc digest of aspartate aminotransferase. Capillary 100 (92) cm x 50 pm, temperature 3O”C, run buffer 50 mA4 sodium phosphate, pH 2.5, electric field 200 V/cm, qectlon 200 mbar x s.

5. Peak Identification

by UV Spectra

All peptides have a similar low UV absorption spectra owing to absorption of the peptide bond (h,, ca. 195 nm). In addition, where peptides contain aromatic amino acids Phe, Tyr, and Trp, this will contribute to their low UV absorption. However, peptides that contain such residues will show distinct spectral differences ca. 280 nm, which reflects the content and composition of the aromatic amino acids in these peptides. Figure 7 shows as an example an overlay of spectra of four peptides taken during separation by CZE from a mixture of about 20 peptides produced by tryptic digestion of recombinant human growth hormone (rHGH; separation shown m Fig, 2). This figure illustrates how these

contarnsTyr

1

Phe

:

------_-_---___________c_

--.-_

no aromatlc amlno acrd

20

\-

:=--

ol----: 250

260

270

280

290

300

nm

Ftg. 7. Overlay of the UV spectra of four different peptides from tryptlc digest measured on-line with a photodiode array detector. Capillary 80 (72) cm x 75 pm with extended light path (3x bubble cell), temperature 35°C run buffer 105 n&I sodmrn phosphate, pH 2.0.

CE of Peptides

spectra can vary for different peptides depending on their amino acid content. Using a computerized spectral library of identified peptides and appropriate software for comparison testing, such spectral differences are sufficient to identify most, but not all peptides (17). If unknown peptides have to be analyzed, their UV spectra can be used to identify whether the sequence contains a tryptophan or tyrosine residue. The second derivative of the tyrosme and tryptophan spectra possessclearly different minima and can therefore be used for identification of the aromatic amino acid content in the peptide (18-20). 6. Micropreparative Peptide Separations and Identification by MALDI-TOF-MS and N-Terminal Sequencing The capability of collecting enough peptide material from CE separations for off-line N-terminal sequencing (21--23), mass and activity determination (24, has created for CE a new dimension in the field of bioanalytical methods. Using a 75-urn id capillary, 0.5-2 pmol of peptide mixtures can be separated. This is more than sufficient for MALDITOF-MS (14), which can easily detect peptides at the femtomole level even in the presence of salt (Fig. 8). Fraction collection from five to eight repetitive runs yielded sufficient material for N-terminal sequencing. The peptides were collected automatically by pressure elution into 20 pL water containing 1% acetic acid. Such automatic fraction collection demands a high degree of migration time reproducibility. With the use of a loo-pm id capillary, fraction collection from a single run was able to provide sufficient material for subsequent off-line N-terminal sequencing. This technique was used to collect fractions from separation of a tryptic digest of the protein GroES (M, 10,700) (Fig. 9) (16). With an injection volume of ca. 90 nL, as calculated by the Hagen Poiseuille equation, and assuming 100% complete tryptic digestion and no loss of peptides during sample preparation, we calculated an injection amount of 560 pmol of the peptide mixture. Loading such large mass onto the capillary can lead to heat-induced precipitation of the sample during analysis. However, captllary thermostatting to 15”C, which reduces Joule heating in the capillary, was able to overcome this problem. Collection was again performed by pressure elution into microvials containing either 20 l.tL 1% acetic acid, 2% trifluoroacetic acid, or 6M

Herold et al.

296 1380

9

13648

I

I

994

1437

,

1961

Fig. 8. Mass spectrum analysis of a peak collected by CE from a rhGH tryptic digest. The measured mass of 1364.8 equals the sequence mass of the peptlde Tl 1. The second peak shows the oxidized mass of the same, onemethionme-containing peptide (ref. 24 with permission).

guanidine hydrochloride. Purity and successful collection of the peptide fractions were confirmed by reinjectlon of an aliquot from the collected fraction (Fig. 9B). The average recovery of the peptides collected by CE was 6040% as estimated from the reinjected fractions. All of Fig. 9. (oppositepage) (A) Electropherogram of a tryptlc digest of bacterial chaperonin GroES protein. (ref. I6 with permission). Capillary 96 (88) cm x 100 pm, temperature15”C,run buffer 105mM sodium phosphate,pH 2.0, voltage gradlent, final electric field 260 V/cm, mJectlon 150 mbar x s. The marked peaks were collected and sequenced. (B) Electropherogram of reinJected peptide fraction number 4,200 mbar x s of the collected fraction direct injected (ref 16 with permission).

CL3 of Peptides

297

A 80

6

60

20

25

30

35

40

time (min)

B 4 Peak 4 reinjected 3

2

1

--20. -

.--40

30

time (min)

Fig. 9.

_----_50

Herold et al. the collected peptides 1-6 were identified by sequencing with a yield of 5-30 pmol (assummg 50% initial yield of the sequencer HP Cl 005A). Peptide separations using MEKC are usually performed using buffers containing 20-80 mA4SDS. Again, using a lOO+m rd caprllary, fraction collection from a smgle run is capable of providing a sufficient amount of peptide for off-line sequencing. In order to exploit fully peptide fraction collectton using MEKC, a sequencing technique that is compatible with SDS-containing samples must be used. The amino acid sequence of collected neuropeptides was successfully determined after fraction collection into electrolyte buffer (50 mM SDS, 20 mM sodium phosphate, pH 9) (data not shown). 7. Applications 7.1, Purity Confirmation Capillary electrophoresis is a very powerful tool for purity confirmation of peptides fractionated by reversed-phase HPLC. In the analysis of synthetic 250 lysme polypeptide, separation from shorter byproducts by reversed-phaseHPLC was not possible (Fig. lOA). However, using CZE, it was possible to separatepartially these impurities (Fig. lOB), although the total amount of impurities was smaller than 0.5% of the total yield (‘5). There are a variety of reported examples in the literature that demonstrate the utility of CZE for peptide purity check and support our view that CZE is the perfect complementary technique to reversed-phase HPLC for such an analysis (1,26-28). 7.2. Peptide Mapping Peptide mapping is one of the mam and most drfficult applicatrons in peptide analysis. It is commonly used for the characterization of recombinant proteins in the biotechnology and pharmaceutical field (29,30). Furthermore, it is a standard procedure prior to sequencing of unknown proteins. Fig. 10. (opposite page) (A) HPLC analysts of the synthetic polypeptide preparation with 250 lysine residues. Column Vydac Cl 8 (1 .Ox 250 mm), Solvent A: H20, 0.05% TFA; Solvent B: acetonitrile, 0.045% TFA, flow rate 50 pL/mm, gradient 0 mm-5% B, 80 mm-55% B, detection 214 nm. (B) CZE analysis of the synthetic polypeptide separation wtth 250 lysme restdues. Capillary 80 (72) cm x 50 urn, temperature 25OC,run buffer 100 mM sodium phosphate, pH 2.0, electric field 375 V/cm, mJectton 100 mbar x s.

299

CE of Peptides mAU

A

350

300 250 200 150

100 50 0

! 20

30

40

50

60

70

Time (mid mAU

B

mAU

1200

14

1000

600

a 600 6 400

200 c

0

c,

5

10

15

20

25

30

35

40

95

Time (mid

Fig. 10.

,

10

10.5

11

115

12

12.5

Herold

et al.

Peptide mapping has been used in clmrcal analysis for the identification and diagnosis of structural Hb variants, which can result m hemoglobinopathies. Tryptic digestion of Hb grves a complex mixture of peptides, which is characteristic of the type of Hb digested. The majority of the 400+ structural Hb variants identified to date are the result of single amino acid substitutions. Therefore, the separation mechanism used must be sensitive to fine differences in peptide number and their physicochemical characteristics. Conventionally, pepttde mapping of Hb trypttc digests is performed using two-drmensronal paper chromatography/electrophoresis, since even gradient elution reversed-phase HPLC cannot provide sufficient resolution or peak capacity to resolve such a digest fully. The followmg example shows the optimization of a CZE separation of peptrdes from digestion of normal HbA and its application in identifying the variant HbC. From the known structure of HbA, tryptic drgestion should give 29 peptides, four of which are insoluble and two consisting only of a single lysine. Therefore, one should expect a mmlmum of 23 peptrde peaks. The effect of pH on the separation is demonstrated in Fig. 11. Twentyeight peaks were partially or fully resolved at pH 2.5, compared with 18 peaks at pH 6.0 and 15 peaks at pH 10.0. At pH 2.5, all peptides were positively charged and their separation was effected by mobility differences that were less obscured by virtue of the very low EOF. Using buffers with pH m the region of pH 1.5-3.0 gave no further enhancement of the separation obtained. Figure 12 shows the effect of varying the operating voltage, using a modular CE instrument, indicating that the use of 20 kV provided the best compromtse between fast separation and good resolution. With the use of automated instrumentation providing temperature control, it was also possible to optimize capillary temperature (II). Usually operating temperaturesbetween 25 and 40°C are used, since at higher temperatures, peptides may precrpitate, and at lower temperatures, the analysis time IS unreasonably extended. After investrgating the use of buffer additives (31) and optimizing further parameters, including kmd of buffer, concentratron of phosphate buffer, and length of capillary, the optimized CZE separation shown m Fig. 13A was obtained. Phosphate buffer at low pH was found to give a more stable baseline and better reproducibihty of peptide resolution. Figure 13B and C shows for comparison the separation of tryptic digests of

301

CE of Peptides pH 2.5

pH 6.00

pH 10.00

a’

Ttme

(mins)

20

Fig. 11. Effect of pH on separatron of a tryptrc digest of HbA. Condrtions: Capillary-70 cm (50 cm eff.) x 50 ym id, detectronnm; load 5 s at 10 kV electrokmetrc; run-25 kV ambient temperature. the isolated a and p globin chains, and the assignment of the peaks from the digest of whole Hb. This allowed the assignment of peaks to the subunit source. The separation obtained in Fig. 13A was reproducibly typical of normal adult HbA.

302

Herold et al,

15kV

1 0

Time

(tnins)

I 20

Fig. 12. Effect of applied voltage on separatton of a tryptic digest of HbA. Conditrons: buffer-50 Mphosphate, pH 2.5; capillary-70 cm (50 cm eff.) x 50 pm id; detection-l 95 nm; load 5 s at IO kV electrokinetic; run-ambient temperature. Figure 14 shows the electropherogram obtained from CZE analysis of a tryptic digest of HbC. HbC is a relatively common Hb variant that causes hemolytic anemia in affected individuals. This variant has a lysine

CE of Peptides

303 A

0 0100

--200

14

200

0 0056 L"

0 0034

-0

0010

,

6

0 0050

1

14

a chain

18 19

0 0017 AU

a

0024

0 0011 -0 0002 -0 0015

00

6

0ongo- C

28

13 chain

16

20 22

0 0037 .

24 I

29 2!7 II

i/i

-0 0002 -0 0015

.

J

Gum--+

6 00

6 40

.!w.i.

