Report
Milos Novotny Department of Chemistry Indiana University Bloomington, Ind. 47405
Microcolumn Separation Methods and Their Ancillary Techniques
From Aug. 25 to Aug. 28, 1982, 24 experts from Japan and the U.S., joined by four participants from other countries and four in dustrial observers, met in Honolulu, Hawaii, to discuss new directions in micro-HPLC and re lated analytical tech niques. The U.S. Japan joint seminar on "Microcolumn Separa tion Methods and Their Ancillary Tech niques," coordinated jointly by Daido Ishii (Nagoya University, Nagoya, Japan) and Milos Novotny, was orga nized to bring together scientists from industry and academia to exchange in formation on the rapidly advancing fields of miniaturized instrumentation in modern chromatography, spectros copy, and electrochemical detection. The first U.S.-Japan joint seminar in the field of chromatography, held in June 1978 in Boulder, Colo., dealt with general aspects of HPLC.
recently reported by Giddings et al. ( i ) . T h e results obtained by both groups have yield ed column efficiencies substantially lower than predicted. There was general agreement that, at least for the time being, mi crobore and packedcapillary columns present a considerably more promising route to micro-HPLC than do open-tubular col umns. In fact, the results obtained by several laboratories with slurrypacked fused-silica or glass capillaries are noteworthy. Y. Hirata and K. Jinno (Toyohashi University of Tech nology, Toyohashi, Japan) and F. J. Yang (Varian Instruments Division, Walnut Creek, Calif.) elaborated on how to pack and handle such columns (2). The reduced plate-height values obtained with such columns ap proached theoretical values for both 3- and 5-μιη particles, providing efficien cies above 100 000 theoretical plates with reasonable analysis times. Appli cations to complex samples such as coal-derived fluids (Hirata and Jinno) and urinary steroid metabolites (Nov otny) were shown. In addition, several applications of microbore columns (3) to the separations of synthetic poly mers were demonstrated by S. Mori (Mie University, Tsu-shi, Japan). Packing small particles into col umns with small diameters was dis cussed. In comparing packing of mi crocolumns and conventional col umns, R. Brownlee (Brownlee Labora tories, Santa Clara, Calif.) described
U.S.-Japan Joint Seminar
At the 1982 seminar, recent ad vances in microcolumn technology were discussed in relation to instru mental design and sampling and de tection in nanoliter volumes. Novel approaches to solute detection are be coming feasible because of the ex tremely low flow rates encountered with microcolumn techniques; these include laser-based detectors, liquid chromatography/mass spectrometry (LC/MS), microcolumn chromatogra phy / F T - I R spectroscopy, flame- and plasma-based detection devices, and electrochemical detectors with mini aturized electrodes.
The first day of the seminar was de voted to a discussion of various minicolumn types. The main column types, i.e., microbore columns, packed capillaries, and open-tubular columns, were discussed in terms of relative ef ficiencies, sample capacity, and speed of analysis. The subject was intro duced by D. Ishii and M. Novotny, who summarized the major develop ments in Japanese and U.S. laborato ries. While the subject of liquid chro matography in open-tubular columns continues to attract attention as "the ultimate solution," the problems of manufacturing columns with i.d. of 10 μπι or less remains formidable. The same is true for designing the neces sary equipment. Eli Grushka (Hebrew University, Jerusalem, Israel) re ported on some recent calculations and preliminary experiments with chromatography in narrow, open channels of rectangular shape; in creased sample capacities without in creased plate heights should be feasi ble following this approach. A similar approach, using the technology of field-flow fractionation channels, was
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his recent experiences with small particles in terms of column efficiency and stability. He noted the column stability problems with 3-fim particles in conventional (2-5 mm i.d.) columns, although several participants felt that the microcolumns prepared with similar particles are quite stable. The presentation by R. Hartwick (Rutgers University, New Brunswick, N.J.) featured the use of microbore columns for high-speed separations. The importance of fast detection and recording devices for this type of work was emphasized. Solute diffusivities in supercritical fluids or dense gases are greater than those encountered in liquids. At the same time, the opposite is true for the mobile-phase viscosities. The presentation by M. Novotny on capillary supercritical fluid chromatography (4,5) emphasized the importance of these phenomena in achieving high separation efficiencies. T. Takeuchi and D. Ishii also presented their results on micro-HPLC using some low-boilingpoint solvents (6) with characteristically high values of solute diffusion coefficients. In addition, such media have advantages in certain detection techniques. The second day of the seminar was devoted to new approaches to miniaturization, evaluation of sampling techniques, existing hardware, and LC/MS. Minimizing