Mass Spectrometry: article from Encyclopedia of Polymer Science and Technology, 4th Edition

MASS SPECTROMETRY Introduction In the past 25 years, mass spectrometrists have found ways to obtain charged polymeric molecules in the gas phase without degrading the polymer molecule. The study of unfragmented polymer molecules in the gas phase offers polymer science one of the most powerful absolute techniques that can be applied to chain characterization. Using mass spectrometry (MS) techniques, we can obtain the molecular mass distribution (MMD) for the whole polymer. Single chain structural information as a function of its molecular mass, for example, repeat units, end groups, and end-group distribution, copolymer composition distributions, and various polymer architectures, are amenable to measurement with various MS methods. Polymer architecture such as the occurrence of ring structure and cyclic structures can be studied. Gas-phase chemical reactions involving polymer chains can be used to study chain structure, that is, copolymer sequencing or chain branching. These hopes have been realized in the past 20 years or so with the advent of soft ionization methods in mass spectrometry, matrix-assisted laser desorption/ionization (MALDI), and electrospray ionization (ESI) methods. These methods place unfragmented high molecular weight polymer molecules in the gas phase. Using MALDI methods, we can examine polymers with molecular mass from about 500 u to polymers with molecular masses of about 1,500,000 u (1). The focus of this article is the soft ionization technique mainly in use for synthetic polymers, MALDI. Another common soft ionization method, the electrospray ionization (ESI) method, has been generally confined to polar biological polymers, which are water soluble. Other ionization methods that have been used on synthetic polymers, mainly laser desorption (LD) and field desorption (FD DESi, etc), are confined to low molecular mass polymers at this time and are discussed in some detail in other references on MS (2,3). The reader should be warned: A quick search of the current literature reveals that MALDI methods or any other soft ionization methods are most commonly used on biopolymers. However, the problems involving the study of synthetic polymers are very different than those encountered with most biopolymers, which are monodispersed—they have only one mass–whereas most synthetic polymers are polydispersed. This is easily seen in Figure 1, which shows a commonly available narrow MMD polystyrene (PS), SRM 2888. Note that even in this narrow MMD, we see signals from masses from 5000 to 9000 u. Thus, we 1 c 2010 John Wiley & Sons, Inc. All rights reserved. Encyclopedia of Polymer Science and Technology. Copyright


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Fig. 1. MALDI TOF MS of polystyrene around 7000u molecular mass. This MALDI spectrum was generated using dithranol as the matrix and AgTFA as the salt. The PS, dithranol, and salt were dissolved in tetrahydrofuran. The solution was than electrosprayed onto the sample plate. The polymer analyzed in reflectron mode with an extraction voltage of about 25 kev.

need a signal calibration as well as mass calibration to obtain an estimate of the polymer mass moments and the polymer MMD. The outline of this article highlights the following: a. An abbreviated history of the work done to successfully place a high molecular mass polymer into the gas phase. b. A description of the most common methods used to place charged high mass synthetic polymer molecules into the gas phase, the MALDI process, as well as the time-of-flight, (TOF) MS system. c. An explanation of four applications of MALDI in synthetic polymers. These applications include quantitation of narrow PD polymers using a calibration method for the signal; studies to look at mass distribution and chemical composition distributions in a copolymer; architectural distributions in a polymer; and the use of size exclusion chromatography (SEC) and MALDI to look at broad MMD polymers.

Brief History of MALDI and Electrospray as Soft Ionization Methods Dole and co-workers introduced electrospray ionization of polymers in 1968 (4) in an effort to determine the MMD of polystyrene by MS. In 1984, Fenn and his colleagues further developed the technique for biopolymers (5) and were able to obtain spectra of poly(ethylene glycols) up to molecular masses of 17,500 u by using ESI (6). In 1992, that same technique allowed Fenn to obtain MS on a poly(ethylene oxide) of about 5,000,000 u (7). Since ESI techniques multiply charge macromolecules, both these studies allowed Fenn and his colleagues to

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obtain spectra on a quadrupole MS with an m/z of less than 2000, where z is the number of charges. However, with many different n-mers in a normal narrow MMD of a synthetic polymers, the effect of z often as high as 40 necessitates resolution in m/z of a small fraction of a mass unit to see the entire MMD. In 1988, Tanaka and co-workers (8) developed a laser-based desorption method that produced ions of high molecular mass for both synthetic polymers and biopolymers. In that same year, Hillenkamp and colleagues (9) developed the laser desorption technique using an organic molecule matrix. This method, now commonly called MALDI, was originally described for biopolymer. However, in 1992, Hillenkamp and colleagues showed that the MALDI technique could be applied to synthetic polymers (10). In 1992 and 1993, the work of Danis and coworkers significantly extended the number and kind of synthetic polymers, which could be analyzed by MALDI (11,12). Also in 1993, Montaudo and colleagues (13) used MALDI on copolymers. MALDI spectra generally yield spectra with z = 1.

Mass Spectrometry of Synthetic Polymers Mass spectrometry comprises three distinct processes: (1) The production of charged gas-phase species from the original analyte. This step involves a way to get the analyte into the gas phase and a way to ionize it. For MALDI, these events occur in the same (single) process (2) The separation of the analytes by mass or, more correctly, by m/z. (3) The detection of the ions. The mass spectrometry of synthetic polymers usually must include a detailed data analysis protocol since the analysis of the spectra of a synthetic polymer with at least 30 peaks is akin to studying a mixture of 30 or so components. We shall consider here the MALDI TOF MS with a microchannel plate detector (see Fig. 2 for the schematic of a linear MALDI TOF MS and Fig. 3 for the schematic of a reflectron MALDI TOF MS). This is currently the instrument most commonly used to analyze synthetic polymers.

