Positively Charged Sol−Gel Coatings for On-Line Preconcentration of

Innovations in sol-gel microextraction phases for solvent-free sample preparation in analytical chemistry. Abuzar Kabir , Kenneth G. Furton , Abdul Ma...
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Anal. Chem. 2004, 76, 218-227

Positively Charged Sol-Gel Coatings for On-Line Preconcentration of Amino Acids in Capillary Electrophoresis Wen Li,† David Fries,‡ Abdel Alli,† and Abdul Malik*,†

Department of Chemistry, University of South Florida, 4202 East Fowler Avenue, Tampa, Florida 33620, and Marine Science, University of South Florida, 140 Seventh Avenue, South, St. Petersburg, Florida 33701

A novel on-line method is presented for the extraction and preconcentration of amino acids using a sol-gel-coated column coupled to a conventional UV/visible detector. The presented approach does not require any additional modification of the commercially available standard CE instrument. Extraction, stacking, and focusing techniques were used in the preconcentration procedures. Sol-gel coatings were created by using N-octadecyldimethyl[3(trimethoxysilyl)proply]ammonium chloride (C18-TMS) in the coating sol solutions. Due to the presence of a positively charged quaternary ammonium moiety in C18TMS, the resulting sol-gel coating carried a positive charge. For extraction, the pH of the samples was properly adjusted to impart a net negative charge to amino acids. A long plug of the sample was then passed through the sol-gel-coated capillary to facilitate extraction via electrostatic interaction between the positively charged solgel coating and the negatively charged amino acid molecules. Focusing of the extracted amino acids was accomplished through desorption of the extracted amino acid molecules carried out by local pH change. Two different methods are described. Both methods showed excellent extraction and preconcentration effects. Preconcentration results obtained on sol-gel-coated columns were compared with the CZE analysis performed on bare fused-silica columns with traditional sample injections. The described procedure provided a 150 000-fold enrichment effect for alanine. The two methods provided acceptable repeatability in terms of both peak height and migration time. Capillary electrophoresis (CE) is a highly efficient separation technique and possesses a number of advantageous features including high overall separation efficiency, short analysis times, and small amounts of reagents or samples required. CE also provides a biocompatible separation environment that is especially suitable for biological molecules including proteins, nucleic acids, peptides, nucleotides, and amino acids. CE separations are often performed using an on-column detection mode to prevent loss of * Corresponding author. Phone: 813-974-9688, Fax: 813-974-3203. E-mail: [email protected]. † University of South Florida, Tampa. ‡ University of South Florida, St. Petersburg.

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separation efficiency due to extracolumn band broadening that usually takes place if an off-column detection cell is used. However, in UV detection (which is the most commonly used detection technique in CE), due to the short path length (equal to the inner diameter of the column), on-column UV detection is characterized by low concentration sensitivity. A series of studies have been undertaken over the past few decades to increase the concentration sensibility in CE. Approaches used to address this problem can be divided into three categories:1 (a) sample preconcentration strategies, (b) alternative capillary geometry and improved optical design, and (c) alternative detection modes. The first technique, commonly called on-line sample preconcentration, is especially attractive since it involves no additional modification of the commercially available standard CE instrument, and it can be easily accomplished by carefully controlling the operation conditions on a standard CE instrument. Stacking is one of the most widely used techniques for sample preconcentration in capillary zone electrophoresis (CZE). In “field amplified sample stacking”,2,3 the velocity of analyte ions is changed by using discontinuous buffers. When a high voltage is applied, a higher electric field is developed in the dilute sample plug than in the more concentrated running buffer because of the higher resistivity of the sample zone. The analyte ions then stack at the boundary between the sample plug and the running buffer, forming a narrow stacked zone.2-9 Discontinuous buffers can be prepared simply by addition of salts into buffer, by dissolving the sample in a low ionic strength buffer, or by adjusting their pH values.8-10 An enhancement factor of more than 100 was achieved for protein samples.6 For zwitterionic solutes such as amino acids, peptides, and proteins, discontinuity in the running buffer pH could be applied to achieve preconcentration and focusing.8 Recently, an on-line focusing of flavin derivatives using dynamic pH junction in CE has been reported11 to achieve a more than 1200-fold improvement in sensitivity relative to the standard (1) Albin, M.; Grossman, P. D.; Moring, S. E. Anal. Chem. 1993, 65, 489A497A. (2) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 1046-1050. (3) Chien, R. L.; Burgi, D. S. Anal. Chem. 1992, 64, 489A-496A. (4) Shihabi, Z. K. J. Chromatogr., A 2000, 902, 107-117. (5) Shihabi, Z. K. Electrophoresis 2000, 21, 2872-2878. (6) Chun, M. S.; Kang, D.; Kim, Y. Microchem. J. 2000, 70, 247-253. (7) Locke, S.; Figeys, D. Anal. Chem. 2000, 72, 2684-2689. (8) Britz-McKibbin, P.; Chen, D. D. Y. Anal. Chem. 2000, 72, 1242-1252. (9) Baker, D. R. Capillary Electrophoresis; John Wiley & Sons: New York, 1995. (10) Wei, W.; Xue, G.; Yeung, E. S. Anal. Chem. 2002, 74, 934-940. 10.1021/ac0301696 CCC: $27.50

