Anal. Chem. 1998, 70, 4353-4360
One- and Two-Dimensional Chromatographic Analysis of Alcohol Ethoxylates Robert E. Murphy*
Analytical Research, Rohm and Haas Company, 727 Norristown Road, Spring House, Pennsylvania 19477 and Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085 Mark R. Schure
Theoretical Separation Science Laboratory, Rohm and Haas Company, 727 Norristown Road, Spring House, Pennsylvania 19477 Joe P. Foley
Department of Chemistry, Villanova University, Villanova, Pennsylvania 19085
Two-dimensional liquid chromatography (2DLC) is an increasingly popular technique which has the potential to provide a far more detailed separation and characterization of alcohol ethoxylates (AE) than has been shown by previously utilized separation techniques. The AE are unique in that these molecules have distributions in both alkyl and ethylene oxide chain lengths. In this paper, we compare the single-column techniques of open-tubular SFC, normal- and reversed-phase HPLC, and the multiple column technique of 2DLC in terms of the efficacy of separation and characterization of the alkyl and ethoxylate distributions in a select group of AE. The combination of normal- and reversed-phase HPLC in a 2DLC system accomplishes the simultaneous alkyl and ethylene oxide distribution analysis. The advantage of using 2DLC over one-dimensional chromatographic techniques is clearly demonstrated in the increased selectivity resulting in the ability to produce the ethylene oxide distributions of each alkyl component in an AE. In addition, 2DLC chromatograms are easier to interpret due to ordering of the chromatograms. Alcohol ethoxylates (AE) are nonionic surfactants which are used in many industrial products and processes. The range of applications varies from stationary phases in gas chromatography, to surfactants in detergents and commercial cleaners, to solubilizers in pharmaceuticals, food, and cosmetics.1 Alcohol ethoxylates are the largest group of nonionic surfactants and this is due to their superior biodegradability over alkylphenol ethoxylates.1 Alcohol ethoxylates are prepared by addition of ethylene oxide (EO) to aliphatic alcohols under base-catalyzed conditions. If the alcohol is technical grade, which is a lower cost mixture of alcohols, then the resulting product has a distribution of alkyl end * To whom correspondence should be addressed: Carlsbad Research Center, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008. (1) Karsa, D. R., Ed. Industrial Applications of Surfactants; Whitstable Litho Ltd., Whitstable, Kent, U.K., 1986. S0003-2700(98)00180-2 CCC: $15.00 Published on Web 09/18/1998
© 1998 American Chemical Society
groups. The specific distribution of both EO and alkyl end groups and the ratio of hydrophobe to hydrophile affect the chemical and application properties. Thus, to characterize these materials for performance and quality control purposes, a technique needs to determine both the alkyl and EO distributions. Many analytical techniques are used to characterize AE. A recent review discusses the use of chromatography, titration, atomic absorption, and mass spectrometry for the analysis of AE.2 Chromatography has the advantage of analyzing AE in complex matrixes and in mixtures. Gas chromatography (GC) is predominately used to characterize AE at low degrees of polymerization.2 Higher distribution oligomers cannot elute from the GC column; hence the distribution will be skewed. Size exclusion chromatography (SEC) can analyze high-mass AE but cannot resolve individual oligomers above a mass of 600.2 Capillary electrophoresis has been used to separate the oligomers of poly(ethylene glycol)s, but the samples have to be derivatized to impart a charge and UV chromophore.3 Thin-layer chromatography can be used to separate AE but does not have the peak resolution of supercritical fluid chromatography or gradient elution HPLC.2 Gradient elution HPLC is most commonly used to characterize alcohol ethoxylates by the number of ethylene oxide units or alkyl chain length.2 The ethylene oxide distributions are typically accomplished by normal-phase LC (NPLC) using aminopropyl silica columns and aliphatic hydrocarbon-alcohol-water mobile phases.2 Reversed-phase LC (RPLC) is generally used to separate AE by the alkyl chain length with C18- or C8-bonded silica and methanol-water mobile phases.2 The use of less hydrophobic RPLC columns (i.e., naked silica) with a reversed-phase gradient results in the oligomeric separation of poly(ethylene glycol)s5 and alkylphenol ethoxylates.6 This technique has been called “pseudo (2) Rissler, K. J. Chromatogr., A 1996, 742, 1-54. (3) Bullock, J. J. Chromatogr. 1993, 645, 169-77. (4) Jandera, P.; Urbanek, J.; Prokes, B.; Churacek, J. J. Chromatogr. 1990, 504, 297-318. (5) Rissler, K.; Fuchslueger, U.; Grether, H. J. J. Liq. Chromatogr. 1994, 17, 3109-3132.