LO 60

1320

15 60

7 16 00

Time (mm)

Fig. 13. Optimized separation of tryptic digest of (A) HbA (B) Hb chain, and (C) Hb p-chain. Conditions: capillary-70 cm (63 cm eff.) x 50 pm Id; detectlon-200 nm; load 5 s hydrodynamic; run-25 kV 25°C residue substituted for glutamic acid at positron 6 in the p chain, whrch introduces an extra cleavage point for tryptic digestion. In the tryptic digest of HbC, the peptide PTl should be absent and two other peptides should be present, i.e., hexapeptide PTId and dipeptide PT,,. Figure 14 compares the separation of HbC and HbA using the same conditions. Peak number 11 was missing from the separation of HbC, and one extra

Herold et al.

304

HbC

HbA

Time

(mm)

Fig. 14. Comparison of tryptic digests of normal HbA and variant HbC. The arrow m the HbC electropherogram indicates the peak corresponding to the variant hexapeptlde PT,,. Conditions: capillary-70 cm (50 cm eff.) x 50 pm id; detection-95 nm; load 5 s at 10 kV electrokmetq run-25 kV ambient temperature.

peak (see arrow) corresponding to the hexapeptide was present. The dipeptide, which has lower absorbance, could not be observed and may have comigrated with another peptide. Such differences were sufficient to establish that the Hb sample was abnormal and to identify it as HbC. In comparison with conventional two-dimensional electrophoresis and paper chromatography or reversed-phase HPLC, Hb fingerprinting by

CE of Peptides

CZE is faster and uses much less sample. Only the two-dimensional method provided higher resolution of the peptides, which would be expected since this technique employed orthogonal separation mechanisms. CZE has been used in combination with LC for analysis of single amino acid substitutions in peptides (27), and this approach has been automated (321, although this is not commercially available. The use of CZE in combination with HPLC for such analyses provides a uniquely powerful analytical tool. 8. Conclusions In the relatively short time of the last three to four years, capillary electrophoresis, particularly CZE, has become the method of choice for analytical separations of peptides in several laboratories. CE has several general advantages compared to other analytical separation techniques. Using suitable microvials, the sample volume can be as small as 5 yL from which nanoliter volumes are injected onto the capillary. This allows both multiple analyses of single samples; this quasi-nondestructive nature allows the sample to be used for other studies. The low sample requirements of CE are especially useful when dealing with expensive or rare peptide preparations, which has been exploited by the development of microtechniques for tryptic digestion requiring only picomole or femtomole amounts of protein (33). Analysis time using CE is faster than in HPLC and usually provides greater resolution of a larger number of peptide fragments. This can be of added benefit in the analysis of large proteins. Unlike gradient elution RP-HPLC, low UV absorption detection can be usedwith CZE, thus exploiting the increased molar absorptivity of the peptide bond at ca. 195 nm and obviating the need for column re-equilibration between analyses. In addition, it is becoming clear that CE has an increasing role as a micropreparative technique. We have demonstrated that peptide peaks can be identified, after collection, by sequencing and mass spectrometric analysis, and collected peptides may also be assessed for biological activity (24). Furthermore, electrospray mass spectrometry can be used on-line for mass identifications with the use of a suitable interface (34,. Using gel-filled capillaries, protein and DNA separation was achieved, which is comparable with or superior to traditional one-dimensional slabgel electrophoresis. An additional advantage here is the easier and more accurate quantification of the peaks (i.e., refs. 35,36).

306

Herold

et al.

The major drawback in usmg CE IS the problem of detection sensitivity. Although mass sensitivity is extremely high owmg to the high peak efficiencies found with CZE, the technique suffers from low concentration sensitivity. This is because UV detection is performed on-capillary with the structure itself forming the detection cell. Therefore, with a very small internal diameter capillary (usually 25-100 urn), the light pathlength is short, and consequently, concentratron detection limits are higher than those obtainable with HPLC. There are two principal ways to counter this problem, at least partially. The path length can be increased by using extended light path capillaries (bubble cell) (37) or by introducing a Z-shape cell (38). Another possibility is stacking or on-column focusing by IEF or ITP. However, these techniques are seldom used routinely. The design of additional detectors is likely to open up new application fields. Presently, other commercially available detectors include laserinduced fluorescence (LIF) detection and interfaces to electrospray MS. The use of LIF detection limits the available excitation wavelengths, and as a consequence, analytes must usually be derivatized to form a suitable fluorophore. As already mentioned (Fig. 8), off-line MALDI-TOF-MS is very powerful for peptrde identification after CE separation. In summary, it should be stated that capillary electrophoresis instruments, m principle, are technically less complex than HPLC pumping systems. Therefore, we expect that the hardware of modern CE mstruments will be at least as reliable as up-to-date HPLC instrumentation. In regard to the application of CE to protein and peptide analysis, control of the EOF and suppression of unspecific adsorption are the most critical points. For CE analysis of peptides, both of these factors have been adequately addressed. We believe that capillary electrophoresis is the perfect complementation to reversed-phase HPLC in peptide and protein chemistry. Acknowledgment We thank Robert Solazzo and Herman Dollekamp for help in the reproducibility and pH effect studies. We appreciate critical comments and support from Martin Verhoef. References 1 Herold, M., Helger, D N , and Grimm, R. (1993) Am Lab (August) 2OJ-20U

CE of Peptides 2 Sampson, R. J., Morttz, L. R., Begg, G. S , Rubna, M. R., and Nice, E C (1989) Anal Blochem. 177,221-236. 3 Perrett, D. and Ross, G (1992) Trends Anal Chem 11, 1X-163 4. Landers, J P., Oda, R. P., Spelsberg, T. C., Nolan, J A., and Ulfelder, K J. (1993) BzoTechntques 14,98-l 1I 5. Schwartz, H. E., Palmten, R. H , and Brown, R (1993) m Capzllary ElectrophoreSES Theory and Practxe (Camillert, P , ed.), CRC, London, UK, 201-254 6 Liu, J P , Cobb, K A, and Novotny, M (1990) J Chromatogr 519, 189-197. 7 Sutchffe, N and Corran, P H (1993) J Chromatogr 636,95-103 8 Yashima, T , Tsuchtya, A., and Morita, 0. (1992) Anal Chem 64,298 I-2984. 9. Mazzeo, J. R , Martmeau, J A , and Krull, I. S. (1991) Anal Blochem 208,323-329 10. Kasicka, V and Pruslk, Z (1991) J Chromatogr 569, 123-174 11 Ross, G A., Ph D thests, Medtcal College of St Bartholomew’s Hospital, Umverstty of London, London, UK (1994) 12. Hetger, D N , Grnnm, R , and Herold, M. (1993) Hewlett-Packard Applzcatzon Note, Pubhcatron number 12-509 l-9062E. 13 McCormrck, R M. (1988) Anal Chem 60,2322-2328 14. Herold, M and Wu, S -L (1994) LC-GC 12,53 l-533 15 Kenndler, E and Schmtdt-Belwl, K (1991) J Chromatogr 545, 397-402. 16 Grtmm, R. and Herold, M (1994) J Capdlary Electrophoresu 1,74-79 17 Steve@ H.-J , Wu, S -L , Chouplek, R , and Hancock, W. S (1990) J Chromatogr 499,221-234.

18. Fell, A. F., Scott, H P , G111,R., and Moffat, A. C. (1983) J Chromatogr 273,3-l 7. 19 Grego, B., Nice, E C , and Simpson, R. J, (1986) J Chromatogr 352,359-368 20 Grnnrn, R , Graf, A., and Hetger, D. N. (1994) J Chromatogr ,679,173-180 21 Camillert, P., Okafo, G N , and Southan, C (1991) Anal Blochem 198,36-42. 22 Schwer, C and Lottsperch, F (1994) Poster No 626 at Szxth international Symposlum on High Performance Electrophoreszs, San Diego. 23 Albin, M , Chen, S.-M., Loure, A., Palraud, C , Colbem, J , and Wtktorowtcz, J (1992) Anal Bzochem 206,382. 24 Lemer, E. A. and Nelson, R. J. (1994) LC-GC 12(7), 336-338. 25 Grimm, R. (1994) Hewlett-Packard Application Note, Pubhcation number 125962-7230E 26. Lemer, E. A., Rrbeiro, J. M C., Nelson, R. J , and Lemer, M. R. (1991) .J. Blol. Chem 266, 11,234-l 1,236. 27. Wheat, T. E., Young, P M , and Astephe, N E (1991) J Lzquzd Chromatogr 14, 987-996. 28. Janim, G. M , Issaq, H J , and Lukszo, J (1994) J High Resol Chromatogr

17,

102,103. 29 Frenz, J., Wu, S -L , and Hancock, W. S (1989) J Chromatogr 480,379-391 30 Nielsen, R G., Rtggm, R M., and Rlckard, E C (1989) J Chromatogr 480, 393-401 3 1. Ross, G. A , Lorkm, P , and Perrett, D. (1993) J Chromatogr. 636,69-79 32. Larman, A. V. Lemmo, Moore, A W , and Jorgenson, J W (1993) ElectrophoreSLY14,439-447

Herold et al. 33. Cobb, K. A. and Novotny, M (1989) Anal Chem 61,2226-223 1 34 Smith, R D , Wahl, J H , Goodlett, D R., and Hofstadler, S. A (1993) Anal Chem 65, A574-A584. 35 Helger, D. N., Cohen, A. S., and Karger, B. L., J Chromatogr 516,33-48 36 Ganzler, K , Greve, K S , Cohen, A S , Karger, B L , Guttman, A , and Cooke, N C. (1992) Anal Chem 64,2665-2671 37 Helger, D. N., Kaltenbach, P , Slevert, H -J P (1994) Diode array detection m capillary electrophoresls. Electrophoresls 15, 1234-1247. 38. Mormg, S R., Pau-aud, C , Albm, M , Locke, S., Thlbault, P., and Tmdall, G W (1993) Am Lab. (July) 32

CHAPTER20

Additional Application Areas of Capillary Electrophoresis Kevin D. Altria 1. Analysis of Small Ions by Capillary Electrophoresis A variety of free solution CE (FSCE) methods have been developed for the determination of low-mol-wt solutes, These samples have included metal ions, small amine compounds, small organic acids, and a range of inorganic anions. Separation mechanisms involve either simple FSCE or addition of a suitable chelating additive to achieve on-column chelation. Detection of these low-mol-wt species has predominantly been achieved by indirect UV detection (see Chapter 7, Section 1l), although several reports detail direct UV detection of ions separated as complexes generated on-column. A wide variety of application areas scanning a number of industries have been reported. Generally, results obtained by the CE method are compared to ion-exchange chromatography (IEC), atomic spectroscopy, or gravimetry. The features of the methods are described as simplicity, speed of analysis, and acceptable sensitivity. This section will cover the application areas in terms of anions and cations subdivided into detection principles of either direct or indirect UV analysis.

1.1. Cations

Detected

by Indirect

UV Detection

Species are generally separated and detected using low-pH electrolytes containing UV-active species, such as imidazole (I) or N-N dimethyl benzylamine (2). From.

Methods m Molecular Biology, Vol 52 Capillary Electrophorem Edtted by K Altna Copynght Humana Press Inc , Totowa, NJ

309

310

Al tria

20

JO

40

TO

60 mh

Ftg. 1. Separation of 16 common metal tons and ammomum Ion. Reprinted with permission from ref. 8. Electrolyte used 11 mh4 lack acid, 2.6 mA4 locrown-6,7.5 mA44-methyl benzylamine, 8% methanol, pH 4.3, peak identlty 1 = NHd+, 2 = K+, 3 = Na+, 4 = Ca*+, 5 = Sr*+, 6 = Mg*+ 7 = Mn*+, 8 = Ba*+, 9 = Cd*+, lO=Fe*+, 11 =Ll+, 12=Co*+, 13=Ni3+, 14=Zn ‘2+ , 15=Pb*+, 16=Cu*+.