Introduction of the Polymer Molecule into the Gas Phase and Ion Formation Since the advent of lasers, it has been hoped that with very short laser pulses of an appropriately chosen wavelength, energy could be transferred into the translational modes of the polymer molecule in such a rapid way that the molecule would obtain enough translational energy to get into the gas phase before it would break apart (fragmentation). Although there had been numerous tries, only small polymers with masses less than 2000 or so could be gotten into the gas phase without significant fragmentation (14,15). These methods of laser desorption or field desorption are described in more detail in a previous encyclopedia article and recent

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Fig. 2. Schematic of linear time of flight matrix assisted laser desorption/ionization instrument.

Fig. 3. Schematic of reflectron time of flight matrix assisted laser desorption/ionization instrument.

books (2,3,16). Since these methods are limited to low molecular mass polymers, they will not be considered here. The MALDI Process. The MALDI process is the ablation of the analyte comixed with an organic small molecule matrix, most commonly an organic acid. The process Karas and Hillenkamp (9) initially used, and which is in common use today, is as follows: A polymer (biological or synthetic) is cocrystallized or comixed with the matrix molecule in the solid phase (details of this process will

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be described later). A short UV or IR laser pulse is used to ablate the matrix and the analyte mixture. The laser energy excites the matrix molecule causing it to vaporize and decompose in a supersonic phase transformation (17). This process excites many modes of the matrix and transfers the energy from electronic or vibration modes into translational modes in the matrix. For MALDI TOF MS, the most commonly used laser is the 337-nm wavelength UV nitrogen laser. Commonly used lasers have a pulse width of about 3 ns and generally perform well in the ablation of the matrices. The laser energy/unit area is a variable that can be adjusted with a variable absorbing neutral density filter. The ablation energy/unit area is usually adjusted to the lowest level that will produce polymer ions. This level keeps the initial energy of the plume lower. Lower initial plume energy results in lower initial velocity distributions of the molecular ions, minimizing broadening. Since fragmentation of the polymer in the gas phase is caused by collisions in the plume, reducing the initial velocity distributions reduces the fragmentation of the polymer. The matrix is selected to absorb most of the energy; few, if any, electronic states of the analyte (the polymer) are excited directly. However, since the analyte is intimately mixed with the matrix, the analyte is carried into the gas phase as a consequence of the phase transformation in the matrix. The analyte, the matrix molecules, clusters of matrix molecules, cations and cation clusters, as well as various combinations of each of these species have been detected in the plume from this laser ablation process (17–19). The biopolymers are charged by the many protons released in the plume and thus charged as a positive ion (3). For synthetic polymers, only those which have labile protons are easily charged in the same way (like polyacrylic acid and polystyrene sulfonate). Most synthetic polymers are charged by the addition of a metal ion. This will be discussed in more detail later in the article. For reasons not completely understood at this time, and regardless of the normal stable charged state of the metal ion, ie, +1,+2, or +3• , the MALDI process generally favors only singly charged n-mers in the gas phase (21). This generalization does not hold for very large molecular mass where multiply charged synthetic polymers can often be seen (1). Once charged, the metal polymer species is then accelerated into the separation region of the MS. Separation Process. Once we have the analyte in the gas phase as an ion, we need to separate analytes on the basis of mass to charge (m/z). Of the various separation techniques available, the most common form of MS separation is the quadrupole mass filter; see, for example, References. 2 and 3. Because this method is limited to m/z less than 2500 u or so, it is not generally used with MS of polymer Fourier transform mass spectrometry (FTMS) is an ion cyclotron resonance method (2,3). This method is currently limited to masses less than 20,000 u. Thus, it works well for lower molecular mass polymers. It is often used with MALDI of polymers; however, the initial velocity of the analytes in the MALDI plume can cause some intensity difficulties that make the method difficult to quantify. Furthermore, because of the expense of the magnet required to obtain quality data, FTMS is not a commonly employed method. For MALDI of synthetic polymers, the most common mass separation technique is time of flight (TOF) MS. In its simplest form, the ions in the MALDI

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plume are accelerated by a high voltage (often as high as 30 kV but usually 20 kV) for a distance of a few millimeters during which the ions obtain a velocity, v. The accelerated ions drift in an evacuated tube, typically about a meter long, at this velocity. (Some instruments have flight tubes as long as 6 m.) The equation describing this simple process is zeV = 1/2mv2

(1)

where V is the electric potential applied to accelerate an ion of charge ze and mass m. Once in the drift tube the translational energy of the ion is given by the right-hand side of the equation where v is the velocity of the ion. There is a correction for the velocity of the particle in equation 1, but if the field is large enough this is a small correction (16–18,22). If the drift tube is long compared to the acceleration region, than v = L/(t – t0 ), where L is the length of the drift tube and t−t0 is the time from some arbitrary zero time, t0 , set by the arrival of the ablating laser pulse. Thus, we have m/z = 2 eV(t − t0 )2 /L2

(2)

m/z = a(t − t0 )2

(3)

or

Equation 3, the equation relating mass and charge to time, is used as the general calibration for a TOF-MS instrument. Some modifications, generally small corrections, are required to be made to equation 2 (16,25). In current instruments, two modifications from a simple time of flight instrument are included. One is the reflectron, a set of ion mirrors that slows the ions down reverses their direction, and by increasing the actual length of the flight path, increases the resolution; see Figure 3 for a schematic of an instrument with a reflectron. Further improvement in the resolution can be obtained by the appropriate choice of reflectron voltages and shape of the voltage fields (16). The overall effect of the reflectron is to make little change in equation 2 for use as a calibration equation. Although the reflectron improves the resolution of the instrument, there can be loss of signal due to ion optics as well as late fragmentation. Fragmentation that occurs while the molecule is in the linear flight tube is considered late fragmentation. (Early fragmentation is fragmentation in the initial plume.) Molecules that fragment in the late fragmentation process will have the proper kinetic energy and arrive at the correct time at the detector in the linear region. Once in the reflectron, these ions are reacclerated with the mass of the charged fragment. Thus they arrive at the reflectron detector at a time reflecting neither the original mass of the polymer nor the mass of the charged fragment. The second modification is called delayed extraction. In earlier instruments, a single high voltage accelerating electric field was on while the plume was being formed by laser ablation. Instruments with delayed extraction have a second field plate located a few millimeters away from the original field plate. The second

