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much higher levels of speed than those observed here could be achieved by further increases in flow rate and field strength. More work is clearly needed to explore this fascinating possibility.
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ACKNOWLEDGMENT
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We would like to acknowledge Lori Kellner for helpful discussions.
LITERATURE CITED
I
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Figure 5. Fractogram of seven latex bead diameters at 38 mL/min and 1400 rpm.
speed in steric FFF, it is not clear how these increases should relate to one another for optimum resolution and speed. Also, the present set of experiments gives no clue regarding the ultimate limits of our high-speed strategy. I t is possible that
(1) Giddings, J. C.; Myers, M. N . Sep. Sci. Techno/. 1978, 73, 637. (2) Giddings, J. C.; Myers, M. N.; Caldwell, K. D.; Pav, J . W. J . Chromafogr. 1979, 785, 261. (3) Caldwell, K. D.; Nguyen, T. T.; Myers, M. N.; Giddings, J. C. Sep. Sci. Techno/. 1979, 74, 935. (4) Peterson, R. E., 11; Myers, M. N.; Giddlngs, J. C. Sep. Sci. Techno/. 1984, 79, 307. (5) Caldwell, K. D.; Cheng, 2.4.; Hradecky, P.; Giddings, J . C. Cell Biophys. 1984, 6 , 233. (6) Giddings, J. C.; Myers, M. N. Sep. Sci. Techno/. 1978, 73, 637. (7) Giddings, J. C. In “Treatise of Analytical Chemistry”; Kolthoff, I . M., Elving, P. J., Eds.; Wiley: New York, 1981; Part I,Chapter 3. (8) Giddings, J. C.; Myers, M. N.; Caldwell, K. D.; Fisher, S. R. In “Methods of Biochemical Analysis”; Glick, D., Ed.; Wiley: New York, 1980; Vol. 26, p 79.
RECEIVED for review October 16,1985. Accepted December 6, 1985. This research was supported by Department of Energy Grant DE-AC01-79EV10244.
Stationary-Phase Phenomena in Capillary Supercritical Fluid Chromatography Stephen R. Springston,’ Paul David, Joette Steger, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington: Indiana 47405 Caplllary supercritical fluid chromatography was used to facllltate physlcochemlcal measurements assoclated with statlonary-phase effects. A technique Is described for determlnatlon of swelling of nonextractable polymer fllms In the presence of a supercritlcal fluid. Appreciable swelllng factors were measured for the stationary-phase SE-30 In supercritical butane, while decreased swelllng was observed when carbon dloxide was used. Addltional swelling measurements In butane at a varlety of temperatures and densltles are reported. Impllcatlons of these phenomena are discussed In terms of kinetic column effects and chromatographic performance.
The basic principles of supercritical fluid chromatography
(SFC)were the subject of numerous investigations throughout the 1960s; however, the analytical potential of the method was not realized a t that time. A major reason for the current “renaissance”in this area has been the introduction of capillary columns (1)in 1981. A recent review in these pages (2)provides an account of the latest analytical developments in capillary SFC. While sufficient evidence of the resolving power and certain detection advantages associated with capillary SFC already exists in the literature ( I - 7 ) , the underlying physicochemical ‘Present address: Department of Chemistry, University of Salt L a k e City, UT 84112.
