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IUPAC Prize for Young Chemists - 2000
Honorable Mention

 

 

Joselito P. Quirino receives one of five Honorable Mention awards associated with the IUPAC Prize for Young Chemists, for his Ph.D. thesis work entitled "On-line concentration of analytes in electrokinetic chromatography"

Current address (at the time of application)

Faculty of Science
Himeji Institute of Technology Kamigori
Hyogo, Japan 678-1297

From April 2000
Department of Chemistry
Stanford University
Stanford, CA, U.S.A. 94305-5080

Tel.: +81-791580171 (Japan)
Fax: +81-791580493 (Japan)
E-mail: [email protected] or [email protected]

Academic degrees

  • Ph.D., Himeji Institute of Technology, Japan; September 1999; Analytical Chemistry
  • M.Sc. Himeji Institute of Technology, Japan; March 1998; Analytical Chemistry
  • B.Sc. University of the Philippines Manila; March 1992; Industrial Pharmacy

Ph.D. Thesis

Title On-line Concentration of Analytes in Electrokinetic Chromatography
Advisers Prof. Shigeru Terabe
Thesis Committee Prof. Shinichi Nakatsuji, Department of Material Science, Faculty of Science; Prof. Tadashi Okuyama, Department of Material Science, Faculty of Science; Prof. Takashi Iyanagi, Department of Life Science, Faculty of Science; Assoc. Prof. Koji Otsuka, Department of Material Science, Faculty of Science; Prof. Takeshi Hirokawa, Department of Applied Physics and Chemistry, Faculty of Engineering, Hiroshima University.

Essay

Scientists in various fields have been delighted at the ability of electrokinetic chromatography (EKC) - a mode of capillary electrophoresis (CE) - to solve many chemical analysis problems and to complement other separation methods like gas chromatography and high performance liquid chromatography. Applications are in the areas of toxicology, environmental pollution, pharmacokinetics, clinical diagnosis, cell composition, to name a few. Notable aspects of EKC include high efficiencies, technical simplicity, applicability to most analytes, requires small amounts of samples and reagents, and miniaturization (EKC on chips). The basic experimental set-up consists of a narrow bore capillary (e.g., 50 mm i.d.), a voltage delivery device (up to 30 kV), a detector equipped with data acquisition and analysis tools, and sample and separation solution reservoirs. In addition to the aqueous buffer used in free solution CE or capillary zone electrophoresis (CZE), a major component called pseudostationary phase (PS) is added. Separation principle in EKC is similar to that of classical chromatography, which is analyte partitioning between the PS (e.g., micelle) and the aqueous phase. The electrokinetic phenomenon, including electrophoresis and electroosmosis, is the means of transporting the PS and analytes inside the capillary. One great concern, however, is the low concentration sensitivity in the ppm levels as a consequence of the short optical pathlength for on-capillary photometric detection and the small volume of sample that can be injected. This hinders application to trace analysis without several time consuming off-line pre-concentration steps.

Powerful laser induced fluorescence and electrochemical detectors can improve detection sensitivity several orders but are not widely applicable and very expensive for an ordinary laboratory. A more practical and easy way is the 'chemical approach' or to concentrate the analytes on-capillary or on-line. This is done by manipulating the composition of the sample and background solutions together with simple injection procedures without alteration of present commercial instrumentation. For example, transient isotachophoresis and sample stacking have provided at most hundred-fold increases in detector response. Here, new and general approaches to concentrate analytes in EKC inside the capillary are then presented. Detection sensitivity with photometric detection was improved from ten to almost a million-fold. The proposed techniques will eventually widen the applicability of EKC in trace analysis. Likewise, sound understanding of the narrowing mechanisms involved opened possibilities for elucidating the behavior of analyte zones in CE.

A novel phenomenon termed as 'sweeping' was introduced [J.P. Quirino, S. Terabe, Science 282, 465 (1998)] which, in theory, predicts an almost unlimited improvement in detection sensitivity for analytes having high affinities toward the PS: 5000-fold improvements have been demonstrated experimentally (equation 1).
(1)

Where Csweep and Cinj are the concentrations of the analyte zones after sweeping and during sample introduction, respectively. The retention factor, k, is directly related to the affinity of the analyte toward the PS. The above equation is useful for both neutral and charged analytes, making the sweeping technique a universal approach to on-line concentration as long as the k is reasonably high. Sweeping results when the PS, which penetrates the sample zone entraps and accumulates the analytes. It is analogous to using a broom (PS) to carefully carry along grains of rice (analyte) scattered on the floor. The sample is prepared in a matrix that is free of the PS and has a conductance similar to or higher than that of the background solution. Environmentally and biologically important compounds such as phenoxyacid herbicides, polycyclic aromatic hydrocarbons, monocyclic aromatic amines, alkyl phenyl ketones, dialkyl phthalates, alkaloids, and steroids have been successfully concentrated using micelles, microemulsions, and a charged cyclodextrin as PS.

Sample stacking techniques for neutral analytes have also been developed. Sample stacking, which was originally developed for the on-line concentration of charged analytes in capillary zone electrophoresis, is defined as the movement of ions across a concentration boundary that separates regions of high and low field strengths. Since the electrophoretic velocity in the high field strength zone (lower conductivity sample zone) is higher compared to the low field zone (higher conductivity separation zone), ions will slow down once it reaches the boundary causing the narrowing of zones. Since neutral analytes are unaffected by an enhanced field, sample stacking was thought to be not amenable for neutral analytes in EKC. However, by careful manipulation of sample matrix and some simple tricks, sample stacking was applied to neutral analytes as well. In EKC, the PS will provide the neutral analytes effective electrophoretic velocities necessary for the approach. Many environmentally relevant phenols, dioxins, alkylphenyl ketones, phenylurea herbicides, and steroids have been successfully concentrated using monomeric and monomolecular micelles. On top of this, sweeping and the different sample stacking techniques developed were found to be orthogonal methods, the sample stacking techniques are more useful for low to moderately high k analytes while sweeping is for high to very high k analytes.

Finally, a novel method that combines two on-line concentration techniques, namely, sample stacking with electrokinetic injection in the CZE mode and sweeping, afforded the detection of positively chargeable analytes in the parts per trillion levels with very high average plate numbers (e.g., 4.4 x 105). This is lowest concentration level reported by direct photometric detection in CE, which translates to detection improvements approaching a million-fold. The main idea is to selectively inject by electrokinetic injection as many molecules as possible from a very dilute sample solution. This creates long concentrated zones of cationic species, which is then focused further by sweeping. Several drugs (e.g., b-adrenergic blockers, tetracycline, and laudanosine), seven environmentally important monocyclic aromatic amines, and two benzidines have been successfully analyzed. At the end, simple, faster, sensitive, reproducible, and cheap real-world trace analysis can be achieved by EKC with the help of the above on-line concentration techniques.


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