Oligomeric Separation of Ionic and Nonionic Ethoxylated Polymers by

In the years since its inception, few applications of CGE to nonbiological synthetic polymers have been reported. CGE has been applied to the separa-...
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Anal. Chem. 1996, 68, 2541-2548

Oligomeric Separation of Ionic and Nonionic Ethoxylated Polymers by Capillary Gel Electrophoresis R. A. Wallingford

Central Technology, Union Carbide Corporation, South Charleston, West Virginia 25303

Capillary gel electrophoresis (CGE) has proven itself as a superior, high-resolution technique for separating proteins, peptides, oligonucleotides, and other naturally occurring molecules. In the years since its inception, few applications of CGE to nonbiological synthetic polymers have been reported. CGE has been applied to the separation of ionic and nonionic ethoxylated surfactants and poly(ethylene glycol) (PEG) oligomers. Oligomer distributions of several sulfated and phosphated alkylphenol ethoxylate surfactants have been baseline resolved with CGE on commercial cross-linked polyacrylamide gel columns. Nonionic surfactants and PEG oligomers were derivatized with phthalic anhydride in order to provide charge and detectability. PEG oligomers ranging from ethylene glycol to species containing more than 120 ethylene oxide units have been resolved. A linear relationship between migration time and molecular weight was found, which indicates that the separation mechanism is not simply based on molecular size but is also influenced by the electrophoretic mobility of the oligomers. The main drawbacks of CGE include relatively long analysis times and somewhat fragile and expensive columns. Polymers based on the polymerization of ethylene oxide are a very important class of compounds which are used in producing consumer products. Two common ethoxylated products are surfactants and poly(ethylene glycol)s (PEGs). Because most ethoxylates are produced through base-catalyzed addition of ethylene oxide monomers, a polydisperse statistical distribution of oligomers is formed. The physical characteristics of an ethoxylated product and its performance in a particular application can often be correlated with information describing the oligomeric distribution. The importance of the correlation of performance with oligomer distribution has spawned much research activity into detailed characterizations of ethoxylated surfactant1-7 and PEG distributions.8-14 (1) Ysambertt, F.; Cabrera, W.; Marques, N.; Salager, J. L. J. Liq. Chromatogr. 1995, 18 (6), 1157. (2) Marques, N.; Anton, R. E.; Usubillaga, A.; Salager, J. L. J. Liq. Chromatogr. 1994, 17 (5), 1147. (3) Wang, Z.; Fingas, M. J. Chromatogr. 1993, 673, 145. (4) Bear, G. R. J. Chromatogr. 1988, 459, 91. (5) Martin, N. J. Liq. Chromatogr. 1995, 18 (6), 1173. (6) Allen, M. C.; Linder, D. E. J. Am. Oil Chem. Soc. 1981, 58, 950. (7) Desbene, P. L.; Desmazieres, B. J. Chromatogr. 1994, 661, 2076. (8) Meyer, T.; Harms, D.; Gmehling, J. J. Chromatogr. 1993, 645, 135. (9) Barka, G.; Hoffman, P. J. Chromatogr. 1987, 389, 273. (10) Rissler, K.; Kunzi, H.-P.; Grether, H.-J. J. Chromatogr. 1993, 635, 89. (11) Escott, R. E. A.; Mortimer, N. J. Chromatogr. 1991, 553, 423. S0003-2700(95)01179-6 CCC: $12.00

