Peptide separation in hydrophilic interaction ... - Wiley Online Library

2084 Hongjing Fu Wenhai Jin Hua Xiao Haiwei Huang Hanfa Zou National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, China

Electrophoresis 2003, 24, 2084–2091

Peptide separation in hydrophilic interaction capillary electrochromatography Separation of small peptides by hydrophilic interaction capillary electrochromatography (HI-CEC) has been investigated. The negative surface charge of a hydrophilic, strong-cation-exchange stationary phase (PolySULFOETHYL A) provided a substantial cathodic electroosmotic flow (EOF). The influence of acetonitrile content, ionic strength, mobile phase pH as well as applied voltage on the migration of the peptides was studied. Possible retention mechanisms of the peptides in HI-CEC were discussed. It was found that hydrophilic interaction between the solutes and the stationary phase played a major role in this system, especially when mobile phases with high acetonitrile content were used. However, an ion-exchange mechanism and electrophoretic mobility also affect the migration of the peptides in HI-CEC. Elution order and selectivity was proved to be different in HI-CEC and capillary zone electrophoresis (CZE), thus revealing the potential of HI-CEC as a complementary technique to CZE for the separation of peptides. Efficiency and selectivity of HI-CEC for the separation of peptides were demonstrated by baseline separating nine peptides in 6 min. Keywords: Capillary electrochromatography / Hydrophilic interaction / Peptides / Separation mechanism / Strong-cation exchange DOI 10.1002/elps.200305462

1 Introduction Since its introduction by Pretorius et al. in 1974 [1], capillary electrochromatography (CEC) has developed into a promising contemporary separation technique, as it embodies many features of both capillary zone electrophoresis (CZE) and micro-high performance liquid chromatography (m-HPLC). In CEC, the electroosmotic flow (EOF) is used to drive the mobile phase through the capillary. This leads to a great enhancement of the separation efficiency, compared to pressure-driven HPLC, because of the flat flow profile of the EOF. Furthermore, the presence of a stationary phase provides chromatographic interaction sites and, therefore, additional selectivity compared to CZE. So far, CEC has demonstrated its potential for separations of small, neutral molecules in the reversed-phase mode on silica-based stationary phase packings. Electrochromatographic separations on silica-based stationary Correspondence: Dr. Hanfa Zou, National Chromatographic R&A Center, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian 116011, China E-mail: [email protected] Fax:+86-411-3693407 Abbreviations: HI-CEC, hydrophilic interaction capillary electrochromatography; HILIC, hydrophilic interaction chromatography; IE-CEC, ion-exchange capillary electrochromatography; ì-HPLC, micro-high performance liquid chromatography; SCX, strong cation-exchange; TEAP, triethylamine phosphate

 2003 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

phases are usually carried out at neutral or alkaline pH, in order to assure a strong cathodic EOF upon application of the electric field. However, in the case of complex amphoteric compounds such as peptides or proteins retention mechanisms under these conditions are much more complicated. Mostly at these pH conditions the uniformity of charge of the analytes is not assured. Therefore, in the case of the negatively charged part of the analytes, electrophoretic migration in the opposite direction to that of the EOF will occur, leading to excessive elution times of these compounds or even the impossibility of loading them on the CEC column. As for the set of analytes with a positive charge overall, electrostatic interactions with the residual silanols on the stationary phase will lead to severe peak tailing and low reproducibility. In addition, the RP-CEC separation may be hampered by bubble formation when used in highly aqueous mobile phases, due to the low wettability of typical reversed-phase packings. In many cases conventional mobile phases and stationary phases in HPLC can be used for separations in CEC. However, stationary phases with strong charge have been designed and prepared specifically for CEC to generate a strong and reliable EOF, as EOF from residual silanol groups on silica material provides a low EOF. Recently, ion-exchange capillary electrochromatography (IE-CEC) has attracted much attention for the separation of peptides, as this kind of stationary phases provides substantial and stable EOF in a much wider pH range. Separa0173-0835/03/12-1306–2084 $17.501.50/0