On-column chelation of metal ions is often employed to improve selectivity. A range of organic acids, such as hydroxyisobutyric acid (HIBA) (3%.5j,ci t ric acid (.5j, and lactic acid (6), have been employed at low-millimolar concentrations. The selective chelation of the metal ions with the particular acid reduces the net mobility of the cation, resulting in an improved separation. These separations have been extensively studied (7,s’ and the separationsachieved have been shown to be dependent on the pH, concentration of complexing agent, concentration of crown ether, and organic solvent content. Figure 1 shows separation of 16 common metal ions and ammomum (at l-5 mg/L) usmg an electrolyte system containing lactate, methanol and crown ether (8). The crown ether is primarily added to achieve separation of NH4+ and K+, but also influences other resolutions. These test methods have been applied to monitoring cations m a range of samples, including mineral water (I), fermentation broths (9), drug samples (IO), and in prenatal vitamin formulations (II). Results obtained (Table 1) from the testing of cation contents m vitamin tablets (11) were

Additional

Application

Areas

311

Table 1 Cation Content in Vitamin Tablets (Results Reported as pg/mL) Tablet 1

Tablet 2

Tablet 3

Species

CE

ICP

CE

ICP

CE

ICP

Ca2+ Fe3+ Zn2+

72 23 1.0

7.8 21 11

7.8 26 1.0

8.4 2.4 1.2

8.0 2.6 1.0

9.0 2.5 11

Data reprmted with permlsslon from ref II

Table 2 Metal Ion Content m Water Samples / Results in ppm (mg/L) Sample no.

Method

Potassium

Sodium

Calcium

HPLC CE Label HPLC CE Label HPLC CE HPLC CE

12 1.6 20 55 3.4 46 50 63 0.5 06

32 3.2 3.6 46 49 69 233 236 53 3.1

47 41 3.8 64 79 100 138 126 15.9 14.5

Magnesium 0.8 1.1 0.6 39 41 44 66 50 33 2.9

Data reprmted with permxsslon from ref !

compared to ICP results. Precisions of l-2% RSD were obtained for peak area and Cl% for migration time. Calibration curves extended three orders of magnitude. Similar performance has been reported in the determination of the sodium and potassium levels in drug salts (10). Levels of metal ions in a range of mineral waters have also been accurately determined by CE (1). Table 2 shows the good agreement among CE, HPLC, and the label claim on the mineral water bottles. Levels of K+, Na+, Ca+, and Mg*+ have been determined in parenteral solutions (12). Low-ppm levels were determined using an electrolyte system containing HIBA. Precisions of ~2% were obtained for iqections of testing containing >30 ppm of the four cations. Good linearities (0.997-0.9997) and recoveries in the region of 90-100% were obtained. Cationic nutrients were determined (13) in foods by CE following micro-

Altria

312 Cr(lll)

I

*

10

15

20

Time (mln)

Fig. 2. Separation of a range of catlons usmg dn-ect UV detectlon. Reprinted with permlsslon from ref. 16 Electrolyte: 20 mM pH 9 0, phosphate contaming 2 mM sodium cyanide. wave digestion of the samples. Results obtained were in line with these achieved by AAS and ion chromatography. 1.2. Cations

Detected

by Direct

W Detection

In these methods, the cation is complexed on-column with ligands, such as EDTA or %hydroxyquinoline, to produce UV-active charged complexes. Typically, the ligand is added into the CE buffer at low-millimolar concentrations. EDTA has been employed for the determination

of the divalent cat-

ions Ca*+, Mg*+, and Co*+, in wheat flour (14). The electrolyte contained 20 mM borax, 5% ethylene glycol, and 2 n-N EDTA. The anionic complexes were detected at 200 nm. Results obtained by this method were in good agreement with those obtained by AAS. A further example of the use of EDTA has been shown in the determmation of alkaline earth ions in water and serum samples (15). Precision values of l-2% RSD were obtamed with good agreement between the CE results and those achieved using EDTA titration. On-line chelation with cyanide ions has also been used to determine a range of metal ions with direct UV detection at 2 14 nm (16). Figure 2 shows a typical separation of a range of cations.

Additional

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Areas

313

An alkaline cyanide solution has also been employed to determine levels of Gold (I) and Silver (I) in ore samples (2 7). The method was employed to monitor levels of silver and gold in various ore samples with good agreement between CE and AAS results. The determination of transition metals separated as 8-hydroxyquinoline-5 sulfonic acid chelates has been reported using fluorescence detection (18) and direct UV detection (19). 1.3. Indirect Detection of Anions This area has received extensive development and application principally by workers from Waters (Milford, MA). Several separation conditions have been reported in which the direction of EOF is reversed and a htgh-mobility ion, such as chromate, is incorporated into the electrolyte to give the background UV signal for indirect detection. The EOF direction is reversed to make it identical to the migration direction of the small anions, thereby reducing analysis times. A range of cationic surfactants have been employed at low-millimolar concentrations, including hexamethonium (1,6-bis-[trimethylammonium]hexane) (20,21), cetyltrimethylammomum bromide (22), Waters OFM Anion-BT (23), and tetradecyltrimethylammonium bromide (IO, 24,251. A range of anionic UV-absorbing species have been employed to provide the background UV signal, including chromate (10,23), 1,2,4,5 benzene tetracarboxylic acid (pyromellitate) (20,24), benzoate (26), napthalene sulfonate (27), hydroxybenzoate, and sorbate (23). Figure 3 shows the effect of varying the UV co-ion on the separation of a range of anions. Chromate gives optimal peak shape and sensitivity for small anions, such as Cl-, S042, whereas benzoate is preferred for medium ions, such as citrate, phthalate, and carbonate. Sorbate gives improved performance for larger anions, such as alkyl sulfonates. A wide range of application areas have been reported, since these methods offer simple and reliable methods of analysis. Applications include determination of organic acids in wine and coffee (281, determination of vinyl sulfonate in water-soluble polymers (29), and profiling of tonic constituents of urine (30). Several samples of water were analyzed by CE and IC for chloride, sulfate, nitrate and fluoride (31). Table 3 shows the good agreement between the two techniques. Typically most applications are performed at low-mid ppm concentrations. However, some applications require significantly higher sensitivity. Electrokinetic injection can improve detection limits IO-fold or

J4 2 “0

11

IO

ii

II

,J AU

,

I

II

I4

r

A-

5 mM Chromate,

pH 8.0

5 mM p-Hydroxybenzoate,

pH 9.37

II IS A

3.0

3.5

5 mM Sorbate, pH 7.64 4.0

4.5

Minutes

Fig. 3. Effect of varying the UV co-ton on the separation of a range of amons Reprinted with perrmssron from ref. 23. Peak identity: l= bromide, 2 = chlortde, 3 = sulfate, 4 = rutrtte, 5 = nitrate, 6 = molydate, 7 = tungstate, 8 = citrate, 9 = phtalate, 10 = carbonate, 11 = ethanesulfonate, 12 = propanesulfonate, 13 = butanesulfonate, 14 = pentanesulfonate, 15 = hexanesulfonate.

Additional

Application

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315

Table 3 Analysis of Water Samples for Anion Content (Results in ppm [mg/L])

Tap water Chloride Sulfate Nitrate Fluoride Well water Chloride Sulfate Nitrate Waste water Chloride Sulfate Fluoride

IC

CE

20.2 14.7 36 N/D

20.0 14 0 35 01

37 7 12 0 32

36 5 114 3.2

83 1 23 8 N/D

83 0 23 1 01

Reprinted with permIssIon from ref 31

greater compared to hydrodynamic injection (32). Good linearities and acceptable precisions were shown (31) using electrokinetic Injection. Typical sample loadings are achieved employing 30-s injections at -5 kV. Electrokinetic injection m conjunction with octanesulfonate addition (75 pm) to the sample solution has been employed (33) to monitor ppb levels of anions in nuclear power plant feed water (Fig. 4). Other applications include analysis of extracts from wood pulping (34), levels of propionate in bread (26), and profiling of explosive residues (35). Injection precisions of -2% RSD were obtamed for repeated injections of 20 ppb sulfate employing citrate as an internal standard. Results obtained for bore water testing were in-line with IC results. CE, gravimetric analysis, and IC were used (20) to determine levels of inorganic anions in detergent samples. Sulfate results in the 2-40 ppm region were statistically compared and were found to be equivalent (correlation coefficients 0.996-0.998). Injection precision was reported as 1.5% RSD. of Anions There have been a few reports m this area principally owing to the limited chromophores possessedby typical analytes. However, the resolution of cis- and trans-isomers, such as fumaric and maleic acid, has 1.4. Direct

Detection

316

Altria

2 x 10“AU

3.0

4.0 Mlnules

50

Fig. 4. Use of electrokmettc inJectton to monitor ppb levels of amons. Reprinted with permission from ref. 31. Peak identity: 3.5 ppb chlorrde (3.07 min), 4.8 ppb sulfate (3.21 min), 6.2 ppb mtrtte (3.24 mm), 5 ppb oxalate (3.33 mm), 1.9 ppb fluorrde (3.78 mm), 5 ppb formate (3.2 min), 3.2 ppb phosphate (3.88 min), 5 ppb acetate (4.57 mm), and 5 ppb propionate (4.91 mm).

been shown with direct UV detection at 214 nm (36). Species such as bromide, nitrate, bromide, and iodide, have sufficient UV activity to allow detection at low UV wavelengths (22). Food additives, such as caprylic, sorbic, benzoic, and propionic acid, have been determined in foods by CE (37) with detection at 190 nm. Ion-pair reagents were employed to obtain the required selectivity. 2. Application

of Chemometric Experimental Designs to Capillary Electrophoresis There are a great many factors that may be varied to optimize a CE

method. Conventionally,

when attempting to assess the impact of each of

these parameters, each factor would be varied sequentially. This univariate approach involves holding all parameters constant, varying the parameter of interest, and measuring method responses, such as resolution, peak efficiency, and analysis time. For example, a sequenceof injec-

Additional

Application

Areas

317

tions may be performed to assessthe impact of pH, followed by an additional injection series to assessthe impact of ionic strength and further sequences to assess the influence of such factors as buffer additives, organic solvent content, temperature, and sample concentration. This step-by-step approach, although widely used, involves a large number of independent analyses, and could be replaced by suitably statrstically designed experimental protocols in which several factors are simultaneously varied. These multivariate approaches have advantages in terms of reductions in the number of experiments and analysis time required. Statistical evaluation of the data can often indicate whether parameters interact, i.e., if both the concentrations of buffer and ion-pair reagent are increased, they will cause an increase m temperature of the solution within the capillary, possibly altering selectivity or migration times. These subtle interactions cannot be assessed in the univariate approaches. Therefore, the appropriate use of experimental designs can be extremely beneficial in capillary electrophoresis. Typical experimental design approaches utilized in CE include Plackett-Burman (38,391, Overlapping Resolution Mapping (4W3), Fractional Factorials and Central Composites (44,451, and a Simplex design (46). The use of chemometrlcs IS well defined in the area of HPLC for method optimization (47-49) and m the evaluation of the robustness of HPLC methods (50-52). These experimental designs are being exploited for similar applications in CE. 2.1. Method Development Using Experimental Designs The instrumental PC-controlled format of CE allows several parameters to be varied within an unattended sequence. For example, several electrolyte compositions could be placed on the autosampler, and the instrument could be preprogrammed to operate at various voltages and temperature. 2.1.1. Full and Fractional