the extra-column sources of band dispersion in microHPLC presents unusual technological demands. Construction of sampling valves and detector cells with small volumes (from fractions of a microliter down to nanoliters in some cases) is mandatory. The general approaches to limiting the sources of extra-column band broadening were discussed by S. R. Bakalyar (Rheodyne, Inc., Santa Rosa, Calif.), who emphasized the importance of flow geometries in addition to volumetric requirements. These points were further reinforced by D. Ishii, who presented examples of design deficiencies and their negative effects on results. The presentations by Japanese instrument company scientists (K. Hibi, Jasco Co., HachiojiCity, Tokyo; and A. Nakamoto, Shimadzu, Nakagyo-ku, Kyoto) were representative of commercial develop-
ments in micro-HPLC instrumentation. Examples from the entire session demonstrated that much work needs to be done to produce reliable high-pressure miniaturized systems, including development of micropumps with gradient elution capabilities and new sample introduction techniques. It has been felt for some time that the availability of microcolumns will be beneficial in coupling HPLC with mass spectrometers because of the very low flow rates in micro-HPLC. Conventional (1-mm i.d.) microbore columns have now been used successfully in several laboratories. J. Henion (Cornell University, Ithaca, N.Y.) described applications (7) of micro-LC/MS to drugs and antibiotics. A conventional quadrupole mass spectrometer was used, while continuous monitoring permitted the acquisition of full-scan chemical ionization spectra from samples of a few nanograms; selected ion monitoring provided picogram sensitivities. An increased mass sensitivity (over conventional HPLC) is a significant attribute of microcolumns in cases where the total sample size is limited. The micro-HPLC/MS techniques described by Henion are relatively uncomplicated, and even aqueous mobile phases are feasible when using microbore columns. Discussion ensued on the subject of solute nebulization (spray) into a mass spectrometer. S. Tsuge (Nagoya University) described the use of a vacuum nebulizing interface (8, 9) that significantly improves the system's versatility. Chemical ionization spectra of various nonvolatile molecules were shown. A. L. Yergey (NIH, Bethesda, Md.), whose presentation concluded the second day of the seminar, reminded participants that development of new ionization techniques remains the single most important factor in determining the future growth of LC/MS. The principles behind the thermospray ionization technique (10) as well as its analytical merits were included in his presentation. It is significant that this "soft" ionization technique permits the use of aqueous and even buffered mobile-phase systems. The thermospray approach was shown to be applicable to the analysis of a wide range of compounds including amino acids and peptides, prostaglandins, antibiotics, phospholipids, nucleotides, and various drugs. The third day of the seminar was devoted to new optical spectroscopic detectors. Although conventional absorbance and spectrofluorimetric detectors have now been successfully miniaturized in several laboratories, the laser-based detection technology is particularly attractive. The introduc-
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tory presentation by B. Josefsson (University of Goteborg, Sweden) described work with fluorescence detectors based on the krypton ion laser (11) and the helium-cadmium ion laser. While the effective cell volumes were estimated to be around a tenth of a nanoliter, sensitivities below 1 0 - 1 5 g were achieved for poly aromatic compounds. The potential of the laser fluorescence detection techniques was further stressed by T. Imasaka (Kyushu University, Fukuoka-shi, Japan), who discussed a time-resolved fluorimetric HPLC detector with a subnanosecond dye laser source and its application to some environmental problems. The lecture by E. S. Yeung (Iowa State University, Ames, Iowa) provided a review of several micro-HPLC detection principles based on lasers that benefit from higher power levels, better collimation, greater monochromaticity, and improved time resolution (12-14). The principles of thermal lens calorimetry (15) and its potential applications to microcolumn work were discussed by J. M. Harris (University of Utah, Salt Lake City, Utah). It appears that this type of detection is ideally suited for supercritical fluid chromatography. The increasing need to characterize the components of complex mixtures separated by HPLC has encouraged the development of optical ancillary techniques. Just as with LC/MS, many of these detection techniques greatly benefit from the low flow rates encountered in micro-HPLC. In terms of element-specific detection, coupling of microcolumns to inductively coupled plasma spectrometers (16) and flame devices (17) has already been demonstrated, but many participants agreed that such studies are merely in their infancy and that considerably more can be accomplished. K. Jinno (Toyohashi University of Technology) described several combinations of micro-HPLC with spectrometric instruments for both atomic and molecular spectroscopic measurements. Various ways of combining microcolumn separation techniques to IR spectroscopy were discussed, including the solute transport approaches and the use of "exotic" solvents. Concerning the latter, the use of certain supercritical fluids as the mobile phases is particularly attractive for IR measurements. The state of the art of optical imaging detectors was reviewed by M. L. McConnell (Chromatix, Sunnyvale, Calif.), and applications of multichannel optical detection were also discussed by N. Suzuki (Tohoku University, Sendai-shi, Japan). Although certain improvements in the technology of the photodiode array and other
