Anal. Chem. 2001, 73, 5116-5125
Characterization of Open Tubular Capillary Electrochromatography Columns for the Analysis of Synthetic Peptides Using Isocratic Conditions M. T. Matyska,† J. J. Pesek,† R. I. Boysen,‡ and M. T. W. Hearn‡
Department of Chemistry, San Jose State University, One Washington Square, San Jose, California 95192-0101, and Australian Centre for Research on Separation Sciences, Centre for Bioprocess Technology, Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria, Australia 3168
In this paper, we report on investigations related to the performance characteristics of two different types of etched chemically (n-octadecyl- and cholesterol-) modified capillaries in the open tubular format of capillary electrochromatography (CEC) for the analysis of synthetic peptides. The results confirm that the nature of the surface chemistry used to modify the capillary wall and type of chemically bonded group employed can affect the selectivity as well as the resolution of peptide samples. The results are consistent with the participation of selective peptide interactions with the bonded phase, although other factors, such as the morphology of the capillary wall surfaces, appear to be also involved. Moreover, several surprising observations related to peptide-specific multizoning effects have been observed. Additional experimental variables that can also be utilized to affect the retention of peptides in this approach to OTCEC include the type and percentage of organic solvent modifier employed in the eluent and the pH of the buffer system. To evaluate the reproducibility of different batches of the n-octadecyland cholesterol-modified capillaries and the stability of the chemically modified surface, the OTCEC selectivity and peak shape behavior of two small basic molecules (serotonin and tryptamine) and two proteins (turkey and chicken lysozyme) were also investigated. Finally, the use of the “bubble” cell technology for creating the detector window has been shown to provide significantly higher detection sensitivity with peptides, as compared with the conventional capillary format. Capillary electrochromatography (CEC) is a rapidly developing method that provides a powerful orthogonal format for the analytical separation of molecules with sizes ranging from small inorganic ions and organic moieties to peptides and proteins.1 CEC is considered to be complementary to other electroseparation techniques, such as capillary electrophoresis (CE), capillary gel electrophoresis (CGE), and capillary micellar electrochromatography (CMEC).2 CEC has features in common with HPLC, except †
San Jose State University. Monash University. (1) Colon, L. A.; Guo, Y.; Fermier, A. Anal. Chem. 1997, 69, 461A. (2) Knox, J. H. J. Chromatogr. A 1994, 680, 3. ‡
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that the transport of the elution buffer and analytes through the capillary occurs via electroosmotic and electrophoretic mobility processes rather than by a pressure-driven flow, as in the case of microbore liquid chromatography. The flat-flow velocity distribution of the electrolyte through the capillary is one of the main advantages of the electrically driven format that is exploited in CEC. As a consequence of this difference in the two flow profiles, peak widths are inherently narrower in CEC than those typically found in microbore HPLC separations. Thus, CEC can be considered a hybrid technique possessing the favorable characteristics of both CE and HPLC, with the separation of solutes occurring via differential electrophoretic mobility, as in capillary electrophoresis, and via solute/bonded-phase interactions, as found in liquid chromatography. A common CEC configuration consists of a fused-silica capillary with an internal diameter of 50-100 µm (typical for CE) and packed with sorbent materials identical or similar to those used in HPLC, that is, a e5 µm ODS-silica.1,3 High efficiencies of 200 000 plates/m or more can be achieved in CEC for neutral compounds driven through the capillary by electroosmosis and separated in terms of their intrinsic chromatographic selectivities. In recent years, development of CEC methods for the analysis of neutral and polar organic analytes has advanced significantly, including reports on the resolution of enantiomers.4 Improvements in instrumentation have also permitted pressure-driven gradient elution to be employed.5 In order for packed CEC to be competitive with CE and HPLC, probably the most challenging problem that must be routinely addressed is the practical necessity to retain the packing material with reproducibly prepared in situ frits while maintaining unrestricted flow through the bed.6-11 Another limitation of packed capillaries is their tendency to form bubbles around the packing (3) Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; Rakestraw, D. J. Anal. Chem. 1995, 67, 2026. (4) Lelievre, F.; Yan, C.; Zare, R. N.; Gareil, P. J. Chromatogr. A 1996, 723, 145. (5) Yan, C.; Dadoo, R.; Zare, R. N.; Rakestraw, D. J.; Anex, D. S. Anal. Chem. 1996, 68, 2726. (6) Rebscher, H.; Pyell, U. Chromatographia 1994, 38, 737. (7) Boughtflower, J.; Underwood, T.; Maddin, D. Chromatographia 1995, 41, 398. (8) Yamamoto, H.; Baumann, J.; Erni, F. J. Chromatogr. 