Factorial

Designs

Factorial designs can be employed to assess the impact of all variables and their interactions, and have, despite the potentially large number of experiments, been employed successfully in HPLC (53,54). For example, to evaluate five factors fully, 243 experiments would be required in a full factorial design. Therefore, fractional factorial designs and Plackett-Burman designs are employed to reduce dramatically the number of experiments, although still maintaining the statistical ability

318

Altria Table 4 Values Employed m Plackett-Burman

Buffer

PH

% SHS 10 10 10 0 0 10 0 0

Design

& ACN

SDS, mM

Buffer, mM

40 50 50 50 40 40 50 40

50 40 50 50 50 40 40 40

20 20 40 20 40 40 40 20

ReprInted with permIssIon from ref 38 The comtcelle mvestlgated was sodturn heptyl sulfate (SHS)

to identify the influence of each parameter and to identify interactions between factors (55). A Plackett-Burman approach has been employed in the optimization of an MECC method for the separation of testosterone esters (38). Five factors (pH, buffer concentration, acetonitrile content, SDS concentration, and comicelle concentration) were investigated in an eight injection sequence. Evaluation of the results indicated that resolution of peaks II and III was decreased with increases in percent ACN and SHS concentration, but improved by increasing pH and SDS concentration. Buffer 5 (Table 4) was shown to be close to the optimum and produced the best separation. A Plackett-Burman experimental design has also been employed in the optimization of a chiral separation by CE (39). The influence of five parameters, each at three levels, on the resolution of clenbuterol enantiomers was assessedin a 15-experiment study with duplicate injections of each experiment. The “main effect” of variation of each parameter was calculated, for resolution, and is shown graphically in Fig. 5 for peak resolution. An increase in response in Fig. 5 indicates an increase in resolution. Conversely, a decrease in percent main effect indicates a poorer resolution. For example, an increase in ionic strength (factor 2) led to an improved resolution. Factor 6 is termed the “dummy” factor and is an indication of the experimental variability involved in the measurement. If the percent main effect for a parameter is larger than the corresponding

Additional

Application

Areas

319

Fig. 5. Percentagemain effect of operating variables on choral resolutron. Reprintedwith permrssionfrom ref. 39. 1 = pH, 2 = ionic strength,3 = cyclodextrrn concentration,4 = methanol content,5 = injection time, 6 = dummy. percent main effect for the dummy, it can be considered to be statistically significant. The minimum value is the lower level of the factor evaluated, i.e., 6 mA4 P-CD, whereas the maximum value is the highest level evaluated, i.e., 16 mA4 P-CD. Operating conditions of pH 4, 100 mA4 electrolyte, 16 mM CD, 0% methanol, and a 1-s injection time were shown to produce the best resolution. This particular combination of factors was not included in the initial design, which highlights the power of this technique. Having established the key factors affecting the method, it may then be appropriate to optimize the method by obtaining response surfaces by employing designs, such as Central Composites (56) and Box-Behnken (57). To date, there have been no reports of the use of Central Composite designs for optimization of CE methods, although their beneficial use in HPLC method development has been shown (58’. Overlapping resolution mapping design schemes have been employed in the optimization of CE methods for the separation of sulfonamides, antimalarial drugs, flavanoids, and plant growth regulators. Overlapping resolution mapping (ORM) schemes have been successfully employed in the optimization of HPLC conditions (.59,60). Similar ORM approaches have been employed in CE for the optimization of ratios of three cyclodextrin types for separation of plant growth hormones (431, optimization of pH and cyclodextrin concentration for seven sulfona-

320

Al tria Table 5 Parameter Ranges Investrgated Durmg Robustness

Parameter Regeneration solutron concentration Regeneratton solutton rmse trme Electrolyte pH Electrolyte concentratton Electrolyte rinse trme Injectton trme Sample concentration (mg/ 10 mL) Applied voltage

Method setting 0 1MNaOH 1.O mm 21 50 mM 2.0 mm 10 s 10mg 10kV

Study Range mvesttgated +O OlM f0 2 f0 2 *5 fO2 +2 +2 _+2

Reprinted with permlsston from ref 44

mides (40), and optimization of both pH and ion-pair reagent for seven antimalarial drugs (42). The only other report to date in this area involves (46) the optimization of an MECC method for the separation of 20 derivatized amino acids using a simplex scheme. Percentage organic solvent, SDS concentration, and pH were simultaneously investigated. An improved, but incomplete separation of the 20 amino acids was obtained after only 10 analyses. 2.2. Robustness Testing Using Experimental Designs Robustness may be defined as the sensitivity of a method to small deliberate change in the method parameters. Appropriate use of experimental designs in robustness testing of HPLC (62) and CE methods has been shown (44,4.5) in a limited number of reports. Few reports have dealt with the robustness testing of CE methods, but these have generally employed an umvariate approach (62). The most appropriate scheme robustness testing may be to use a fractional factorial destgn to identify the key method parameters and an additional design, such as a central composite, to enable method limits to be set together-with system suitability parameters. This approach has been employed (44) in the evaluation of the robustness of a CE method used to determine levels of impurities in a basic drug. Initially, a fractional factorial design of 36 experiments was conducted to obtain the effect of eight parameters on resolution, percent peak area, and migration times. The parameters investigated and ranges used are given in Table 5.

Additional

Application

Areas

321

Figure 6A shows the separation obtained at the method settings. The effect of each parameter on the various measured responses can be deduced by appropriate statistical examination of the data. For example, Fig. 6B shows a graphical representation (Pareto plot) of the “size of effect” of each of the parameters investigated on the migration time of the main peak. In this example, a parameter is deemed to have a sigmficant influence on the response if the size of effect is >2. Therefore, in Fig. 6B, voltage is shown to have a significantly negative effect (i.e., an increase in voltage reduces migration time). The overall analysis of the data set indicated that the only significant factors identified were sample concentration, injection time, voltage, and pH. Increases in injection time or sample concentration had a detrimental effect on resolution of the impurity denoted 11 from the mam peak. Voltage and pH had more pronounced effects on several responses and were therefore assessedin a further central composite design. A representation of the design is given in Fig. 7. The voltage range employed was 8-12 kV, whereas the effect of pH was assessed between 1.8 and 2.3 in a 16-analysis sequence. Analysis of the results obtained from these injections confirmed operation at the method conditions of 10 kV and pH 2.1 was the most suitable. An additional experimentally designed robustness evaluation has been performed on a CE method employed for the determination of potassium (4.5). The method employs an electrolyte comprised of formic acid and imidazole with indirect detection at 214 nm. Sodium is employed as an internal standard. A fractional factorial design was used to screen the effect of seven parameters on resolution and migration time. The system suitability criteria was set as baseline resolution of potassium and sodium. The parameters investigated were formic acid concentration, imidazole concentration, temperature, sodium concentration, potassium concentration, rinse time, and voltage. The separation was shown to be robust within the ranges examined. The significant factors of voltage and formic acid were further assessedin the range 3-7 kV and 3.5-4.5 rnM formic acid. The advantages of employing experimental designs in method development and robustness testing are widely acknowledged. Features of experimentally designed studies include reductions in the number of experiments and overall time, and the generation of data that may be statistically analyzed to provide information on the principal factors

Altria

322 yr

A

ma," peak 14 50-

14.00I4 13.50-

12.50I2 12.0 0-I

I 0.00

I IO 00

13

I 2000

I 30 00

I 4000

6

-2i In]

time

lSMlPl-31 Phosptmle

I I I

-0218 0 663 -0034 10 10

PH

Rlnsc

e1ectr

VoltageWho

10

Size

15

of

Fig. 6.

effect

88

Additional

Application

Areas

323 *

pH18,lOkV

*

pH 2 0, 12kV

.++-+

*

l

pH2 3,lOkV

0 *

pH 2 0,8kV

Fig. 7. Central composite design.

affecting the separation and any subtle interactions between parameters. The design of commercial CE instrumentation coupled with PC-controlled instruments allows several operating parameters to be evaluated in unattended overnight sequences. Several commercial computer software packages are available to generate the designs and to analyze statistically the CE data. Undoubtedly, the area of chemometrics will greatly influence the future direction of CE, since expert systems will inevitably become available, which will greatly simplify the process of method development and method robustness evaluation. 3. Agrochemicals The application of CE to the analysis of agrochemicals is logical in that their chemical structures and physical characteristics are similar to conventional pharmaceuticals, which are widely analyzed by CE (see Chapter 18). The application areas are also similar to those exploited in pharmaceutical analysis, and include assay, impurity determination, chiral determinations, and trace level determinations. However, to date, relatively few reports have appeared on this subject (63-69). Fig. 6. (previouspage) (A) Separation obtained at the method settings. Separation conditions: electrolyte: same as method settmgs m Table 5. (B) Pareto plot for size of effect of parameter variation on migration time. Reprinted with permission from ref. 44.

Altria

324

YIN

,

Fig. 8 Separation of four alkylphosphonic acids wrth indirect UV detection. Reprinted with perrmssron from ref. 6.5.Separation conditions: 100 mM borate buffer contammg 10 mM benzoic acid. Indirect detection at 254 nm. Alkylphosphonic acids are widely employed as herbicides and pesticides. However, their analysis represents a challenge owing to their limited chromophores. Operation at a pH of 6.0 with an appropriate UVabsorbing agent provides adequate resolutron and sensitivity. Figure 8 shows separation of four alkylphosphomc acids (6.5). Good precision in terms of injection (peak height) and migration times was obtained. Correlation coefficients of >0.999 were obtained. The limit of detection was 5 pg/mL. A range of 10 chlorophenoxy acids were baseline-separated employmg MECC conditions within a 9-min analysis time (66). Injection repeatability was 1% for migration times and 2% for peak areas. The separation of eight sulfonylurea herbicides has been achieved (64) using an ammonium acetate:acetonitrile 75:25 electrolyte adjusted to pH 5.0 with acetic acid. The baseline-resolved components were detected by both UV and a mass spectrometer (MS) (64). An MS-MS arrangement was also employed to obtain spectral data for the peaks, which were matched against library spectra of authentic samples. Levels of sulfonylureas were spiked into soil extract samples and were determined by the method. To date, only one report has focused on the application of CE to industrial samples (63). In this paper, the use of CE was shown to monitor impurity levels in phenoxyacid herbicides. A range of herbicides and

Additional

Application

Areas

325

related impurities were separatedusing a lithium acetate buffer at pH 4.8 with detection at 200 nm. Various cyclodextrins were added to appropriately modify selectivity. Figure 9 shows separation of a test mixture (Fig. 9A) and a production batch (Fig. 9B) employing dimethyl-P-CD as electrolyte modifier. Certain impurities are chirally resolved under these conditions, Detection levels of CO.1% were possible for the impurities. Good precision of peak area (I. 1-l .4% RSD) and migration time (0.3-0.5% RSD) was achieved with linearities of CO.9998. Chiral analysis of herbicides produced as single enantiomers was performed. The CE method gave good agreement with results generated by HPLC test methods. The aqueous stability of metsulfuron has been monitored by CE (67). Three major degradation products were produced following storage for 4 d. Peak identification was confirmed by GC-MS. Another important analytical application in agrochemical analysis is the determination of pesticide residues. Levels of the sulfonylureas, chlorsulfuron, and metsulfuron have been determined in tap water at subppb levels (68). Samples were prepared following solid-phase extraction and analyzed using a pH 9.0 borate buffer with detection at 214 nm. Recoveries of over 90% were demonstrated. In a similar paper, four herbicides were determined by MECC in tap and drinking water (67). Sample preparation involved a lOOO-fold sample enrichment followmg solid-phase extraction (67). Good (80-95%) recoveries were shown in the range l-5 p.g/L range. Levels of paraquat and diquat were determined in crop water by both HPLC and CE (69). Similar detection limits were obtained for the two techniques. 4. Carbohydrates Carbohydrate analysis representsa particular challenge to CE in that the principal analytes possess no readily ionizable groups or chromophores. Three approaches have been suggested to overcome those difficulties: 1. Indirect detectionat htgh pH; 2. Borate complexation with detectionat 195nm; and more commonly 3. Separationof chargedcarbohydratederivatives. Operation at pH values of approx 12 ensures the ionization of the weakly acidic hydroxyl groups on underivatized saccharides (70). Sorbic acid was added at 6 mM to provide the background UV signal for indirect detection.

