Capillary Electrochromatography with Novel Stationary Phases. 3

Jun 24, 1999 - In this investigation, the potentials of porous and nonpo- rous octadecyl-sulfonated-silica (ODSS) microparticles were demonstrated in ...
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Anal. Chem. 1999, 71, 3277-3282

Capillary Electrochromatography with Novel Stationary Phases. 3. Retention Behavior of Small and Large Nucleic Acids on Octadecyl-Sulfonated-Silica Minquan Zhang, Changming Yang, and Ziad El Rassi*

Department of Chemistry, Oklahoma State University, Stillwater, Oklahoma 74078-3701

In this investigation, the potentials of porous and nonporous octadecyl-sulfonated-silica (ODSS) microparticles were demonstrated in the capillary electrochromatography (CEC) of small (e.g., nucleotides and dinucleotides) and large (e.g., transfer ribonucleic acids (t-RNAs)) nucleic acids. The ODSS stationary phase comprised two layers: a hydrophilic sulfonated (permanently charged) sublayer and an octadecyl top layer. While the sublayer is to provide a relatively strong electroosmotic flow, the octadecyl top layer is to ensure the retentivity and selectivity required for the separation of the analytes. Mono-, di-, and triphosphate nucleotides were best separated when a small amount of tetrabutylammonium bromide was added to the mobile phase. The tetrabutylammonium bromide functioned as an ion-pairing agent and consequently allowed the rapid separation of 12 different nucleotides. It is believed that the dynamic complex exchange model explains the basis of retention in ion pair reversed-phase CEC. Eight different dinucleotides, which have similar mass-to-charge ratios, separated very well by CEC. These solutes exhibited similar migration times (i.e., little or no separation) in capillary zone electrophoresis (CZE). Similarly, t-RNAs that did not separate by CZE were well resolved in CEC with nonporous ODSS. This demonstrates that CEC is very suitable for the separation of solutes that have similar mass-to-charge ratios but differ in their hydrophobicity.

Capillary electrochromatography (CEC) is emerging as a promising microseparation technique of high resolving power 1-4 whose full potential has not been exploited yet in the separation and determination of a wide range of compounds. For CEC to become an important microseparation technique, improvements * Corresponding author: (tel) (405) 744-5931; (fax) (405) 744-6007; (e-mail) [email protected]. (1) Dittmann, M. M.; Wienand, K.; Bek, F.; Rozing, P. R. LC-GC 1995, 13, 800-814. (2) Colon, L. A.; Reynolds, K. J.; Alicea-Maldonado, R.; Fermier, A. M. Electrophoresis 1997, 18, 2162-2174. (3) Cikalo, M. G.; Bartle, K. D.; Robson, M. M.; Myers, P.; Euerby, M. R. Analyst 1998, 123, 87R-102R. (4) Rathore, A. S.; Horva´th, C. J. Chromatogr., A 1997, 781, 185-195. 10.1021/ac990306s CCC: $18.00 Published on Web 06/24/1999