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imaging detectors are urgently need ed, it seems that miniaturization of such devices will be feasible in the near future. The final part of the seminar con cerned capillary electrophoretic tech niques and the use of electrochemical detectors in micro-HPLC. Capillary high-voltage electrophoresis (18) is a novel technique for separation of charged species in complex mixtures that bears much technological resem blance to capillary HPLC. The lecture by J. W. Jorgenson (University of North Carolina, Chapel Hill, N.C.), described the technical aspects of cap illary electrophoresis carried out at voltages in excess of 30 000 V. Several applications to relatively small mole cules were shown, but application to macromolecules has been considerably less successful. It was generally agreed that this area of separations has been relatively unexplored and deserves further attention. Most electrochemical detectors are concentration-sensitive devices, and their miniaturization for HPLC thus appears attractive. In addition, microelectrodes (19) are likely to provide the ability to vary parameters of a de tector during the elution of a chromato graphic peak, and thus enhance the selectivity. The use of voltammetric detectors in micro-HPLC was dis cussed by M. Goto (Nagoya Universi ty), who demonstrated several appli cations of these devices to detection (20) of electrochemically active biolog ical compounds. The presentation by S. Rokushika (Kyoto University, Kyoto, Japan), dealt with the widely used technique of ion chromatogra phy; he has shown that in certain ap plications the use of microbore col umns can be useful for industrial and environmental problems. The use of ion-selective electrodes as detectors for HPLC was the subject of the final presentations by N. Ishibashi (Kyushu University, Fukuokashi, Japan) and W. Simon (Swiss Fed eral Institute of Technology, Zurich, Switzerland). Substantial enhance ment of sensitivity can be achieved through miniaturization of the ion-se lective electrode (21). Simon, who. used open-tubular HPLC in conjunc tion with a drastically miniaturized electrode situated at the capillary out let, demonstrated that femtomole sensitivities are feasible. It was point ed out that the electrode technologies at the microscopic scale have been used in electrophysiological investiga tions, but analytical chemists have barely started to take advantage of them. Acknowledgment Funding for the seminar was pro vided by the U.S. National Science
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Foundation and the Japan Society for Promotion of Science. References (1) Giddings, J. C; Chang, J. P.; Myers, M. N.; Davis, J. M.; Caldwell, K. D. J. Chromatogr. 1983,255,359-79. (2) Yang, F. J. J. Chromatogr. 1982,236, 265-77. (3) Ishii, D.; Takeuchi, T. J. Chromatogr. 1983, 255, 349-58. (4) Novotny, M.; Springston, S. R.; Pea den, P. Α.; Fjeldsted, J. C; Lee, M. L. Anal. Chem. 1981,53, 407-11 A. (5) Peaden, P. Α.; Fjeldsted, J. C; Lee, M. L.; Springston, S. R.; Novotny, M. Anal. Chem. 1982,54, 1090-93. (6) Takeuchi, T.; Watanabe, Y.; Matsuoka, K.; Ishii, D. J. Chromatogr. 1981, 216, 153-59. (7) Henion, J. D.; Maylin, G. A. Biomed. Mass Spectrom. 1980, 7, 115-21. (8) Tsuge, S.; Hirata, Y.; Takeuchi, T. Anal. Chem. 1979,57, 166-70. (9) Yoshida, H.; Matsumoto, K.; Itoh, K.; Tsuge, S.; Hirata, Y.; Mochizuki, K.; Nokubutan, N.; Yoshida, Y. Z. Anal. Chem. 1982,311, 674-80. (10) Blakley, C. R.; Vestal, M. L. Anal. Chem. 1983,55,750-54. (11) Folestad, S.; Johnson, K.; Josefsson, B.; Galle, Β. Anal. Chem. 1982,54, 92529. (12) Woodruff, S. D.; Yeung, E. S. Anal. Chem. 1982,54,1174-78. (13) Sepaniak, M. J.; Yeung, E. S. J. Chro matogr. 1981,2i;, 95-102. (14) Kuo, J. C; Yeung, E. S. J. Chroma togr. 1982,229,293-300. (15) Leach, R. Α.; Harris, J. M. J. Chroma togr. 1981,2/8, 15-19. (16) Jinno, K.; Tsuchida, H. Anal. Lett. 1982,15 (A5), 427-37. (17) McGuffin, V. L.; Novotny, M. J. Chromatogr. 1981,278,179-87. (18) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981,53,1298-1302. (19) Wightman, R. M. Anal. Chem. 1981, 53,1125-34 A. (20) Goto, M.; Sakurai, E.; Ishii, D. J. Chromatogr. 1982,238,357-66. (21) Manz, Α.; Simon, W. J. Chromatogr. Sci. 1983,27,326-30.
Milos Novotny is professor of chemis try at Indiana University. A native of Czechoslovakia, he received his un dergraduate education in chemistry and earned a doctoral degree in bio chemistry at the University of Brno. In addition to microcolumn liquid chromatography, Novotny's research interests include capillary gas chro matography, HPLC, and GC/MS, as well as biomedical and environmental applications of these methods.