124

0

120

0

A

0

F CPA

t 116

0 b

I -KPP

OP

A

HO

CP

Ir

109

5

’

B

Pi

108 0 E 106

5

t t

Pechn.

WCPP

H

HO

i-ktCPP YCPA

A 100

5 --Iryw*h*iJ

99OL

' 2 0

3

"

' 3.0

5

I

' * 4 0

e

I

" 5.0

I 6.0

I Tim0

I

I

I, 1.0

I

,I,,,,,,,,, 8.0

9.0

10.0

11.0

12

0

Iminutes)

Fig. 9. Separation of a test mixture: (A) a production batch (B) and employmg dlmethyl-@-CD as modifier Reprinted with permission from ref. 63. Separation condltlons: 20 g/L mM dimethyl+-cyclodextrin m 30 mM hthium acetate (PH 4.Q detection: 200 nm.

326

Altria

Many carbohydrates can complex m solution with borate ions, which permit their separation as anions, The complexation 1sfavored at higher temperature and higher borate concentrations (72). Underivatized carbohydrates were resolved using 60 mM borate at 60°C. A number of derivatizing agents have been suggested, and the selection should be made on the functionality present on the carbohydrate of interest. For example, CBQCA is a useful choice for carbohydrates containing amino groups (72). Perhaps the most appropriate of all the available derivatives is 8-amino-napthalene- 1,3,6-trisulfonic acid (ANTS) (73). ANTS derivatives can be prepared relatively simply and separated over a wide pH range, since the ANTS molecule possessesthree acidic groups with varyingp&s. The presence of the napthalene group ensures good UV and fluorescence sensitivity. Aminobenzoate derivatives are a useful alternative (74), particularly for ketoses. Separation of aminobenzoate derivatives (Fig. 10) has been performed at pH 10.0 with electrolytes containing high levels of borate (74). 5. Vitamin Analysis This area has yet to receive extensive development, although the applicability of CE to the analysis of vitamins has been clearly demonstrated (7.5-80). This is somewhat surprrsmg given the extensive development and application of CE to analysis of pharmaceuticals. The range of vitamins can be broadly subdivided into water- or oilsoluble. It is possible to perform separation of water-soluble vitamins using simple free solution CE, whereas use of MECC is mandatory for the analysis of oil-soluble vitamins. The most detailed study on vitamin analysis used both FSCE and MECC to analyze a range of vitamins m commercial vitamin preparations (7.5). Table 6 shows the FSCE and MECC results compared to results generated by the USP HPLC method. Acceptable precision data of
Additional

Application

329

Areas

12 13

1. 0

5

10

15

min

Fig. 10. Resolution of ammobenzoate derivatives of mono- and ohgo-saccharides. Reprinted with permisslon from ref. 74. Separation condltlons: 175 mA4 borate, detection at 305 nm, peak identlty: R = reagent, 1 = 2-deoxy-oribose, 2 = maltotriose, 3 = rhamnose, 4 = celloblose, 5 = xylose, 6 = nbose, 7 = lactose, 8 = glucose, 9 = arabinose, 10 =fucose, 11 = galactose, 12 = mannuromc acid, 13 = glucuromc acid, 14 = galacturonic acid.

6. Biomedical Applications The clinical application of CE has been an important research area that is only now becoming recognized as a useful routine technique (81). Two recent reviews by Xu (82) (169 references) and Deyl et al. (83) (98 references) cover the current biomedical applications of CE. The strategies for monitoring drugs in biofluids have been covered by Thormann et al. (84,. Other activities include diagnosis of metabolic disorders by biofluid analysis (85), patients phenotyping (86), and protein analysis (87,88).

Sample pretreatments (84) are similar to those involved in HPLC, such as deproteinization with acetonitrile (89), ultracentrifugation (84), or

Al tria

330 Crossvabdatton

Table 6 of Vrtamm Assay Results by FSCE, MECC, and HPLC Results as % label claim

Sample

Analyte

FSCE

Bl (15 mg) 1187f 17 PP (50 mg) 1108f3 1 B2 (15 mg) 990+22 110.9f3 3 B6 (10 mg) 117.2 + 4.0 Bl (10 mg) SY~P (5 mL) 111.2Ik 14 PP (20 mg) 115.4f 1.5 ~32 (1 w) 109.9 zk 1 4 B6 (5 mg> 122 0 f 2.2 Soft capsule Bl (10 mg) PP (30 mg) 111 3 + 1.8 112 I +3 1 B2 (7 mg) 1086k 1.8 B6 (5 mg> Repnnted wtth pernnsslon from ref 75 n/a = not analyzed Tablet

MECC

HPLC

1236_+26 108.0 X!I1 2 994+2 1 113 7* 17 112.4 3~ 1 3 1094+09 119.3 + 2.9 106 2-1: 3.1 126.6 21 1 7 1086k 17 1149k 16 108 2 Z!I 1.6

1238+36 108752 1 1039+07 1124+3 2 111.6+ 16 1115+34 117.2 + 2 2 1132+39 n/a n/a n/a n/a

solid-phase extraction (84). However, the rugged nature of the CE capillary format can also allow direct mjection of biofluids, such as serum (90) and urine (86), with obvious savings in analysis time and cost of consumables. The majority of direct injection analyses are performed by MECC, since the SDS micelles strongly interact with the sample proteins causing the proteins to be eluted later, and the small solutes of interest are then visible free from protein interfaces. Figure 11 shows that antiepileptic drugs can be directly monitored in patient serum as the proteins migrate after the peaks of interest (90). The performance of CE methods in clinical assays has been assessed by many workers and general comments indicate that CE is not as sensitive as HPLC, but has benefits in terms of simplicity and possible sample pretreatment reductions. Validation parameters, such as linearity, recoveries, and precision, show acceptable performance (81,83,86). Crossvalidation of CE results with other techniques, such as HPLC (86) and immunoassays (90,91), show that CE is capable of generating accurate results. Table 7 compares levels of creatinine and uric acid as determined by both an enzymatic method and by MECC (92). Despite the interest and research focus in this area, increased routine application may require

Additional

Application

Areas

331 Proteins

Phenobarbital 1,. 5

.,

10

15

.

2( 1

Fig. 11. Separation of antieplleptic drugs by direct qectlon of patient serum. ReprInted with permission from ref. 90. Separation conditions: 75 n-&I SDS, 6 mA4 borax, 10 mA4 phosphate, 220 nm, 35°C.

Comparison

Table 7 of Levels as Determmed by MECC and Enzymatic Methods Creatmine, yglmL

Plasmasample I 2 3 4 5 6 7 8

Uric acrd, pg/mL

Enzymatic

MECC

5 7 4 6 7 1 8 4

5 7 4 5 7 N/R 8 4

Enzymatic 28 50 38 27 66 12 56 60

MECC 32 51 N/R 32 66 11 58 60

Reprinted with permIssIon from ref 92 N/R = no result obtamed

further advances in sensitivity by instrument improvements sample introduction procedures.

or by better

7. Alternative Detection Systems (Including CE-MS) Currently, all commercial CE systems incorporate a UV-absorbance detector. Some systems are modular, or partially modular, and alternative detection systems can be employed. The principal detection alternatives to UV-absorbance detectlon that are commercially available are fluorescence and mass spectrometry. Other less-developed detector

Altria

332

options include electrochemical (93,94) and conductivity detection (9.5). An increasing number of commercial instruments are also available with UV-absorbance photo-diode array facilities. Diode array detectors have been shown to be of use in peak identification and peak purity assessments (96,9 7). 7.1. CE-Mass

Spectrometry

There has been a great deal of activity in the area of interfacing CE to mass spectrometers, and the advances to date have recently been reviewed (98). A number of interfaces have been successfully demonstrated, include continuous-flow fast atom bombardment (99), electrospray (100, 10 I), and ionspray (102). Generally, reports have indicated that CE-MS is less sensitive than UV-absorbance detection, which may have limited a more substantial exploitation of this detection mode. However, the significant potential sensitivity advances offered by ion-trap MS (103) or the use of on-line stacking in CE (104) may lead to an alleviation of this issue. 8. Amino Acid Analysis Much of the early development work in CE was performed using derivatized amino acids as test solutes. A range of derivatization agents are avaliable for this purpose (Table 8). The vast majority of separations have been achieved employing SDS-based MECC conditions with the addition of organic solvent (105). Figure 12 shows the highly efficient resolution of 23 dansylated amino acids using 102 mMSDS, pH 9.2, electrolyte and a temperature of 10°C (105). Underivatized amino acids have not been widely analyzed, since they generally possess very limited chromophores. However, native amino acids have been separated and monitored by indirect detection, employlng 1 mM sodium salicylate at pH 9.7 (11.5). 9. Particulates,

Bacteria, and Dyes 9.1. Analysis of Particulates CE has been successfully applied to several separations of large polymeric species, such as polysterene latex particles (I I6) and Jeffamine polymers having molecular weights up to 2100 (II 7). Separations are performed in free solution with resolutions owing to size differences. Silica gel sols, which are used in the preparation of HPLC packing mate-

Additional

Application

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333

Table 8 Derwatlzatlon Reagents Employed m Amino Acid Analysis Derwatwe

Reference no

PTH FITC CBQCA TBQCA Dns OPA NDA

106 107,108 109,rro Ill 112,105 113 113,114

PTH (phenythlohydantom), FITC (fluorescem lsothlocyanate), CBQCA (3-[4-carboxybenzoyll-2-qumolmecarboxaldehyde), Dns (Dansyl), OPA (o-phthaladehyde), TCQCA (3-[4-tetrazolebenzoyll-2-qumolmecarboxaldehyde), NDA (napthalene dlaldehyde)

0

10

12

14

16

7.0

22

mln

Fig. 12. Resolution of 23 dansylated amino acids usmg MECC at 10°C Reprmted with permlssion from ref. 105. Separation conditions: 20 mA4borax, 100 mMSDS, 10°C, 214 run. rial, have also been characterized by CE (118) using pH 9.0 buffers and detection at 190 nm. Silica sol colloids up to 500 nm diameter were separated (118).

of Bacteria by CE Mixtures of various viable bacteria, such as Enterococcus, Streptococcus, and Staphylococcus strains, were resolved by CE (129) using Tris/Borate/EDTA buffer. The pH of this buffer is high, and the bacteria 9.2. Separation

and Isolation

were resolved as anions. UV detection at 190 or 200 nm, together with

use of a loo-pm capillary, allowed sufficient sensitivity. Preparative CE was performed to collect specific bacteria from mixtures. Positive iden-

Al tria

334

tification of collected fractions was achieved by several techniques, including metabolic fermentation. Purities of recovered fractions were >99%. The authors concluded that CE could afford the microbiologist a new tool for studying the composition and distribution of microorganisms in mixed populations. It 1s noted that these separations were conducted on homemade equipment and that sophisticated commercial equipment may offer significant advantages in terms of improved performance and sensitivity.