tions of peptides and basic tricyclic antidepressants by IE-CEC on strong cation exchange (SCX) stationary phases were reported by several authors [2–4]. Anionexchange CEC for protein separation has also been studied [5]. Also so-called mixed-mode stationary phases consisting of a combination of charge-carrying groups (ion-exchange) and nonionic functionalities have been developed for the separation of peptides in CEC [6–9]. In addition, CEC with polymeric open-tubular columns and monolithic stationary phases have been applied for separations of peptides and proteins [10–16]. Hydrophilic interaction chromatography (HILIC) is an alternative mode of chromatography where separations are performed in aqueous-organic mobile phases on polar stationary phases. This mode is similar to normalphase liquid chromatography in that polar compounds are more retarded than nonpolar compounds. However, it is not conducted in pure organic mobile phase, but water serves as the strong solvent and the retention of analytes can be effectively manipulated by varying the concentration of organic modifier in the mobile phase. HILIC has been demonstrated to be an efficient method for the separation of polar compounds including amino acids, peptides, nucleic acids, pharmaceuticals, and carbohydrates [17–22]. We have presented the use of HILIC stationary phases for the separation of polar compounds by hydrophilic interaction capillary electrochromatography (HI-CEC) [23]. The results showed that HI-CEC was a powerful technique for the separation of highly polar compounds. The present work is aimed at the systematic study of peptide separations by HI-CEC. In order to get insight into the retention mechanisms of peptides in the HI-CEC mode, the influence of the acetonitrile content, the ionic strength, the mobile phase pH as well as the applied voltage on the migration of peptides has been studied.

2 Materials and methods 2.1 Instrumentation and materials All CEC and CZE experiments were performed on a Beckman P/ACE 5510 instrument (Beckman, Fullerton, CA, USA). The m-HPLC experiment was carried out on an Agilent CE (Agilent, Waldbronn, Germany) instrument, equipped with a DAD UV detector. A Waters 510 pump (Waters, Milford, MA, USA) was used to pack the capillary columns. Fused-silica capillaries (50 mm ID, 365 mm OD) were obtained from Yongnian Optic Fiber Plant (Hebei, China). PolySULFOETHYL A (5 mm, 300 Å) and PolyHYDROXYETHYL A (5 mm, 300 Å) were gifts from PolyLC (Columbia, MD, USA).

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2.2 Chemicals and buffers Stock solutions were prepared by dissolving each peptide into acetonitrile/water (50/50 v/v) with its concentration of 1 mg/mL and stored at 47C. Then it was diluted with mobile phase by ten times or mixed each other for sample injection. Acetonitrile was of chromatographic grade; all the other reagents used were of analytical reagent grade. Ultrapure water used for preparing solutions was produced by a Milli-Q water system (Millipore, Bedford, MA, USA). Stock solutions of triethylamine phosphate (TEAP, 1 M in phosphate), were prepared by addition of triethylamine to a concentrated solution of phosphoric acid until the desired pH was obtained. The pH was measured before mixing the buffer with the organic modifier. The TEAP stock solution was filtered through a 0.45 mm membrane. Mobile phases were prepared by addition of the required volume of acetonitrile and TEAP buffer to a volumetric flask, followed by addition of water to 1 mL below the mark. Then the flask was placed in an ultrasonication bath for 5 min. Water was then added to the mark after the solution reached room temperature. Before the run, the mobile phase was degassed further in an ultrasonic bath for 30 min.