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reverse phase”,6 but we prefer normal phase with aqueous solvents2 since retention is in order of increasing number of ethylene oxide units, thus more normal phaselike. With the advent of evaporative light-scattering detection (ELSD),7 AE can be analyzed by gradient elution HPLC without derivatization. The quantitative determination of AE distributions can be accomplished with the ELSD and gradient elution HPLC, excluding the lowmass oligomers since they are volatile.8 HPLC in combination with matrix-assisted laser desorption/ ionization time-of-flight mass spectroscopy (MALDI-TOF MS) is a powerful tool used to identify the alkyl and EO components of an AE.9 In the analysis of polydisperse materials, MALDI-TOF MS suffers in the quantitation of distributions because of mass discrimination10,11 whereby lower mass oligomers are preferentially ionized. Experimental parameters in MALDI-TOF MS such as instrument design10 and the sample preparation and matrix11 can affect the distribution analysis of polymers. Open-tubular supercritical fluid chromatography (OTSFC) is useful for the high-resolution analysis of both the alkyl and ethylene oxide chain lengths simultaneously.12,13 Alcohol ethoxylates made from more than two alcohols result in peak overlap, which could only be resolved with MS detection.12 Two-dimensional liquid chromatographic systems have been used for several years to characterize and separate biomolecules, polymers, and other complex mixtures.14-16 In so-called “comprehensive” two-dimensional liquid chromatography (2DLC), sequential aliquots from the first-dimension effluent are sampled “on-line” by the second-dimension separation system.15 The resulting data is a matrix, usually represented as a contour plot with each chromatographic separation along an axis. This technique is very useful for the higher resolution analysis of multiple fused peaks from the first-dimension column which are resolved in the second dimension when orthogonal separation systems can be found. Two-dimensional liquid chromatography has previously been utilized to separate poly(ethylene glycol)s. Using liquid chromatography under critical conditions (LCCC) in combination with SEC of the collected fractions (in the “off-line” mode), 2DLC has been used for the analysis of poly(ethylene oxide-block-propylene oxide) copolymers17 and alcohol ethoxylates.18-21 In additional (6) Ibrahim, N. M. A.; Wheals, B. B. J. Chromatogr., A 1996, 731, 171-177. (7) Charlesworth, J. M. Anal. Chem. 1978, 50, 1414. (8) Trathnigg, B.; Kollroser, M. J. Chromatogr., A 1997, 768, 223-238. (9) Pasch, H.; Rode, K. J. Chromatogr., A 1995, 699, 21-29. (10) Schriemer, D. C.; Li, L. Anal. Chem. 1997, 69, 4176-4183. (11) Schriemer, D. C.; Li, L. Anal. Chem. 1997, 69, 4169-4175. (12) Pinkston, J. D.; Bowling D. J.; Delaney, T. E. J. Chromatogr. 1989, 474, 97-111. (13) Geissler, P. R. J. Am. Oil. Chem. Soc. 1989, 66, 685-689. (14) Erni, F.; Frei, R. W. J. Chromatogr. 1978, 149, 561-569. (15) Bushey, M. M.; Jorgenson, J. W. Anal. Chem. 1990, 62, 161-167. (16) Kilz, P.; Kruger, R. P.; Much, H.; Schulz, G. Chromatographic Characterization of Polymers: Hyphenated and Multidimensional Techniques, Advances in Chemistry Series 247; American Chemical Society: Washington, DC, 1995; Chapter 17. (17) Pasch, H.; Brinkmann, C.; Much, H.; Just, U. J. Chromatogr. 1992, 623, 315-322. (18) Trathnigg, B.; Thamer, D.; Yan, X.; Maier, B.; Holzbauer, H. R.; Much, H. J. Chromatogr., A 1994, 665, 47-53. (19) Trathnigg, B.; Kollroser, M. Int. J. Polym. Anal. Charact. 1995, 1, 301313. (20) Dohmeier, D. M. 21st International Symposium on High Performance LiquidPhase Separations and Related Techniques Conference Abstracts, Birmingham, U.K., 1997; poster PX-10/B.