9.3. Determination

of Dyes by CE

Currently HPLC IS predominantly employed in the separation and determination of levels of cationic, anionic, and neutral dyes, and dye intermediates. CE has been shown to be of use in this area (120,121). Many dyes have two or three membered ring structures and are water soluble, making them very suitable for analysis by CE.

Notes Added

in Proof

1. Analysis of small ions by capillary electrophoresls: An optrmized separatlon has been reported (122) that allows simultaneous determmatlon of ammonium, alkali, alkalme-earth, and various transition metals using an electrolyte containing lmidazole, crown ether, methanol, and HIBA Low ppb detection levels were possible with electrokinetic mJectlon. Laserinduced indirect fluorlmetrlc detection of cations has been reported (123); the electrolyte employed contained fluorescem sodium and EDTA, and low-mid ppb levels of metal ions could be detected using pressure mJectlon. Recent advances in the determination of anions has centered on the optlmlzation of electrolyte systems, For example, the use of p-aminobenzoate as an electrolyte additive has been shown (124) to be useful for the detection of orgamc acids. 2,6-napthalene dicarboxylic acid has been employed as a UV absorber and has been shown (125) to be a considerable improvement over pthalate. Migration time drifts can occur using the standard electrolyte containing chromate because of electrolyte depletion. This problem can be overcome (126) by the addition of 1 r&k! 5,Sdiethylbarblturate to the electrolyte. 2. Experimental design: Multivariate regression analysis has been employed to study the effect that various ratios of EDTA and borate concentrations have on the migration time of metal complexes (127). A central composite design has been utilized for optimization of electrolyte composition (122). A full factorial design was then used to measure the mam effects of several parameters on the EOF velocity.

Additional

Application

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335

Carbohydrate analysis: A recent survey (128) has been published concernmg the application of CE to carbohydrate analysis. It comprehensively reviews the derivative types available and a range of appltcations. Another recent report (129) dlscussed the use of tags, such as ANTS Underlvatized carbohydrates have been separated using NaOH electrolytes with electrochemical detection (130). Indirect UV detection using a pH 12.3 electrolyte containing sorbic acid has been used (131) to assay the carbohydrate content m fruit Juices and good agreement with HPLC data was obtained 4. Vitamin analysis: Surpnsmgly, there contmues to be relatively few reports of analysis of vitamins by CE. One notable exception is the work concernmg the determmatlon of vitamin A in dried blood spots. Laser-based fluorescence measurements allowed (232) a detection limit of 3 pg/L to be obtained for retinol. The analysis could be conducted from one or two drops of blood. 5. Biomedical apphcations: The number of biomedical apphcatlons of CE contmues to expand rapidly. Some particular examples are discussed covering both applications and methodology approaches. Theophylline and metabolites have been determined in urine (133) using solld-phase extraction pretreatment. A variety of ephedrine alkaloids were determined m urine with direct sample injection (234), and levels of free and total 7-hydroxy-coumarin were determined (23.5) in both urine and serum samples. The use of SDS solution as a rinse solution between analyses of blosamples has been shown to be more effective (136) than conventional rinsing regimens. The different approaches to quantifying drug m human serum followmg direct sample Injection have been compared (23 7) 6. CE-MS: The apphcatron of CE-MS combinations as separation-detectlon systems continues to grow. For example, peptides (138) and DNA fragments (139) have been detected by MS followmg their separation by CE. 3.

References 1. Beck,W. andEngelhardt,H. (1992) Capillary electrophoreslsof orgamcand morganlc cationswith mdlrect UV detection.Chromatographza 33,3 13-3 16. 2. Chen, M. andCassldy,R M. (1993) Separationof metal ions by capillary electrophoresis.J Chromatogr 640,425-43 1 3. Weston,A., Brown, P. R., Heckenberg,A , Jandlk, P., and Jones,W. R. (1992) Effect of electrolyte composltionon the separationof morgamc metal catlonsby capillary ion electrophoresls.J Chromatogr 602, 249-256. 4. Shi,Y. andFritz, J. S.(1993) Separationof metal ionsby capillary electrophoresls with a complexlng electrolyte.J Chromatogr 640,473-479. 5. Weston,A., Brown, P. R., Jandlk, P , Jones,W R., andHeckenberg,A. L (1992) Factors affecting the separationof inorganic metal cations by capillary electrophoresis.J. Chromatogr 593,289-295.

Altria 6 Jackson, P E and Haddad, P (1993) Capillary electrophoresis of inorganic ions and low-molecular-mass iomc solutes TRAC 12,231-238. 7 Quang, C and Khaledi, M G (1994) Prediction and optimisatton of the separation of metal cations by capillary electrophoresis with indirect UV detection J Chromatogr 659,459-466 8 Shi, Y. and Fritz, J S (1994) New electrolyte systems for the determmation of metal cations by capillary zone electrophorests J Chromatogr 671,42%-435 9 Backmann, K , Boden, J., and Haumann, I. (1992) Indirect fluorimetric detection of alkah and alkaline earth metal ions m capillary zone electrophoresis with cermm (III) as carrier electrolyte. J Chromatogr 626,259-265 10. Altrta, K D., Goodall, D M , and Rogan, M M (1994) Quantitative determmatton of drug counter-ton stoichiometry by capillary electrophoresis Chromatographla 38,637-642 11 Swartz, M E. (1993) Capillary electrophoretic determmation of morgamc tons in prenatal vitamin formulation J. Chromatogr 640,44 l-444 12 Koberda, M., Konkowskt, M , Youngberg, P., Jones, W. R , and Weston, A (1992) Capillary electrophoretic determination of alkali and alkaline-earth cations in various multiple electrolyte solutions for parenteral use J Chromatogr 602,235-240 13. Morawski, J , Alden, P , and Sims, A. (1993) Analysis of cationic nutrients from foods by ion chromatography J Chromatogr. 640,359-364 14 KaJiwara, H , Sato, A , and Kaneko, S. (1993) Analysis of calcmm and magnesium ions in wheat flour by capillary zone electrophoresis BIOSCL Blotech, Bzochem 57, lOlO,lOll. 15 Motomizu, S , Oshima, M., Matsuda, S -Y., Obata, Y., and Tanaka, H (1992) Separation and determmatton of alkaline-earth metal ions as UV absorbing chelates with EDTA by capillary electrophoresis. Determmation of calcium and magnesium m water and serum samples. Anal Scz 8,619-624 16 Buckberger, W , Semenova, 0 P., and Timerbaev, A R. (1993) Metal ton captllary zone electrophoresis with direct UV detection. separation of metal cyanide complexes JHRCC 16, 153-156. 17 Aguilar, M., Farran, A , and Martinez, M. (1993) Determmatton of gold (1) and silver (1) cyanides m ores by capillary zone electrophoresis. J Chromatogr 635, 127-13 1 18 Swaile, D. F and Sepamak, M J (199 1) Determmatton of metal ions by capillary zone electrophoresis with on-column chelation usmg 8-hydroxyquinoline-%sulfomc acid. Anal Chem 63, 179-184 19. Timerbaev, A. R , Buchberger, W , Semenova, 0 P., and Bonn, G K (1993) Metal ion capillary zone electrophorests with direct UV detection determination of transition metals using a 8-hydroxyqumoline-5-sulphomc acid chelating system. J, Chromatogr 630,379-389 20 Pretswell, E. L , Morrisson, A R., and Park, J S. (1993) Compartson of capillary zone electrophoresis with standard gravimetric analysis and ion chromatography for the determination of inorganic amons in detergent matrtces. Analyst 118,1265--1267. 21 Harrold, M. P., Wojtusik, M. J , Riviello, J , and Henson, P. (1993) Parameters mfluencmg separation and detection of anions by captllary electrophorests, J Chromatogr 640,463-47 1

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Application

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22. Kaneta, T., Tanaka, S., Taga, M , and Yoshrda, H (1992) Migration behaviour of inorganic amens m micellar electrokmetrc capillary chromatography using a catiomc surfactant. Anal Chem 64,798-801 23. Jones, W. R. (1993) Method development approaches for capillary ion analysts. J. Chromatogr. 640,387-395. 24. Kelly, L. and Nelson, R. J. (1993) Capillary electrophoresrs of organic acids and anions J. Liquid Chromatogr 16,2 103-2 112 25. Buchberger, W. and Haddad, P. R (1992) Effects of carrier electrolyte composrtion on selectrvrty m capillary zone electrophoresrs of low-molecular-mass anions. J Chromatogr 608,.59-64. 26. Ackermans, M. T., Ackermans-Loonen, J C. J M., and Beckers, J L (1992) Determmatton of propronate in bread using capillary zone electrophorests J Chromatogr 627,273-279 27. Jackson, P. E. and Haddad, P. (1993) Capillary electrophoresrs of inorganic ions and low-molecular-mass tonic solutes. TR4C 12,231-238. 28. Tinfdall, G. W., Wilder, D. R., and Perry, R. L. (1993) Optrmtsmg dynamic range for the analysts of small ions by capillary zone electrophoresrs. J Chromatogr 641, 163-167. 29. Ryder, S. (1992) Determination of sodmm vinyl sulphonate m water-soluble polymers using capillary zone electrophoresis J Chromatogr. 605, 143-147. 30 Wildman, B. J., Jackson, P E., Jones, W. R., and Alden, P G. (1991) Analysis of anion constituents of urine by inorganic caprllary eiectrophoresrs. J. Chromatogr. 546,459-466 3 1. Romano, J. P and Krol, J (1993) Capillary ion electrophoresrs, an envuonmental

method for the determmatron of amons in water J. Chromatogr 640,403-4 12. 32. Jackson, P. E and Haddad, P. R. (1993) Optrmrsation of inJection technique m capillary electrophoresrs for the determmatron of trace levels of anions in envrromental samples. J Chromatogr. 640,481-487. 33 Jandrk, P and Jones, W. R (1991) Optimisatron of detection sensitivity m the capillary electrophoresis of inorganic anions. J. Chromatogr 546,43 l-443 34. Salomon, D R. and Romano, J. (1992) Applications of capillary ion electrophoresis in the pulp and paper industry. J Chromatogr. 602,2 19-225. 35 Hargadon, K. A. and McCord, B. R. (1992) Explosive residue analysis by caprllary electrophoresis and ion chromatography J. Chromatogr. 602,241-247 36. Chadwick, R. C and Hsreh, J C. (1991) Separation of cis and trans double bond isomers using caprllary zone electrophoresis Anal Chem 63,2377-2380 37. Ng, C. L., Lee, H. K., and LI, S F Y. (1992) Analysis of food additives by ronpairing electrokmetic chromatography. J. Chrom Sci 30, 167-l 70. 38. Vindevogel, J. and Sandra, P. (1991) Resolutron optrmisatron in mrcellar electrokinetic chromatography: use of Plackett-Burman statistical design for the analysis of testosterone esters. Anal Chem. 63, 1530-l 536. 39. Rogan, M. M., Altna, K D., and Goodall, D. M. (1994) Plackett-Burman experimental design m chiral capillary electrophoreus. Chromatographia 38,723-729. 40. Ng, C. L., Lee, H. K., and Li, S F Y. (1993) Systematic optimisatron of capillary electrophoresis of sulphonamides. J. Chromatogr 598, 133-I 38.