© 1999 American Chemical Society

in instrumentation design, column technology, and understanding of the underlying phenomena are prerequisite. A critical area that is under intensive investigation is the development of novel stationary phases with strong electroosmotic flow (EOF) that yield efficient and rapid separations.5-11 Very recently, we introduced new column design9 and stationary phases8,10,11 and demonstrated their utility in the separation of nucleosides and their bases as well as mono- and oligosaccharides. A novel octadecyl-sulfonated-silica (ODSS) with a relatively strong EOF, which has already shown promise in CEC separations,10,11 is further exploited here in the separation of small (e.g., mono- and dinucleotides) and large nucleic acids (e.g., transfer ribonucleic acids (t-RNAs)). The ODSS stationary phase comprised a relatively hydrophilic and charged sublayer covalently attached to the silica support and a nonpolar top layer of octadecyl functions chemically bonded to the sublayer. While the sulfonated sublayer is to yield a strong EOF, the nonpolar top layer is to provide the retentivity and selectivity required for achieving chromatographic separations. To the best of our knowledge, this is the first investigation regarding the retention behavior of monoand oligonucleotides in CEC. EXPERIMENTAL SECTION Apparatus. A P/ACE 5010 capillary electrophoresis system from Beckman Instruments Inc. (Fullerton, CA) equipped with a UV detector and a data-handling system comprising an IBM personal computer and P/ACE software was used for the CEC and CE studies. A Shandon column packer from Keystone Scientific (Bellefonte, PA) was employed for the CEC capillary column packing. Chemicals and Materials. Zorbax silica, with a 10-µm average particle diameter, 150-Å average pore diameter, and 165 m2/g specific surface area was donated by Mr. Klaus Lohse from BTR Separation (Wilmington, DE). Nucleosil silica with a 5-µm average particle diameter, 120-Å average pore diameter, and 200 m2/g (5) Guo, Y.; Colo´n, L. A. Anal. Chem. 1995, 67, 2511-2516. (6) Liao, J.-L.; Chen, N.; Ericson, C.; Hjerte´n, S. Anal. Chem. 1996, 68, 34683472. (7) Palm, A.; Novotny, M. V. Anal. Chem. 1997, 69, 4499-4507. (8) Yang, C.; El Rassi, Z. Electrophoresis 1998, 19, 2061-2067. (9) Yang, C.; El Rassi, Z. Electrophoresis 1999, 20, 18-23. (10) Zhang, M.; El Rassi, Z. Electrophoresis 1998, 19, 2068-2072. (11) Zhang, M.; El Rassi, Z. Electrophoresis 1999, 20, 31-36.

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specific surface area was purchased from Alltech Associates, Inc. (Deerfield, IL). Nonporous silica having 2-µm average particle diameter was synthesized in-house according to our wellestablished procedures.12 Zorbax SAX of 5-µm mean particle diameter, 70-Å mean pore diameter, and 300 m2/g specific surface area was from DuPont (Wilmington, DE). HPLC grade acetonitrile and 2-propanol were from Baxter (McGaw Park, IL). Sodium phosphate monobasic was obtained from Mallinckrodt (St. Louis, MO). The main reagents for the synthesis of the stationary phases were from Aldrich Chemical Co. (Milwaukee, WI). Nucleotides including cytidine 5′-mono-, di-, and triphosphate (CMP, CDP, and CTP, respectively); uridine 5′-mono-, di-, and triphosphate (UMP, UDP, and UTP, respectively); adenosine 5′-mono-, di-, and triphosphate (AMP, ADP, and ATP, respectively); and guanosine 5′-mono-, di-, and triphosphate (GMP, GDP, and GTP, respectively) were from Sigma Chemical Co. (St. Louis, MO). Dinucleotides such as cytidylyl(3′f5′)cytidine (CpC), cytidylyl(3′f5′)uridine (CpU), cytidylyl(3′f5′)adenosine (CpA), cytidylyl(3′f5′)guanosine (CpG), uridylyl(3′f5′)cytidine (UpC), uridylyl(3′f5′)guanosine (UpG), uridylyl(3′f5′)adenosine (UpA), adenylyl(3′f5′)guanosine (ApG), adenylyl(3′f5′)uridine (ApU), guanylyl(3′f5′)uridine (GpU), guanylyl(3′f5′)adenosine (GpA), and guanylyl(3′f5′)guanosine (GpG) were also from Sigma. t-RNAs specific for glutamic acid (t-RNAGlu), valine (t-RNAVal), lysine (t-RNALys), phenylalanine (t-RNAPhe), and alanine (t-RNAAla) were all from Sigma. All solutes were dissolved in water at a concentration level of ∼5 × 10-4 M. Stationary Phases and Column Packing. The ODSS stationary phase was made in a three-step process which was described previously.10,11 The ODSS stationary phase comprised a primary sulfonated, relatively hydrophilic, and charged sublayer and a top retentive layer of octadecyl functions. A slurry packing technique was used to pack the capillary columns. Acetone was used to prepare the suspension of ODSS or SAX stationary phases, and 2-propanol was used as the packing solvent. Fused-silica capillaries of 360 µm o.d. × 100 or 50 µm i.d. from Polymicro Technologies, Inc. (Phoenix, AZ) were used as the separation capillaries in CE and CEC, respectively. Before packing the capillary column, a moderately strong sintered porous frit was made at the outlet of the fused-silica capillary by first tapping the capillary end into bare 5-µm silica moistened with deionized water and then by heating the capillary tip over a Bunsen burner for ∼1 min. After the capillary column was packed with ODSS stationary phase, the column was flushed with deionized water for about 30 min to 1 h. Thereafter, the packed column was cut to desired length, and the inlet retaining frit was made in the same way as the outlet frit. Last, the column was washed with acetonitrile and mounted to a Beckman capillary cartridge. In our work, whole packed capillary columns were produced with a detection window at 6.5 cm from the outlet end of the column. The detection window was made before packing by burning off the polyimide coating of capillary with thermal wire stripper. The column preparation was described in details in recent work from our laboratories.8-11 RESULTS AND DISCUSSION Retention Behavior of Nucleotides. Figure 1 shows the separation of 12 mono-, di-, and triphosphate nucleotides obtained (12) Yu, J.; El Rassi, Z. J. Liq. Chromatogr. 1993, 16, 2931-2959.