© 2004 American Chemical Society Published on Web 11/27/2003

injection method in CE. When a large volume of sample is introduced into the separation column for stacking, the solute zone is as wide as the length of the sample plug. Several techniques had been developed to achieve a narrow stacked sample band for further analysis. Chien and Burgi2,3 developed a method for stacking from a very large sample volume. First, a large volume of sample prepared in a dilute buffer was introduced into the column. A negative voltage was then applied at the capillary ends to obtain electroosmotic flow (EOF) directed toward the capillary inlet. Under these conditions, the sample matrix was gradually pushed out of the capillary by EOF and the anions were stacked at the boundary between sample solution and the background electrolyte. The resulting sample zone was narrow, and highefficiency separation capabilities in CE were preserved. This method was also used to determine some quaternary ammonium herbicides in spiked drinking water.12 Another focusing technique in CE is capillary isoelectric focusing (CIEF).13 This method utilizes the differences in the isoelectric points (pIs) of analytes. The separation capillary is filled with a solution of ampholytes. If an electric field is applied across such a capillary, a pH gradient is generated along its length, and the zwitterionic analytes in the subsequently injected sample begin to migrate through the capillary under the applied field. Each of the analytes will lose its net charge when it reaches the location in capillary where the pH of the ampholyte equals to the pI of the analyte. In the absence of EOF, focused discrete neutral analyte zones line up inside the capillary at locations corresponding to their pI values. CIEF is widely used for the analysis of analytes having different pI values.14-19 For example, an enhancement factor of 500 was achieved for polypeptide mixtures resulting from digestion of proteins, even though the components of the resulting mixture had very small differences in isoelectric points (∆pI ∼ 0.01).14 Capillary isotachophoresis (CITP)20 can also be employed for sample stacking. CITP is accomplished in a capillary by injecting the sample between two discrete buffer plugs: a leading buffer with a higher mobility ion and a terminating buffer having a lower mobility ion than the charged analytes. When an electric field with constant current is applied, the ions inside the sample are distributed into narrow and concentrated zones between leading and terminating buffer based on the differences in their mobilities. CITP has been used to on-line preconcentrate and separate inorganic,21,22 organic,23,24 and biomolecules.25 (11) Britz-Mckibbin, P.; Otsuka, K.; Terabe, S. Anal. Chem. 2002, 74, 37363743. (12) Nu´ez, O.; Moyano, E.; Puignou, L.; Galceran, M. T. J. Chromatogr., A 2001, 912, 353-361. (13) Hjerten, S.; Zhu, M.-D. J. Chromatogr. 1985, 346, 265-270. (14) Shen, Y.; Berger, S. J.; Anderson, G. A.; Smith, R. D. Anal. Chem. 2000, 72, 2154-2159. (15) Spanik, I.; Lim, P.; Vigh, G. J. Chromatogr., A 2002, 960, 241-246. (16) Clarke, N. J.; Naylor, S. Biomed. Chromatogr. 2002, 16, 287-297. (17) Hiraoka, A.; Tominaga, I.; Hori, K. J. Chromatogr., A 2002, 961, 147-153. (18) Cifuentes, A.; Moreno-Arribas, M. V.; de Frutos, M.; Diez-Masa, J. C. J. Chromatogr., A 1999, 830, 453-463. (19) Righetti, P. G.; Couti, M.; Gelfi, C. J. Chromatogr., A 1997, 767, 255-262. (20) Martin, A.; Everaerts, F. Proc. R. Soc. London, A 1970, 316, 493-514. (21) Tu, C.; Lee, H. J. Chromatogr., A 2002, 966, 205-212. (22) Church, M. N.; Spear, J. D.; Russo, R. E.; Klunder, G. L.; Grant, P. M.; Andresen, B. D. Anal. Chem. 1998, 70, 2475-2480. (23) Toussaint, B.; Hubert, Ph.; Tjaden, U. R.; Van der Greef, J.; Crommen, J. J. Chromatogr., A 2000, 871, 173-180. (24) Shihabi, Z. K.; Electrophoresis 2002, 23, 1621-1617.