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field plate is energized for a brief time, keeping the plume trapped for a short time, hundreds of nanoseconds, between the two field plates. The second field is then suddenly dropped to zero potential, accelerating the ions from the contained plume. This brief containment of the analyte plume allows it to come to some modest equilibration, decreasing the range of initial translational velocities and thus improving the resolution of the final spectra (22,24). Detectors. Several detectors are available for TOF MS instruments. An optimum detector should show no ion velocity dependence and should work for even the lowest velocities. In addition, the detector should not saturate or should have a sufficiently rapid recovery rate on the time scale of the smallest collection time interval. At this time, no current detector satisfies these requirements. Watson (2) has discussed many of the most common detectors. Byrd and McEwen (26) have discussed differences in signals from two detectors using the same TOF instrument. Using equimass blends of narrow PMMA standards to simulate a wide polydispersity polymer, they showed that different detection systems produced different apparent MMD for the polymer blend using the same sample preparation method and the same analyzer conditions. They found that the differences arose from detection mechanisms, saturation effects in the detector, and signal to noise problems. We will describe only one detector, the microchannel plate (MCP) detector, since it is the most commonly used on instruments employed for the analysis of synthetic polymers. The MCP detector is made up of an assembly of lead glass capillaries coated on the inside with electron-emissive materials and fused together. The capillaries are biased by a high voltage. Ions strike the inside wall, creating secondary electrons that amplify each ion impact signal with a gain of 103 or 104 . Higher gains are obtained with two MCP detectors in series. Matrices for the MALDI Process. The matrix is crucial to the MALDI process. It is generally believed that how the polymer is placed in the matrix has a significant influence on the success of the MALDI process. Liquid matrices have been used in a number of applications for biopolymers but only in a few applications for synthetic polymers. Solid matrices are most common for both biopolymers and synthetic polymer work. The earliest Karas and Hillencamp (9) experiments that described the MALDI technique employed solid matrices. To use solid matrices, usually solutions of polymer, matrix, and cationizing salt are mixed. The solvent is then allowed to evaporate from the solutions deposited onto a sample surface The mass proportion ratios of the matrix:polymer:salt in the final solid mixture cover the range of 5:1:2 to 2000:1:1. These proportions are often dependent on molecular mass of the polymer (1). Hanton and Owens (27) have discussed the choice of matrix compounds for synthetic polymers with respect to the polarity of the polymer. They suggest, as a general rule, trying to match matrix polarity with the polarity of the polymer so that both are soluble in a common solvent. Since the MALDI sample preparation requires intimate mixing of matrix, analyte, and salt, this choice will assure the best comixing, by their view, of the final solid mixture of the analyte and the matrix. They offer the following table for the appropriate choice of matrix and polymer in terms of solvent strength.

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Polymer

Thiourea Polyethylene glycol Dihydrobenzoic acid Polyproplylene oxide Cyano-hydrobenzoic acid Polyvinyl acetate Ferulic acid Polytetramethylene glycol Indol acrylic acid Polymethylmethacrylate Dithranol Polystyrene Retinoic acid Polybutadiene Diphenyl butadiene Polydimethylsiloxane Hydrophobic

This list offers a good preliminary list for the choice of various matrices for these polymers. Neilen’s (25) review of MALDI of synthetic polymers has an extensive reference list of polymers and the appropriate matrix to use with them. The NIST MALDI Web site for synthetic polymers http://polymers.msel.nist.gov/maldirecipes/ lists MALDI recipes and the related references for many synthetic polymers. From this and the earlier references, a method for MALDI of a specific synthetic polymer can be developed. Maier and co-workers (28) have presented a statistical design to looking at compounds for matrices for synthetic polymers and from this design proposed and tested five new matrices for synthetic polymers. Choice of Salts. Synthetic polymers generally obtain a charge by a different mechanism than biopolymers. Most biopolymers obtain a charge by addition or loss of a proton from the parent molecule. For synthetic polymers like PS sulfonic acid, this mode of charging is also available. However, most synthetic polymers do not contain labile protons. These synthetic polymers that are polar or contain double bonds can be cationized. The polar polymers molecules require a metal ion, often an alkali like Na+ or K+ , to attach to the polymer n-mers to obtain a charge. The Na and K metal ions are usually found in the matrix itself or introduced though the glassware used in sample preparation. For example, with PEO or PMMA, the common matrices used for these polymers have enough Na and K so that one commonly sees ions with both Na and K attachment without the addition of any Na or K salt. One can also add salts to the sample preparation solutions. For polymers with double bonds, polybutadiene or polystyrene, for example, one normally has to add Ag or Cu salts. These metal salts are found to work best, although other salts also give spectra (29). Polymers without polarity or double bond, for example, polyethylene or polypropylene, have difficulty being cationized. Only a few papers report MALDI spectra on these saturated hydrocarbon polymers (30–33) and these work for only very low molecular masses and show serious fragmentation. An effort to chemically attach charges to PE has been successful in increasing the range of molecular mass attainable from MALDI, but there is still serious fragmentation (34–37). Other materials have been used to cationize the polymers. Poehlein and coworkers (32) have used metallocenes (ferrocene, nickelocene, and cobaltocene) as