1992, 593, 313. (9) Yan, C.; Schaufelberger, D.; Erni, F. J. Chromatogr. 1994, 670, 15. (10) Rebscher, H.; Pyell, U. J. Chromatogr. 1996, 737, 171. 10.1021/ac010384r CCC: $20.00
© 2001 American Chemical Society Published on Web 09/28/2001
material or at the frit surface, resulting in an unstable baseline, variable migration times, perturbation of the electrical field profile, and increases in electrical resistance that lead to very low current (or stoppage of current) in the system. To prevent bubble formation, a number of options are available. Thus, the eluent can be thoroughly degassed, and pressure can be applied to one or both buffer vials;12,13 either low concentrations of electrolytes or high proportions of organic solvent, or both, can be used in the eluent;14,15 the buffer used can contain zwitterionic species;16 or surfactants can be added to the eluent.17 Packing of CEC capillaries can be more difficult than with microbore HPLC columns because of the narrow inner diameter of the capillary and the use (typically) of smaller diameter particles (e 5 µm). Basic compounds are particularly difficult to analyze by CEC with nonend-capped n-alkylsilica sorbents becaause of the presence of silanol groups, which are necessary to generate an adequate electroosmotic flow in order to move the solutes through the capillary. Various applications of packed CEC systems for the separation of basic compounds have been reported, but in these cases, it has often been found to be necessary to add a competitive base, such as triethylamine,18 triethanolamine18 or hexylamine, to the mobile phase.19 A promising approach to solve some of the problems encountered with packed capillaries involves the use of open tubular CEC (OTCEC). The concept of OTCEC is not new, having been utilized in a number of different formats for almost 20 years. The earliest work involved 30-µm soda-lime capillaries that were treated with NaOH followed by attachment of an n-octadecylsilane coating.20 These capillaries were subsequently used for the separation of hydrocarbon samples. The same coating was used with 10-25µm capillaries for the separation of polycyclic aromatic hydrocarbons (PAHs).21 Polymer coatings are one way to increase the phase ratio in OTCEC and to promote solute/bonded-phase interactions. Thus, polymethacrylate coatings have been employed with 25-µm-i.d. capillaries for the separation of benzoates with efficiencies greater than 250 000 plates/m.22 Capillaries coated with a copolymer of N-tert-butylacrylamide and 2-acylamido-2-methyl1-propane sulfonic acid (AMPS) have also been used to separate various mixtures of PAHs, parabens, and ketones.23 The presence of AMPS in the polymeric coating created a stable electroosmotic flow, because this moiety has a negative charge, which favors typical CEC operating conditions. Another method for increasing (11) Hennessy, T.; Huber, M. I.; Unger, K. K.; Hearn, M. T. W. In Chromatography Protocols for Protein Analysis: A Laboratory Manual, Simpson, R., Ed.; Cold Spring Harbour Laboratory Press: New York, 2001, in press. (12) Choudhary, G.; Horvath, Cs. J. Chromatogr. A 1997, 781, 161. (13) Deng, Y.; Zhang, J.; Tsuda, T.; Yu, P. H.; Boulton, A. A.; Cassidy, R. M. Anal. Chem. 1998, 70, 4586. (14) Knox, J. H.; Grant, I. H. Chromatographia 1991, 32, 317. (15) Seifer, R. M.; Kraak, J. C.; Kok, W. Th.; Poppe, H. J. Chromatogr. A 1996, 775, 165. (16) Boughtflower, R. J.; Underwood, T.; Paterson, C. J. Chromatographia 1995, 40, 329. (17) Seifer, R. M.; Kok, W. Th.; Kraaak, J. C.; Poppe, H. Chromatographia 1997, 46, 131. (18) Gillott, N. C.; Barrett, D. A.; Nicolas-Shaw, P.; Euerby, M. R.; Johnson, C. M. Anal. Commun. 1998, 35, 217. (19) Lurie, I. S.; Conver, T. S.; Ford, V. L. Anal. Chem. 1998, 70, 4563. (20) Tsuda, T.; Nomura, K.; Nakagawa, G. J. Chromatogr. 1982, 248, 241. (21) Bruin, G. J. M.; Tock, P. P. H.; Kraak, J. C.; Poppe, H. J. Chromatogr. 1990, 517, 557. (22) Tan, Z.; Remcho, V. T. Anal. Chem. 1997, 69, 581. (23) Sawada, H.; Jinno, K. Electrophoresis 1999, 20, 24.