Recently, with the development in microchip-based capillary electrophoresis,26-28 sample stacking techniques have also been used in the microfluidic CE devices.29,30 A few orders of magnitude in sample enrichment have been obtained by stacking in microchipbased CE.29 Quirino and Terabe31-38 introduced the concept of sample sweeping. Sample sweeping is accomplished in micellar electrokinetic chromatography (MEKC), in which micelles act as a pseudostationary phase and “sweep” the analytes from the long injected sample plug and convert it into narrow zone(s), thereby preconcentrating the analyte(s) from the wide band of originally injected dilute sample. Sweeping makes it possible to preconcentrate neutral analytes. A millionfold sensitivity increase has been reported with a combination of stacking and sweeping effects.31 Landers et al.39-41 used a different strategy to preconcentrate neutral analytes in MEKC. Contrary to preparing sample in a dilute, low-conductivity electrolyte commonly used in the sample stacking process, one of their methods involved the use of a highconductivity sample matrix, which enables the micelles to be focused before they enter the sample zone.40 Their method also solves the problem for the preconcentration of samples having high salt content, which is frequently met in real-life situations. Cao et al.42 recently reported the stacking of ionizable analytes in a high-salt sample matrix by means of a transient moving chemical reaction boundary method (tMCRBM). Sample got stacked in the tMCRBM generated between two phases (a weak acid of the running buffer and a weak base of sample matrix). The mechanism is dependent on the zwitterionic properties of the analytes that change their net charges based on the pH. The high-salt concentration in the sample matrix slows down the migration velocities of analytes producing a narrow, stacked zone. Solid-phase extraction (SPE) is another important sample preconcentration strategy. With this method, multiple column volumes of sample can be injected since the analytes are adsorbed on the stationary phase. SPE can be coupled to capillary electrophoresis system, where the preconcentrated samples get separated on the CE column.43,44 The extraction also could be accomplished by an ion-exchange procedure.45-47 (25) Shihabi, Z. K.; Friedberg, M.J. Chromatogr., A 1998, 807, 129-133. (26) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1935-1941. (27) Deng, Y.; Zhang, H. Henion, J. Anal. Chem. 2001, 73, 1432-1439. (28) Deng, Y.; Henion, J.; Li, J.; Chang, Y.-S. Anal. Chem. 2001, 73, 639-646. (29) Yang, H.; Chien, R.-L. J. Chromatogr., A 2001, 924, 155-163. (30) Lichtenberg, J.; Verpoorte, E.; de Rooij, N. F. Electrophoresis 2001, 22, 258271. (31) Quirino, J. P.; Terabe, S. Anal. Chem. 2000, 72, 1023-1030. (32) Quirino, J. P.; Terabe, S. Anal. Chem. 1999, 71, 1638-1644. (33) Quirino, J. P.; Terabe, S. Science 1998, 282, 465-468. (34) Kim, J.-B.; Quirino, J. P. J. Chromatogr., A 2001, 916, 123-130. (35) Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 791, 255-267. (36) Quirino, J. P.; Terabe, S. J. High Resolut. Chromatogr. 1999, 22, 367-372. (37) Quirino, J. P.; Terabe, S. Anal. Chem. 1998, 70, 1893-1901. (38) Quirino, J. P.; Terabe, S. J. Chromatogr., A 1997, 781, 119-128. (39) Palmer, J.; Landers, J. P. Anal. Chem. 2000, 72, 1941-1943. (40) Palmer, J.; Munro, N. J.; Landers, J. P. Anal. Chem. 1999, 71, 1679-1687. (41) Palmer, J.; Burgi, D. S.; Munro, N. J.; Landers, J. P. Anal. Chem. 2001, 73, 725-731. (42) Cao, C.-X.; He, Y.-Z.; Li, M.; Qian, Y.-T.; Gao, M.-F.; Ge, L.-H.; Zhou, S.-L.; Yang, L.; Qu, Q.-S. Anal. Chem. 2002, 74, 4167-4174. (43) Strausbauch, M. A.; Landers, J. P.; Wettstein, P. J. Anal. Chem. 1996, 68, 306-314. (44) Veraart, J. R.; Gooijer, C.; Lingeman, H. J. High Resolut. Chromatogr. 1999, 22, 183-187.

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In the present study, we developed a novel sample preconcentration technique by using a positively charged sol-gel coating. Sol-gel columns offer many advantages over conventional columns for the separations in GC,48,49 HPLC,50,51 and CE.52,53 In addition, sol-gel-coated capillaries have been employed for the extraction and preconcentration of a wide variety of polar and nonpolar analytes in solid-phase microextraction analysis.54,55 The sol-gel coatings often possess a porous structure and provide higher surface areas, which, in turn, provide efficient analyte extractions from solution.54 In the current study, we combined principles of capillary microextraction55 with those of stacking and focusing techniques to increase the sample concentration sensibility in CZE. On-line capillary microextraction was accomplished by using a positively charged sol-gel coating in a CZE separation column. EXPERIMENTAL SECTION Equipment. All sample preconcentration and CZE experiments were performed on a Bio-Rad BioFocus 3000 capillary electrophoresis system (Bio-Rad laboratories, Hercules, CA) equipped with programmable, multiwavelength UV/visible detector. The BioFocus 3000 operating software system (version 6.00) was used to collect and process the CZE data. A Barnstead model 04741 Nanopure deionized water system (Barnstead/Thermodyne, Dubuque, IA) was used to prepare deionized water, ∼17 MΩ. A homemade gas pressure-operated capillary filling/purging device53 was used for coating the fused-silica capillary. A Microcentaur model APO 5760 centrifuge (Accurate Chemical and Scientific Corp., Westbury, NY) was used for centrifugation of the sol solutions. A Fisher model G-560 Vortex Genie 2 system (Fisher Scientific, Pittsburgh, PA) was used for thorough mixing. A Chemcadet model 5984-50 pH meter (Cole-Palmer Instrument Co., Chicago, IL) equipped with a TRIS-specific pH electrode (SigmaAldrich, St. Louis, MO) was used to measure the buffer and sample pH. Chemicals and Materials. Fused-silica tubing of 50-µm i.d. was purchased from Polymicro Technologies (Phoenix, AZ) for the preparation of sol-gel-coated columns. Sample vials (600 µL), HPLC grade methylene chloride, methanol, and acetonitrile were purchased from Fisher Scientific (Pittsburgh, PA). Tetramethyl orthosilicate (99+%) and trifluoroacetic acid (99%) were purchased from Aldrich (Milwaukee, WI). Tris (hydroxymethyl)aminomethane hydrochloride (reagent grade) and amino acids (DL-alanine, DLasparagine, DL-phenylanlaine, DL-tryptophan) were purchased from (45) Breadmore, M. C.; Boyce, M.; Macka, M.; Avdalovic, N.; Haddad, P. R. Analyst 2000, 125, 799-802. (46) Breadmore, M. C.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2001, 73, 820-828. (47) Breadmore, M. C.; Palmer, A. S.; Curran, M.; Macka, M.; Avdalovic, N.; Haddad, P. R. Anal. Chem. 2002, 74, 2112-2118. (48) Wang D.; Chong, S. L.; Malik, A. Anal. Chem. 1997, 69, 4566-4576. (49) Shende, C.; Kabir, A.; Townsend, E.; Malik, A. Anal. Chem. 2003, 75, in press. (50) Chen, Z.; Uchiyama, K.; Hobo, T. J. Chromatogr., A 2002, 942, 83-91. (51) Guo, Y.; Colon, L. A. Anal. Chem. 1995, 67, 2511-2516. (52) Hayes, J. D. Malik, A. Anal. Chem. 2001, 73, 987-996. (53) Hayes, J. D.; Malik, A. J. Chromatogr., B 1997, 695, 3-13. (54) Chong, S. L.; Wang, D.; Hayes, J. D.; White, B. W.; Malik, A. Anal. Chem. 1997, 69, 3889-3898. (55) Bigham, S.; Medlar, J.; Kabir, A.; Shende, C.; Alli, A.; Malik, A. Anal. Chem. 2002, 74, 752-761. (56) Malik, A.; Electrophoresis 2002, 23, 3973-3992. (57) Liu, Q.; Lin, F.; Hartwick, R. A. J. Chromatogr. Sci. 1997, 35, 126-130.