Altria 4 1 Ng, C. L , Ong, C P , Lee, H K , and LI, S F Y (1992) Systematic opttmrsatron of mlcellar electrokmetrc chromatographrc separation of flavanords. Chromatographla 34, 166-172 42. Ng, C L., Toh, Y. L , Lr, S. F Y., and Lee, H. K (1993) Captllary electrophoresrs of btologtcally important compounds. opttmlsatron of separation condmons by the overlapping resolution mapping scheme J Lrqurd Chromatogr 16, 36533666. 43 Yeo, S. K , Ong, C. P., and Lr, S. F Y. (1991) Optrmrsatron of high-performance capillary electrophoresrs of plant growth regulators using the overlapping resolution mapping scheme Anal Chem 63,2222-2225 44 Altrta, K D. and Filbey, S D. (1994) The applrcatron of experimental design to the robustness testing of a method for the determmatton of drug related tmpurmes by capillary electrophoresrs Chromatographla 39,306-3 10. 45. Frlbey, S. D and Altria, K D. (1994) Robustness testing of a capillary electrophorests method for the determmatron of potassium content in the potassium salt of an acidic drug. J Capdlary Electrophoresls 1, 190-195. 46 Castagnola, M., Rossettr, D. V , Casstano, L., Rabmo, R., Nocca, G , and Gtardma, B (1993) Opttmrsatton of phenylhydantomammo acid separation by micellar electrokmettc capillary chromatography. J Chromatogr 638, 327-334 47 Vanbel, P F , Gilhard, J A., and Tilqum, B. (1993) Chemometric opttmrsatton m drug analysis by HPLC: a crmcal evaluation of the quality criteria used m the analysis of drug purity. Chromatogruphm 36, 120-l 24 48 Andersson, A M , Karlsson, A , Josefson, M., and Gottfries, J. (1994) Evaluatton of mobile phase additives m LC-systems using chemometrrcs. Chromatographla 38,715-722. 49 Rrghezza, M. and Chretren, J R. (1993) Factor analysis of experimental design m chromatography. Chromatographza 38, 125-129. 50 Mullholland, M. and Waterhouse, J. (1988) Investrgatron of the limttatrons of saturated fractional factorial experimental desrgns, with confounding effects for an HPLC ruggedness test Chromatogruphza 25, 769-774. 5 1 Mullholland, M (1988) Ruggedness testing in analytical chemistry TRAC 7, 383-389 52 Berrrdge, J. C. (1989) Chemometrrcs and method development in hrgh-performance hqurd chromatography. Part 2 sequential experimental designs Chemometrlcs Intell Lab Syst 5, 195-207 53. Lmdberg, W and Johannson, K. (1981) Apphcatron of stattstrcal optimrsatron methods to the separation of morphine, codeme, noscapine and papaverme m reversed-phase ion-pair chromatography. J Chromatogr. 211,20 l-2 12 54. Ahmad, S. U , Lane-Cam, C. A , and Bolton, S M. (1990) Factorial design in the study of the effects of selected liquid chromatographic conditions on resolution and capacity factors J Liquid Chromatogr 13,525 55 Plackett, R C. and Burman, J. P (1946) The desrgn of optimum multtfactorral experiments. Blometrrca 23,305-325. 56. Demmg, S. L. and Morgan, S. L (1983) Teaching the fundamentals of expenmental design. Anal Chum Acta. 150, 183-198.

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57 Box, G. E P., and Hunter, J S. (1978) m Statlsttcsfor Experiments, An Introduction to design, Data Analysis and Model Bulldmg, Wiley, New York, pp 291453. 58. Tucker, R. P., Fell, A. F., Berndge, J C., and Coleman, M W (1992) Computeraided models for optimisation of eluent parameters m chnal hquid chromatography Chtrality 4,3 16-322 59 Yao, Y C., Lee, H. K , and LI, S F Y. (1993) Opttmrsatron of mobile phase composition for HPLC separations of mtroaromattcs using overlappmg resolutron mapping. J Llq Chromatogr 16,2223-2225 60. Glajch, J. L , Kirkland, J S , Squire, K M., and Mmor, J M (1980) Optimization of solvent strength and selectivity for reversed-phase hqurd chromatography using an inter-active mixture-design statrstrcal technique J Chromatogr 199, 57-79 61 Mullholland, M and Waterhouse, J (1987) Development and evaluation of an automated procedure for the ruggedness testing of chromatographtc conditions m high-performance hqurd chromatography J Chromatogr 395,539-55 1 62. Thomas, B R. and Ghodbane, S. (1993) Evaluation of a mixed micellar electrokinetic capillary electrophoresis method for validated pharmaceutical quahty control J. Liquid Chromatogr 16, 1983-2006. 63 Nielen, M W. F (1993) (Enantto-) separation of phenoxy acid herbicides usmg capillary zone electrophoresis J Chromatogr 637, 8 l-90 64 Garcia, F. and Hemon, J (1992) Fast capillary electrophoresis-ion spray mass spectrometric determinatton of sulfonylureas. J Chromatogr 606, 237-247 65 Pianettr, G. A , Tavema, M., Baillet, A , Mahuzler, G., and Baylocq-Ferrrer, D (1993) Determmatton of alkylphosphomc acids by capillary zone electrophoresrs using indirect UV detection J Chromatogr 630,37 l-377 66 Wu, Q., Claessens, H. A , and Cramers, C. A (1992) The separation of herbicides by micellar electrokmetic capillary chromatography Chromatographta 34,25-30. 67. Dinelh, G., Bonetti, A , Catizone, P., and Gallettt, G C. (1994) Separation and detection of herbicides m water by mrcellar electrokmetrc capillary chromatography J Chromatogr 656,275-280 68. Dmelli, G , Vicar-i, A , and Catrzone, P (1993) Use of captllary electrophoresrs for detection of metsulfitron and chlorsulfuron m tap water. J. Agrlc Food Chem 41,742-746

69 Camerro, M C., Puignou, L , Galceran, M. T. (1994) Comparison of capillary electrophoresis and reversed phase ion-pair high-performance hqurd chromatography for the determination of paraquat, diquat and drfenzoquat. J. Chromatogr 669,2 I7-224 70. Vorndran, A. E , Oefner, P. J , Scherz, H , and Bonn, G. K (1992) Indirect UV detection of carbohydrates m capillary zone electrophorests. Chromatographta 33, 163-l 68. 71. Hoffstetter-Kuhn, S , Paulus, A , Gassman, E., and Wrdmer, H. M. (1992) Influence of borate complexation on the electrophoretrc behaviour of carbohydrates m capillary electrophoresis. Anal Chem 63, 154 l-l 547 72. Lm, J., Shirota, O., and Novotny, M. (1991) Capillary electrophoresis of amino sugars with laser-induced fluorescence detection. Anal Chem. 63,4 13-4 17.

340

Altria

73 Chtesa, C and Horvath, C S. (1993) Captllary zonal electrophoresis of maltoohgosacchartdes dertvattsed wtth 8-ammonapthalene-1,3,6,-trtsulphonic acid. J Chromatogr

645,337-352

74. Grill, E , Huber, C , Oefner, P , Vordran, A., and Bonn, G. (1993) Captllary zone electrophorests of p-aminobenzotc acid derivatives of aldoses, ketoses and uranic acids Electrophoresrs 14, 1004-1010 75 Boonkerd, S , Detaevernter, M. R , and Michotts, Y (1994) Use of capillary electrophoresis for the determmation of vttamms of the B group m pharmaceuttcal preparations. J Chromatogr 670,209-2 14 76 Ong, C P , Ng, C. L , Lee, K H., and Li, S F. Y (199 1) Separation of water and fat-soluble vttamins by mtcellar electrokmetic chromatography J Chromatogr 547,419428 77. FuJlwara, S , Iwase, S., and Honda, S. (1988) Analysts of water-soluble vttamins by mtcellar electrokmettc capillary chromatography J Chromatogr 447, 133-140 78 Ntsht, H., Tsumagart, N , Kaktmoto, T , and Terabe, S (1989) Separation of water-soluble vitamins by mtcellar electrokmettc chromatography J Chromatogr 465,33 1 79. Kenndler, E , Schwer, C., and Kaniansky, D (1990) Purtty control of riboflavm5’-phosphate (vttamm B, phosphate) by capillary zone electrophorests J Chromatogr

508,203

80. Kobayasht, S , Ueda, T , and Ktkumoto, M. (1989) Photodtode array detectton m htgh-performance captllary electrophoresis. J Chromatogr 480, 179-l 84 81. Thormann, W , Moltem, S., Caslavska, J , and Schmutz, A (1994) Clinical and forenstc applications of capillary electrophorests. Electrophoresis 15,3-l 2 82. Xu, Y. (1993) Capillary electrophoresis Anal Chem 65,425R-433R 83 Deyl, Z , Tagltaro, F , and Mtksik, I. (1994) Btomedtcal applications of captllary electrophorests. J Chromatogr 656,3-27 84. Thormann, W , Lienhard, S , and Wernly, P. (1993) Strategies for the momtormg of drugs in body fluids by micellar electrokmettc capillary chromatography J Chromatogr.

636,137-148

85 Tagltaro, F , Moretto, S , Valentmi, R , Gambaro, G , Anatoh, C., Moffa, M , and Tato, L (1994) Captllary zone electrophorests determination of phenylalanine m serum-a raptd, mexpenstve and simple method for the dtagnosis of phenylketonurta. Electrophoreszs 15,94-97 86. Lt, S., Frted, K., Warner, I W., and Lloyd, D. K. (1993) Determination of dextromethorpan and dextrorphan in urine by capillary zone electrophorests, apphcatton to the determmation of debrtsoqum-oxidation metabolic phenotype Chromatographta

35,216222.

87 Chen, F. T. A and Sternberg, J C. (1994) Charactertsation of protems by captllary electrophoresis m fused stltca columns-revtews on serum-proteins analysts and apphcatton to immunoassays. Electrophoresls 15, 13-2 1. 88. Guzman, N. A , Moschera, J., Iqbal, K., and Mahck, A. N. (1992) Effect of buffer constituents on the determmatton of therapeuttc proteins by capillary electrophoresis. J Chromatogr 608, 197-204.