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Figure 1. Typical electrochromatogram of mono-, di-, and triphosphate nucleotides. Capillary column, packed with 10-µm ODSS stationary phase, 20.5/27 cm × 100 µm i.d.; mobile phase, hydroorganic eluent containing 9.75 mM phosphate, 3.25 mM tetrabutylammonium bromide and composed of 35% (v/v) acetonitrile and 65% (v/v) aqueous sodium phosphate, pH 6.50; running voltage, 20 kV, electrokinetic injection, 1.0 kV for 2 s. Solutes: 1, CMP; 2, UMP; 3, AMP; 4, GMP; 5, CDP; 6, UDP; 7, ADP; 8, CTP; 9, GMP; 10, UTP; 11, ATP; 12, GTP.

under optimal conditions as far as the pH and the composition of the mobile phase are concerned. The separation was facilitated by incorporating 3.25 mM tetrabutylammonium bromide, an ionpairing agent, which has previously found use in reversed-phase ion-pair chromatography (RP-IPC).13 In CEC, the technique is referred to as reversed-phase ion-pair capillary electrochromatography (RP-IPCEC). The basis of retention in RP-IPC as well as in RP-IPCEC with reversed-phase columns (e.g., ODSS) is still controversial. There are three different models to explain retention:13 (i) ion-pair model, (ii) dynamic ion-exchange model, and (iii) dynamic complex exchange model. In model i, the solute is thought to associate with the ion-pairing agent of opposite charge in the mobile phase to form an ion pair that will bind to the stationary phase via nonpolar interactions with the alkyl ligands of the stationary phase, as follows:

Xm- + Pm+ T (X-,P+)m0 T (X-,P+)s0

(1)

where the subscripts m and s stand for mobile and stationary phases, respectively, X- is the solute, and P+ is the ion-pairing agent. According to eq 1, at equilibrium, the stronger the interaction of the nucleotide with the tetrabutylammonium bromide the more retarded the analyte by the stationary phase. In the case of nucleotides, the solutes are separated via their differential electromigration as well as their differential interactions with the stationary phase. While the ion-pairing agent reduces the net negative charge of the solute and brings about its faster migration toward the detection end, the binding of the ion pair to the stationary phase retards the migration of the solute. The net result of these two opposing phenomena is a relatively fast analysis (13) Melander, W. R.; Horva´th, C. In High-Performance Liquid Chromatography; Horva´th, C., Ed.; Academic Press: New York, 1980; Vol. 2; pp 113-319.

with high selectivity as shown in Figure 1 where the separation of 12 nucleotides is achieved in less than 15 min. As can be seen in Figure 1, the monophosphated nucleotides elute first, followed by the diphosphate nucleotides, and then the triphosphate nucleotides elute last. The elution of some monophosphate nucleotides was obtained at retention times either very close to (e.g., UMP) or before (e.g., CMP) the dead time of the column, which was ∼3.08 min (i.e., an EOF of 1.11 mm/s). This indicates that CMP and UMP form a strong interaction with the ion-pairing agent (i.e., tetrabutylammonium bromide), which is moving much faster than the EOF, and their individual ion pairs with tetrabutylammonium ions are little retarded (if at all) by the ODSS stationary phase. The ion-pair model can explain the observed retention behavior of the nucleotides, but it fails to account for any possible adsorption of the ion-pairing agent to the stationary phase to form a dynamic anion exchanger column. In model (ii), the ion-pairing agent is thought to dynamically coat the stationary phase, thus transforming it to an in situ ionexchanger. The solute will then compete with the mobile phase counterions for cationic sites on the surface of the stationary phase, as follows: Xm-