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cationizing agents for PS and PEO. These cations have the advantage that they are soluble in many organic solvents and also do not have the isotopic splitting seen with Ag, which often complicates the interpretation of the MALDI spectra. Choice of Sample Preparation Methods for Solid Matrices. Sample preparation is the process of taking the components of the recipe and getting them to lay down on the surface of the MALDI target so that each component is well mixed with the other components on the length scale of a small fraction of the diameter of the laser beam on the target. The reader should recall the target is the surface, which is placed in the vacuum and from which the sample, and the other components of the recipe are laser desorbed into the plume. If the plume is not representative of the sample and does not have a consistent representation of the recipe components, then the MS will not either. For the plume to be representative of the composition of the analyte, the region being ablated into the plume must be representative of the analyte. King and Owens (36) also have found that the intimate dispersion of the salt in the matrix is important. They find that the salt may not be well dispersed, and this often causes a loss of signal. The matrix is often an organic acid; by making the salt of the matrix acid and mixing this with the parent matrix acid, increased polymer signal is obtained. Too much salt, however, leads to no polymer signal since matrix salt alone is not a good matrix for the polymer. Some of the currently used methods of laying the mixture of polymer matrix and salt on to the target are described below. Hand Spotting. In hand spotting a solution of matrix salt and polymer is made with a solvent, which will evaporate quickly (more than one solvent is often necessary to get all these three in solution). The target is then spotted using a small amount of solution, often as between 1 and 10 µL, from a pipet. The small volume allows the solvent to evaporate before the species have a chance to separate into too large distinct “patches.” This process favors a more intimately mixed solid phase. The presumption is made that the polymer and the salt are well dispersed in the final matrix mixture to achieve a one-to-one representation of the polymer MMD in the solution to the polymer MMD in the gas phase. Yet, the matrix is commonly crystalline and the polymer may be either semicrystalline, like PEO, or glassy, like atactic PS. Kinetic processes occurring during the loss of solvent from the solution of the mixture of matrix, salt, and polymer must occur either to cocrystallize the polymer with the matrix and salt or to embed the polymer in the defect structure of the organic matrix. To obtain the correct representation of the MMD in the MS, each n-mer in the MMD must occur in the MS in proportion to its appearance in the original MMD. This means that the n-mer in the MMD of the polymer must be in each target region in its correct proportion. One may have to put down more than one spot to get enough material down to look at it for hundreds of shots. Some experimentalists drag the pipet over the target to obtain a thinner, more consistent film. Some authors have suggested that once a spot has been laid down, a spatula be used to mash together the spot to encourage more intimate mixing of the species in the spot. A special nanoliter device has been used to limit the volume of the matrix analyte salt solution (38). This seems to improve the spectra. In another version of hand spotting, the matrix or matrix and salt are in one solvent and the polymer in a nonsolvent for matrix. The matrix is laid down and the solvent allowed to evaporate away, and

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then the polymer is put on top of the matrix (or matrix and salt mixture) and its solvent allowed to evaporate away. This layering method to given a layer of polymer in intimate contact with the matrix or matrix–salt and will often show good spectra. The drawback to all forms of hand spotting is that even with care in the use of solvent and droplet size, phase separation between the species is possible leading to “sweet spots,” regions where the spot seems to give a lot of signal compared to no signal in other spots. Electrospraying. In electrospraying sample preparation for MALDI, a solution of matrix salt and polymer t is sprayed on the target using the instrument described in Ref. (39) where the target distance and field are adjusted so small droplets almost dry hit the target. In this method, the solution of polymer matrix and salt are in each original droplet and thus the dried droplet which hits the target are representative of the original solution. In a fashion similar to the handspotting, there has been some success with spraying the matrix first and then over spraying with a polymer. (40). Some studies have found that some polymers degrade when they undergo electrospray sample preparation (41). Nebulizing. In nebulizing sample preparation for MALDI, a solution of matrix salt and polymer t is sprayed on the target using a nebulizer where the target distance and flow of the nebulizing gas are adjusted so small droplets almost dry hit the target. As in electrospray sample preparation method, in the nebulizing method, the solution of polymer matrix and salt are in each original droplet and thus the dried droplet which hits the target are representative of the original solution. In a fashion similar to the handspotting, some authors have had success with spraying the matrix first and then overspraying with a polymer (42). Nebulizing has been successful in using polymer solvent systems that need high temperatures to keep the polymer in solution (42). Solvent-Free Grinding Method. The three materials, polymer, matrix, and salt, are ground together. A variety of grinding methods have been discussed (43). Simple grinding in a mortar and pestle is possible although the grinding of the soft polymer with the soft matrix is often difficult, and the cleanup from this is difficult. Grinding in a cold mill allows one to make the matrix and polymer harder and more easily ground. Hanton and Paress have suggested using a small glass vial and two small beads (often copper BBs) which are than vibrated (44). They further studied the morphology of the solvent-free MALDI methods and have shown that the average particle size of the matrix is on the order of hundreds of nanometers (45). After grinding together, the materials are pressed on the target with a spatula. A clean gas is then sprayed onto the surface to assure that none of the ground material, which has not stuck to the target, does not fall into the vacuum system when the target is placed into the sample chamber. Often grinding yields spectra better than those obtained from handspotting. Furthermore, grinding allows one to look at polymers that are otherwise intractable, that is, a polymer that will not dissolve in any solvent (46–48). Paste Method. In this method, all the materials matrix polymer and salt are ground together in the correct final ratios used in the solvent-free grinding method, described above. A small amount of solvent, usually for the polymer, is added and a paste created, which is then spread on the target (49).