Utah,
processes have not been fully elucidated. The validity of the Golay theory (8) for open tubular columns was assumed in the initial optimization studies (9);however, under various circumstances, departures from this theory may be encountered (10,II). In the carefully designed, miniaturized systems that are currently available, meaningful column studies can be conducted to quantify solute mass-transfer processes. Better understanding of these processes, in turn, may provide us with directions for the design of more effective instrumentation and improved chromatographic conditions for analytical purposes. Consequently, recent investigations in our laboratory have focused on the following processes: (a) mobile-phase mass-transfer effects under different chromatographic conditions ( I I , 1 2 ) , (b) hydrodynamic processes that promote enhanced solute mass transfer (13), and (c) masstransfer phenomena in the stationary phase. Whereas mobile-phase processes are treated elsewhere (11, 12), certain stationary-phase effects will be described in this report. According to the Golay theory (8), the stationary-phase contribution to the plate height, C,, is strongly dependent on the film thickness, dF
d~’ c, = 3(12k+ k ) 2 D,
(1)
where k is the solute capacity factor and D, is the solute diffusion constant in the stationary phase. In capillary gas chromatography (GC), this contribution is often neglected for
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columns with film thicknesses of a few tenths of a micrometer or less. However, recent interest in thick-film columns for increased sample capacity (14-18) has stimulated studies of chemically immobilized polymeric layers with thicknesses up to a few micrometers. Understandably, such GC columns provide only moderate efficiencies (a trade-off for increased sample capacity), in rough agreement with theory. An interestingly different situation occurs in open tubular liquid chromatography (LC). As the mobile-phase and the stationary-phase diffusivities become relatively close to each other, only small losses in efficiency are expected, even when the stationary-phase film approaches 25% of the column diameter (19). While such a phase ratio presently appears impractical for technological reasons, the potential importance of thick-film columns is once more underscored. Although only limited data on the solute diffusivities in polymeric stationary phases have been published (20-22,18), a recent study by Cramers et al. (28) on D, values in GC failed to find any measurable differences between mechanically coated and chemically immobilized (cross-linked) siloxane polymers. According t o molecular kinetic theory (23, 24), several factors may influence solute diffusivity in the liquid phase: temperature, viscosity, and its "molecular enviroment" (for example, as expressed by the association factor of the Wilke-Chang equation (25)). While attempting to evaluate solute diffusion characteristics in chemically immobilized stationary-phase layers for capillary SFC, we have observed that these films can actually swell to a considerable degree. While some solubility of the mobile phase in the stationary phase was expected from earlier suggestions (26,27),we have now developed a more quantitative approach to evaluate the extent of mobile-phase penetration. Both this procedure and actual swelling measurements are described below. Additionally, several consequences of polymer swelling in SFC are also discussed. Although many common solvents are known to induce swelling of polymers, including polysiloxanes (28-32), the subject has been somewhat neglected in chromatography (obviously, we are excluding organic gels used in ion-exchange or size-exclusion modes of LC from this discussion). The silicone polymers, which satisfy demands for high-temperature stability and efficient columns in GC, will not change their volumes appreciably under typical GC conditions. In SFC, however, as the mobile-phase pressure is increased and densities approach those of the liquids, some volume changes are expected in the stationary phase. This article illustrates how thick-film columns greatly accentuate the effects of phase swelling and are associated with numerous important consequences for the nature of SFC separations. E X P E R I M E N T A L SECTION Chromatographic Apparatus. The basic SFC system used in this study has been described previously ( I , 3,11,12). Samples were introduced into fused silica capillaries through a 200-nL sample loop (high-pressure valve from Valco Instruments, Houston, TX) connected directly to the column. On-column fluorescencedetection (3) was accomplished by using a Fluoromat FS 950 detector (Kratos Analytical Instruments, Westwood, NJ). Similarly, for on-column UV detection, a UVIDEC-100-IVvariable-wavelengthdetector (JASCO, Inc., Tokyo, Japan) was employed. A Varian 4100 syringe pump was modified to maintain the adjusted pressure within 0.4 atm. Temperature was maintained with a precision of f0.25 "C. Glass capillaries of various lengths (50 pm id.) were drawn on a Hupe-Busch glass drawing machine (Karlsruhe, West Germany); these served as pneumatic restrictors (1) at the end of the analytical capillary column, past the point of detection. The following capillary columns were prepared by coating commerical fused silica tubing (products of either Spectran Corp., Sturbridge, MA, or Polymicro Technology, Phoenix, AZ): 322 f 0.5 pm i.d., with film thicknesses of 3.1 and 8.0 fim SE-30
methylsilicone phase; 320 f 0.5 Fm i.d., coated with 2.4- and 6.0-fim films of the same phase; and 320 f 0.5 pm i.d., coated with SE-54 phenylmethylsilicone phase. The column lengths ranged from 7 to 15 m. One column from each capillary spool was left uncoated to measure the holdup time of a nonretained solute. The stationary phases, SE-30 and SE-54 (Alltech Associates, Deerfield, IL),were coated as a solution in a 1:1mixture of pentane and methylene chloride. Filling was easily accomplished by first filling the column with the solvent mixture followed by the much more viscous coating solution. To prevent a severe coating solution concentration gradient at the solution-solventinterface,low filling pressures were used (