Mm- + Pm+ T (M-,P+)m0 T (M-,P+)s0 798 (X-,P+)s0 + Mm- (2)

Figure 2. Effect of ion-pairing agent (i.e., tetrabutylammonium bromide) concentration on the retention time of monophosphate nucleotides. Mobile phase, hydro-organic eluent containing 20 mM phosphate, tetrabutylammonium bromide and composed of 35% (v/ v) acetonitrile and 65% (v/v) aqueous sodium phosphate, pH 6.50; running voltage, 20 kV, electrokinetic injection, 1 kV for 2 s. Curves: 1, CMP; 2, UMP; 3, AMP; 4, GMP.

where M- is the mobile-phase counterion. As in model i, triphosphate nucleotides are more retarded than diphosphate nucleotides and the latter are more retained than the monophosphate nucleotides. However, the dynamic ion-exchange model, which neglect the possible formation of an ion pair between solute and the ion-pairing agent in the mobile phase, fails to explain the relatively short analysis time observed in Figure 1 as well as the elution of some monophosphate nucleotides very close to (e.g., UMP) or before (e.g., CMP) the dead time of the column. Model iii is a combination of models i and ii as follows: the solute is considered to undergo ion-pair formation and binding to the stationary phase as in (i). This stationary-phase-ion-pair complex is thought to dissociate, thus releasing the bound solute to the mobile phase and allowing a mobile-phase counterion to bind in its place. The solute thus released will then exchange by a displacement mechanism with the mobile-phase counterion bound to the ion-pairing agent in the stationary phase, a process that yields a solute-ion-pairing agent-stationary-phase complex and releases the counterions to the mobile phase, as follows:

ion-pair dissociation dissociation, Mm-

(X-,P+)s0 798 (M-,P+)s0 + Xm- (3) ion exchange Xm- + (M-,P+)s0 T (X-,P+)s0 + Mm- (4) Model iii can explain both the elution order and the relatively short analysis time. Therefore, it is the most acceptable model for the mechanism of retention in this case. To better understand the underlying retention, five nucleotides, CMP, AMP, UMP, GMP, and IMP, were electrochromatographed on two separate capillary columns. One column was packed with

Figure 3. Typical electrochromatogram of eight dinucleotides. Capillary column, packed with 5-µm ODSS stationary phase, 20.5/ 27 cm × 100 µm i.d.; mobile phase, hydro-organic eluent containing 12 mM phosphate and composed of 30% (v/v) acetonitrile, 40% methanol, and 30% (v/v) aqueous ammonium phosphate, pH 6.0; running voltage, 20 kV; electrokinetic injection, 1.0 kV for 2 s. Solutes: 1, CpC; 2, CpU; 3, UpC; 4, CpA; 5, ApU; 6, CpG; 7, GpU; 8, UpG.

the ODSS stationary phase and the second column was packed with a SAX silica-based stationary phase mixed with 10% (w/w) ODSS. On the ODSS column, elution was performed with a hydroorganic eluent at 35% (v/v) acetonitrile containing 20 mM aqueous phosphate, pH 6.50, and 2 mM tetrabutylammonium Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

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Figure 4. Capillary electrophoresis of XpA dinucleotides at 0, 5, 10, and 15 mM Mg2+, in (a-d), respectively. Capillary, fused silica, 50/57 cm × 50 µm i.d.; electrolytes, 10 mM sodium acetate, 75 mM ammonium acetate, pH 5.0, at various MgCl2 concentrations; running voltage, 15.7 kV.

