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(11) Morl, A.; Katayama, Y.; Hlgashldate, S.;Kimura, S. J . Neurochem. 1079, 32. 643. (12) Bloomfield, D. K.; Cangiano, J. L. C u r . Ther. Res. 1060, T I , 727. (13) Fed. Reglst. 1078, 4 3 , 60018. (14) Beyer, W. F.; Gleason, D. D. J . Pharm. Scl. 1975, 6 4 , 3420. (15) Kaiser, D. G.; KO,H.;VanGlessen, G. J.; Zieserl, J. F.; Marks, D.; Kenny, M. D. “Abstracts of Papers”, 174th Natlonal Meeting of the American Chemical Soclety; Chicago, IL, 1977; Amerlcan Chemical Soclety: Washlngton, DC, 1977; COMP 14.
(16) Kaiser, D. G., The Upjohn Company, unpublished work.
RECEIVED for review August 25, 1981. Accepted March 29, 1982. The work reported in this manuscript was presented in part at the 27thAPS Meeting, APhA Academy of Pharmaceutical Sciences, Kansas City, MO, 1979.
Instrumental Aspects of Capillary Supercritical Fluid Chromatography Paul A. Peaden, John C. Fjeldsted, and Milton L. Lee” Departmenf of Chemistry, Brigham Young University, Provo, Utah 84602
Stephen R. Sprlngston and Milos Novotny Department of Chemistry, Indiana University, Bloomington, Indiana 47405
The bask Instrument components requlred for caplllary supercrltlcal fluld chromatographyinclude a high-pressure pump with pressure programmer, a small-volume sample Inlet system, a constant temperature oven, and a small-volume detector. The major stresses applied to the hstrumentatlon are the hlgh pressures and temperatures required to maintain the mobile phase at or above Its crltlcal polnt. Of the dlfferent sample introductlon systems studied, spllt Injection presently appears to be the most effectlve. By use of the described hstrumentatlon, a plate helght of 0.30 mm was obtalned for pyrene ( k = 0.50) on a 100-hm caplllary column contalnlng a bonded poly(methylphenylslloxane)statlonary phase.
Since the first description of supercritical fluid chromatography (SFC) in the separation of metal porphyrins ( I ) , much research has been done to explore the potential advantages of this analytical technique. The unique feature of SFC is that the mobile phase is subjected to pressures and temperatures near its critical point. Under these conditions ita density approaches that of a liquid, while at the same time, solute diffusion coefficients are approximately 2 orders of magnitude greater than those found in liquids. A supercritical fluid possesses solvating properties similar to a liquid, and solute diffusivities intermediate between a gas and a liquid. Therefore, with the favorable mass transfer properties of SFC, higher efficiencies can be obtained in shorter analysis times than can be achieved with capillary liquid chromatography, and comparable efficiencies ( 40 plates/s) to high-performance LC are achievable. In addition, SFC demonstrates the ability to analyze relatively nonvolatile and thermally labile solutes which cannot be analyzed by gas chromatography or, in many cases, even by liquid chromatography. The density of a supercritical fluid is largely determined by temperature and pressure. When operating at a constant temperature above the critical temperature of the fluid, liquid formation is prevented and the mobile phase density can be easily controlled by adjusting the pressure. Hence, pressure programming (2, 3) gradually increases the mobile phase density and decreases solute retention. This effect is analogous to temperature programming in gas chromatography and gradient elution in liquid chromatography. N
The advantages of using capillary columns in SFC ( 4 , 5 ) are similar to those of usigg capillary columns in liquid or gas chromatography. The low pressure drop across an open bore tube allows higher efficiencies to be achieved than obtainable with a packed column, simply because the column can be made much longer. This low pressure drop is additionally beneficial in capillary SFC because the density of the mobile phase is more uniform throughout the length of the column. Larger pressure drops, and hence density gradients, have been previously shown to be disadvantageous in packed column SFC (6, 7). Another advantage of using capillary columns is the absence of the plate height contributions due to alternate solvent flow paths as are found in packed columns. The discovery of new chromatographic methods has traditionally been followed by the development of suitable instrumentation for their proper utilization. New instrumentation was developed for packed column SFC after it first appeared, and a number of papers have been published on this subject (8-12). Analogous to past trends in which modifications in equipment were needed in converting from packed to capillary columns in gas and liquid chromatography, capillary SFC also requires its own unique instrumentation. The major instrumental modifications are a result of the stresses of high pressure and temperature required to maintain the mobile phase at or above its critical point and the low tolerance of extracolumn volume in sample introduction and detection systems. This has resulted in the use of split injection systems and on-column detection in this study, both of which are new to SFC. This paper describes the successful development of workable instrumentation for capillary SFC. Particular emphasis is placed on sample introduction and detection systems which provide minimum band broadening and maximum sensitivity. Although a number of different mobile phases can be used (13) and are presently under study, this paper was limited to our initial studies employing n-pentane. EXPERIMENTAL SECTION Instrumentation. A general schematic of the total chromatographic system is shown in Figure 1. Each of the principal parts of the SFC system is explained below. Id all cases, n-pentane was used as the mobile phase. A pressure-controlled system was obtained by using a Varian 8500 syringe pump modified for pressure control as published by
0003-2700/82/0354-1090$01.25/00 1982 American Chemical Society
