Anal. Chem. 2000, 72, 3038-3042
Coupling Photochemical Reaction Detection Based on Singlet Oxygen Sensitization to Capillary Electrochromatography Jim Dickson, Matt Odom,† Frank Ducheneaux, Jackie Murray, and Robert E. Milofsky*
Department of Chemistry, Fort Lewis College, Durango, Colorado 81301
Despite the impressive separation efficiency afforded by capillary electrochromatography (CEC), the detection of UV-absorbing compounds following separation in capillary dimensions remains limited by the short path length (575 µm) through the column. Moreover, analytes that are poor chromophores present an additional challenge with respect to sensitive detection in CEC. This paper illustrates a new photochemical reaction detection scheme for CEC that takes advantage of the catalytic nature of type II photooxidation reactions. The sensitive detection scheme is selective toward molecules capable of photosensitizing the formation of singlet molecular oxygen (1O2). Following separation by CEC, UV-absorbing analytes promote groundstate 3O2 to an excited state (1O2) which reacts rapidly with tert-butyl-3,4,5-trimethylpyrrolecarboxylate, which is added to the running buffer. Detection is based on the loss of pyrrole. The reaction is catalytic in nature since one analyte molecule may absorb light many times, producing large amounts of 1O2. The detection limit for 9-acetylanthracene, following separation by CEC, is ∼6 × 10-9 M (S/N ) 3). Optimization of the factors effecting the S/N for four model compounds is discussed. Over the past decade, developments in biological, pharmaceutical, and combinatorial technologies have increased the demand for high-efficiency separations.1 These demands have been met, in part, by electrophoretic separation methods including capillary zone electrophoresis (CE), capillary gel electrophoresis (CGE), and micellar electrokinetic capillary chromatography (MEKC) which have been used for the separation of small inorganic and organic ions as well as macromolecules including proteins and DNA restriction fragments.2-5 More recently, capillary electrochromatography (CEC) has emerged as a powerful variation of * Corresponding author: (tel) 970-247-7467; (fax) 970-247-7567; (e-mail) milofsky•
[email protected]. † Present address: Department of Chemistry, Colorado State University, Fort Collins, CO 80523. (1) Czarnik, A. W. Anal. Chem. 1998, 70, 378A-386A. (2) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (3) Joregenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 209-216. (4) Beale, S. C. Anal. Chem. 1998, 70, 279R-300R. (5) Larive, C. K.; Lunte, S. M.; Zhong, M.; Perkins, M. D.; Wilson, G. S.; Gokulrangan, G.; Williams, T.; Afroz, F.; Schoneich, C.; Derrick, T. S.; Middaugh, C. R.; Bogdanowich-Knipp, S. Anal. Chem. 1999, 71, 394R396R.
3038 Analytical Chemistry, Vol. 72, No. 14, July 15, 2000
electrokinetic separations.6-8 The ability to separate hydrophobic molecules in the absence of surfactants makes CEC more attractive than MEKC, particularly for detection schemes where impurities in surfactants lead to high background signals. Despite the inherent advantages of CEC, detection of most molecules at the nano-to-picoliter volume level remains a significant obstacle with respect to widespread acceptance of CE-based separations.9 For example, the detection of even the strongest chromophores (using analyte preconcentration by stacking) by UV absorbance is limited to 10-6-10-7 M by the short path length (5-75 µm) through the column.10,11 Nonetheless, UV absorbance remains the most commonly used detection method in CE.4,9 Several attempts have been made to develop selective and sensitive detection strategies for CEC including laser-induced fluorescence detection (LIF) of PAHs,12 mass spectrometry of DNA adducts13 and peptides,14 and NMR for chemical, pharmaceutical, biological, and environmental samples.15-16 For analytes that lack a strong chromophore, detection in CE has been carried out by electrochemical methods,17,18 chemiluminescence,19-23 phosphorescence,11,24 and energy transfer.25-27 Many of these (6) Jorgenson, J. W.; Lukacs, K. D. J. Chromatogr. 1981, 218, 209. (7) Colon, L. A.; Guo, Y.; Fermier, A. Anal. Chem. 1997, 69, 461A-467A. (8) Dadoo, R.; Zare, R. N. Anal. Chem. 1998, 70, 4787-4792. (9) Culbertson, C. T.; Jorgenson, J. W. Anal. Chem. 1998, 70, 2629-2638. (10) Kok, S. J.; Koster, E. H. M.; Gooijer, C.; Velthorst, N. H.; Brinkman, U. A. Th.; Zerbinati, O. J. High Resolut. Chromatogr. 1996, 19, 99-104. (11) Kuijt, J.; Brinkman, U. A. T.; Gooijer, C. Anal. Chem. 1999, 71, 13841390. (12) Yan, C.; Dadoo, R.; Zhao, H.; Zare, R. N.; Rakestraw, D. J. Anal. Chem. 1995, 67, 2026-2029. (13) Ding, J.; Vouros, P. Anal. Chem. 1997, 69, 379-384. (14) Schmeer, K.; Behnke, B.; Bayer, E. Anal. Chem. 1995, 67, 3656-3658. (15) Wu, N.; Peck, T. L.; Webb, A. G.; Magin, R. L.; Sweedler, J. V. Anal. Chem. 1994, 66, 3849-3857. (16) Pusecker, K.; Schewitz, J.; Gfrorer, P.; Tseng, L.; Albert, K.; Bayer, E. Anal. Chem. 1998, 70, 3280-3285. (17) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1987, 59, 1762-1766. (18) Holland, L. A.; Lunte, S. M. Anal. Commun. 1998, 35, 1H-4H. (19) Dadoo, R.; Colon, L. A.; Zare, R. N. J. High Resolut. Chromatogr. 1992, 15, 133-135. (20) Ruberto, M. A.; Grayeski, M. L. Anal. Chem. 1992, 64, 2758-2762. (21) Wu, N.; Huie, C. W. J. Chromatogr. 1993, 634, 309-315. (22) Sokolowski, A. D.; Vigh, G. Anal. Chem. 1999, 71, 5253-5257. (23) Yeung, E. S. In Advances in Chromatography; Brown, P. R., Grushka, E., Eds.; Marcel Dekker: New York, 1995; Vol. 35, Chapter 1. (24) Forbes, G. A.; Nieman, T. A.; Sweedler, J. V. Anal. Chim. Acta 1997, 347, 289-293. (25) Milofsky, R. E.; Malberg, M. G.; Smith, J. M. J. High Resolut. Chromatogr. 1994, 17, 731-732. (26) Milofsky, R.; Spaeth, S. Chromatographia 1996, 42, 12-16. (27) Milofsky, R.; Bauer, E.; J. High Resolut. Chromatogr. 1997, 20, 638-642. 10.1021/ac0000978 CCC: $19.00
© 2000 American Chemical Society Published on Web 06/03/2000
schemes provide an added degree of selectivity and, in some instances, up to 3 orders of magnitude improvement in sensitivity over conventional UV absorbance detection. Unfortunately, several of these detection schemes require postcolumn reactors which, in CE/CEC, are difficult to fabricate and lead to zone broadening. Because of the simplicity, ease of implementation, wide applicability, low cost, and commercial availability of UV detectors for CE/CEC, we are interested in developing methods to improve detection limits (LODs) using UV absorption without sacrificing the high efficiency of CEC separations. There are a number of ways of improving UV absorbance detection in CE including the use of rectangular capillaries,28 z-cells29 and bubble cells.30-33 Such approaches have been demonstrated to improve the LOD by up to 20 times with minimal zone broadening (