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Sigma (St. Louis, MO). N-Octadecyldimethyl[3-(trimethoxysilyl)propyl]ammonium chloride and phenyldimethylsilane were purchased from United Chemical Technologies, Inc. (Bristol, PA). Preparation of Sol-Gel Open Tubular CZE Columns with Positive Surface Charge. Sol-gel open tubular C18-TM columns with positive surface charge were prepared according to procedures described by Hayes and Malik.52 Briefly, the sol solution was prepared by dissolving appropriate amounts of two sol-gel precursors [tetramethoxysilane (TMOS) and N-octadecyldimethyl[3-(trimethoxysilyl)propyl] ammonium chloride (C18-TMS)], a deactivation reagent [phenyldimethylsilane (PheDMS)], and a sol-gel catalyst [trifluoroacetic acid (TFA) (containing 5% water)]. A 1-m-long piece of a previously cleaned and hydrothermally treated fused-silica capillary was first sealed at one end using an oxyacetylene flame. The capillary was then filled with the prepared sol solution from the open side, creating a pressurized gas pocket at the sealed end. The filling process was carried out using a homemade filling device53 operated under 40 psi helium pressure. Because of the presence of a pressurized gas pocket at the sealed end of the capillary, the inner surface in this part of the capillary remained untouched by the sol solution. After a 20-min in-capillary residence time, the filling gas pressure was released to allow the pressurized gas pocket to expel the sol solution from the capillary through its open end. The sealed end was the cut open and the capillary was further purged with helium from the previously sealed end, allowing a segment of the capillary at this end to remain untouched by the sol solution. The capillary was then coated, leaving the initially sealed end an uncoated segment (∼25 cm), which was used to create an optical window for on-column UV detection. This step was followed by sealing both ends of the capillary and conditioning it in a GC oven at 150 °C for 2 h. Following thermal treatment, the sealed capillary ends were cut open. The column was purged under 40 psi helium pressure for an additional 30 min and then sequentially rinsed with 100% acetonitrile, deionized water, and the desired running buffer. The UV detection window was created by burning the outside polyimide coating on the undisturbed section of the capillary Preparation of Samples. DL-Alanine, DL-asparagine, DL-phenylanaline, and DL-tryptophan were dissolved in deionized water to make the test samples. The pH values were adjusted by the addition of 0.1 M sodium hydroxide solution. Procedures for Extraction and Preconcentration. After the sol-gel-coated column was installed on the CE system, it was first filled with the running buffer by pressure. The inlet end of the column was then inserted into the sample vial. Samples were hydrodynamically injected for 3 min at 100 psi. Under these conditions, the pH of the sample solution was kept above the pI of the test amino acid to impart a net negative charge to the solute amino acid. Electrostatic interaction between the positively charged sol-gel coating and negatively charged amino acid molecules led to their extraction on the sol-gel column. To show the extraction effect of the sol-gel coating, the sample solution was removed from the column either by purging with deionized water or by reversed electroosmotic flow. After this, the inlet end of the capillary was returned back to the buffer reservoir, and a high electric field was applied. Safety Precautions. The presented work involved the use of various chemicals (e.g., organic solvents, acids, and bases) that

Table 1. Names and Structures of All Sol-Gel Reagents Used in the Fabrication of Columns

can be hazardous to human health and the environment. Handling of these and other chemicals must be done based on instructions provided in the materials safety data sheets. This is especially important in handling organic solvents such as methanol and acetonitrile that may have adverse health effects due to improper handling. Use of safety goggles and protective gloves, working under fume hoods, and other safety procedures should be strictly followed in handling these solvents. Appropriate safety measures should also be implemented during high-voltage operation of the capillary electrophoresis system (e.g., use of an automatic locking mechanism) to isolate the high-voltage areas of the CE system while the system is in operation. RESULTS AND DISCUSSION Table 1 lists the names and structures of sol-gel solution ingredients used in the present study. The sol-gel co-precursor, C18-TMS, possesses a number of important structural features. The methoxysilyl groups in the C18-TMS and in TMOS are solgel active, and they participate in the formation of the sol-gel polymeric network through hydrolysis and polycondensation reaction.52,53,56 To generate a positively charged coating surface, poly(diallyldimethylammonium chloride),57 chitosan,58 and cryptandcontaining polysiloxane59 have been used by different research groups. In the present study, C18-TMS was used. Its quaternary amine moiety is responsible for the positive charge on the solgel coating, which not only provides the basis for electrostatic (58) Sun, P.; Landman, A.; Hartwick, R. A. J. Microcolumn Sep. 1994, 6, 403407. (59) Huang, M.; Yi, G.; Bradshaw, J. A.; Lee, M. L. J. Microcolumn Sep. 1993, 5, 199-205. (60) (a) Mathews, C. K.; van Holde, K. E.; Ahern, K. G. Biochemistry, 3rd ed.; Benjamin Cummings: San Francisco, CA, 2000; p 128. (b) Reference 60a, p 46.

