Anal. Chetn. 1990, 62, 1299-1301 (7) Cody, R. 8.; Burn&, R. C.; Cassadya, C. J.; Freiser, B. S. Anal. Chem. 1982, 54, 2225-2228. (8) McLafferty, F. W.; Amster, I.J.; Fvbng, J. J. P.; Loo, J. A,; Wang, B. H.; Williams, E. R. In FoukK Transform Mass Spectrotnoby;Buchanan, M. V., Ed.; American Chemical Society Symposium Series, No. 359; American Chemical society: Washington, w , 1987; PP 116-126. (9) Wang, B. H.; Williams, E. R.; Henry, K. D.; M-fferty, F. w.; shahnowitz, J.; Hunt, D. F. of the 37th ASMS Conference on Mass Spectrcunsby and A M ToprcS, May, 1989 Mlaml, FL ; Amer~ ~ Lansing, MI, 1989; pp ican ~ ~ ~ i for e tMy~ S Ss ~ c t r o m t East 236-237.
1299
(10) Kaminsky, M. Atmfc and Ionic Impact W m o m n a on Metal Surfaces; Academic Press: New York, 1965.
R E C E ~for D review January 17, 1990. Accepted March 20, 1990. Partial support of this research from the National Science Foundation (CHE-89-11685) is gratefully acknowledged, as is suppod for purchase of the 7-T FTMS-2OOO under Grant GM-30604 from the National Institutes of Health.
Capillary Supercritical Fluid Chromatography at Pressures above 400 atm T. L. Chester,* D. J. Bowling, D. P. Innis, and J. D. Pinkston The Procter & Gamble Company, Miami Valley Laboratories, P.O. Box 398707, Cincinnati, Ohio 45239-8707
Selectlvfty and effkkncy advantages often occur In capillary supercrltlcal fluld chromatography (SFC) when the column temperature Is ralsed. However, the slopes of the mobile phase dendy-pressure Isotherms drop wlth Increasing temperature. Commonly used SFC pumps (and other chromatographic components) specify a somewhat arbltrary pressure Umtt, often around 400 atm. Thk results In a severe reduction of the accerolble denslty range (and moblle-phase strength range) when higher temperatures are chosen. The potential of uslng capMary SFC wRh pressures up to 560 atm to extend the range of eluted solutes Is demonstrated wlth several chromatograms. Eventually, the permlsslble temperature ranges for typical SFC columns must be determined In the presence of high-pressure moblle phase. Then the upper pressure lknlt required, correspomllng to the maxknum denslty desked wlth each moMle phase at Its upper temperature Ihnlt, can be Specified.
INTRODUCTION Supercritical fluid chromatography (SFC) can elute much less volatile solutes than gas chromatography (GC). Viewed as a nonideal gas mobile phase, the supercritical fluid enhances the volatility of solutes compared to the volatility observed in ordinary gaseous mobile phases like helium or hydrogen (I). From another, simple, point of view, this enhanced partitioning of sample components into a supercritical fluid mobile phase can be considered due to the solvation property of supercritical fluids and added to the partitioning due to “normal” solute volatility (2). The strength of this solvation effect depends on the density of the supercritical fluid at any specified temperature. Rigorous descriptions of the partition process in SFC have appeared in the literature (3, 4 ) . Regardless of how it is viewed, a supercritical fluid mobile phase is more capable than an ordinary gas of competing with the stationary phase for solute at a given temperature, thereby lowering retention in SFC compared to GC. The two practical consequences of this behavior are an increase in the eluate molecular weight range (or extension of the analysis scope to less volatile solutes) for SFC compared to GC and the ability to achieve a given degree of retention at much lower temperatures in SFC compared to GC, if desired (5). Thus, SFC 0003-2700/90/0382-1299$02.50/0
can be used advantageously in situations where solutes have insufficient volatility for conventional GC (for example, ref 6) or when solute thermal instability requires a low column temperature ( 7 , 8 ) . There are several reasons why SFC is not routinely performed at the lowest possible temperature for a given mobile phase: (1)The binary diffusion coefficients of solutes in the mobile phase increase with increasing temperature (9). The mobile phase optimum velocity depends directly on the value of the solute diffusion coefficient and increases as the temperature is raised. This translates directly into shorter analysis times or more efficient separations within a maximum analysis time when optimum velocities are exceeded. (2) The viscosity of supercritical COP, the most common supercritical fluid mobile phase, generally tends to decrease with increasing temperature over the range of 37-500 “C (IO). This results in lower pressure drops and more uniform solvent strength over the length of the column. (3) The selectivity of the system is temperature dependent and can be tuned by temperature adjustment after the column and mobile phase selection have been made ( 2 , I I ) .The separate control over selectivity and mobile phase strength afforded by independent adjustment of temperature and pressure (or program rate) is a powerful advantage of SFC not yet widely practiced. As we consider raising the temperature of SFC separations, we cannot ignore what will happen to the mobile phase density-pressure behavior. Plots of density-pressure isotherms for C02 are shown in Figure 1at several temperatures. With increasing temperature the isotherms become less sigmoidal and more linear. But, more importantly, the maximum slope of the isotherms and the density at a given pressure both decrease with temperature. If the chromatograph has an arbitrary pressure limit, then the maximum density available may decrease with increasing temperature (depending on the specific circumstances). Many SFC systems, both commercial and research instruments, are unable to attain pressures much higher than 400 atm. This probably results from early work with C 0 2 as mobile phase when it was common to use rather low column temperatures-% to 45 OC was fairly typical-combined with the pressure capabilities of commonly available pumps at that time. It is clear from Figure 1 that pressures above 400 atm are not particularly valuable at these temperatures since the density rises quickly with pressure and approaches what ap0 1990 American Chemical Society
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Flgure 1. Density-pressure Dsothermsfor CO, at several temperatures. The 40,70, 100, and 150 "C data are from ref 12. The 200 and 300 "C data were Interpolated from ref 13.