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studies,18,19 the separation of AE was facilitated by LCCC and collected fractions were further analyzed to determine the EO mass fraction using SEC. Furthermore, NPLC coupled with RPLC in the comprehensive mode with immiscible mobile phases was recently reported20 for the analysis of phenol ethoxylates and derivatized alcohol ethoxylates. More recently, our laboratory has studied the effect of the sampling rate on two-dimensional resolution in comprehensive 2DLC (RPLC/SEC) for the composition and size analysis of alcohol ethoxylates.21 The use of 2DLC with NPLC and RPLC separation dimensions should increase the selectivity and resolution over one-dimensional LC and 2DLC with RPLC and SEC21 and allow the separation of all the components of a multiple-distribution AE. The use of NPLC instead of SEC in the 2DLC system will result in the separation of individual EO oligomers rather than the single peak observed in the lower resolution size separation. If each dimension of a 2DLC can separate an individual chemical distribution of the AE, then 2DLC has the selectivity to analyze each distribution along an axis independently. This increased selectivity should also make interpretation of AE distributions easier since each oligomer class is aligned along an axis. In this paper, we report on the use of comprehensive 2DLC using NPLC and RPLC as the separation dimensions for separating the ethylene oxide and alkyl distributions of several alcohol ethoxylates and show the advantages over one-dimensional LC and OTSFC. The mobile-phase conditions of the first and second dimension were constant throughout our experiments for comparison of the two-dimensional retention times of the different alcohol ethoxylates examined. The mobile phases were developed for optimum resolution of Neodol 25-12 since it contained the largest number of components. The EO distribution is obtained on a silica column with a water-acetonitrile gradient, whereas the alkyl distribution is accomplished on a C18 column with isocratic methanol-water. The mobile phases used in the two dimensions are miscible, resulting in the injection of the entire first-dimension effluent into the second dimension for increased sensitivity and ease of use. This 2DLC analysis scheme will be shown to allow the complete separation and ordering of the AE distributions for easier interpretation and characterization. EXPERIMENTAL SECTION Chemicals. The solvents (acetonitrile, heptane, methanol, methylene chloride, 2-propanol) were HPLC grade and purchased from J. T. Baker (Phillipsburg, NJ), except for the in-house water, which was purified by a Milli-Q system (Millipore Corp., Milford, MA). The alcohol ethoxylates Neodol 25-12 (Shell Chemical Co., Houston, TX), Neodol 25-7 (Shell), and Novel II 1412-70 ethoxylate (Condea Vista Co., Houston, TX) were analyzed without further purification. Poly(ethylene glycol)-600 (PEG-600) was purchased from Aldrich Chemical Co. (Milwaukee, WI). The number following the PEG abbreviation is the molecular weight and the numbers after the Neodol and Novel names are commercial designations. The exact molecular composition of each component, as provided by the manufacturer, is given in Table 1. The samples for OTSFC analyses were dissolved in methylene chloride at 5% (w/w) concentration. The samples for amino NPLC analyses (21) Murphy R. E.; Schure, M. R.; Foley, J. P. Anal. Chem. 1998, 70, 15851594.