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Mass Axis Quantification. Mass axis calibration of most TOF instruments is usually done with biopolymers of known molecular masses. These biopolymers are selected because they typically provide a single major peak whose mass is known accurately; thus, mass axis quantification is quite straightforward. Calibration usually can be done using three of these biopolymers. Collecting data with 2 ns time intervals, one can get better than single mass unit resolution on an instrument with a 1.5-m flight tube in reflectron mode at about 7000 Da. Calibration of the mass axis can also be done by combining a single biopolymer with a homopolymer calibrant. The oligomeric masses, mj , with n repeat units of mass r and masses of the end group, mend , of the polymer calibrant are given by mj = nr + mend + msalt

(4)

where n is the number of repeat units in the n-mer of the polymer and the msalt refers to the mass of the metal cation adducted to the polymer n-mer. Thus, calibration of the mass axis using a polystyrene calibrant reduces to determining n for one of the peaks; this is accomplished through use of the biopolymer mass as follows: The main peak from the biopolymer is assigned to its mass. The biopolymer peak will either lie between the masses of two n-mers of the PS calibrant, or exactly correspond to the mass of an n-mer. If it is at exactly the same mass as one of the n-mers of the PS calibrant, equation 4 can be used to find the degree of polymerization, n, for the n-mer. If the peak of the biopolymer lies between the masses of two n-mers of the PS calibrant, equation 4 can be used to find n1 , the mass of the n-mer whose mass is less than that of the repeat unit lower than the mass of biopolymer. Additional calibration points can be found by selecting PS peaks at intervals between 5 and 10 repeat units less than and greater than n1 and compute masses from equation 1. Generally, a total of four or five calibration points are selected. Signal Axis Quantification. For synthetic polymers, signal axis quantification is of utmost importance to obtain a good representation of the MMD or molecular composition distribution (MCD) or to compute the moments of the MMD to compare with classical polymer characterization methods like light scattering, nuclear magnetic resonance, or membrane osmometry. The signal axis may be in error due to a variety of reasons. Many of the problems have been discussed in our earlier chapter on MS in the Encyclopedia of Polymer Science. A detailed model of the MALDI TOF MS instrument and process for signal intensity seems unlikely at this time. However, the work of Yan and co-workers (50), Chen and He (51), and Zhu and co-workers (52) described previously implicitly assume that there is a point in the parameter space of the instrument and the sample preparation where we can assume that the signal intensity, Si , for an oligomer of mass mi is linearly proportional to ni , the number of polymer molecules at that oligomer mass. Guttman and co-workers (53) recently discussed an approach to quantitation of the signal axis using this approach in the production of the MMD of the SRM 2881.

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Mathematically, the equation for this is given by Si = ki ni

(5)

for ni < n0 From the work of Goldschmidt and Guttman (54) for PS in retinoic acid or dithranol, there are large regions of the linearity in the overall signal to the ratio of polymer to matrix where there is a large molar excess of Ag salt for PS of molecular mass around 8000 Da. From that work, we also know that if we get to a high polymer-to-matrix ratio, we see that the curve becomes nonlinear and approaches saturation at n = n0 . This cutoff seems to be a weak function of molecular mass. Now if we assume in the above equation that ki is a slowly varying function of i (hence of mi ) then we may make a Taylor’s expansion around a mass peak near the center of the MMD, termed M 0 . The center of the mass spectrum is used to assure that the function is changing as little as possible over the entire width of the MMD. Then Si = k0 ni + Q(mi − M0 )ni

(6)

Here Q and k0 are functions of M 0 as well as of all the experimental conditions: the instrument parameters, the sample concentrations, and the sample preparation method. exp One can obtain an experimental moments like Mn as 

exp Si mi Si (7) Mn = with the result that  Mnexp = Mno

o (1 + (Q/k0 )(Mw − M0 ) (1 + (Q/k0 )(Mno − M0 )

 (8)

where Mn is the experimentally measured Mn◦ . and where the true mass average relative molecular masses are given by exp

0 Mw

=




 m2i ni




i

mi ni

(9)

i

and Mn0

=


i

 mi ni




ni

(10)

i

All higher moments may be obtained in a similar way and have a similar form. If one allows M 0 = Mn◦ , one obtains from equation 8   Mnexp = Mno 1 + (Q/k0 )Mno (PD − 1)

(11)

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We see that the deviation of the number average molecular mass moment measured by MALDI--TOF MS from the true mass moment is a function of the polydispersity (PD) (arising from that moment). This is a result found by Muntaudo and co-workers (55) and Zhu and co-workers (52). As described in the next section, Guttman and co-workers (53) used the gravimetrically mixing polymers to obtain estimates of Q/k0 ,

Applications of MALDI TOF MS Narrow MMD Homopolymers. Generally, most laboratories obtained reasonable agreement for narrow MMD polymers between moments of MMD obtained from MALDI and those obtained by classical methods, eg, light scattering to obtain Mw and end group analysis or osmometry to get Mn or SEC (see also LIGHT SCATTERING or OSMOMETRY). Montaudo and co-workers have shown good agreement between classical methods and MALDI for polymers with Mw /Mn < 1.2 ((55). Lloyd and co-workers (56) in their study of PMMA by SEC and MALDI support this view. Li and co-workers (52) have made a careful study of PS of molecular mass 5050, 7000, and 11,600 u and blends of the three. Using the PS 5050 and 11,700 u, they studied changes in the MMD of the polymer of 7000 u by blends and found they could detect no systematic uncertainties within 0.5% in the MMD or the moments of the MMD. A MALDI-TOF-MS interlaboratory comparison was conducted by NIST among 23 laboratories using MALDI TOF MS on a well-characterized polystyrene to determine the reproducibility in determining the molecular mass distribution (57). An example of a MALDI-TOF MS spectrum from this polymer is given in Figure 1. NMR characterization of Mn was found to give 7050 ± 400 u. The Mw was found to be 7300 ± 600 u by light scattering. By compiling all the returned data using all protocols into one preliminary analysis, it was found that MALDI mass spectrometry returned an Mn of 6600 ± 100 u and an Mw of 6700 ± 90 u. The uncertainty values given following the ± represent only type A standard uncertainties (statistical uncertainty). These Mw and Mn obtained from MALDI were below the Mw and Mn of the classical methods. The statistical uncertainty in the mass spectrometry measurements was very small, indicating that from laboratory to laboratory, reproducibility was extremely good. A 2008 interlaboratory study by the National Institute of Advanced Industrial Science and Technology of Japan (AIST) showed that instrument settings and measurement protocols have more influence on the MMD than does the operator (58). Using the above work, an indication that MALDITOF MS is a robust method but has a small but systematic uncertainty, Guttman and co-workers (53,59) used mixtures of three octyl-initiated narrow PS at 6k, 9k, and 12k along with a butyl initiated at 9k to calibrate the signal axis using the development on calibration of the signal axis described previously. From this, they estimated the values of Q/k0 for the system and thus were able to obtain the true MMD for the narrow polymer from MALDI and estimation of the systematic uncertainty of the method. Broad MMD Homopolymers and the Use of Size Exclusion Chromatography. Early in the studies on MALDI of synthetic polymers, it was