© 1996 American Chemical Society

Characterization of the distribution of polydisperse systems is commonly performed by gel permeation chromatography (GPC), which can provide weight- and number-average molecular weights as well as polydispersity index.1,15 More detailed characterizations of oligomer distributions are made possible by employing the superior resolving power of high-performance liquid chromatography2-11 (HPLC), supercritical fluid chromatography (SFC),11,12 capillary zone electrophoresis (CZE),16-18 and, for low molecular weight ethoxylates, gas chromatography13,14 (GC). Because most polyethoxylated products are nonvolatile, HPLC and SFC have become the preferred techniques for resolving oligomer distributions. HPLC and SFC can often provide resolution of the individual polyethoxylated oligomers, which allows for detailed comparisons to be made between different products or different lots of the same product. Calibration of the detector response allows the intensities of the oligomer peaks to be employed to calculate weight- and number-average molecular weights and polydispersity values with potentially higher accuracy than is obtained by GPC. A major difficulty encountered in the HPLC separations of PEGs is detection of the oligomers. Because PEGs contain no easily detected moiety, detection has been traditionally limited to refractive index (RI) detectors8 and low UV detection.9,11 Barka and Hoffmann9 employed UV detection at 190 nm and gradients of acetonitrile and water on a C8 column to produce good resolution of PEG oligomers from ethylene glycol up to about 110 ethylene oxide units. An alternative is to employ precolumn derivatization10 of the PEGs to add a chromophore or fluorophore to render the oligomers detectable by conventional UV/visible or fluorescence detectors. RI detection limits the analyst to isocratic mobile phases, which in turn limits the resolution and analysis speed. Conversely, precolumn derivatization techniques allow the use of gradient elution to enhance resolution and decrease analysis times. Additionally, the evaporative light scattering detector10 allows the use of mobile phase gradients and provides detection of underivatized PEG oligomers. Capillary gel electrophoresis19-23 (CGE) is a specialized form of capillary electrophoresis which has found application exclusively in the bioanalysis arena. CGE usually employs a fused silica (12) Hagen, H. M.; Landmark, K. E.; Greibrokk, T. J. Microcolumn Sep. 1991, 3, 27. (13) Rasmussen, H. T.; Pinto, A. M.; DeMouth, M. W.; Touretzky, P.; McPherson, B. P. J. High Resolut. Chromatogr. 1994, 17, 593. (14) Lipsky, S. R.; Duffy, M. L. J. High Resolut. Chromatogr. 1986, 9, 725. (15) Yau, W. W.; Kirkland, J. J.; Bly, D. D. Modern Size-Exclusion Liquid Chromatography; John Wiley & Sons, Inc.: New York, 1979. (16) Bullock J. J. Chromatogr. 1993, 645, 169. (17) Chen, S.; Pietrzyk, D. J. Anal. Chem. 1993, 65, 2770. (18) Zweigenbaum, J. Chromatogram 1990, 11, 9.

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capillary that is filled with an immobilized and cross-linked polyacrylamide gel. The gel contains a network of pores, which facilitate resolution of ions migrating through the gel based on differences in molecular size. Because diffusion of separated ions is very small in the gel, separation efficiencies for CGE are often in the millions of theoretical plates. CGE has the distinction of producing the highest separation efficiencies achieved to date, with 10-20 million plates being reported for large biomolecules.20 Cross-linked polyacrylamide gel-filled capillary columns for CGE are available commercially from several vendors. Capillary zone electrophoresis has been applied to the oligomeric resolution of surfactants16-18 and PEGs,16 but no reports of the application of CGE to these types of synthetic polymers have been found. Although CGE has been exclusively a biochemist’s tool, there is no fundamental reason why this technology could not be applied to synthetic ionic polymers. To the author’s knowledge, no applications of CGE (with cross-linked gels) to separations of synthetic polymers have been reported in the literature thus far. This report describes the application of CGE with commercial cross-linked polyacrylamide gel columns to the resolution of the oligomer distributions of ionic and nonionic ethoxylated surfactants and PEGs. Methodology is discussed for rendering the PEG oligomers both ionic and detectable by UV absorption and for converting nonionic surfactants into ionic species. EXPERIMENTAL SECTION Capillary Electrophoresis System. All CGE experiments were performed on a Dionex CES-1 capillary electrophoresis system. The gel-filled columns were obtained from J&W Scientific under the trade name µ-PAGE. µ-PAGE-3 and µ-PAGE-5 columns with and without urea and having inner diameters of 75 µm were employed for this study. Gel columns without urea are a special order item from J&W Scientific but are no more expensive than the standard urea-containing columns. µ-PAGE-3 columns contained a gel with 3% T/3% C composition, while the µ-PAGE-5 columns contained a gel of 5% T/5% C composition. Column lengths of 45 or 50 cm were employed throughout this study. µ-PAGE buffer (Tris-borate pH 8.3, with or without 7 M urea) obtained from J&W Scientific was employed as the operating buffer. The columns were conditioned by operating at -100 V/cm for 5 min, followed by ramping the field strength to -250 V/cm over 30 min and holding at -250 V/cm for 5 min. Injections of samples were performed electrokinetically at either -5000 or -10 000 V and for up to 90 s. Detection was performed by oncolumn UV absorbance at 275 or 280 nm. The distance from the center of the detection window to the grounded end of the capillary was 4.9 cm. Data acquisition and analysis was performed with the Beckman CALS system using an acquisition rate of either 7.5 or 15 points/s and no averaging. Derivatization Procedure. Polyethylene glycol (PEG) and nonionic ethoxylated surfactant samples were obtained locally. Derivatization was performed with a solution of phthalic anhydride and imidazole in pyridine. The derivatizing solution was prepared by weighing 14.8 g of phthalic anhydride (Aldrich) into a 100 mL (19) Cohen, A. S.; Najarian, D. R.; Paulus, A.; Guttman, A.; Smith, J. A.; Karger, B. L. Proc. Natl. Acad. Sci. U.S.A. 1988, 85, 9660. (20) Karger, B. L.; Cohen, A.; Guttman, A. J. Chromatogr. 1989, 492, 585. (21) Liu, J.; Shirota, O.; Novotny, M. V. Anal. Chem. 1992, 64, 973. (22) Guttman, A.; Cohen, A. S.; Heiger, D. N.; Karger, B. L. Anal. Chem. 1990, 62, 137. (23) Lausch, R.; Scheper, T.; Reif O.-W.; Schlosser, J.; Fleischer, J.; Freitag, R. J. Chromatogr. 1993, 654, 190.