the amount of stationary phase in OTCEC is to use sol-gel technology. By including n-octadecyl moieties in the silane reagents, a thin film can be formed on the inner wall of the capillary, enabling mixtures of nonpolar compounds to be separated in the CEC column.24 The approach described in this paper utilizes a technology developed several years ago25 that involves, first, etching the inner wall of the fused capillary and, second, chemical modification of the new surface via silanization/hydrosilation reactions.26 Previous studies have shown that this particular format of OTCEC is well-suited for the separation of basic compounds.26-32 The investigations described in the present paper extend these observations and document a number of additional advantages of OTCEC procedures for the analysis of synthetic peptides. MATERIALS AND METHODS OTCEC Capillaries. Two types of OTCEC capillaries were used in this study: a C18- and a cholesterol-modified surface capillary produced by etching and chemical modification of Agilent Technologies (Waldbronn, Germany) fused-silica capillaries, i.d. ) 20 µm, with a “bubble cell” at the detection window. The process to produce the etched and hydride-modified capillaries, as well as the C18-modified capillaries, was based on methods previously described.26 A number of changes in the manufacturing procedure were made to these methods, including 1. The 20-hour drying procedure that previously used nitrogen gas was substituted by a washing step using acetone with drying of the capillary for 0.5 h under nitrogen; and 2. The solvents used for washing the hydride-modified capillary were changed from tetrahydrofuran (THF)-water followed by THF to dioxane, followed by toluene. Attachment of cholesteryl-10-undecenoate to the hydridemodified capillaries was carried out as described previously.32 Before use, each capillary was conditioned overnight in the presence of methanol then was manually washed via a syringe with Milli-Q water and, finally, several volumes of the running buffer (degassed with helium for 30 min) was forced through the capillary. The compositions of the buffer systems used in the mobile phases were as follows: buffer 1, 60 mM phosphoric acid and 38 mM Tris, pH ) 2.14; buffer 2, 50 mM Tris and 125 mM boric acid, pH ) 7.3; and buffer 3, 20 mM Tris/HCl, pH ) 7.5. Peptide Samples. The synthesis of TRAP-1 and other peptides was achieved according to Fmoc/Boc methods described elsewhere33-38 and characterized by ESI-MS, circular dichroism (24) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511. (25) Pesek, J. J.; Matyska, M. T. J. Chromatogr. A 1996, 736, 255. (26) Pesek, J. J.; Matyska, M. T.; Sandoval, J. E.; Williamsen, E. J. Liq. Chromatogr. 1996, 19, 2843. (27) Pesek, J. J.; Matyska, M. T. J. Chromatog. A 1996, 736, 313. (28) Pesek, J. J.; Matyska, M. T.; Mauskar, L. J. Chromatogr. A 1997, 763, 307. (29) Pesek, J. J.; Matyska, M. T. J. Liq. Chromatogr. Relat. Technol. 1998, 21, 2923. (30) Catabay, A. P.; Sawada, H.; Jinno, K.; Pesek, J. J.; Matyska, M. T. J. Capillary Electrophor. 1998, 5, 89. (31) Pesek, J. J.; Matyska, M. T.; Cho, S. J. Chromatogr. A 1999, 845, 237. (32) Matyska, M. T.; Pesek, J. J.; Katrekar, A. Anal. Chem. 1999, 71, 5508. (33) Jong, A.; Boysen, R. I.; Hearn, M. T. W. Submitted. (34) Munozbarroso, I.; Durell, S.; Sakaguchi, K.; Appella, E.; Blumenthal, R. J. Cell Biol. 1998, 140, 315-323. (35) Boysen, R. I.; Jong, A.; Hearn, M. T. W. Biophys. J., 2001, in press. (36) Fields, G. B.; Noble, R. L. Int. J. Peptide Protein Res. 1990, 35, 161-168. (37) Keah, H. H.; O’Bryan, M.; de Kretser, D. M.; Hearn, M. T. W. J. Peptide Res. 2001, 57, 1-10.
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Q
38
D
37
K
36
L
35
Y
34
R
33
E
32
V
31
A
30
a