Figure 1. Schematic representation of the 2D HPLC instrument used for this study. Table 1. Alkyl and EO Composition of AE Analyzed
a
AE
alkyl components
av no. of EO
PEG 600 Novel II 1412-70 Neodol 25-7 Neodol 25-12
none C12 and C14 C12, C13, C14, and C15 C12, C13, C14, and C15
14 10a 7 12
Narrow distribution.
were dissolved in 2-propanol at 1% concentration. The samples for RPLC, silica NPLC, and 2DLC (NPLC/RPLC) analyses were dissolved in water at 2% concentration. Chromatographic Equipment. The supercritical fluid chromatograph, model 501, was from Lee Scientific (Salt Lake City, UT) and is equipped with a flame ionization detector operating at 300 °C. A carbon dioxide density gradient was utilized. The density was initially 0.25 g/mL for 5 min and then linearly programmed to 0.50 g/mL at 0.0025 g/mL per min. The oven temperature was initially 100 °C for 5 min and then linearly programmed to 200 °C at 1 °C/min. A timed split injection of 0.05 s was used. The column was a Dionex (Salt Lake City, UT) biphenyl phase, 50 µm i.d. × 10 m with 0.10-µm film thickness and frit restrictor. The one-dimensional RPLC and NPLC experiments were conducted with a Perkin-Elmer (Norwalk, CT) model 410 pump, ISS-100 autosampler, and SEC-4 solvent chamber. The solvents were purged with helium for 15 min before pressurizing. The injection volume was 25 µL. The evaporative light scattering detector (Sedex, model 55, Richard Scientific, Novato, CA) was set at 40 °C, 2.2 bar of house nitrogen, and a gain of 8. Two normal-phase columns were used. The amino NPLC column was a Supelcosil LC-NH2 (Supelco, Bellefonte, PA),
150 × 4.6 mm, 3-µm particles, 100-Å pores, and run at a flow rate of 0.5 mL/min. The solvent gradient for the LC-NH2 column was initially 95/5/0 (heptane-2-propanol-water) and then programmed with a concave gradient (-3 gradient on 410 pump) to 66.5/32/1.5 in 100 min. The silica NPLC column was a Zorbax SIL (MAC-MOD Analytical, Chadds Ford, PA), 150 × 3 mm, 3-µm particles, 70-Å pores, and run at a flow rate of 0.2 mL/min. The solvent gradient for the silica column was initially 90/10 (wateracetonitrile) then programmed with a concave gradient to 20/80 in 100 min. The column used for one-dimensional RPLC analysis was a Zorbax SB18, 150 × 3.0 mm, 5-µm particles, 80-Å pores, and run at a flow rate of 1 mL/min. The solvent gradient for the SB18 column was initially 50/50 (methanol-water), then linearly programmed to 100/0 in 30 min, and held for 10 min. The equipment used for the 2DLC experiments utilizes NPLC as the first dimension and RPLC as the second, configured as shown in Figure 1. The 2DLC system used the same equipment in the first dimension as the one-dimensional experiments, with the addition of a DuPont (Wilmington, DE) model 880 pump, and a Valco eight-port valve (model C8W with high-speed switching accessory and dual 50-µL loops) for collecting the first-dimension effluent with subsequent injection into the second dimension. The injection volume onto the first dimension column was 10 µL. The solvent gradient used with the silica NPLC column was initially 100/0 (water-acetonitrile) and then programmed with a concave gradient to 20/80 in 300 min with a flow rate of 0.05 mL/min. The second dimension was a RPLC column from Perkin-Elmer (Pecosphere 3, C18 column, 33 × 4.6 mm, 3-µm particles, 100-Å pores) with a flow rate of 1.5 mL/min. The second dimension was run isocratically at 95/5 (methanol-water). Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
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Figure 2. Open-tubular SFC chromatograms of Novel II 1412-70 (a) and Neodol 25-12 (b). The inset shown in (b) gives an expanded view of the chromatograms from 60 to 70 min.