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pears to be a limiting density around 1.1 g/mL. However, much higher pressures are required to approach this density when temperatures are higher. For example, the density a t 40 "C and 400 atm is 0.96 g/mL, or about 79% of the density of liquid C02 (1.21 g/mL at -50 "C and 300 atm). At 100 "C a density of only 0.94 g/mL is reached a t 800 atm. Temperatures around 150 "C are fairly common in the SFC literature. But, with a 400-atm pressure limit the maximum density is just 0.61 g/mL at that temperature. This limit in available density range will lead to situations where a particular solute cannot be eluted a t the desired temperature. Retention in packed-column SFC is generally much stronger than in capillary SFC with the packings and columns available today. Therefore, higher densities are usually necessary in packed-column SFC than in capillary SFC for eluting a particular solute. This has prompted the use of higher pressures in packed-column SFC and the availability of some commercial packed-column SFC instruments with 10000 psi (680 atm) capability. However, the need for higher pressures in capillary SFC has not yet been widely realized. Here we present several examples of capillary SFC chromatograms requiring unusually high pressure to illustrate the need for this capability.
EXPERIMENTAL SECTION Work was performed with a home-built capillary SFC system based on a model 8500 pump (Varian, Walnut Creek, CA) modified for pressure control and programmed with an external computer and a Model 5700 GC (Hewlett-Packard,Avondale, PA) equipped with a flame ionization detector. The system was used as previously described ( 6 , I I )except that the pressure monitor circuit within the pump was modified to produce a signal of 5 mV/atm rather than 10. The software was appropriately changed and a maximum pressure limit of 560 atm (corresponding to 97% of the specified pump pressure limit) was dialed into the system via the hardware pressure-limit potentiometer on the pump. Direct injection with a room-temperature inlet tube (14) was used. The injection valve, a Model ECI4W.1 (Valco, Houston, TX),has a maximum specified pressure of 5000 psi (340 atm) with the factory preload adjustment. However, we found no outport or crossport leaks up to 560 atm and made no further adjustment of the preload assembly. The mobile phase was SFC-grade COP (Scott Specialty Gases, Plumsteadville, PA). The column was a 10-m x 50-pm-i.d. SB-biphenyl-30 (Lee Scientific, Salt Lake City, UT). Safety considerations must always be kept in mind with the use of high-pressure equipment. The valve body and fittings are rated at or above loo00 psi (680atm) at room temperature. Some lowering of the specification is appropriate for the two unions located inside the oven. However, these specifications pertain only to the slippage of tubing and the Occurrence of leaks. Much higher pressures would be necessary for destruction of the fitting. No slippage of fused-silica tubes occurred in the course of this work. Even if a fused-silicatube had become completely detached from a fitting, it would not pose a serious safety risk (except possibly as an eye hazard when the oven door is open) because
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Flgue 2. Chromatograms of trimethylsilyl deriiathres of poly(ethylene glycol) 1000 (upper chromatogram) and poly(ethy1ene glycol) 2000 (lower chromatogram). The column temperature was 120 "C.
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Figure 3. Trimethylsilyl derlvatiie of "E15" ethoxylated allyl alcohol. The column temperature was 100 "C. This chromatogram was as expected.
of the very small volume of compressed mobile phase present in the oven. In any case, the 560 atm pressure limit we adopted leaves a generous safety margin. Ordinary safety precautions include wearing safety glasses while working on the system and not opening the oven door with the system at elevated pressure except when necessary for leak checking.