I R A Q L Q K I G W V I H Q E
V D H A
V I Q Q
E N H Q
F R Q H
G H L
Q L
G Q
L
T
Amino Acid
P P P P P P C D H I N N N N N A M W Q A R R R R A R L S H R L L L A L L I N Q L L L A L L L T H H L F A F F F F M H K N S S S S S S Q H M NN
The synthetic peptides contained free amino and carboxyl termini, and cysteine residues were protected by the acetamidomethyl (acm) group
L
29 28 27 26 25 24 23 22 21 20 19
residue number
18 17 15 15 14 13 12 11 10 9 8 7 6 5 4 3 2
Sequencea
Peptide TRAP-1 TRAP-2 TRAP-3 TRAP-4 TRAP-5 TRAP-6 BF12 HT1 HT8 TMD38
(38) Boysen, R. I.; Hearn, M. T. W. J. Peptide Res. 2001, 57, 19-28.
1
RESULTS AND DISCUSSION Validation of the Stability and Reproducibility of the C18and Cholesterol-Modified OTCEC Capillaries. An important factor in the use of etched chemically modified capillaries with biologically related samples is the stability and reproducibility of the capillary surface. To evaluate the etched n-octadecyl capillaries used in the present investigations, four batches prepared over a period of two years were tested. Each of these batches was taken through the same manufacturing process, that is, the same etching, silanization, and hydrosilation methods were employed. Although evaluation of reproducibility is based on testing of the final surface modified capillaries, the results nevertheless reflect any variation(s) that may have occurred during any of the steps in their fabrication. Besides the results discussed below for the peptides, two other types of samples were used for this study: (i) a mixture of two small basic compounds, serotonin and tryptamine, and (ii) a mixture of two basic proteins, chicken and turkey lysozyme. The results with these latter two sets of analytes in terms of selectivity and peak efficiency obtained with these different batches of modified capillaries are shown in Table 2. All measurements of reproducibility have small variation coefficients among the four batches of surface-modified capillaries, indicating that their fabrication is controllable within acceptable limits. The RSD for the selectivity of the test samples was ∼0.5%. The high efficiency and symmetry of the peaks for these test analytes suggest that coverage of the etched surface provided by the silanization/hydrosilation process results in a reproducible surface having minimal undesirable adsorption effects. Previous studies have shown that achievement of separation factors with these basic analytes on the etched n-octadecyl-modified capillaries used in
Table 1. Peptide Codes and Amino Acid Sequences of the Various Synthetic Peptides Employed in This Investigationa
(CD), HPCZE ,and analytical RP-HPLC procedures. The sequences of these synthetic peptides are shown in Table 1. The synthetic peptides were purified by semipreparative RP-HPLC on a C18 column (Tosoh TSK ODS-120T column (300- × 21.5-mm i.d) packed with 10-µm particles) using a linear gradient from buffer A (0.1% TFA in water) to 75% buffer B (0.09% TFA in 60% (v/v) acetonitrile/water) over 90 min. The analytical RP-HPLC of the peptides was carried out on a C18 column (TSK C-18 columns (150 × 4.6 mm; particle size, 5 µm) with the same mobile-phase compositions but utilizing a gradient time of 60 min. Electrospray ionization mass spectroscopy was used to confirm peptide identity in terms of molecular mass and amino acid sequence by employing a Platform LC mass spectrometer from Micromass, Manchester, U.K., using the Masslink software. The scan range was set between 100 and 2500 amu, and samples were injected via a manual injector at a rate of 10 µL/min. Serotonin, tryptamine, and turkey and chicken lysozyme were obtained from Sigma-Aldrich (Milwaukee, WI). Instrumentation. The CEC studies related to the separations of the peptides were carried out using a Beckman P/ACE 5510 series capillary electrophoresis instrument (Beckman, Fullerton, CA) equipped with a UV detector. The system was interfaced with a Pentium computer utilizing the System GOLD software (version 8.1) for instrument control and data collection. The stability and reproducibility studies were carried out on an Applied Biosystems (Foster City, CA) model 270 HT capillary electrophoresis system.