bromide. On the SAX column, elution was performed using the same mobile phase as that used for the ODSS column but in the absence of the ion-pairing agent. The order of elution on the ODSS column was CMP, UMP, AMP, GMP, and IMP, while that on the SAX column was CMP, AMP, UMP, GMP, and IMP. This difference indicates that the retention in the presence of an ionpairing agent on the ODSS column is not occurring solely by an ion-exchange mechanism with an in situ anion exchanger (i.e., as would be predicted by the dynamic ion-exchange model). As can be predicted from any of the three models, increasing the concentration of the ion-pairing agent increases the retention times of the nucleotides on the ODSS column; see Figure 2. Dinucleotides. Figure 3 shows the separation of eight dinucleotides of the CpX, UpX, ApX, and GpX types under optimal conditions as far as the ionic strength, the pH of the aqueous phase, and the percent organic modifier in the mobile phase are concerned. To better understand their electrochromatographic behavior, the dinucleotides were analyzed by CZE using a running electrolyte of 10 mM sodium acetate and 75 mM ammonium acetate, pH 5.0. As we reported earlier,14,15 and as can be seen in Figure 4a, the XpA dinucleotides were very poorly resolved due to little differences in their charge-to-mass ratios. Magnesium ion is known to complex with the phosphate groups of the nucleic acids.16 As can be seen in Figure 4, 15 mM Mg2+ was required to bring about the separation of the XpA dinucleotides. However, the analysis time became relatively high (∼42 min) to separate three XpA dinucleotides. The substantial increase in the migration time upon increasing the concentration of Mg2+ in the electrolyte is due

primarily to the decrease in the EOF. In fact, the EOF decreased from 0.4 mm/s in the absence of Mg2+ to 0.2 mm/s at 15 mM Mg2+ in the running electrolyte. In general, the zeta potential of the silica surface is most markedly reduced by ions carrying a charge opposite in sign to that of the silica surface, and of these, polyvalent ions (e.g., Mg2+) are the most effective.17 Similar behavior was observed with the XpG dinucleotides; see Figure 5, which also required the addition of Mg2+ to bring about the separation of the XpG solutes. It is obvious from the above results that CEC is an important technique for separating solutes that possess similar charge-tomass ratios but differ in their hydrophobicity. Solutes of this kind tend to comigrate in CZE, a fact that necessitates the use of complexing electrolytes to modify the electrophoretic mobility of the analytes and bring about their separation. Transfer Ribonucleic Acids. t-RNAs are relatively large molecules containing between 73 and 93 ribonucleotide residues with a molecular mass of ∼24 kDa.18 t-RNAs have about the same charge-to-mass-ratios, a fact that makes them very difficult to separate in CZE. In fact, this is shown in Figure 6a where four different t-RNAs are not separated. By adding 7 mM Mg2+, a marginal separation was obtained with an analysis time exceeding 50 min; see Figure 6b. Four different t-RNAs, namely, t-RNAGlu, t-RNALys, t-RNAVal, and t-RNAPhe, were well separated by CEC with the novel nonporous ODSS stationary phase; see Figure 7. Due to the relatively large molecular sizes of the t-RNAs, a nonporous stationary phase should be in principle preferred over a porous stationary phase. This is because large molecular mass solutes diffuse slowly in

(14) Mechref, Y.; El Rassi, Z. Electrophoresis 1995, 16, 2164-2171. (15) Nashabeh, W.; El Rassi, Z. J. Chromatogr. 1992, 596, 251-264. (16) El Rassi, Z.; Horva´th, C. J. Chromatogr. 1985, 326, 79-90.

(17) Morris, C. J. O. R.; Morris, P. Theoretical aspects of electrophoresis, 2 ed.; John Wiley & Sons: New York, 1976; pp 705-760. (18) Stryer, L. Biochemistry; 4th ed.; W. H. Freeman and Co.: New York, 1995.

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Figure 5. Capillary electrophoresis of XpG dinucleotides at 0, 5, 10, and 15 mM Mg2+, in (a-d), respectively. Conditions are as in Figure 4.