The sampling time into the second dimension was 1.0 min. The first dimension flow rate was reduced, as compared to the one-dimensional experiments, to 0.05 mL/min. This results in approximately four injections onto the second dimension column per first dimension peak width, which has been shown to give high two-dimensional resolution without sampling-phase artifacts.21 Data Processing. Data were collected from the ELS detector with a Turbochrom data acquisition system (PE Nelson, Cupertino, CA) using an acquisition rate of 4 Hz. The 2D data are collected as one Turbochrom file and processed with a FORTRAN program which produces an IEEE floating point format binary matrix file. The matrix is read by Spyglass Transform (Fortner Research, Savoy, IL) and presented as a contour plot using a bilinear interpolation algorithm for 2D presentation and analysis. RESULTS AND DISCUSSION Supercritical Fluid Chromatographic Analysis. The OTSFC analyses of Novel II 1412-70 and Neodol 25-12 are shown in Figure 2a and b, respectively. The supercritical fluid chromatograms separate the AE by both EO and alkyl distributions simultaneously. The Novel II 1412-70 sample is fully resolved as shown in Figure 2a, resulting in the appearance of two distributions. The C14 end group is the major component in Novel II 141270, which will be further studied in the 2DLC section. Retention 4356 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
of AE in OTSFC with different end groups has been previously shown to be in order of increasing molecular weight;12 thus the C12 end group should elute prior to the C14 end group of a single EO length. The assignments displayed in the inset of Figure 2b, where the chromatograms shown in Figure 2a and b are expanded in the 60-70 min range, show this retention order for the data given in the figure. The C12 and C14 end group assignments of the Novel II 1412-70 peaks were given by a priori knowledge that the C14 end group is present in greater quantity and by comparison to the Neodol 25-12 retention times. The C12 and C14 end groups of each ethylene oxide oligomer are well separated, but the C12 and previous C14 end groups are not as well separated. The Neodol 25-12 sample (Figure 2b) results in a parallel series of peaks but is not fully resolved. There is no distinct appearance of the four distributions present. The alkyl distribution is repeating on every fourth peak; thus, the C12 and C15 are coeluting as assigned in the inset. In one study of AE separations,12 Neodol 45-7 (containing C14 and C15 end groups) was able to be separated out to 20 oligomers with OTSFC. However, in the same study, Neodol 91-6 (containing C9, C10, and C11 end groups) was only able to be separated out to five oligomers before coelution.12 Three alkyl end groups appear to be the limit for the OTSFC resolution of AE for low average numbers (less than five) of EO, but for higher average numbers of EO, the limit is approximately two alkyl end groups. Thus, OTSFC appears to be most useful for resolving AE with two or less alkyl end groups; however, OTSFC does not appear to have the efficiency needed to accommodate more peaks in the separation space. One-Dimensional HPLC Analysis. Of all of the normalphase packings which have been utilized to separate AE, the aminopropyl-bonded stationary phases have been shown to give the best separation of AE.4 The one-dimensional NPLC separations of Novel II 1412-70 and Neodol 25-12 are shown in Figure 3a and b, respectively. The amino NPLC column results in baseline separation of Novel II 1412-70 oligomers and better resolution than OTSFC (Figure 2a). The OTSFC analysis has some peak tailing and could be the cause for the decreased resolution. The inset shown in Figure 3b gives the end group assignments as was done in the OTSFC case, and elution for each EO subunit now occurs with the C14 end group eluting prior to the C12 end group. Hence, there is a reversal of the retention times of the distribution maximums, as compared to the alkyl elution order seen by OTSFC. The origin of this effect may be due to solubility effects of the alkyl chain in the alkyl solvent, some form of steric limitation or size exclusion effect of the analyte near the stationary phase, very subtle polarity effects, or a combination of all of these. The NPLC separation of Neodol 25-12, shown in Figure 3b, results in an alkyl distribution for each EO oligomer. There appears to be an optimum in the resolution around the tenth set of alkyl distributions eluting at ∼50 min. The alkyl distributions eluting after 55 min start to coelute and are indistinguishable. Similar to OTSFC, NPLC can resolve alkyl distributions when the EO number is low to medium; however, NPLC cannot resolve both alkyl and EO distributions of Neodol 25-12 throughout the EO range of interest.