interaction between sol-gel coating and analytes in samples but also supports reversed electroosmotic flow in the CZE column. In addition, the octadecyl chain, like a pendant group, is capable of providing the chromatographic interactions. Mechanism of Extraction on Positively Charged Sol-Gel Coating. The mechanism of extraction in a CZE column with a positively charged sol-gel C18-TMS coating is based on the electrostatic interaction. The structures of amino acid test solutes used in the current study together with their disassociation constants are shown in Table 2. Because of their zwitterionic properties, amino acids can bear a net positive charge, a negative charge, or be electrically neutral in different pH environments. At pH values above its isoelectric point, an amino acid will possess a net negative charge. Because of the electrostatic interactions, the negatively charged species get extracted by a positively charged C18-sol-gel coating on the inner surface of the CZE columns. The direction and magnitude of EOF in the positively charged C18-sol-gel column will be determined by two competing factors: (a) anodic EOF generated from the positively charged C18sol-gel coating and (b) cathodic EOF resulting from the deprotonated residual silanol groups. In the current study, EOF in all the C18-sol-gel columns was characterized by the neutral marker, DMSO. The C18-sol-gel columns generated a strong anodic EOF. In addition, the C18-sol-gel-coated inner surface of the fused-silica capillary possesses a roughened texture,52 responsible enhanced surface area and a favorable environment for solute-stationary phase interaction. Unlike Quirino and Terabe,31-35 who used sweeping of the analytes from the dilute sample solutions by employing micelles, we used an acidic buffer to elute the extracted amino acid analytes from the sol-gel surface coating and collect them in the form of Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

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Table 2. Structures and Some Physical Properties of Analytes in Current Study

a

Data were calculated according to the procedure described in ref 60b.

a compressed zone. This was accompolished in free solution CE, without requiring any micellar solution. Extraction and Preconcentration of Amino Acids by SolGel Columns. Based on reported stacking and sweeping methods,2,3,8,12,13,31-38 the maximum volume of the sample that could be injected into the CE system is the volume of the column itself. In the case of very dilute samples, even such a sample volume may not be enough to enrich a detectable amount of the analyte after preconcentration. In the sample preconcentration technique described in this work, the sample volume that can be used for analyte enrichment is not limited to one column volume. It allows for the injection of multiple column volumes of sample. By continuously passing the sample solution for an extended period through the sol-gel-coated capillary with enhanced surface area and appropriate surface charge, the analytes can be extracted from a large volume of the sample. The pH of the sample solution should be carefully chosen, so that the zwitterionic analyte of interest assumes a net electric charge that is opposite to the surface charge on the sol-gel-coated column. The extracted analytes can be further focused into a narrow band by manipulating the buffer pH. Figure 1 shows the enrichment of the tryptophan sample (pI ) 5.9) through extraction by the sol-gel coating and postextraction focusing of the extracted analytes. After an extended period of sample injection (3 min), the sample matrix was pushed out of the column by a flow of deionized water (A) or the running buffer (B). After this, a high voltage was applied and CZE was performed using an acidic buffer (50 mM Tris-HCl, pH 2.22). No sample peak could be detected, if a voltage was applied after rinsing the column with the low-pH buffer (Figure 1B) capable of washing the extracted analytes off the sol-gel coating. On the other hand, when the sample matrix was removed by deionized water (pH 7) (Figure 1A), the extracted amino acid molecules remained attached to the positively charged capillary surface due to 222 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

Figure 1. Extraction of tryptophan on the sol-gel column. Experimental conditions: sol-gel-coated column 75 cm × 50 mm; the effective length of the column is 70.4 cm; mobile phase 50 µM TrisHCl (pH 2.22); injection 3 min at 100 psi; running voltage V ) -15 kV; wavelength of UV detector 200 nm; sample 10 µM tryptophan (pH 7.67). After the injection, the sample matrix was removed by (A) water 3 min at 100 psi, (B) mobile phase 3 min at 100 psi, and (C) A blank run for trace A.