2.3 Column preparation and separation conditions CEC columns were packed in-house by a slurry packing technique as reported in literature [24, 25]. Columns used for CEC were 27 cm long with a packed length of 6.5 cm. Before a CEC experiment, the columns were flushed with mobile phase for 30 min using a syringe. The columns were then conditioned in the CEC instrument with the mobile phase for at least 2 h. The applied voltage was first ramped from 0 to 5 kV over 10 min and then operated at 5 kV. The temperature was kept at 257C and the detection wavelength was set at 214 nm. The separation voltage was set at 5 kV if not otherwise stated. All CEC experiments were performed in the isocratic elution mode. The injection was performed electrokinetically by applying a voltage of 5 kV for 5 s. The column used for m-HPLC was 32 cm long with an effective length of 8.5 cm. The preparation of the column is same as that of CEC. The m-HPLC experiment was carried out by applying a pressure of 10 bar to the outlet (the short end when referring to the Agilent CE instrument) and the injections were performed by applying a pressure of 10 bar for 6 s at the same side. Temperature and detection wavelength were the same as those in CEC experiments. The column used for capillary electrophoresis (CE) was 27 cm long with an effective length of 6.5 cm. The operation voltage was at 5 kV and its ramp time was

CE and CEC


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0.17 min. Temperature, detection wavelength, and injection mode were the same as during the CEC experiments.

of less than 0.8% and the RSD of the void time of about 0.5%. The column efficiencies for the six peptides varied from 183 000 to 293 000 plates/m, when a mobile phase with a relatively high buffer concentration, 100 mM TEAP (pH 2.8), was chosen.

3 Results and discussion In CEC, due to the simultaneous presence of electrophoretic mobility and chromatographic interactions, the characteristic retention factor (k 0 ) used in chromatography is no longer valid for the description of the migration process of ionic compounds. The electrochromatographic retention factor (k*) has been defined as follows [26, 27]: k

k 0 mep =meo 1 mep =meo

(1)

where k 0 is the actual capacity factor caused by chromatography alone in CEC, and meo and mep are the electroosmotic and electrophoretic mobilities, respectively. However, for practical measurements the retention factor can also be expressed as follows [26]: k* = (tr 2 t0)/t0

(2)

where tr is the migration time of a solute, and t0 is the migration time of a neutral and chromatographically unretained solute. Equation (2) also includes the contribution of the electrophoretic mobility component to the k* value, and analytes that migrate before the EOF will have a negative k* value, and analytes migrating after the EOF with no chromatographic retention will have a positive value. In this work, toluene was selected as the t0 marker, as it was the most hydrophobic in the tested solutes and, therefore, eluted first in HI-CEC because of the minimal hydrophilic interaction with the polar stationary phase [23].

3.1 Effect of the organic modifier concentration in the mobile phase The content of organic modifier in the mobile phase has a great influence on the resolution and selectivity of polar compounds in HILIC. Hydrophilic interactions are promoted by increasing the organic modifier content. Therefore, the influence of acetonitrile content on the retention of peptides in HI-CEC was investigated. Nine peptides including one acidic peptide, Gly-Asp, were selected as test solutes. In order to elute the peptides in a reasonable time, a mobile phase with a relatively high buffer concentration, 100 mM TEAP (pH 2.8), was chosen. The effect of the acetonitrile concentration in the range between 30% and 80% v/v on the retention of the peptides is shown in Fig. 1. The retention of the solutes increases with increasing acetonitrile content and changes dramatically at an acetonitrile content above 60% v/v. The electrochromatograms of nine peptides obtained at different concentrations of acetonitrile in the mobile phases are shown in Fig. 2. As the acetonitrile content increases, the retention times and the resolution of the solutes increase. This is the result of enhanced hydrophilic interactions between the solutes and the hydrophilic stationary phase. The

PolySULFOETHYL A, a stationary phase frequently used in HILIC [17, 19] has been chosen as the stationary phase for the HI-CEC experiments. This stationary phase is prepared by chemically bonding a polypeptide, poly(2-sulfoethyl aspartamide), on a silica gel matrix and proved to be very hydrophilic, particularly compared to other silicabased and nonsilica-based matrices [28]. Moreover, the sulfonic groups on the surface of the stationary phase produce substantial EOF in a wide pH range. The major advantage of CEC is its high column efficiency due to the flat flow profile of the EOF. In order to test the performance of the PolySULFOETHYL A column, peptides including Ala-Ile, Gly-Leu, Gly-Phe, Gly-Met, GlyVal, Gly-Tyr were selected to investigate the repeatability of the migration time and the efficiency of HI-CEC with a mobile phase containing 100 mM TEAP buffer (pH 2.8) and 80% v/v acetonitrile in ten consecutive runs. The repeatability proved to be very high, with relative standard deviations (RSDs) for the migration times of the peptides