RESULTS AND DISCUSSION Figure 2 shows chromatograms of poly(ethy1ene glycol) samples with average molecular weights of lo00 and 2000. In both cases the samples were prepared by dissolving in a 5:3 mixture of N,O-bis(trimethylsily1)trifluoroacetamidewith 1% trimethylchlorosilane (BSTFA/TMCS) and pyridine. The oven temperature was only 120 "C in these examples. Even a t this relatively low temperature it is clear that SFC instruments limited to maximum pressures in the vicinity of 400 atm would not be able to elute some of these peaks (with the stationary phase and phase ratio used here). Figures 3 and 4 are chromatograms of ethoxylated allyl alcohol derivatized in 1:l BSTFA/TMCS and pyridine (15). The "E15" material in Figure 3 looks as it was expected, but the "E16" material in Figure 4 was apparently ethoxylated beyond target during synthesis. The chromatogram clearly shows the sample distribution peaking a t E27, and materials out to E41 are visible. The series was only about half eluted when the pressure reached 400 atm. Even though we did not exceed 560 atm, these few examples demonstrate the value of even such a modest pressure range improvement in capillary SFC. Many of the eluted peaks
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would provide COz densities of at least 0.94 g/mL. But a t 300 OC considerably higher pressures would be necessary. Quipment capable of operating at pressures approaching 4O00 atm is conceivable, especially for capillary SFC with its small volume requirements. Finally, attention must be paid not only to the pumping system but also to the injector, connedors, and even the tubing used as the pressure range is increased to ensure reliable and safe operation.
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ACKNOWLEDGMENT The authors thank M. Harvey of Valco Instruments for helpful discussion regarding the use of their products at high pressures. Registry No. Poly(ethy1ene glycol), 25322-68-3;Poly(ethy1ene glycol) allyl ether, 27274-31-3.
Flgure 4. Trimethylsllyl derivative of "E 16" ethoxylated allyl alcohol. Conditions were exactly as in Figure 3. (See text for explanation.)
would have gone undetected with a conventional pressure limit. In addition to the capability of eluting more strongly retained peaks, it is also often of great importance to demonstrate that no additional peaks of significance exist in a chromatogram when the last peak seen is eluted within the conventional pressure range. In such a case, all available elution strength is necessary to add as much certainty as possible to the analysis. This is especially important when separating components of an unknown sample. Regardless of the type of column chosen, the full density range (or, a t least, some large fraction of it) needs to be available to the analyst over the permissible temperature range for the column in use for maximum flexibility in optimizing the separations. Thus, the pressure range of the equipment should be matched to the temperature limits of the column and to the mobile phase density-pressure behavior, if possible. New research is necessary to determine the temperature limits of typical SFC columns in the presence of very-high-pressure mobile phase and to determine if any practical advantages exist with these temperature-pressure-stationary phase combinations. Once these temperature limits are known, the minimum upper pressure limit corresponding to, for example, 80% of the limiting density of mobile phase at that temperature can be specified as an instrument design criterion. For temperatures up to 150 "C a pressure limit of 1000 atm
LITERATURE CITED (1) Sie, S. T.; Van Beersum, W.; Rljnders G. W. A. Sep. Sci. 1988, 7 ,
459-490. (2) Chester, T. L.; Innis, D. P. HRC CC, J . H@ Resolut. Chrmtogr. Chromatogr. Common. 1985, 8 , 561-566. (3) Schoenmakers, P. J. J . Chromatogr. 1984, 375, 1-18. (4) Yonker, C. R.; Smith, R. D. J . Mys. Chem. 1988, 92, 1664-1667. (5) Chester, T. L.; Burkes, L. J.; Delaney, T. E.; Innis, D. P.; Owens, 0. D.; Pinkston, J. D. I n Supercritic81Fhrid Exb8Ctlon and Chromatogmphy, Techniques 8nd AppUcathms; Charpentier. B. A., Sevenants, M. R., Eds.; ACS Symposium Series 250; American Chemical Society: Washington, DC, 1988;pp 144-160. (6) Chester, T. L.; Innis, D. P.; Owens, G. D. Anal. Chem. 1985, 57,
2243-2247. (7) Fjeklsted, J. C.; Kong, R. C.; Lee, M. L. J . Chrometop. 1983, 279, 449-455. (8) Chester, T. L.; Pinkston, J. D.; Innis, D. P.; Bowling, D. J. J . M i c m lumn Sep. 1989, 7 , 182-189. (9) Lauer, H. H.; McManningili. D Board, R. D. Anal. Chem. 1883, 55, 1370-1375. . . . . ..
(IO) Micheis, A.; Botzen. A.; Schuurman, W. Mysica 1957, 23, 95-102. (11) Chester, T. L. J . Chromatogr. 1984, 299, 424-431. (12) Newitt, D. M.;Pai, M. U.; Kuloor. N. R.; Huggill, J. A. W. In 7Bemodynamic Functions of Gases; Din, F., Ed.; Buiterworths: London, 1956; VOl. 1, pp 102-134. (13) Encyclopedia des Gaz; Elsevier: Amsterdam, 1976;pp 338-339. (14) Chester, T. L.; Innis, D. P. Presented at the Twenty-Eighth Annual Conference on the Practlce of Chromatography presented by ASTM
Committee E-19,Houston, TX, October 1989. (15) Pinkston, J. D.;Bowling, D. J.; Chester, T. L.; Delaney, T. E.; Innis, D. P. Presented at the I989 Symposium/Workshop on Supercritical Fluid chromatography, Snowbird, UT, June 1989.
RECEIVED for review December 15,1989. Accepted March 2, 1990.