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89 Shihabi, Z. K. (1993) Serum phenobarbital assay by captllary electrophoresis J. Llqurd Chromatogr l&205!?-2068 90. Schmutz, A. and Thormann, W. (1993) Determinatton of phenobarbital, ethosuximide, and primrdone m human serum by mleellar electrokmettc capillary chromatography with direct sample inJection Therapeutic Drug Monztorzng, 15, 310-316 91 Caslavska, J., Ltenhard, S., and Thormann, W. (1993) Comparatrve use of three electrokinettc capillary methods for the determmatton of drugs m body flutds Prospects for rapid determmatton of mtoxications. J Chromatogr. 638, 335-342 92 Miyake, M , Shrbukawa, A , and Nagawaka, T (1991) Simultaneous determmatron of creatinine and uric acid m human plasma and urine by mrcellar electrokinetrc chromatography. JHRCC 14, 18 l-l 85 93. Kuhr, W G (1990) Capillary electrophoresis. Anal Chem 62,403R-4 11 R 94 Yik, Y F and Lr, S. F. Y. (1992) Captllary electrophorests with electrochemical detection TRAC l&325-332 95. Avdalovtc, N , Pohl, C A , Rocklm, R D , and Stillain, J. R (1993) Determmation of cations and anions by capillary electrophorests combined wrth suppressed conductivity detectron. Anal. Chem. 65, 1470-1475 96 Kobayashi, S , Ueda, T., and Krkumoto, M. (1989) Photodtode array detection m high-performance captllary electrophoresis. J Chromatogr 480, 179 97. Beck, W., Van Hoek, R., and Engelhardt, H. (1993) Applicatton of a drode-array detector m capillary electrophorests. Eiectrophoresls 14, 540-546 98. Niessen, W. M A., TJachen, U. R , and Van der Greef, J (1993) Caprllary electrophoresis-mass spectrometry J Chromatogr 636, 3-l 9. 99 Deterding, L. J., Moseley, M A., Tomer, K. B., and Jorgenson, J W (1991) Nanoscale separations combrned with tandem mass spectrometry. J Chromatogr 554,73-82. 100. Remhold, N J., Tmke, A. P , Tjaden, U. R., Niessen, W. M A, and Van der Greef, J. (1992) Captllary isotachophoretic analyte focussing for capillary electrophorests with mass spectrometrtc detection using electrospray tonizatton. J Chromatogr 627,263-27 1 101. Smtth, R. D., Wahl, J. H., Goodlett, D. R , and Hofstadler, S. A. (1993) Capillary electrophoresis I mass spectrometry. Anal. Chem 65,574A-584A 102. Johansson, I. M , Pavelka, R., and Henion, J. D. (1991) Determinatton of small drug molecules by capillary electrophorests-atmospheric pressure tomzatton mass spectrometry. J. Chromatogr 559,5 15-528 103 Kostamen, R., Lasonder, E , Bloemhoff, W., Vanveelen, P A., Welling, G W., and Brums, A. P. (1994) Charactertsation of a synthetic 37-residue fragment of a monoclonal antibody against herpes virus by capillary electrophoresrs/electrospray (tonspray) mass spectrometty and 252Cf plasma desorption mass spectrometry. Biol. Mass Spectrom 23,346352 104. Lamoree, M. H., Reinhold, N J., Tjaden, U. R., Niessen, W. M. A., and Van Der Greef, J. (1994) On-line tsotachophoresis, for loadabihty enhancement m capillary zone electrophorests/mass spectrometry of P-agonists. Blol. Mass Spectrom. 23,339-345.

342

Altria

105 Skoclr, E , Vmdevogel, J , and Sandra, P. (1994) Separation of 23 danyslated ammo acids by mlcellar electrokinetlc chromatography at low temperatures Chromatographza

39,7-10

106 Terabe, S., Ishlhama, H , Nlshl, H , Fukuyama, F , and Otsuka, K. (1991) Effect of urea addltlon m mlcellar electrokmetlc chromatography. J Chromatogr 545,359 107 Waldron, K C , Wu, S , Earle, C W , Harke, H R., and Dovlchl, N J (1990) Capillary zone electrophoresls separation and laser-based detection of both fluorescem thiohydantom and dlamethylammoazobenzene thiohydantom derivatives of amino acids. Electrophoresv 11,777-780. 108 Wu, S and Dovlchl, J N. (1992) Capillary zone electrophoresls separation and laser-induced fluorescence detection of zeptomole quantities of fluorescem thlohydantoin derivatives of ammo acids. Talanta 39, 173-l 78. 109 LIU, J , Hsleh, Y , Wlesler, D., and Novotny, M (1991) Design of 3-(4carboxybenzoyl)-2-qumolmecarboxaldehyde as a reagent for ultrasensltlve determination of primary ammes by capillary electrophoresls using laser fluorescence detectlon. Anal Chem 63,408-412. 110 Toulas, C and Hernadez, L (1993) Apphcatlons of a laser-induced fluorescence detector for capillary electrophoresls to measure attomolar and zeptomolar amounts of compounds. LC GC lo,47 l-476 111 Camillen, P , Dhanak, D , Druges, M , and Okafo, G. (1994) High sensmvlty detection of ammo acids usmg a new fluorogemc probe. Anal Proc. 31,99-102 112 Ong, C P , Ng, C L , Lee, H K , and LI, S F Y (1991) Separation of Dns-amino acids and vitamins by mlcellar electrokmetlc chromatography J Chromatogr. 559,537-545

113. Remhoud, N J , Tjaden, U. R , and Van der Greef, J. (1994) Automated on-caplllary Isotachophoretlc reactlon cell for fluorescence derlvatlsatlon of small sample volumes at low concentrations followed by capillary zone electrophoresls .I Chromatogr

673,255-266.

114. Nickerson, B and Jorgenson, J W. (1988) High speed capillary zone electrophoresls with laser induced fluorescence detection JHRCC 11,533,534. 115. Kuhr, W G. and Yeung, E. S (1988) Indirect fluorescence detection of native ammo acids in capillary electrophoresls Anal Chem. 60, 1832-1834 116 Jones, H. K, and Ballou, N. E (1990) Separations ofchemlcally different particles by capillary electrophoresls Anal Chem. 62,2484-2490 117. Amankwa, L. N., Scholl, J , and Kuhr, W. G. (1992) Characterlsatlon of the ollgomerit dispersion of poly(oyxalkylene)diamine polymers by precolumn derlvatlsatlon and capillary zone electrophoresls with fluorescence detectlon. Anal Chem. 62,2189-2193

118. McCormick, R M. (199 1) Characterlsatlon of slhca sols using capillary zone electrophoresls. J Llquld Chromatogr 14,939-952 119. Ebersole, R. C. and McCormick, R. M (1994) Separation and isolation of viable bacteria by capillary zone electrophoresls &o/technology 11, 1278. 120. Burkmshaw, S M , Hinks, D., and Lewis, D M (1993) Capillary zone electrophoresis in the analysis of dyes and other compounds m the dye industry and dyeusing industries J Chromatogr 640,4 13-4 17

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12 1. Hmks, D and Lewrs, D M (1993) Capillary electrophoresis of dyes Chromatogr Anal. AugJSept., 9-11. 122 Yang, Q., Smeyers-Verbeke, J , Wu, W , Knots, M. S., and Mussart, D. L. (1994) Sunultaneous separatton of ammotuum and alkali, alkahne earth and transrtion metal tons m aqueous-orgamc media by captllary ton analysis. J. Chromatogr. 688,339-349 123 Desbene, P L., Morm, C J., Desbene Monvemay, A. M , and Groult, R. S (1995) Utihsation of fluorescein sodium salt m laser-induced indirect fluorimetric detectton of tons separated by capdlary zone electrophorests. J Chrovnatogr 689, 135-148. 124. Roder, A and Bachmann, K (1995) Simultaneous determmatton of organic and inorganic anions m the sub-pmol range m ram water by capillary electrophoresis J Chromatogr 689,305-311 125. Dabek-Zlotorynska, E. and Dlouhy, J F. (1994) Captllary zone electrophoresls wtth indnect UV detectton of orgamc anions using 2,6-napthalene dlcarboxylic acid J Chromatogr. 685, 145-153 126. Oefner, P. J (1995) Surface-charge reversed capillary zone electrophoresis of inorganic and organic amens. Electrophoresis 16,46-56. 127 Timerbaev, A. R and Semenova, 0. P (1995) Theoretical esttmatton of captllary zone electrophoresis behavtour of metal complexes using multivartate regression analysts. J Chromatogr A 690, 141-148 128. Oefner, P., Chtesa, C., Bonn, G., and Horvath, C (1994) Developments in capillary electrophorests of carbohydrates J Cap Electrophor 1, 5-26 129. Mechref, Y., Ostrander, G K , and Elrassi, Z. (1995) Capillary electrophorests of carboxylated carbohydrates. 1. Selecttve precolumn derivattzatron of gangliosrdes with UV absorbing and fluorescent tags J Chromatogr 695, 83-95 130 Ye, J and Baldwin, R. P. (1994) Determinatton of carbohydrates, sugar acids and aldrtols by capillary electrophorests and electrochemtcal detection at a copper electrode J Chromatogr 687, 141-148 131 Klockow, A, Paulus, A, Ftguerredo, V., Amado, R., and Wtdmer, H. M (1994) Determinatton of carbohydrates m fruit juices by capillary electrophoresrs and hrgh-performance hquid chromatography J. Chromatogr 680, 187-200 132. Sht, H., Ma, Y. F., Humphrey, J. H., and Craft, N E (1995) Determmatton of vttamm A m dned blood spots by high-performance capillary electrophoresis with laser-excited fluorescence detection. J Chromatogr. B. 665, 89-96. 133. Zhang, Z. Y , Fasco, M. J., and Kammsky, L. S (1995) Determmatlon of theophylline and its metabohtes in rat liver mlcrosomes and human urine by caprllary electrophoresrs. J. Chromatogr B. 665,201-208. 134. Chicharro, M , Zapardrel, A., BernIeJo, E , Perex-Lopex, J A., and Hernandez, L. (1995) Direct determmatron of ephedrme alkaloids and epinephrme in human urine by capillary zone electrophorests. J. Lzq Chromatogr 18, 1363-l 38 1. 135 Bogan, D. P., Deasy, B., Okennedy, R., Smyth, M. R., and Fuhr, U (1995) Determination of free and total 7-hydroxycoumarin content m urine and serum by capillary electrophoresis J Chromatogr B 663,37 l-378 136. Lloyd, D. K. and Watzig, H. (1995) Sodium dodecyl sulphate IS an effective between-run rinse for capillary electrophoresrs of samples m biologtcal matrices. J. Chromatogr B 663,400-405.

Al tria 137 Zhang, C.-X and Thormann, W (1994) Determmatlon of drug levels m human serum by mlcellar electrokmetlc capillary chromatography with direct sample InJectIon using different quantttatlon strategies. J Cup. Electrophor 1,208-2 18 138 Amankwa, L N , Harder, K , Jink, F , and Aebersold, R (1995) High-sensltlvlty determmatlon of tyrosme-phosphorylated peptldesby on-line enzyme reactor and electrospray lomsatlon massspectrometry Pvot Scz 4, 113-125 139 Janning, P , Schrader, W , and Lmscheld, M (1994) A new massspectrometrx approach to detect modlficatlons m DNA. Rapzd Comm Mass Spectrom. 8,1035-1040