electrostatic attractive forces between negatively charged solute ions and the positively charged capillary surface. Desorption of the extracted amino acid and its focusing into a narrow zone was accomplished by using a high electric field (V ) -15 kV) and a low-pH buffer, as illustrated in Figure 2. The whole procedure consisted of three steps: (a) extraction, (b) removal of the sample matrix and (c) desorption and enrichment of the extracted analyte

acids than the preconcentrated ones. From Figure 3, it is evident that the sample is greatly preconcentrated when analyzed on a sol-gel column. For example, the sol-gel column preconcentrated a 10 µM tryptophan sample and gave a peak height of more than 6 mAU in Figure 3d (A). While with the uncoated column and with conventional mode of hydrodynamic injection, no peak was obtained for 10 µM tryptophan in Figure 3d (C). Using the uncoated capillary, we also ran a tryptophan sample of 1000 times higher concentration (10 mM). As a result, a peak with a little more than 10 mAU in height was obtained shown in Figure 3d (B). Based on these results, the limit of detection values (LOD, S/N ) 3) were calculated and the results are listed in Table 3. We can see that, with the C18-sol-gel-coated column and the presented preconcentration method, the LODs of these amino acids were lowered significantly. The most effective preconcentration result was obtained for sample alanine. Its limit of detection value (S/D ) 3) was reduced from 10.2 mM on an uncaoted column to 139 nM on the sol-gel-coated column with the preconcentration method, which corresponds to an enrichment factor of more than 73 000 times. To calculate the sensitivity enhancement factor (SEF), we employed peak heights as well as the peak areas using the following equation.31

Figure 2. Preconcentration and focusing of zwitterionic analytes on a positively charged sol-gel column. (1) Sample was passed through the column under helium pressure (e.g., 100 psi for 3 min). (2) Sample matrix was removed from the column by water under helium pressure (e.g., 100 psi for 3 min). 3(3) Application of high voltage (e.g., V ) -15 kV). (4) The acidic running buffer desorbed the extracted analytes and carried them to the detector.

using a low-pH running buffer and a high electric field. In the first step, the column was filled with sample solution. Negatively charged analytes were extracted on the positively charged inner surface of the sol-gel column. This process was followed by the removal of sample matrix by deionized water. In the third step, a high negative voltage (-15 kV) was applied between the ends of the sol-gel capillary, using pH 2.22 Tris-HCl (50 mM) as the running background electrolyte. The cathode was on the inlet side and anode on the outlet side. Under the effect of an electric field, an anodic EOF was generated in the CZE capillary with positively charged sol-gel coating. Once the acidic running buffer (50 mM Tris-HCl pH 2.22) came into contact with the front of the extracted solute zone, it reversed the net charge of the amino acid molecules, providing a repulsive mechanism for their desorption from the capillary surface. EOF moved the desorbed analyte molecules forward, gradually desorbing more and more amino acid molecules and focusing them into a narrow zone. Figure 3 shows the electropherograms for four amino acids preconcentrated from a 10 µM solution by a positively charged sol-gel column using an extended injection time (3 min). To show the enrichment effect, two samples of the same amino acid at two different concentration levels were analyzed on an uncoated fusedsilica column with the identical dimensions using conventional injections. One of the samples had the same concentration as the one used in the preconcentration experiment, and the other sample had at least 1000-fold higher concentrations of the amino

SEF ) peak parameter obtained with preconcentration × peak parameter obtained without preconcentration dilution factor The SEFs for each analyte are presented in Table 4. The SEF values were different with respect to different samples. In addition, the observation that the migration time of the sample running in an uncoated column was much longer than that of the obtained in sol-gel-coated column is due to the different electroosmotic flow in coated and uncoated columns. When a low-pH acidic buffer is used as the mobile phase in an uncoated column, a significant portion of the deprotonated silanol groups on the fused-silica surface gets protonated by the acidic mobile phase, resulting in a decreased of EOF. Based on the experiment with a neutral marker, DMSO, when the pH value of the running buffer was 2.22, the electroosmotic mobility in the untreated fusedsilica column was 1.02 × 10-4 cm2/V‚s. On the other hand, an acidic running buffer practically did not influence the positive charge on the sol-gel surface of the column. This is explained by the fact that dissociation of the quaternary amine group anchored to the surface coating practically remains unaffected by this pH change. Based on the result of the neutral marker, DMSO, the electroosmotic mobility obtained on the sol-gel-coated column using the same buffer was reserved and had a value of 4.02 × 10-4 cm2/V‚s., which is ∼4 times greater than the EOF in the uncoated column under identical operating conditions. Preconcentration of Tryptophan with the Removal of Sample Solution by Reversed EOF. Remarkably high sample enrichment factors were achieved using the sample preconcentration method described above. However, it might be possible to further improve this SEF, if we consider the following. Deionized water was used during the sample matrix removal step after extraction for an extended period of time. Undoubtedly, this led to the elution and, therefore, loss of a portion of the analytes Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

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Figure 3. Effect of sol-gel column on sample preconcentration. The concentrations of the analytes are listed in Table 3. Sample pH values were 7.7. Electropherograms A in (a-d) were obtained using sol-gel-coated column (75 cm × 50 µm). The effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH 2.22). Hydrodynamic injection for 3 min at 100 psi. UV detection at 200 nm. Sample matrix was removed by water for 3 min at 100 psi, running voltage V ) -15 kV. Electrophoregrams B and C in (a-d) were performed with an uncoated column (75 cm × 50 µm) with the same mobile phase. The effective length of the column is 70.4 cm. Hydrodynamic injection for 10 s psi. running voltage V ) +15 kV. UV detection at 200 nm.

extracted on the sol-gel column. To prevent this loss, the following experiment was designed. The procedure is illustrated schematically in Figure 4. The sample was passed through the sol-gel column for an extended injection period (3 min at 100 psi). Most of the anions were extracted by the positively charged sol-gel coating. Next, a high voltage (+15 kV) was applied with the anode in the inlet side and the cathode in the outlet end. In this stage, a number of processes occurred. One of them is that 224 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

the anions in sample solution migrated to the anode while cations migrated toward the cathode side by electrophoretic flow. In addition, the electroosmotic flow was generated toward the capillary inlet. EOF, being significantly stronger than the electrophoreitc flow of the ions, forced the sample matrix to move toward the capillary inlet and go out of the capillary from the inlet end. At the same time, because the running buffer was acidic, it eluted extracted samples from the coating and took them toward