Table 2. Reproducibility Data for Four Batches of Etched n-Octadecyl-Modified Capillariesa batch
Rserotonin/tryptamine
plates/m (serotonin)
Rlysozymes (chicken/turkey)
1 2 3 4
1.06 1.06 1.07 1.07
141 000 140 000 139 000 142 000
1.09 1.10 1.10 1.10
a
Buffer 1: 60 mM phosphoric acid and 38 mM Tris, pH 2.14.
Figure 1. Separation of tryptamine and serotonin on two etched cholesterol-modified capillaries: (A) batch no. 1, and (B) batch no. 2. Conditions: capillary i.d., 50 µm; L, 50 cm; length to detector window, l, 25 cm; V, 25 kV; electrolyte, buffer 1 (60 mM phosphoric acid and 38 mM Tris), pH 2.14; current, i, 18 µA.
this study are not possible with unmodified and unetched capillaries.29,31 The stability, as well as the separation reproducibility, of the etched n-octadecyl capillaries was checked by measuring the selectivities and efficiencies for the same analytes after prolonged use, as reported in Table 2. Each of the four surface-modified capillaries was subjected to between 200 and 400 injections, with no statistically measurable change in the three parameters. A more limited evaluation of the etched cholesterolmodified capillaries was also undertaken. Figure 1 shows the separation of tryptamine and serotonin on two etched cholesterolmodified capillaries that came from two different fabrication batches. The R-values were 1.11 and 1.12, respectively. These selectivities, as well as the efficiencies and peak shape, were maintained after more than 200 injections with these cholesterolmodified capillaries, and similar peak symmetry coefficients were obtained. These results are similar to those that were obtained on cholesterol-modified capillaries used for the analysis of proteins and small basic molecules.32 On the basis of these observations, the performance of these OTCEC capillaries was evaluated using different synthetic peptides. Separation of Synthetic Peptides with OTCEC Systems. Previous studies with etched, chemically modified OTCEC capillaries have indicated that considerable potential exists for the analyses of biomolecules and pharmaceuticals. One of the possible disadvantages of the open tubular approach to CEC is the low loadability of the capillary and, hence, lower detection sensitivity in comparison to packed CEC capillaries. Figure 2 illustrates both the usefulness of the OTCEC procedure per se and a means to overcome limitations associated with low detection sensitivity, which is an increasingly important consideration for the analysis
of low-abundance biosamples. Figure 2A shows the separation of a synthetic TRAP-3 peptide sample using an etched n-octadecylmodified 20-µm-i.d. capillary that utilizes the Agilent Technologies “bubble cell” for optical detection. This result can be compared to the electrochromatogram (Figure 2B) obtained using a similar capillary prepared and operated under identical experimental conditions but without a bubble cell in the optical detection path. It is obvious from this comparison that the bubble cell technology offers one solution to overcoming low detection sensitivities that can limit the effectiveness of the open tubular approach. The enhancement obtained in this particular example was almost an order of magnitude in detectability of this low-molecular-weight peptide sample. High selectivity of OTCEC systems with polar analytes separated under isocratic