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Table 1. Description of Ionic and Nonionic Ethoxylated Surfactants Studied

surfactant designationa

n

R

AP3S AP7P AP40P AP30

∼3 ∼7 ∼40 ∼30

OSO3-Na+ OPO3H2 OPO3H2 H

a AP, alkylphenol; S, sulfate; P, phosphate. R′, alkyl side chain (same for all surfactants listed above).

volumetric flask and diluting to the mark with pyridine (Sigma). To a second 100 mL volumetric flask was added 2.05 g of imidazole (Aldrich), followed by dilution to the mark with the previously prepared phthalic anhydride/pyridine solution. The resultant derivatizing solution had a composition of 1 M phthalic anhydride and 30 mM imidazole in pyridine solvent. Samples of PEG were derivatized by weighing about 0.1 g of PEG into a Waters 4 mL vial and adding 1 mL of phthalic anhydride derivatizing solution. (Less than 0.1 g sample size was necessary for PEGs lower in molecular weight than PEG 1000). The mixture was then heated in an aluminum heating block at 100 °C for 60 min to form the phthalate derivatives. The sample was removed from the heating block and allowed to cool. To quench the reaction mixture and convert unreacted phthalic anhydride to phthalic acid, 2 mL of deionized water was added to the sample mixture, followed by heating in an aluminum heating block to 50 °C for 30 min. The final dilution for CGE analysis was prepared by transferring 500 µL of the reaction mixture into a clean 4 mL vial, along with 2 mL of deionized water. For a 0.1 g sample size, the final concentration of the phthalate derivative was about 7000 µg/mL. RESULTS AND DISCUSSION I. Separation of Ionic Ethoxylates. Our first investigations into the use of CGE as a characterization tool for ethoxylated surfactants concentrated on anionic surfactants based on the alkylphenol hydrophobe. This was done because such surfactants contain an easily ionized functionality, and the alkylphenol provides a good chromophore for detection. Table 1 describes the structures of the ionic surfactants discussed in this paper. AP3S is a 3 mol ethylene oxide derivative of alkylphenol that has been sulfated. Anionic ethoxylated surfactants can be easily separated into nonionic and ionic species by capillary zone electrophoresis, as is shown in Figure 1a. In addition, for low molecular weight anionic ethoxylated surfactants such as AP3S, the distribution of anionic oligomers can be easily separated. The first peak in the electropherogram for AP3S represents the nonionic species, primarily unsulfated alkylphenol ethoxylate oligomers. Since the nonionic oligomers have no electrophoretic mobility, they are not separated but rather elute as a single peak. The peaks eluting later in the electropherogram represent the sulfated oligomer distribution. The most intense peak has been identified as the 2 mol ethylene oxide adduct, which indicates a distribution of oligomers ranging from 1 to 8 mol of ethylene oxide. This identification has been verified by comparison with a sulfated 1.5 mol ethylene oxide adduct of alkylphenol, which provided a distribution of oligomers known to start at 1 mol of