Table 3. Sample Extraction and Preconcentrations Effecta Aa sample

Bb

Cb

concn, LOD, nM concn, LOD, µM concn, LOD, nM µM (S/N ) 3) mM (S/N ) 3) µM (S/N ) 3)

alanine asparagine phenylalanine tryptophan

10 10 10 10

139 98 141 115

100 50 10 10

10,170 864 195 203

10 10 10 10

n/ac n/a n/a n/a

a A: column (75 cm × 50 µm) with a positively charged sol-gel coating; the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH 2.22). Samples had been injected hydronamically for 180 s at 100 psi. Running voltage V ) -15 kV. Wavelength of UV detector 200 nm. b B and C: uncoated column (75 cm × 50 µm); the effective length of the column is 70.4 cm; mobile phase 50 mM TrisHCl (pH 2.2). Samples had been injected hydronamically for 10 s*psi. Running voltage V ) +15 kV. Wavelength of UV detector 200 nm. c n/a, not applicable.

Table 4. Sensitivity Enhancement Factors Obtained by C18-Sol-Gel Coated Columns Using Amino Acids as Test Solutesa SEF by method 1

SEF by method 2

sample

by height

by area

by height

by area

alanine asparagine phenylalanine tryptophan

55 374 3 596 995 928

61 048 1 817 1 730 1 496

153 770 16 773 11 248 6 326

66 782 21 427 63 469 10 754

a

Operation conditions are as same as shown in Figure 3 and Figure

5.

the inlet of the column. During this process, the analyte was focused at the boundary of the sample solution and the running buffer. The current was observed carefully to decide the time when the voltage polarity needed to be reversed. While the column was filled with sample solution, the current was low due to the low conductivity of the dilute sample solution. With more and more sample matrix was pushed out of the column, more and more running buffer filled in the column, and the current increased because of the higher conductivity of the media filling the column. Just before the current soared up quickly, the polarity of the voltage was reversed. The focused sample zone was carried by the resulting electroosmotic flow toward the outlet of the capillary and detected by the UV/visible detector. It can be noted that instead of mechanically rinsing the column with deionized water, a reversed electroosmotic flow was applied in conjunction with a low-pH buffer. This moved the liquid sample matrix from outlet to inlet of the column. Unlike the water-rinsing procedure described in the previous section, this method prevented the loss of analytes that have already been extracted on the sol-gel column. The sample preconcentration results obtained by this method are shown in Figure 5. The results show that, with this method, (hereafter referred as method 2), the sample preconcentration effect is more significant even compared with the results obtained by the method we described in the previous section (hereafter referred as method 1). This indicates that, in method 1, when the sample matrix was removed by water, some analytes were eluted by the water. However, with method 2, the sample matrix was pushed out of the capillary by the reversed electroosmotic flow. The amount of lost analytes was greatly reduced.

Figure 4. Method 2 for the preconcentration of zwitterionic analytes on the positively charged sol-gel column. (1) Sample was passed through the column under helium pressure (e.g., 100 psi for 3 min). (2) High voltage was applied (V ) +15 kV), which caused the redistribution of ions inside the column. (3) Under the reversed EOF, the sample matrix was removed from the capillary, and the positively charged analytes were focused at its anodic end. (4) The polarity of the electric field was switched to V ) -15 kV, and the focused analytes were carried to the detector.

Figure 6 represents experimental data showing the preconcentration of alanine by method 2 (trace A), which was compared with the blank run (trace B). This experiment was designed to verify whether the peaks obtained by the described preconcentration methods were artifacts of system peaks. The absence of such a peak in the blank run clearly indicates that the peak in (A) is not a system peak and confirms the real possibility of performing sample preconcentration using the described methods. Unlike method 1 (which includes extraction and focusing operations only), method 2 includes an additional step allowing electrophoretic migration of the extracted charged analytes. This provides a real opportunity to achieve separation of the extracted analytes by method 2. Figure 7 highlights this point and illustrates the practical utility of method 2 by providing an example of online preconcentration and separation of two amino acids: tryptophan and asparagine. As can be seen in Figure 7, the two preconcentrated amino acids are more than baseline separated with a wide gap between them. The LOD (S/N ) 3) by method 2 was calculated and is listed in Table 5. Comparing the data listed in Table 3 and Table 5, we can see the LODs (S/N ) 3) for the same concentration samples were greatly reduced with method 2. For example, with tryptophan Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

225

Figure 5. Effect of sol-gel column on sample preconcentration by method 2. The concentrations of the analytes are listed in Table 5. Sample pH values were 7.7. Electropherograms A in (a-d) were obtained using a sol-gel-coated column (75 cm × 50 µm); the effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH 2.22). Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V ) -15 kV. UV detection at 200 nm. Electrophoregrams B and C in (a-d) were performed with an uncoated column (75 cm × 50 µm) with the same mobile phase; the effective length of the column is 70.4 cm. Hydrodynamic injection for 10 s psi. Running voltage V ) +15 kV. UV detection at 200 nm.