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hoped that MALDI could be used successfully for both narrow and broad MMD. Soon it was discovered that for a variety of reasons the correct MMD of broad MMD polymers could not be obtained in one step by MALDI-TOF-MS. Montaudo and co-workers (60) found that they got good agreement between MALDI and SEC for MMD moments for polymers with polydispersities less than Mw /Mn = 1.1–1.2. Polymers with higher polydispersities regularly showed incorrect moments of the MMD (1,57,60–72). SEC is the most commonly used chromatography technique for analysis of polymers. Two common approaches have been used to obtain a MMD from the combination of MALDI and SEC. In one case, one fractionates the polymer using SEC collecting each fraction and obtaining the MALDI on each fraction. One then must combine the data from each MALDI spectra to create the MMD on the polymer. This is done by summing each MALDI chromatogram used in obtaining the moments to obtain Mn and Mw and the polydispersity (73). In another method, fractions are taken from the SEC at well-defined elution volumes. A MALDI analysis of these fractions is made and the peak of the mass MMD, M p , is computed from the MALDI data on the fraction. The M p so obtained is then used to calibrate the SEC. The calibrated SEC is then run to get the exact MMD of the polymer (74). A number of authors have used this technique. Two limitations of this technique should be mentioned. The first problem arises because one must have relatively narrow fractions in each MALDI fraction used for this to work. Thus, few linear or lightly branched homopolymers or random or alternating copolymers meet this criterion. Block copolymers in a solvent such that one block is extended and other is collapsed (with very different hydrodynamic volumes) may result in mass broadening too. This will make it difficult to assign a mass to the broadened fraction by MALDI and thus to get a good calibration for the SEC columns. The second problem comes from the assignment of the total mass of the material in each SEC fraction. For a copolymer, for example, a single detector may not be enough to assign the total mass of different groups at a given molecular mass. Two detectors at least may be required for copolymers. The work of Puglisi and co-workers (75) also shows a different phenomenon. In this case, poly(bisphenol A carbonate) (PC) terminated with hydroxyl groups undergoes self-association by hydrogen bonding in solution and these molecular aggregates remain in the SEC column. In the application of the SEC MALDI method, self-aggregation is suppressed in the sample preparation for the MALDI analysis and thus a very broad molecular mass for each MALDI appears. This is an extreme case of where SEC alone cannot provide a narrow MMD for MALDI. SEC alone will not necessarily work in this case. Owing to ring closure and higher order branching, chains with the same hydrodynamic volume in the SEC have significantly different molecular mass (76,77) A number of papers discuss placing a MALDI MS instrument in line with a continuous chromatographic process. For example in a series of papers, Murray and Russell (78) developed a method, which they called aerosol MALDI. In this method, elutant from an SEC is mixed with matrix prior to pneumatic nebulization, which sprays directly into a mass spectrometer. MALDI is performed on aerosol particles using a 355-nm laser. The solvent is evaporated from the aerosol particles by passing the aerosol through a heated tube. This technique has been used successfully with PEG of mass 1000 u and polypropylene glycol (PPG) also of

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15

mass 1000 u. Mass resolution with this method is less than with normal MALDI. In addition, the problem with data analysis is overwhelming. Baseline determinations, identification of peaks, and quantification are all problems. Furthermore, a wealth of data has to be dealt with. A number of review articles have been published on interfacing MALDI with various chromatographic processes. Gusev has critically discussed both offline uses of MALDI, like the one described above (79), and inline use of MALDI where many MALDI spectra are taken continuously. SEC is a separation method of polymers, which separates polymers on the basis of the polymer size alone. Only the interaction of a polymer that excludes it from the wall of the column support is operative in normal SEC. An attractive interaction between the polymer in solution and the solid portion of the porous medium can substantially affect molecular separation. The attractive interaction counterbalances the exclusion effect until a compensation point is obtained, “the adsorption theta point,” where the excluded volume effect between the polymer and the flow matrix vanishes (80,81). Selective molecular separation is lost at this compensation point. For an even greater attractive polymer matrix interaction, the polymer becomes adsorbed onto the surface of the matrix so that the higher molecular weight polymers exhibit an inhibited flow through the porous media. The change from an excluding interaction to an attractive interaction with the porous media can be affected by changing the solvent strength by use of mixed solvents. For block copolymers, one can choose a solvent system that will have one block at the polymer compensation point and another block in the exclusion regime. Falkenhagen and co-workers (82) have used this chromatographic method to examine the polyethylene oxide (PEO) co-polyethylene (PE) block system. The solvent is chosen so that the PEO is at its critical condition, ie, there is no separation with respect to PEO, but there is separation with respect to PM. MALDI TOF MS was used as a detector on this system. Each separate PM peak, was continuously transferred onto a MALDI target. The fractionation was made into separate PM peaks. Within each PM peak, there is a MMD of PEO. This allowed the authors to estimate both the MMD and molecular composition distribution (MCD) for the whole polymer. End Groups. Polymer end groups play an important role in determining polymer properties. This is increasingly important as the molecular mass of the polymer decreases. Low molecular mass polymers with well-defined end group functionality are used as prepolymers for many important final polymer products, ie, polyurethanes, epoxies, UV cure adhesives, and various other thermosets (see Thermosets). For example, polyurethanes are commonly made by a three-step processing in which the activity of the end groups is important in determining the final product (see Polyurethane). Bulk functionality of prepolymers can be determined by NMR, UV-vis spectroscopy, or titration methods. The SEC data from multiple detectors can be used sometimes to obtain estimates of functionality, but this often is not available due to the lack of difference of detector signal between the end group compared to the central groups. Even if this is possible, SEC does not allow one to distinguish between mono-from multi-functional end groups. Furthermore, the calibration of SEC at low molecular masses is, at best, difficult. MALDI-TOF MS offers a unique

