Anal. Chem. 1999, 71, 4223-4231
Packed Column Supercritical Fluid Chromatography/Mass Spectrometry for High-Throughput Analysis. Part 2 Manuel C. Ventura, William P. Farrell, Christine M. Aurigemma, and Michael J. Greig*
Alanex Corporation, 3550 General Atomics Court, San Diego, California 92121-1194
A supercritical fluid chromatograph was previously interfaced to a mass spectrometer (SFC/MS) and the system evaluated for applications requiring high sample throughput using negative-mode atmospheric-pressure chemical ionization (APCI) (Ventura et al. Anal. Chem. 1999, 71, 2410-2416). This report extends the previous work demonstrating the effectiveness of SFC/MS, using positive ion APCI for the analysis of compounds with a wide range of polarities. Substituting SFC/MS for LC/MS results in substantial time saving, increased chromatographic efficiency, and more precise quantitation of sample mixtures. Flow injection analysis (FIA) also benefits from our SFC/MS system. A broader range of solvents is compatible with the SFC mobile phase compared with LC/MS, and solutes elute more rapidly from the SFC/MS system, reducing sample carryover and cycle time. Our instrumental setup also allows for facile conversion between LC/ MS and SFC/MS modes of operation. In recent years, supercritical fluid chromatography (SFC) has been exploited as an alternative to high-performance liquid chromatography (HPLC) because of its superior selectivity and speed. Similarly, SFC/MS has been demonstrated by several groups as a viable replacement for LC/MS in the analysis of small molecules.2-10 Its applications to industries requiring high sample throughput become obvious as its characteristics are explored. As previously described,1 many advantages of SFC relative to HPLC arise from the lowered intramolecular energy of interaction between mobile-phase molecules.11 SFC mobile phases, generally (1) Ventura, M. C.; Farrell W. P.; Aurigemma, C. A.; Greig, M. J. Anal. Chem. 1999, 71, 2410-2416. (2) Combs, M. T.; Ashraf-Khorassani, M.; Taylor, L. T. J. Chromatogr. 1997, A785, 85-100. (3) Matsumoto, K.; Nugata, S.; Hattori, H.; Tsuge, S. J. Chromatogr. 1992, 605, 87-94. (4) Thomas, D.; Sim, P. G.; Benoit, F. Rapid Commun. Mass Spectrom. 1994, 8, 105-110. (5) Scalia, S.; Games, D. E. Org. Mass Spectrom. 1992, 27, 1266-70. (6) Via, J.; Taylor, L. T. Anal. Chem. 1994, 66, 1385-95. (7) Jedrzejewski, P. T.; Taylor, L. T. J. Chromatogr. 1995, A703, 489-501. (8) Pinkston, J. D.; Baker, T. R. Rapid Commun. Mass Spectrom. 1995, 9, 108794. (9) Pinkston, J. D.; Baker, T. R. Proceedings of the 46th ASMS Conference on Mass Spectrometry and Allied Topics; Orlando, FL, 1998; p 1157. (10) Berger, T. A.; Wilson, W. H. Anal. Chem. 1993, 65, 1451-55. (11) Berger, T. A. Packed Column SFC, 1st ed.; The Royal Society of Chemistry: London, 1995; Chapter 1. 10.1021/ac9902906 CCC: $18.00 Published on Web 08/26/1999
© 1999 American Chemical Society
consisting of condensed CO2 mixed with a polar liquid modifier, have much lower viscosities and higher diffusivities than HPLC mobile phases.12 Van Deemter plots (mobile-phase linear velocity vs column plate height) show the optimal linear velocity (µ0) of SFC mobile phases is up to 5-fold higher than HPLC, meaning a flow rate of 1 mL/min in HPLC may be increased to 5 mL/min with SFC without adversely affecting the separation.10 Chromatographic resolution is also enhanced not only by adjustment of modifier concentration and composition as with HPLC, but also by varying mobile-phase conditions such as flow rate, temperature, and pressure. Polar modifiers with appropriate additives can stretch the SFC polarity window to include strong organic acids and bases.13 Additionally, lower supercritical fluid viscosities relative to liquids enable the use of longer columns in SFC while maintaining optimal separation conditions.10,11,14 Diffusion coefficients are an order of magnitude higher in supercritical fluids than in liquids. Therefore, the number of theoretical plates
N ) 5.54(tR/Wh)2
where tR is retention time and Wh is peak full width at halfmaximum, generated per unit time, increases approximately 3-fold, contributing to SFC’s increased resolving power relative to HPLC.11,14 Thus, for columns of equal peak capacity
nc ) (N1/2)/4
efficiency is improved due to the increased chromatographic speed of SFC relative to HPLC. SFC is also readily interfaced to API sources designed for LC/ MS, and its advantages can thus be exploited for the demands of high-throughput combinatorial library analysis.15-20 Using the 3:2 (12) Berger, T. A. Packed Column Supercritical Fluid Chromatography Course Manual; Lake Tahoe, NV, 1998; p A-24. (13) Berger, T. A. Packed Column SFC, 1st ed.; The Royal Society of Chemistry: London, 1995; Chapter 4. (14) Schleimer, M.; Schurig, V. In Analysis with Supercritical Fluids: Extraction and Chromatography; Wenclawiak, B., Ed.: Springer-Verlag: Berlin, 1992; pp 134-150. (15) Edlund, P. O.; Henion, J. D. J. Chromatogr. Sci. 1989, 27, 274-282. (16) Morgan, D. G.; Norwood, D. L.; Fisher, D. L.; Moseley, M. A. III. Proceedings of the 44th ASMS Conference on Mass Spectrometry and Allied Topics; Portland, OR, 1996; p 183.
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splitting tee previously described,1 mobile-phase flow rates as high as 10 mL/min were successfully employed without loss of mass spectral sensitivity or adverse affects to the vacuum system. Similar flow rates in LC/MS often result in prohibitively high backpressure for most columns, loss of coherent mass spectral signal due to incomplete vaporization of the solvent, and overloading of the mass spectrometer vacuum system. The efficiency of positive APCI with SFC/MS can be enhanced by the introduction of methanol, water, and other gas-phase proton-generating solvents to the mobile-phase modifier without adversely affecting SFC chromatography.3,4,21-23 We describe a system not only capable of rapid change between SFC/MS and LC/MS modes of operation, but also between autosamplers which inject from individual vials or multiple 96-well plates. The SFC/MS system was also used for mass spectrometric flow injection analyses. This was accomplished by diverting the mobile phase from the injector directly into the detectors bypassing the column. Again, the higher flow rates used in SFC dramatically reduce cycle time and sample carryover. An extensive variety of sample solvents may be introduced without affecting the high-pressure mobile-phase system of the SFC. This makes flow injection analysis with the SFC/MS system a more “userfriendly” open-access method, requiring less preparation time for analysts seeking quick confirmation of compound identity. The results presented show that separation and mass identification of components in combinatorial library mixtures can be achieved more rapidly and with greater resolution while maintaining mass spectrometric integrity using SFC/MS compared with LC/MS. One example of a complex mixture consisting of standard and synthetic compounds was examined using both methods. The LC/MS and SFC/MS methods used in our labs are not the fastest methods possible. This is because extremely rapid methods (