a

b

Figure 1. (a) CZE separation of AP3S. Capillary, 75 µm × 80 cm, fused silica; buffer, 6 mM Na2B4O7/10 mM NaH2PO4/20% acetonitrile/ 30% methanol at pH 7; applied potential, 30 kV; injection, hydrodynamic, 20 s at 50 mm; detection, 206 nm; sample concentration, ∼1500 ppm in operating buffer. (b) CGE separation of AP3S. Column, µ-PAGE-3 with 7 M urea, 75 µm i.d. × 50.2 cm; buffer, µ-PAGE with 7M urea; injection, electrokinetic at -5 000 V for 5 s; field strength, -249 V/cm; detection, 230 nm; sample concentration, 1930 µg/mL in water.

ethylene oxide (data not shown). Because the sulfated species are anionic and their electrophoretic mobilities oppose electroosmosis, the elution order for the oligomers is from high molecular weight to low molecular weight. The separation of the components of AP3S is achieved easily by CZE under a variety of conditions. Zweigenbaum18 reported oligomeric resolution of a similar sulfated ethoxylate surfactant by CZE under slightly different conditions, but with very similar results. CZE provides a great deal of information, including the anionic oligomer distribution as well as quantitative data concerning the concentrations of ionic and nonionic alkylphenol ethoxylates in the product. The first CGE data obtained in our lab for ethoxylated surfactants were for AP3S. Figure 1b shows the separation of AP3S by CGE with a 3% T/3% C column containing urea. Urea is typically present in gel columns as a denaturing agent for the separation of proteins, but it is not necessary for surfactants analysis. After our initial work with columns containing urea, J&W Scientific indicated that gel columns without urea could be obtained on special order. The 3% gel column produced an excellent separation of about eight sulfated oligomers and provided a separation efficiency of ∼200 000-250 000 theoretical plates. Based on the separation obtained for AP3S by CZE (Figure 1), the oligomers in Figure 1b have been identified as ranging from 1 to 8 mol of ethylene oxide. In contrast with CZE separation, the sieving mechanism of

Figure 2. CGE separation of AP7P. Column, µ-PAGE-3 with 7 M urea, 75 µm i.d. × 50.2 cm; buffer, µ-PAGE with 7 M urea; injection, electrokinetic at -5000 V for 5 s; field strength, -249 V/cm; detection, 230 nm; sample concentration, 1940 µg/mL in water.

separation present in CGE causes the lower molecular weight oligomers to elute first, followed by increasingly larger species. CGE provides an excellent high-resolution separation of the ionic oligomers of AP3S; however, for this relatively low molecular weight surfactant, CZE can provide similar resolution and efficiency in less time. Additionally, CZE allows resolution and detection of a single peak for the unreacted, nonionic alkylphenol ethoxylate starting material. Thus, for AP3S, CZE has the potential to provide more information than CGE and with similarly high-resolution. Figure 2 shows a separation of the ionic components of AP7P obtained on a 3% C/3% T column containing urea. CGE separated and detected about 15 ionic oligomers in this sample. As in the case of AP3S, resolution of the oligomers was excellent, and efficiencies ranged from about 200 000-250 000 theoretical plates. The oligomers have not been positively identified with respect to the number of moles of ethylene oxide contained, but the distribution probably starts with the 1 mol product and ranges to the 15 mol adduct. Thus far, CGE data have been shown for two ethoxylated surfactants based on 3-7 mol of ethylene oxide; however, for these relatively low molecular weight surfactants, CZE provides a somewhat more attractive alternative. In addition to providing baseline resolution of the ionic oligomers, CZE can also provide quantitative data concerning the levels of nonionic and ionic species present, which can be useful for characterizing these materials. In contrast, CGE separation of these low molecular weight surfactants provides excellent separation of the anionic oligomers but cannot yield any information concerning the nonionic content of the product. Thus, for lower molecular weight anionic ethoxylated surfactants, CZE is generally preferred over the more expensive and more time consuming CGE technique. However, as the molecular weight of the surfactants is increased, CGE becomes necessary to provide resolution of the oligomers. AP40P is a phosphated alkylphenol ethoxylate containing 40 mol of ethylene oxide. Compared to the other surfactants described in this report, AP40P has a significantly higher molecular weight. As is shown in Figure 3a, the AP40P anionic oligomers are unresolved by CZE. Most of the oligomers elute as a large, unresolved envelope, with only a few of the lower molecular weight oligomers being resolved at later migration times. Although CZE under the conditions noted cannot easily Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