Figure 1. Effect of acetonitrile concentration on the k* value in HI-CEC. Experimental conditions: column packed with 5 mm PolySULFOETHYL A, 27 cm (packed length, 6.5 cm)650 mm ID; separation voltage, 5 kV; injection, 5 kV65 s. Mobile phases, acetonitrile concentration from 30% to 80% v/v in 100 mM TEAP buffer (pH 2.8). Solutes: (1) Ala-Ile; (2) Gly-Leu; (3) Gly-Phe; (4) Gly-Met; (5) Gly-Val; (6) Gly-Tyr; (7) Gly-Thr; (8) Gly-Ser; (9) Gly-Asp.



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Figure 2. Electochromatograms of peptides by HI-CEC with different acetonitrile content in the mobile phase. Other conditions and tested solutes are as in Fig. 1.

nine tested peptides were baseline-separated when the acetonitrile concentration was above 80% v/v. The retention of the test peptides with the composition Gly-X was also measured on a PolyHYDROXYETHYL A stationary phase, a hydrophilic stationary phase without surface charges, however, this case in the m-HPLC mode. The separation selectivity of the peptides on the column was compared to that in HI-CEC on the PolySULFOETHYL A column. As shown in Fig. 3, without Gly-Asp, a good linear relationship (r = 0.9915) between the log k’ in m-HPLC and log k* in HI-CEC was observed. This result suggests that the retention of the solutes is governed by hydrophilic interaction, which increases with polarity of solutes. The acidic peptide Gly-Asp is eluted last in HI-CEC whereas it is eluted before Gly-Thr and Gly-Ser in m-HPLC. This is because of the involvement of electrophoretic migration. At low pH, most peptides are positively charged and, therefore, accelerated from the anode to the cathode by electrophoretic mobility. However, Gly-Asp, an acidic peptide, has a lower electrophoretic mobility than other peptides, and is eluted later.

Figure 3. Plots of k* of peptides in HI-CEC vs. k 0 of peptides in m-HPLC. Experimental conditions: column packed with 5 mm PolyHYDROXYETHYL A for m-HPLC, 32 cm (packed length, 8.5 cm)650 mm ID; mobile phase containing 80% v/v acetonitrile in 100 mM TEAP, pH 2.8. Other conditions for CEC experiments and tested solutes are as in Fig. 1.

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The effect of organic modifier concentration in the mobile phase on the EOF was also investigated under the same conditions as in Fig. 1. An increase of the acetonitrile concentration in the mobile phase results in an increase in EOF. This tendency is similar to that in RP-CEC with acetonitrile as organic modifier, which may be the result of a decrease in the viscosity of mobile phase [29].

3.2 Effect of ionic strength in the mobile phase Electrostatic interactions will inevitably take place between the positively charged peptides and the negatively charged packing surface. Ionic strength in the mobile phase, therefore, should have a strong influence on the separation of peptides in HI-CEC, which was studied by changing the TEAP buffer concentration from 25 to 200 mM in the mobile phase, but keeping the eluent pH at 2.8 and the acetronitrile content at 60% v/v. It was observed that the EOF decreased from 14.1 to 12.4 cm2/kV?min by increasing the buffer concentration from 25 to 200 mM. Generally, in ion-exchange chromatography the retention of solutes decreases with increasing ionic strength in the mobile phase. This tendency was confirmed in stronganion-exchange CEC, where the electrochromatographic retention factor (k*) decreased with increasing buffer concentration [3]. A linear relationship between log k* of the peptide and the logarithm of the buffer concentration (log [c]) was observed in strong-cation-exchange CEC on a SCX stationary phase [2]. In this work, the effect of the ionic strength of the mobile phase on the retention of peptides in HI-CEC has been investigated as shown in Fig. 4. The retention of the peptides also decreased with increasing ionic strength of the mobile phase. However, a linear relationship between log k* and log [c] as observed in SCX-CEC experiment was not obtained. Probably besides the ion-exchange interaction the difference in hydrophilicity between the Spherisorb-SCX and the PolySULFOETHYL A particles plays an important role. The increase in hydrophilicity of the mobile phase by increasing salt concentration, as has been observed in a study on HILIC separations of dipeptides [17], leads to a decrease of hydrophilic interactions between the peptides and the stationary phase. This is more pronounced on the hydrophilic PolySULFOETHYL A stationary phase. Finally, electrophoretic migration also contributes to the retention of peptides because peptides tested here were positively charged at the pH used. However, as seen in Fig. 4, the elution order of the peptides, which follows the hydrophilicity of the peptides, remains unchanged in all ionic strength conditions. Figure 5 shows typical chromatograms of the separation of the peptides in HI-CEC by using the eluent of 60% v/v acetronitrile in 25, 100, and