Figure 6. Capillary electrophoresis of t-RNAs. Capillary, fused silica, 50/57 cm × 50 µm i.d.; electrolyte, 20 mM sodium acetate, pH 5.0, containing 0 mM MgCl2 in (a) or 7.0 mM MgCl2 in (b); running voltage, 25 kV.

and out of the pores (i.e., high mass-transfer resistances), a fact that usually leads to band broadening of the solutes. However, a nonporous stationary phase offers a relatively low sample loadability. The pH of the running electrolyte influenced the retention of the four t-RNAs under investigation. Increasing the pH from 3.0 to 6.0 and then to 7.0 resulted in a slight decrease in the retention time. The EOF velocity changed very marginally when going from pH 3.0 to 6.0 and then to 7.0. However, the resolution between t-RNAGlu and t-RNAVal decreased substantially especially when going from pH 6.0 to pH 7.0. This is due primarily to the ionization of the phosphate residues of the t-RNAs molecules,

which leads to stronger repulsion of the t-RNAs from the ODSS stationary phase. Also, there were some changes in solute elution order when the pH of the running electrolyte was changed. At pH 3.0, the order of elution was t-RNAGlu < t-RNALys < t-RNAVal < t-RNAPhe. At pH 6.0, the order changed to t-RNAGlu < t-RNAVal < t-RNALys < t-RNAPhe. This order became t-RNAVal < t-RNAGlu < t-RNALys < t-RNAPhe at pH 7.0. Figure 7b shows the separation of t-RNAs using methanol instead of acetonitrile as the organic modifier. The analysis time increased from 12.6 to 19.0 min as a result of this change while other elution conditions were kept unchanged. Due to the Analytical Chemistry, Vol. 71, No. 15, August 1, 1999

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modifier. The eluent strength of methanol is less than that of acetonitrile,19-21 and the viscosity of the methanolic mobile phase (i.e., 1.70 cP) is higher than that of the acetonitrile-containing mobile phase (i.e., 0.95 cP).13 These two effects lead to an increased retention time as the solute is more retarded by the stationary phase and the EOF is decreased when methanol is used instead of acetonitrile. CONCLUSIONS The multilayered ODSS stationary phase proved very useful for the separation of small and large nucleic acid fragments. Twelve mono-, di-, and triphosphate mononucleotides were separated in ∼14 min by IP-RPCEC in the presence of small amounts of tetrabutylammonium bromide in the mobile phase. The dynamic complex exchange model, which is a hybrid of the ion-pair model and dynamic ion-exchange model, is believed to explain the retention mechanism of the nucleotides. Eight different dinucleotides, which have similar mass-to-charge ratios, showed similar migration times (i.e., little or no separation) in capillary zone electrophoresis (CZE). These eight dinucleotides, which differed in their hydrophobic character, separated very well by CEC in ∼22 min. Similarly, t-RNAs that did not separate by CZE were well resolved in CEC with nonporous ODSS, thus demonstrating that CEC is very suitable for the separation of charged solutes that have similar mass-to-charge ratios but differ in their hydrophobicity. ACKNOWLEDGMENT We gratefully acknowledge the financial support by the Cooperative State Research, Education and Extension Service, U.S. Department of Agriculture under Agreements 96-35201-3342 and 98-35102-6529. Figure 7. Capillary electrochromatography of four t-RNAs. Capillary column, packed with 2-µm nonporous ODSS stationary phase, 20.5/ 27 cm × 100 µm i.d.; running voltage, 20 kV, electrokinetic injection, 1 kV for 2 s; mobile phase in (a), hydro-organic eluent containing 15 mM phosphate and composed of 40% (v/v) acetonitrile and 60% (v/ v) aqueous sodium phosphate, pH 6.0; mobile phase in (b) as in (a) but containing 40% (v/v) methanol. Solutes: 1, t-RNAGlu; 2 t-RNAVal; 3, t-RNALys; 4, t-RNAPhe.

increased retention time, the peaks were more broadened with methanol than those obtained with acetonitrile as the organic

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Received for review March 22, 1999. Accepted April 30, 1999. AC990306S (19) El Rassi, Z.; Lee, A. L.; Horva´th, C. In Separation Processes in Biotechnology; Asenjo, J. A., Ed.; Marcel Dekker: New York, 1990; pp 447-494. (20) El Rassi, Z. In Carbohydrate Analysis: High Performance Liquid Chromatography and Capillary Electrophoresis; El Rassi, Z., Ed.; Elsevier: Amsterdam, 1995; pp 41-101. (21) El Rassi, Z. In Handbook of HPLC; Katz, E., Eksteen, R., Schoenmakers, P., Miller, N., Eds.; Marcel Dekker: New York, 1998; pp 463-482.