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a

b

Figure 4. Derivatization of nonionic ethoxylates with phthalic anhydride.

Figure 3. (a) CZE separation of AP40P. Capillary, 75 µm × 80 cm fused silica; buffer, 6 mM Na2B4O7/10 mM NaH2PO4/20% CH3CN/ 30% methanol, pH 7; injection, gravity, 25 mm at 25 s; applied potential, 30 kV; detection, UV at 206 nm. (b) CGE separation of AP40P. Column, µ-PAGE-3 without urea, 75 µm i.d. × 50.1 cm; buffer, µ-PAGE without urea; injection, electrokinetic at -5000 V for 5 s; field strength, -220 V/cm; detection, 230 nm; sample concentration, 20 730 µg/mL in water.

resolve the anionic oligomer distribution, it can still provide useful information concerning the nonionic/ionic levels. The failure of CZE to easily resolve the anionic oligomer distribution of AP40P provided the impetus for investigating CGE for such separations. The power of CGE for separation of anionic surfactant oligomers is illustrated in Figure 3b, which shows a separation of AP40P by CGE on a 3% T/3% C gel column. CGE was successful in baseline resolving and detecting more than 54 oligomers of AP40P. If we assume that the most intense peak in the electropherogram of AP40P corresponds to the 40 mol ethylene oxide adduct, then the CGE separation resolved oligomers with up to 58 mol of ethylene oxide. The price for the high-resolution of CGE is time, with about 75 min required for this separation of AP40P under the conditions noted. Shorter columns of different gel compositions might allow similar resolution on a faster time scale, but this has not yet been attempted due to the minimum column length usable with the Dionex instrument (∼45 cm). Also, higher field strengths have the potential of shortening analysis time; however, we have observed significantly decreased column lifetime when operating above the manufacturer’s recommended limit of 250 V/cm. Field strengths of 300 V/cm or greater are commonplace in the literature, but information concerning column lifetime at such field strengths has not generally been provided. 2544 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

II. Separation of Nonionic Ethoxylates. The feasibility of CGE for resolving ionic ethoxylate oligomers has been demonstrated by the preceding surfactant separations. However, many ethoxylates, including surfactants, poly(ethylene glycol)s, and others, are not ionic but could benefit greatly from the power of CGE. To perform CGE on nonionic ethoxylates, the molecules must first be rendered ionic so that they can be migrated in a potential field. For PEGs, it is also important to provide a chromophore to allow detection. We have found that derivatization of nonionic ethoxylates with phthalic anhydride16,24,25 can provide both charge and chromophore for these products. The derivatization of nonionic ethoxylates with phthalic anhydride is shown schematically in Figure 4 and is described in the experimental section. CGE Separation of Phthalate-Derivatized Nonionic Surfactant Oligomers. Nonionic ethoxylated surfactants based on alkylphenol contain a good chromophore for detection but do require that charge be placed on them in order for migration in a potential field to be possible. An example of the data that can be obtained for nonionic surfactants such as AP30 (see Table 1), a 30 mol alkylphenol ethoxylate, is shown in Figure 5. The large peak eluting at ∼7-11 min corresponds to phthalic acid, which is a result of hydrolyzing the unreacted phthalic anhydride. The phthalic acid peak will be present in the electropherograms of any products derivatized with phthalic anhydride. The oligomers of AP30 were well resolved using a 3% T/3% C gel column. We have applied this methodology to 40 and 70 mol ethylene oxide surfactants with good results, although the higher molecular weight surfactants suffer from low sensitivity and lengthy analysis times (data not shown). CGE Separation of Phthalate-Derivatized PEG Oligomers. Derivatization of PEGs with phthalic anhydride provides two ionizable groups, and thus two charges on each oligomer. This increased charge allows for larger ethylene oxide species to be (24) Standard Test Methods for Chemical Analysis of Alcohol Ethoxylates and Alkylphenol Ethoxylates; American Society for Testing and Materials: Philadelphia, PA; Methods D4252-D4289. (25) Wellons, S. L.; Carey, M. A.; Elder, D. K. Anal. Chem. 1980, 52, 1374.