Figure 4. Effect of the eluent ionic strength on the k* values of peptides in HI-CEC. Experimental conditions: mobile phase, 60% v/v acetonitrile in TEAP buffers with varied concentration from 25 to 200 mM (pH 2.8). Other conditions and tested solutions are as in Fig. 1.

200 mM TEAP buffer (pH 2.8), respectively. It can be seen that the peak shape of the peptides as well as their resolution was obviously improved in mobile phases with higher buffer concentration. This is the reason why the mobile phase with relatively high ionic strength was chosen for the investigation of the effects of acetonitrile content and pH of the mobile phase on the retention of the peptides.

3.3 Effect of pH The influence of the pH on the retention of the peptides has been studied at pH 2.8, 4, 5, and 6, keeping the acetonitrile content constant at 80% v/v. The results are shown in Fig. 6. As can be seen, substantial EOF was produced in a wide pH range due to the complete ionization of sulfonic groups on the surface of the stationary phase. However, an increase in the EOF was observed when the pH of mobile phase was raised. This may be attributed to the increased number of negatively charged silanol groups at higher pH values. Figure 6 also illustrates the dependence of k* of some peptides on the pH values of the mobile phase. The retention of the peptides is remarkably dependent on the pH of the mobile phase owing to their amphoteric property. Three mechanisms, i.e., hydrophilic interaction, ion-exchange mechanism and electrophoretic migration, may contribute to the migration process. At low pH value, the peptides are positively charged and, therefore, both hydrophilic as well as electrostatic interaction contribute to the retention of the peptides, while electrophoresis accelerates the solutes towards the cathode. The combination of these mechanisms



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Figure 6. Effect of pH on the electroosmotic mobility (meo) and the k* values of peptides in HI-CEC. Experimental conditions: mobile phase, 80% v/v acetonitrile in 100 mM TEAP buffer with pH 2.8, 4, 5, and 6. Other conditions and solutes are as in Fig. 1.

charged, leading to electrostatic repulsion from the stationary phase of the same sign. However, the negatively charged peptides tend to migrate against the EOF, and thereby the migration times at higher pH are relatively long. Another possible reason for the long retention time of peptides is that the hydrophilicity of the negatively charged peptides is stronger than that of the neutral peptides, and, therefore, the hydrophilic interaction between the peptides and the hydrophilic stationary phase becomes stronger. So hydrophilic interaction and electrophoretic mobility are the main mechanisms for the migration of peptides in this system at relatively high pH.