as the test sample, method 2 allowed to lower the LOD to 24.5 nM from 115 nM that was achieved by method 1. This is an enhancement in sensitivity of more than 5 times. Compared with the results obtained from a bare fused-silica column, the sensitivity enhancement factors were calculated and are shown in Table 4. It is shown that the best results for both preconcentration methods belong to the same amino acid, alanine. This can be explained by its smaller size compared with other amino acid samples. According to Beer-Lambert law, the amount of absorbed light is proportional to the product of sample concentration and its molar absorptivity coefficient. Since the inner surface area of the column is constant, the smaller the analyte, the more amino acids can be extracted on the same area of the sol-gel column. After they are desorbed from the column, the smaller molecule possesses a 226 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

higher concentration. From Table 4, we note two important points. (a) Both methods greatly increased the detection sensitivity, and (b) method 2 is more effective compared with method 1 because it reduced the sample loss during the sample matrix removal step. The reproducibility of the sample preconcentration methods was examined by a series of experiments and shown in the terms of the relative standard deviation (RSD) of migration time and peak height. Table 6 shows the experimental and calculation results. Quite good repeatability in migration times was obtained with both preconcentration methods for all test analytes. The RSD values in terms of migration time are no more than 3.7%. RSD values in the range of 3.8-28% were obtained for peak height repeatability. The presented data reveal that, in both cases, method 2 provided significantly better repeatability than method 1. The

Figure 6. Comparison between the effect of sol-gel column on sample preconcentration by method 2 and a blank run with the same method. Trace A is 10 µM alanine. Sample pH value was 7.7. Trace B is a blank run. Sol-gel-coated column (75 cm × 50 µm), the effective length of the column was 70.4 cm; mobile phase 50 mM Tris-HCl (pH 2.22). Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V ) -15 kV. UV detection at 200 nm. Table 5. Sample Extraction and Preconcentrations Effect Aa sample alanine asparagine phenylalanine tryptophan

Bb

Cb

concn, LOD, nM concn, LOD, µM concn, LOD, µM µM (S/N ) 3) mM (S/N ) 3) µM (S/N ) 3) 10 10 10 10

60.7 47.3 23.3 24.5

100 50 10 10

10,170 864 195 203

10 10 10 10

n/ac n/a n/a n/a

A: column (75 cm × 50 µm) with a positively charged sol-gel coating; the effective length of the column is 70.4 cm; mobile phase 50 mM Tris-HCl (pH 2.22). Samples had been injected hydronamically for 180 s at 100 psi. Running voltage V ) -15 kV. Wavelength of UV detector 200 nm. b B and C: uncoated column (75 cm × 50 µm); the effective length of the column is 70.4 cm; mobile phase 50 mM TrisHCl (pH 2.22). Samples had been injected hydronamically for 10 s psi. Running voltage V ) +15 kV. Wavelength of UV detector 200 nm. a n/a, not applicable. a

sample matrix removal procedure in method 1 probably caused the inferior reproducibility for some solutes in method 1. CONCLUSION For the first time, we have shown an on-column extraction and preconcentration effect offered by a positively charged sol-gel column in capillary zone electrophoresis. Using a positively charged sol-gel coating, a 150 000-fold enrichment effect was obtained for alanine. The newly developed methods do not limit the volume of the injected sample and they do not require modification of a standard CE system to achieve the preconcentration effect. They allow large-volume injection of the sample for an extended period of time and are very effective in enriching trace concentrations of zwitterionic solutes. A large sensitivity

Figure 7. Effect of sol-gel column on an amino acid mixture sample preconcentration by method 2. Sample pH values were 7.7. Electropherograms were obtained using sol-gel-coated column (75 cm × 50 µm); the effective length of the column was 70.4 cm; mobile phase 50 µm Tris-HCl (pH 2.22)/ACN 50/50. Hydrodynamic injection for 3 min at 100 psi. Sample matrix was removed by reversed EOF. Running voltage V ) -15 kV. UV detection at 200 nm. Electropherogram A was obtained with a preconcentrated and separated amino acid mixture, in which peak a is tryptophan and peak b is asparagine. Electropherogram B was obtained with a blank. Table 6. Repeatability Data for the Preconcentration by C18-Sol-Gel-Coated Column Using Amino Acids as Test Solutes method 1a

method 2b

av RSD RSD av RSD av tR RSD (%) height (%) av tR (%) height (%) analytes (min) (n ) 4) (mAU) (n ) 4) (min) (n ) 4) (mAU) (n ) 4) alanine asparagine phenylalanine tryptophan

4.37 4.36 4.44

2.77 1.32 1.11

5.80 5.73 7.57

4.45

2.27

6.61

a

27.70 17.67 13.81 12.08 3.83 14.43 9.18

9.56

1.62 0.95 1.73 3.70

20.80 26.53 94.04

10.10 9.45 5.91

86.26

15.93

b

Experimental conditions: same as in Figure 3. Experimental conditions: same as in Figure 5.

enhancement factor on the order of 105 was obtained in the study. Further sensitivity enhancement should be possible in a number of ways by (a) performing the preconcentration step without the removal of sample buffer, (b) using thicker sol-gel coatings or monolithic beds, and (c) derivatizating the amino acids with proper derivatization reagents. ACKNOWLEDGMENT This work was partly funded by a subcontract from a grant by the U.S. Naval Office (N00014-98-1-0848).

Received for review April 24, 2003. Accepted September 30, 2003. AC0301696 Analytical Chemistry, Vol. 76, No. 1, January 1, 2004

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