Figure 3. Amino NPLC chromatograms of Novel II 1412-70 (a) and Neodol 25-12 (b). The inset shows an expanded view of the chromatograms from 28 to 38 min.
In an effort to better resolve the EO and alkyl distributions individually, silica NPLC and RPLC were investigated. Panels a and b of Figure 4, respectively, show the results of these onedimensional HPLC analyses of Neodol 25-12. The EO distribution is determined on a silica column using a reversed-phase gradient (Figure 4a). This separation was attempted using other grades of silica (Zorbax Rx-SIL, Supelcosil LC-Si, Micra NPS) resulting in worse resolution, suggesting that the Zorbax SIL has a different treatment or structure that improves the selectivity over the other silicas. Similar results were shown for the analysis of alkylphenol ethoxylates which could only be separated on Spherisorb silica using acetonitrile and water.6 A Partisil silica column was used to separated dibenzo-crown ethers with a water-to-methanol gradient in order of crown size or number of EO. It has been previously shown5 using a Spherisorb silica column that waterto-acetonitrile gradients give better resolution over water-tomethanol gradients for the analysis of poly(ethylene glycol)s. It was proposed5 that methanol shields residual silanol groups better than acetonitrile; thus the ethylene oxide can interact more strongly with the surface when acetonitrile gradients are used. The separation of only the alkyl distribution is accomplished using an octadecyl-bonded silica column and reversed-phase gradient, as shown in Figure 4b. Thus, by using two onedimensional techniques, the bulk alkyl and bulk EO distributions can be determined independently when the appropriate columns and solvent systems are used. This multitechnique method is commonly used to characterize materials by one particular
Figure 4. One-dimensional HPLC chromatograms of Neodol 2512: silica NPLC column (a) and C18 RPLC column (b).
distribution at a time and takes the analyst twice the amount of time due to preparing samples, solvents, and equipment twice. Moreover, the results are sometimes inconclusive due to poor resolution from overlapping distributions. As will be shown in the next section, much more information can be obtained if we perform this type of analysis using a comprehensive twodimensional system rather determining these distributions separately. Two-Dimensional HPLC Analysis. The analysis of Neodol 25-12 resulted in a EO distribution of ∼20 peaks by NPLC (Figure 4a) and an alkyl distribution of 4 peaks by RPLC (Figure 4b). The resolution in the RPLC separation is high, and the analysis time can be decreased by using a shorter column with a smaller particle size and a higher percentage of organic modifier (as described in the Experimental Section). The choice of RPLC as the second dimension in 2DLC was due to the fact that the alkyl distribution analysis can be performed in 1 min using isocratic conditions. To maintain the EO distribution resolution, the NPLC column had to be run using a gradient, which made it more difficult for use as a second dimension. As described in a previous publication,21 2DLC method development is usually done on the second dimension first and then the first dimension is set to accommodate the number of samples per peak and injection volume into the second dimension. Consequently, in this work, the 2DLC method utilized NPLC as the first dimension and RPLC as the second dimension. The first dimension flow rate was reduced to 50 µL/min to accommodate a 50-µL injection into the second dimension every minute. Thus, the first dimension was Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
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Figure 5. Two-dimensional HPLC (NPLC/RPLC) chromatogram of Novel II 1412-70 with the corresponding chemical structure and average EO as supplied by the manufacturer.