conditions has been noted previously.30-32 Similar versatile attributes are also evident for the OTCEC analysis of synthetic peptide mixtures. Illustrative of this selectivity behavior is the electrochromatogram (Figure 3) obtained using an etched cholesterol-modified capillary for a partially fractionated sample of the highly hydrophobic peptide BFL2 obtained following cleavage of the synthetic product from the solid-phase Wang resin and diethyl ether precipitation. The RP-HPLC profile obtained for this crude synthetic product obtained using a standard linear gradient condition from buffer A to 75% buffer B over 60 min with an analytical TSK C-18 column (150 × 4.6 mm; particle size, 5 µm) exhibited only a broad peak. The results shown in Figure 2A,C also reveal a further selectivity feature associated with the OTCEC separation of synthetic peptides in comparison to the corresponding RP-HPLC separation profile. Electrospray mass spectroscopic analysis of the TRAP-related peptides, purified by semipreparative RP-HPLC and analyzed on TSK C-18 and Agilent C8 columns, confirmed that they were predominantly, that is, > 95%, one molecular weight species of the correct amino acid sequence. It can be seen, however, that the synthetic TRAP-3 peptide was resolved into several component peaks under the isocratic OTCEC conditions that were employed, but under gradient-elution RP-HPLC, only a single peak was observed. Importantly, the closely related analogues, TRAP-1, -2, -4, -5, and -6, or the 38-mer synthetic R-helical polypeptide TMD38, when separated under identical OTCEC conditions, did not show the same behavior as TRAP-3. Although multizoning interconversion of polypeptides and proteins has often been observed in RP-HPLC as significantly broadened peaks, the presence of relatively sharp peak species in the OTCEC separation of TRAP-3 suggests that a novel peptide-specific retention mechanism may be operating. Such behavior has been predicted to occur with polypeptides in packed CEC from theoretical studies on the roles of molecular and submolecular forces that control selectivitity.39 Because the presence of contaminating compounds in the TRAP-3 sample can be excluded on the basis of the mass spectroscopic and chromatographic data, other explanations need to be sought for the multizoning behavior observed in the OTCEC electrochromatgrams of these peptides but not the other five TRAP-analogues or TMD38. This multiple peak behavior cannot be accommodated in terms of the participation of cis/trans isomerization for the TRAP peptides, because the proline residue occurs at the free (39) Walhagen, K.; Huber, M. I.; Hennessy, T.; Unger, K. K.; Hearn, M. T. W. Submitted.
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Figure 2. Analyses of the TRAP-3 synthetic peptide sample. (A) By OTCEC with an etched C18-modified capillary with bubble cell for detection. Conditions: capillary i.d., 20 µm; L, 70 cm; l, 63 cm; V, 25 kV; i, 3.1 µA; electrolyte, buffer 1 (60 mM phosphoric acid and 38 mM Tris), pH 2.14, + 20% (v/v) methanol. (B) By OTCEC with etched C18-modified capillary and no bubble cell. Other conditions same as (A). (C) By RP-HPLC. Column, TSK-ODS 120T column (150 × 4.6 mm i.d.); mobile phase, linear gradient of 0-100% B over 60 min. Solvents: A, 0.1% TFA in water; B, 0.09% TFA in 60:40 (v/v) acetonitrile/water. Flow rate, 1 mL/min. Detection for OTCEC and for HPLC at 214 nm. Table 3. Elution Ordera of TRAP Peptides on Etched Chemically Modified Capillaries pH 2.14b
Figure 3. Separation of the synthetic peptide sample on etched cholesterol-modified capillary. Conditions: i.d., 20 µm; L, 68 cm; l, 61 cm; V, 25 kV; electrolyte, buffer 1 (60 mM phosphoric acid and 38 mM Tris), pH 2.14; i, 10 µA; detection at 214 nm.