a

Figure 5. CGE separation of OP30 nonionic surfactant. Column, µ-PAGE-5 with no urea, 75 µm × 50 cm; buffer, Tris-borate at pH 8.3; injection, -5000 V at 15 s; potential field, -300 V/cm; detection, 275 nm.

b

a

b

Figure 6. (a) Electropherogram of PEG 600 on a 3% T/3% C column. Column, µ-PAGE-3 with no urea, 75 µm × 45 cm; buffer, Tris-borate at pH 8.3; injection, -10 000 V at 60 s; potential field, -243 V/cm; detection, 275 nm; concentration, ∼1300 µg/mL. (b) Electropherogram of PEG 600 spiked with triethylene glycol.

migrated through and resolved by the gel. Figure 6a shows an electropherogram of phthalate-derivatized PEG 600 (the number refers to the approximate number average molecular weight) obtained with a 3% T/3% C gel-filled capillary column. This electropherogram exhibits baseline resolution of 23 PEG oligomers. The efficiency of the gel-filled column is very good, with the oligomer peaks exhibiting from 100 000 to 300 000 theoretical plates. Figure 6b is an electropherogram of the same PEG 600 sample spiked with a small amount of triethylene glycol in an attempt to identify the oligomers. It can be seen in Figure 6b that the third

Figure 7. Electropherogram of two samples of PEG 1000 as phthalate derivatives. Column, µ-PAGE-3 with no urea, 75 µm × 45 cm; buffer, Tris-borate at pH 8.3; injection, -10 000 V at 60 s; potential field, -243 V/cm; detection, 275 nm.

peak eluting after phthalic acid corresponds to triethylene glycol. This suggests that the first peak after phthalic acid corresponds to ethylene glycol, and the second is diethylene glycol. Thus, it appears that this PEG 600 sample contains oligomers ranging from 1 ethylene oxide group (ethylene glycol, MW ) 62) up to 23 ethylene oxide groups (MW ) 1031). Figure 7 shows electropherograms obtained for two different sources of PEG 1000 when separated by CGE as phthalate derivatives. These data illustrate the utility of CGE for discerning differences between different sources of product on a molecular composition level. The electropherograms indicate significant differences in the molecular weight distribution for the two sources of PEG 1000. Source A (Figure 7a) is relatively clean in the low molecular weight region, while the electropherogram for source B shows the presence of considerable low molecular weight oligomers (Figure 7b). Source A showed the presence of oligomers ranging from 1 to about 40 ethylene oxide units, while source B showed the presence of oligomers from 1 to about 35 units. These differences in composition, which are easily detected and quantified by CGE, can have a dramatic effect on the performance of PEG 1000 in some applications. Figure 7b illustrates the previously mentioned problem concerning the need to have a large molar excess of phthalic anhydride to force the reaction to tag both ends of the PEG oligomers. Notice the distribution of small peaks eluting after the main PEG oligomer distribution. It is thought that this distribution corresponds to singly tagged PEG/phthalate derivaAnalytical Chemistry, Vol. 68, No. 15, August 1, 1996

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a

b

Figure 8. Electropherogram of PEG 3350 (a) and PEG 4600 (b) as phthalate derivatives. Column, µ-PAGE-3 with no urea, 75 µm × 50 cm; buffer, Tris-borate at pH 8.3; injection, -5000 V at 60 s; potential field, -300 V/cm; detection, 275 nm.