not run at optimum flow conditions, but the analysis of Neodol 25-12 on the silica column at 0.5, 0.2, and 0.05 mL/min gave very similar results (not shown). The two-dimensional HPLC analysis of AE can be used to determine the alkyl and EO distribution simultaneously. In 2DLC, the axes correspond to retention along each chromatographic column. The data are displayed as a contour for easier viewing. The ELSD intensity is represented by different colors. The colors range from white, yellow, orange, blue, and then black which increase in intensity from lowest to highest, respectively. Use of RPLC as the second dimension allows for each peak separated in the first dimension, by their polarity, to be further separated by their hydrophobicity. This results in the EO distribution along the NPLC axis and the alkyl distribution along the RPLC axis because the EO distribution does not appear to be significantly separated by the RPLC column and the alkyl distribution does not appear to be significantly separated by the NPLC column. Figure 5 shows the 2DLC chromatogram of Novel II 1412-70. Two distributions with different alkyl end groups are evident along the RPLC axis. The C14 end group, eluting at 0.75 min in the RPLC axis, is the major component since it is represented as a darker color in the contour plot. From these data, the EO distribution with each alkyl end group is similar and ranges from 125 to 160 min, with a center of the distribution at ∼140 min. The low-intensity peaks that appear at the beginning of each RPLC dimension are due to system peaks. In addition, these peaks appear in 2DLC runs when peak amplitudes are comparable to the analytical peak heights. Shown in Figure 6 is the 2DLC analysis of Neodol 25-12. Four distributions with different alkyl end groups are clearly seen. The EO distributions have a similar center of distribution, but the C15 end group (eluting at 0.90 min in the RPLC dimension) appears to have a narrower EO distribution. This is due to using isocratic RPLC in the second dimension which results in broader peaks of lower intensity for later-eluting components. Thus, the C15 end group has a lower ELSD intensity relative to the other end groups. The EO distributions are very similar and will be shown in a later section for comparison. The EO distributions range from 120 to 4358 Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
Figure 6. Two-dimensional HPLC (NPLC/RPLC) chromatogram of Neodol 25-12 with the corresponding chemical structure and average EO as supplied by the manufacturer.
Figure 7. Two-dimensional HPLC (NPLC/RPLC) chromatogram of Neodol 25-7 with the corresponding chemical structure and average EO as supplied by the manufacturer.
180 min in the NPLC dimension, with a center of distribution at ∼150 min. This EO distribution elutes later and is much broader than that of the Novel II 1412-70 distribution. This result is expected since Neodol 25-12 has a larger and broader EO distribution than Novel II 1412-70. The narrower distribution of Novel II 1412-70 is expected since the manufacturer sells this product specifically for the narrower distribution. The slant in the 2D chromatograms was attributed to the first-dimension concave gradient. Figure 7 shows a 2DLC chromatogram of Neodol 25-7. The early-eluting EO oligomers of Neodol 25-7 are not as well resolved as the later oligomers, since the first-dimension gradient was optimized for Neodol 25-12 oligomers. The alkyl distribution is similar to Neodol 25-12. The EO distribution ranges from 112 to 160 min in the NPLC dimension, with the center of the distribution at ∼135 min. Figure 8 is a 2DLC chromatogram of PEG-600. This sample was added to show where an AE without an alkyl end group elutes.
Figure 8. Two-dimensional HPLC (NPLC/RPLC) chromatogram of PEG-600 with the corresponding chemical structure and average EO as supplied by the manufacturer.