C-terminus. None of these peptides contains Asp-Pro structures, and therefore, hydrolytic cleavage of peptide bonds is most unlikely under the rather innocuous OTCEC electrolyte conditions. Moreover, the TRAP-related peptides lack Asn-Gly or AspGly sequences and, hence, would not be able to undergo β-rearrangement to the R-aminosuccinimido structure, a conclusion that is consistent with the absence of a M+-18 molecular ion mass species in the ESI-MS of these synthetic peptides. Thus, the origin of this peptide-specific effect has to be attributed to a combination of other causes, including the possibility that in this OTCEC format, three processes could happen to the TRAP-3 peptide: (i) The TRAP-3 peptide undergoes multiple, reversible equilibrium binding processes with regions of the etched cholesteryl10-undecenoate-modified capillary that have higher or lower coverage of the steroid-like ligand on the capillary wall surfaces. This behavior would lead to multiple peaks due to regional microheterogeneities at the atomic scale on the capillary surface and, thus, multiple distribution phenomena. It can, however, be noted that the reproducibility of production of etched cholesteryl5120 Analytical Chemistry, Vol. 73, No. 21, November 1, 2001
pH 7.3b
C18d
cholesterole
C18d
cholesterole
TRAP-4 TRAP-1 TRAP-6 TRAP-3 TRAP-2 TRAP-5
TRAP-6 TRAP-4 TRAP-3 TRAP-2 TRAP-1 TRAP-5
TRAP-2 TRAP-3 TRAP-4 TRAP-6 TRAP-1 TRAP-5
TRAP-4 TRAP-3 TRAP-1 TRAP-6 TRAP-2 TRAP-5
a Elution order is from shortest to longest migration time. b Mobile phase ) 80:20 buffer 1/methanol; buffer 1: 60 mM phosphoric acid and 38 mM Tris, pH 2.14. c Mobile phase ) 80:20 buffer 2/trifluoroethanol; buffer 2: 50 mM Tris and 125 mM boric acid, pH 7.3. d Capillary i.d., 20 µm; L, 78 cm; length to detector window, l, 71 cm. e Capillary i.d., 20 µm; L, 68 cm; length to detector window, l, 61 cm.
10-undecenoate-modified capillaries has been shown to be high in the present investigations with serotonin, tryptamine, and chicken or turkey lysozyme, as well as previously with nonpolar and basic organic compounds.30,31 The occurrence of multiple peaks for an essentially homogeneous peptide sample suggests that regional bonded surface micro-heterogeneities, evident by scanning electron (SEM) and atomic force field (AFM) microscopy, solid-state cross-polarization magic angle spinning (CP-MAS) NMR, and photoelectron (ESCA) spectroscopic procedures as chemically modified protrusions32 that radially extend from the wall of the OTCEC capillary can be probed in a peptide-specific manner. These chemically modified protrusions arise from the dissolution and redeposition of silica material during the etching process prior to the silanization/hydrosilation reactions. (ii) The TRAP-3 peptide exists in different solvated charged states or, much less likely because of its molecular size, in different conformational states that are resolvable under the chosen OTCEC conditions. (iii) The TRAP-3 peptide undergoes a peptide-specific monomer-dimer-oligomer self-association phenomenon under the chosen OTCEC conditions, with these monomer-multimer species resolvable as a result of subtle differences in their effective
Table 4. Elution Order and Migration Time of Peptides and Neutral Marker at pH 7.5 Using Buffer 3a C18-modified capillary order time (min) cholesterol-modified capillary order time (min) charge on peptides at pH 7.5 a
DMSO 6.0