Figure 5. Electrochromatograms for separation of peptides by HI-CEC with 60% v/v acetonitrile in 25, 100, and 200 mM TEAP buffer (pH 2.8). Other conditions and tested solutes are as in Fig. 1.

resulted in the moderate retention of peptides at low pH. The retention of the peptides almost stays the same with increasing pH of the mobile phase from 2.8 to 5.0. This can be attributed to the fact that the peptides are deprotonated and change to the neutral form gradually, whereafter electrostatic interaction and the hydrophilic interaction as well as the electrophoresis mechanism lose importance. When the pH value of the mobile phase was further increased, peptides gradually became negatively

In order to gain insight in the retention mechanisms, separation of peptides in CEC was compared with that in CZE by using an untreated fused-silica capillary (effective length/total length = 27/6.5 cm) under otherwise identical conditions. Five peptides were baseline-separated with an elution order of Gly-Leu,Gly-Met,Gly-Tyr,GlySer,Gly-Thr in HI-CEC by using a mobile phase of 100 mM TEAP buffer (pH 5.0) containing 80% v/v acetonitrile, which is much different from the elution order of Gly-Tyr,Gly-Leu=Gly-Met,Gly-Thr,Gly-Ser in CZE. Separation of charged solutes in CZE is only based on the difference of mass-to-charge ratios; therefore, it is difficult to separate the solutes with similar mass-to-charge ratios. However, good separation of such kinds of charged solutes can be achieved by CEC, due to the contribution of the chromatographic mechanism. For example, baseline separation of isomeric peptides of Ser-Leu and Leu-Ser was achieved in HI-CEC with a separation factor of 1.2. However, these peptide isomers could not be resolved in CZE under the same conditions.

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3.4 Effect of applied voltage In CEC, the mobile phase is driven by EOF generated by an applied electric field. The influence of the electric field on EOF, column efficiency, and the retention of neutral compounds have been extensively studied, but the influence of the electric field on retention of ionic compounds has seldom been reported. Kitagawa and Tsuda [30] reported that the electric field could induce a variation of the distribution coefficient in CEC packed with anionexchange resin, and result in the alternation of retention factors of ionic compounds. Ye et al. [31] investigated the effect of the electric field on the retention of acidic compounds in strong-anion-exchange CEC. They observed an increase of the k* values of acidic compounds with increasing applied voltage. The influence of the applied voltage on the migration behavior of peptides in the HI-CEC is shown in Fig. 7. It can be seen that the retention of all peptides decreases with increasing applied voltage, although the elution order remains the same. The decrease of the retention may result from the generated Joule heat in the system. Figure 8 shows a typical chromatogram of a separation of nine peptides at an applied voltage of 15 kV. Separation was achieved in 6 min.

Figure 7. Effect of the applied voltage on k* of peptides in the HI-CEC. Experimental conditions: mobile phase, 80% v/v acetonitrile in 100 mM TEAP buffer (pH 2.8); applied voltages varied from 3 to 15 kV. Other conditions and solutes are as in Fig. 1.

Figure 8. Electrochromatogram for separation of peptides with applied voltage at 15 kV in HI-CEC. Mobile phase: 80% v/v acetonitrile in 100 mM TEAP (pH 2.8). Other conditions and solutes are as in Fig. 1.

process. However, the hydrophilic interaction dominated the migration behavior of peptides in HI-CEC. In order to promote the hydrophilic interaction between the polar solutes and the hydrophilic stationary phase, the organic modifier content should be kept high. In order to efficiently separate peptides, mobile phases with relatively high ionic strength and low pH value were adopted. The retention of the peptides was found to be reduced by applying a high separation voltage. The potential of HICEC for the separation of peptides was demonstrated by successfully separating nine peptides under different conditions. As elution order and selectivity are different in HI-CEC and CZE, HI-CEC can provide a complementary technique to CZE for the separation of peptides. Financial support from the National Natural Science Foundation of China (No. 20075032) is gratefully acknowledged. We would like to thank Dr. A. J. Alpert at PolyLC Inc. for the donation of PolySULFOETHYL A and PolyHYDROXYETHYL A stationary phases and carefully reading the manuscript. Received December 6, 2002

5 References 4 Concluding remarks The influence of the organic modifier content, the ionic strength, pH, and the applied voltage on the migration of peptides was studied in CEC on hydrophilic SCX stationary phases. It was found that three mechanisms, i.e., hydrophilic interaction, ion-exchange mechanism, and electrophoretic migration, contributed to the migration

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