Poly(ethylene glycol)s are commonly analyzed as an impurity in AE; thus, this type of molecule needs to be resolved from the main component. It is interesting to note how the EO oligomers of PEG-600 are well separated in this sample, whereas Neodol 25-7 eluting at the same time in the NPLC dimension is not as well resolved. Also, the NPLC retention of PEG-600 ranges from 80 to 170 min, averaging at 125 min, which is much earlier than any of the AE. The PEG-600 has an average EO number of 14, which we would expect to elute after Neodol 25-12 in the NPLC dimension, but it elutes earlier. This result suggests that the first dimension is not only separating by number of ethylene oxide units but also by a mixed mechanism which also takes into account the alkyl chain length. Similar results were seen previously6 with a similar column and gradient. Although poly(ethylene glycol) was not analyzed in this work,6 nonylphenol ethoxylates eluted after octylphenol ethoxylates, suggesting an alkyl contribution to retention. Since AE have both a hydrophobic and a hydrophilic section, the alkyl portion may be interacting with the “hydrophobic” siloxane functionality and the ethylene oxide portion may be interacting with the “hydrophilic” silanol functionality of the silica, as postulated in another study22 where the separation of dibenzocrown ethers was reported. After the 2DLC chromatograms are collected, the data may also be presented as EO distributions. Figure 9 is a plot of the EO distributions of the four alkyl components of Neodol 25-12. The EO distributions were constructed by averaging the NPLC runs across the listed RPLC times on the chromatograms. This average is constructed by summing the intensity values across the columns of the data matrix and then dividing by the number of columns summed. Individual NPLC runs may be extracted from the 2DLC data but are not as representative as the averaged distribution. The EO distributions of the four components are similar, but resolution decreases with increasing alkyl content of the end group at the (22) Bij, K. E.; Horvath, C.; Melander, W. R.; Nahum, A. J. Chromatogr. 1981, 203, 65-84.
Figure 9. One-dimensional NPLC chromatograms extracted for each alkyl component from 2DLC analysis of Neodol 25-12 in Figure 6. Times listed are the range of chromatograms extracted and then averaged from the 2DLC chromatogram to produce each NPLC chromatogram.
low end of the distribution. The resolution decrease is probably due to alkyl dominance in the retention mechanism at low EO as the silica NPLC column is unable to distinguish between a small number of ethylene oxide units with a large alkyl end group. These distributions were not corrected for the variation in intensity as a function of EO chain length. However, this effect has been studied previously8 and found not to have a significantly large effect for large EO chain lengths detected with ELS detection. CONCLUSIONS The use of 2DLC (NPLC/RPLC) for the analysis of higher alkyl AE is clearly superior to one-dimensional NPLC and OTSFC. This 2DLC system is capable of simultaneously separating AE into alkyl and EO components. The retention times in the NPLC dimension of the AE examined correspond fairly well with the number of ethylene oxide units. The PEG-600 retention in the NPLC dimension does not correlate with the AE, which suggests a mixed mechanism on the silica column with a reversed-phase gradient. Software reconstruction of the EO distributions allows the analysis of each alkyl component independently in a single run. The analysis of each EO distribution of a multiple alkyl AE is the first such study to our knowledge and should facilitate better characterization protocols. One-dimensional normal-phase HPLC may provide sufficient resolution for less complicated AE (i.e., Novel II 1412-70 and Brij 35), but 2DLC offers the selectivity to display the EO distribution of each end group independently, which is not easy or unambiguous to extract from one-dimensional data. Two-dimensional HPLC is a powerful technique not only Analytical Chemistry, Vol. 70, No. 20, October 15, 1998
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to separate materials but also as an aid in identification, characterization, analytical trouble shooting, synthesis optimization, and quality control. This technique should improve the analysis of polymers, surfactants, combinatorial libraries, and other industrial and biological samples that are too complicated for one-dimensional techniques alone. Two-dimensional HPLC should be used in situations where one-dimensional separations give inadequate resolution or selectivity or when higher peak capacity is needed. The use of two-dimensional HPLC could also be used in situations where one-dimensional data have sufficient resolution but inter-
pretation of fingerprints would be aided by the use of two retention mechanisms, for example, the analysis of unsaturated triglycerides on RPLC and silver-impregnated silica for the hydrophobicity and functional class (bond order) separation. In our particular application, we do not have an absolute method of calibration because the alkyl chain length influences the retention of the EO oligomers. However, mass spectrometry would be an ideal third dimension. The automated combination of two-dimensional chromatography and spectroscopy23,24 is the next step toward the future of simultaneous separation and identification of very complicated samples.
(23) Opitek, G. J.; Lewis, K. C.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 1518-1524. (24) Opitek, G. J.; Jorgenson, J. W.; Anderegg, R. J. Anal. Chem. 1997, 69, 22832291.
Received for review February 16, 1998. Accepted July 20, 1998.
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AC980180J