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probe of the end-group functionality as a function of the molecular mass of the polymer itself. For polymers of molecular masses below 10 ku or so, current commercially available MALDI TOF MS instruments can differentiate end groups differing by a few mass units. With the sensitivity of FTMS, one can achieve single mass unit end group identification up to 20 ku (3). In a paper by Maloney and co-workers (83), PMMA with a variety of end groups was analyzed and it was shown that the various end groups can be identified. Jackson and co-workers (84,85) have also examined a variety of PMMAs of industrial relevance and were able to distinguish many end groups for polymers with molecular masses of 16 ku. Liu and co-workers (86) have applied MALDI to the analysis of poly(alkylthiophenes) (PAT), a rigid rod-type polymer. In particular, they were able to determine the end-group compositions of the PAT samples. The samples showed large variations in the presence of each end-group pair as a function of preparation conditions. Such end-group monitoring is necessary for the development of new materials from these polymers. Furthermore, for the polymers with Br end groups, they found some end-group fragmentation as a function of laser power, with more fragmentation at higher laser powers. Puglisi and co-workers (75) examined the end groups in the poly(bisphenol A carbonate) study described in Section 5.1. They were able to distinguish among a variety of end groups up to 16 ku for the mass of the n-mer. This work was done with the MALDI acting as a SEC detector. Copolymers. MALDI TOF MS of copolymers offers the opportunity to obtain a MMD and molecular composition distribution (MCD). Generally, to obtain a MCD the MS can only be done on low mass copolymers. Even at lower molecular masses where one can obtain isotope resolution, this is generally difficult because the number of n-mers separated by only a few mass units quickly becomes very high for copolymer composition near 50–50. If the mole faction of one of the copolymers is small, it is possible to obtain single or nearly single n-mer resolution for the MMD of the copolymer. A simple example of low mole composition copolymer is the study of Guttman and co-workers (69). MALDI TOF MS was used to study the molecular composition distribution. One can obtain the number of α-methyl styrene (α-MeSty) repeat units in SRM 1487, a narrow MMD poly(methyl methacrylate) NIST standard reference material of about 6300 g/mol. Here, the major copolymer is MMA and α-MeSty is the minor component, only a mole fraction of a few percents. The α-MeSty is, in fact, part of the initiator for this polymer so we have either a “MMA styrene MMA” triblock copolymer or a “styrene MMA” diblock with the α-MeSty block length containing from 0 to 6 α-MeSty repeat units. With so few α-MeSty, the entire copolymer MMD and composition can be determined. In a study of a much more complex composition distribution, Wilczek-Vera and co-workers (87–89) have examined the copolymer systems, polystyrene-blockpoly(α-MeSty) and poly(α-MeSty)-block-poly(4-vinyl pyridine). In the work of Wilczek-Vera and co-workers (88) on poly(α-MeSty)-block-poly(4-vinyl pyridine), polymers with different mole ratios of α-MeSty and 4-vinyl pyridine were studied. The composition of the maximum peak was used to start the analysis. The compositional assignment of this maximum peak was made from the NMR data and/or the initial chemical composition of the polymer. The rest of the peaks were

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17

assigned by a procedure that references the composition of the main peak. From this analysis, the two-dimensional distribution of the di-block copolymer was obtained. Such use of collateral data shows that MS alone may not be enough to obtain such a complex copolymer distribution. However, with the copolymer composition distribution, a richer understanding of the copolymer chemistry can be obtained. In their study on the copolymer systems polystyrene-block-poly(α-MeSty), Wilczek-Vera and co-workers (88) were able to confirm that the copolymer composition agrees with the predicted Zimm–Schulz distribution (88,89). Using an analysis method similar to that used previously on the poly(α-MeSty)-block-poly (4-vinyl pyridine) system, the MMD of both parts of the copolymer was determined. The data analysis method was claimed to verify the random coupling hypotheses. Wilczek-Vera and co-workers (85) confirmed the Freyss and co-workers (90) hypothesis that the polydispersity of individual blocks is higher than the polydispersity of the whole polymer. That is, block copolymers with narrow MMD have broad complex chemical composition distribution. Montaudo and Samperi (91) have studied the composition and sequence distribution of poly(butyleneadipate-co-butyleneterephthalate). They compared the intensity of the MS peaks with theoretical intensities obtained using a Bernoulli distribution of the sequences. This result is in excellent agreement with NMR analysis of the bulk polymer. MALDI to Elucidate Polymer Chemistry. Perhaps one of the most profitable future uses of MALDI will be for elucidation of polymer chemistry. Examining a reaction not yet complete or looking for polymer side products differing only by an end group will yield important reaction kinetics information. Unlike NMR, UV, or FTIR, where one can perhaps find the presence of differing end groups or internal species for the overall MMD, MALDI can identify these species and show how they can appear as a function of molecular mass. A caveat to all this work is the matrix attachment studies of Goldschmidt and Guttman (18). Without careful studies of the linear and reflectron modes for each reaction examined and the effect of matrix and attached ion on the resultant spectra, one has to consider many of the conclusions from these studies tentative. One can often determine the polymer chemistry by examining the polymer products during, or at the end of a reaction. Much of what has been discussed in the copolymer section relates to this. The work of Wilczek-Vera and co-workers (87–89) describes the distribution from which the chemistry can be elucidated. Schulz and co-workers (92,93) had extensively studied the course of the anionic reaction of PMMA initiated by α-MeSty with a Na counterion. They concluded that dimer and tetramer α-MeSty difunctional anion initiators are the dominant species in the initiation reaction. (m = 2 or 4). Arnould and co-workers (94,95) used MALDI-TOF-MS to examine the products from anionic polymerizations using hydrocarbon solutions with styrene initiated by sec-butyl-Li to t-C4 H9 O(CH2 )3 Li and extended by isoprene. Several electrophilic reagents affected termination. The results showed unusual rearrangements to yield some end groups not normally expected. From this observation, they were able to suggest new mechanisms for the termination reaction of the polymer. Pollack and Morgan (96) have used MALDI TOF MS to study the preparation of linear polysiloxanes with hydride end-capped and cyclic polysiloxanes from