tives. Since such species would have only one ionizable group, their electrophoretic mobilities would be roughly half those of the doubly tagged oligomers, and they would therefore elute later than the doubly tagged species. Calculation based on the amount of sample derivatized indicates that there was a ∼5× molar excess of phthalic anhydride relative to the hydroxyl end group concentration for the experiment shown in Figure 7b. A 10× molar excess of phthalic anhydride is preferred and generally eliminates single end tagging. The mobility of the singly tagged PEG oligomers was low enough that some of these species were observed eluting at the beginning of the next electropherogram (Figure 7a). Figure 8 shows electropherograms obtained for higher molecular weight PEG samples, including PEG 3350 (Figure 8a) and PEG 4600 (Figure 8b), and illustrates the ability of CGE to separate these higher molecular weight phthalate derivatives. Separation efficiencies produced ranged from 200 000 to 400 000 theoretical plates. Signal-to-noise ratio (S/N) is lacking for both PEGs shown in Figure 8. Increased S/N could be obtained by injecting more sample (i.e., longer time) or by decreasing the sample dilution; however, a point will be reached at which overloading severely diminishes the resolution obtained. Sensitivity for the phthalate/PEG derivatives could be improved by using a lower UV wavelength; however, 230-240 nm appears to be the useful UV cutoff for polyacrylamide gel columns, mainly because the gel absorbs appreciably at lower wavelengths. Baseline 2546 Analytical Chemistry, Vol. 68, No. 15, August 1, 1996

Figure 9. Electropherogram of a mixture of PEG 1000, 3350, and 4600 as phthalate derivatives. Column, µ-PAGE-3 with no urea, 75 µm × 50 cm; buffer, Tris-borate at pH 8.3; injection, -10 000 V at 90 s; potential field, 300 V/cm; detection, 275 nm.

stability at 230 nm has been problematic, probably resulting from gel decomposition. Several research groups have begun to produce more UV-transparent gels for CGE that allow the use of more energetic detection wavelengths26 and thus provides increased sensitivity. PEG 4600 represents the highest molecular weight PEG that we have been able to resolve thus far. Even for PEG 4600, the analysis time at 300 V/cm field strength was about 100 min, which is rather lengthy by today’s standards for separations. Field strengths above 250 V/cm are not recommended by the manufacturer for routine use, as the gel columns tend to degrade rapidly at such high field strengths. Performing the separation of PEG 4600 at a more suitable 250 V/cm would require an analysis time of ∼120 min. To further illustrate the power of CGE for separating PEG oligomers, Figure 9 shows an electropherogram obtained for a blend of PEG 1000, 3350, and 4600. Over 120 oligomers were detected in this separation, which illustrates the range over which PEG oligomers can be baseline resolved by CGE. Barka and Hoffman successfully resolved PEG oligomers from 1 to 110 ethylene oxide units using HPLC with UV detection at 190 nm in roughly the same time frame as the CGE experiments presented in this paper. Both HPLC with low UV detection and CGE of phthalate derivatives suffer from decreasing sensitivity with increasing molecular weight. Bullock16 described CZE separation of PEG oligomers that were derivatized with phthalic anhydride. Excellent resolution of low molecular weight PEG oligomers was demonstrated; however, CZE could not baseline resolve the higher (>60 EO units) oligomers. This observation parallels our experiences with ionic surfactantsslow molecular weight species are easily resolved by CZE, but higher molecular weight oligomers (i.e., >15 mol of ethylene oxide) are difficult to resolve. CGE extends the molecular weight range over which ionic surfactant and PEG oligomers can be resolved. Although separation efficiency for CZE separations of PEG oligomers was significantly lower than that obtained by CGE, CZE is certainly less expensive than CGE and can provide good results for low molecular weight PEGs. Since no UVabsorbing gel is employed in CZE, lower detection wavelengths can be employed, thereby producing greater detection sensitivity (26) Hjerten, S.; Srichaiyo, T.; Palm, A. Biomed. Chromatogr. 1994, 8, 73.

than has been achievable in CGE. As was the case for ionic surfactants, CZE with the electrolyte systems developed by Bullock is adequate for resolving low molecular weight PEGS (