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Fig. 4. The central portion of the full mass spectrum of a polysilsequioxane prepolymer showing the characteristic shape of a condensation polymer. The major repeats show the changing number of RSiO3/2 with a repeat of 188.12 u.

the ring opening anionic polymerization reaction with hexamethylcyclotrisiloxane and octamethylcyclotrisiloxane. The PDMS reaction was followed by MALDI. The incorporation of the hydride end-cap and the creation of the cyclics was studied as a function of time. Hawkridge and Gardella (97), using MALDI and SIMS, have further studied this reaction; they find that MALDI provides more reproducible relative intensities of the n-mers than does SIMS. Wolf and co-workers (98) studying the chemistry of the creation of an poly(lactide)-block-poly(HEMA) block copolymer used MALDI and NMR to confirm the complete terminal functionalization, an important step in creating the block copolymer. Kona and co-workers (99) looked at the epoxidation of polydienes following two different chemistry pathways and found the degree of epoxidation for each of the polymers of different polydispersities and molecular masses using MALDI. Polymer Architecture Elucidated by MALDI. Although MS is a massdetermining device, MS studies in combination with an understanding of polymer chemistry allows researchers to make deductions on the architecture of polymer molecules. In this section, we shall describe a study of trifunctional polymerizations similar to those studied by Flory in the early 1940s (see, eg, Ref. 100, where the structure of the condensation polymerization of a multifunctional polymer was studied). Here, however, instead of a carbon-based backbone a Si-based backbone was studied. The prepolymers of the polysilsesquioxanes were studied by MALDITOF MS by Wallace and co-workers (101,102). Polysilsesquioxanes are threedimensional polymers with a repeat unit of the form [RSiO3/2 ] where each silicon

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Fig. 5. (a) A single-repeat unit detail of the full mass spectrum is shown. The distance between peaks in a single major cluster is 18 u, indicative of the intermolecular loss of water. (b) The schematic of the polymer ms shows the relation of each peak of the MS to the degree of intermolecular polymer condensation, the important polymer architecture change in this system. The 1, 2, 3, and 4 refer to the number of closed loops in the chain

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is coordinated with three oxygen atoms. After a low temperature sol–gel synthesis and processing, silsesquioxanes are then fully cross-linked to form a fourfold coordinate structure. One important unknown in the processing of silsesquioxanes is the “degree of condensation” of the prepolymer silsesquioxanes before it is converted into the final monolith. That is, how many of the silicon atoms are three fold coordinated with bridging oxygen atoms and how many have terminal silanol (SiOH) groups? In the polymerization process to create the prepolymer, a condensation reaction of two silanol groups to create one Si O Si bridge with the elimination of water occurs. In the prepolymer, these terminal silanol groups control the reactivity of the polymer in subsequent processing steps that result in a cross-linked monolith. Before the use of MALDI MS only the average number of terminal silanol groups in the whole sample could be measured by infrared spectroscopy. Condensation of SiOH groups out of the growing prepolymer silsesquioxane molecule can lead to the formation of intramolecular Si O Si bridges with the loss of water. This elimination reaction is easily identified via high-resolution time-of-flight mass spectrometry. Figure 4 shows the overall spectrum, whereas Figure 5 shows the detail of a single low-mass oligomer from the overall spectrum. The maximum possible mass of an oligomer with n repeat units occurs when every silicon atom has one silanol group in addition to one R-group and two bridging oxygen atoms. However, the highest intensity peak generally does not occur at the maximum possible mass. Instead, lower mass peaks are more intense. These peaks correspond to the loss of water as a pair of SiOH groups react. This indicates that intramolecular reactions are occurring. Each loss of 18 u closes a loop in the molecule moving it away from a highly branched linear structure toward a closed polyhedron. In the limit of the loss of all silanol groups, a fully condensed polyhedral structure results. For example, for n = 8 the fully condensed structure is a cube with Si atoms at each corner and Si O Si linkages for the cube edges. Thus, the structure and chemical architecture of each n-mer can be deduced from MALDI TOF MS. In another determination of architecture by MALDI, Puglisi and co-workers (75) investigated the appearance of cyclics and linears in the poly(bisphenol A carbonate) study described above in Section 5.1. They were able to distinguish between the linear and cyclic species. From their MALDI studies, a separate SEC calibration line was obtained for the cyclic and the linear species. This work was done with the MALDI acting as a SEC detector.

Conclusions In this article, we have discussed some of the current uses of the MS soft ionization technique used for synthetic polymers, MALDI -TOF-MS. Recent advances show the ability of the method to look at complex architectures never before analyzed in polymers. Furthermore, the beginnings of quantitation of the MMD and molecular composition distribution of copolymers using MALDI TOF MS was discussed.. It is expected that the ability to create, study, and use beams of charged polymer molecules will spawn new characterization methods as well as new technology applications.

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CHARLES M. GUTTMAN Polymers Division, National Institute of Standards and Technology (NIST) Gaithersburg, MD, USA