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mL of water was added, drop by drop, while the mixture was rapidly stirred. Refluxing was continued for an additional 15 min after which the ethanol was removed with a rotary evaporator. Approximately 1.0 mL of chloroform was added to redissolve the residue, with subsequent evaporation using a rotary evaporattor, at least three times in succession in order to remove any dissolved HC1. Before being coated, glass capillary columns were treated with A silicon tetrachloride by dynamically coating at the rate of 10 cm/s, sealing the ends, and heating for 15 h at 300 "C. Unreacted Sic& was then flushed out of the columns by purging with dry nitrogen at 220 "C for 2 h. Coating was carried out dynamically with a 15% v/v solution of the polymer in methylene chloride. A plug of the coating solution, equal to approximately one-fifth of the column length, was pushed through the column at a linear velocity of 1 cm/s --.-I I under nitrogen pressure. After being dried with a nitrogen purge Figure 1. Schematic diagram of the instrumentation used in capillary at room temperature, the polymer was further polymerized and SFC: (A) pump, (5) injector, (C) solvent preheating coil (hot injector conditioned by temperature programming the column from 60 only), (D) analytical column, (E) fluorescence detector, (F) restrictor, to 330 "C at 2 "C/min under nitrogen flow and holding the upper (G) oven. temperature for 25 h. Analysis of Coal Tar and Carbon Black Extract. A coal tar sample was analyzed by SFC on a 20 m X 0.11 mm i.d. capillary Van Lenten and Ilothman (14). The chromatographic oven was column containing a bonded poly(methylphenylsi1oxane) staa Hewlett-Packard 5700A gas chromatograph without its contionary phase as described above. The mobile phase was nventional iinjection port or detector. A Berkin-Elmer 204-A pentane at 210 "C,and rcom temperature split sampling was used. spectrofluorimeter was mounted above the oven and used for The pressure was held constant at 18 atm for 30 min after insolute detection. A heated stainless steel block designed to hold jection, followed by a linear rise to 36 atm during a 2-h period. the end of the capillary column in proper alignment for on-column The fluorescence detector excitation and emission wavelengths detection was used in place of the conventional fluorescence were set at 300 and 360 nm, respectively, for the first 66 min. The detector flow cell, emission wavelength was then changed to 400 nm for the reSeveral different sample introduction systems were evaluated mainder of the chromatogram in order to obtain better sensitivity in this study. For on-column injection, a Carlo Erba on-column for the larger compounds. A 0.10-mm split was placed between injector was used. Sampling with heated sample loops was acthe excitation source and the capillary column in the detector to complished by using Valco CFSV-4HTAX-N6Ovalves with 0.5-~L reduce the background signal. and 0.2-pL volumes (Valco, Houston, TX). A 1-m coil of 0.76 mm The methylene chloride extract of a carbon black was chroi.d. stainless steel tubing was positioned inside the oven to preheat matographed on a 60 m X 0.13 mm i.d. capillary column coated the mobile phase before it reached the valve. The valve was with SP 2340, using n-pentane as the mobile phase. The column mounted approximately 3.8 cm from the wall, inside the oven, temperature was 210 "C, and the pressure was programmed froim with the handle extended to the outside. Room temperature split 27 to 40.3 atm at 5 atm/h. Injection was accomplished without sampling was accomplished using a Valco CFSV-4-HPa 0.2-bL splitting using a heated 0.2-wL valve and 1,2,4-trichlorobenzerte valve. Thisi valve was connected with a short piece of 1 rnm i.d. as the solvent. Excitation and emission wavelengths were 335 stainless steel tubing to a in. Swagelok tee. The column was and 450 nm, respectively. connected by inserting one end through the tee and 1 mm i.d. tubing and butting it up next to the valve. Selected lengths of RESULTS AND DISCUSSION 50 Mm i.d. Pyrex tubing were connected to the other arm on the tee to provide for split control. On-column fluorometric detection (4, 17, 28) was chosen In order to maintain column pressure, a restrictor consisting in order to minimize band broadening often obtained when of a length of 35 p,m or 50 Mm i.d. Pyrex tubing was connected connecting a capillary to other ancillary equipment. This was at the end of the column immediately after the detector. The convenient since the column only needed to be straightened length of the restrictor determined the column flow rate and was and carefully positioned in the detector light path. Effective chosen appropriately for the analytical column diameter being detector cell volumes of 0.71,0.31, and 0.08 KLwere achieved used. by illuminating a 1cm length of the column on the 0.30,0.20, A more convenient, alternative restrictor system consisted of and 0.10 mm i.d. capillaries, respectively. Calculating the a shorter capillary restrictor at the end of the column, which in turn was connected to a tee. This tee was connected to a highincrease in peak widths for a 20 m long column based on the pressure nitrogen regulator (variable to 1500 psi) and to a uecond above figures and assuming a plate height equivalent to the restrictor. This second restrictor must be short so that the given column diameter, values of 3.2%, 4.0%, and 5.6% are pressure in the tee does not increase above that of the nitrogen obtained for the 0.30, 0.20, and 0.10 mm i.d. columns, rehead pressure applied. If this were to occur, the mobile phase spectively. would back up to the nitrogen regulator. With this assembly, Proper alignment of the column with the excitation source mobile phase velocities in the column were easily controlled by and the emission detection system was quite important to the adjustment of the nitrogen head pressure. detector sensitivity. With Pyrex capillaries, the shortest usable Column Preparation. Pyrex glass capillary columns conexcitation wavelength was approximately 280 nm. The taining a bonded poly(methylphenylsi1oxane) stationary phase similar to that reported by Blomberg and Wannman (15) for gas background noise was significantly reduced by using a slit of chromatography were used in this study. This phase was a approximately the same width as the inside diameter of the polymeric bonded phase providing greater film thickness than column to direct the excitation beam through the cross section the typical high-performanceLC surface bonded phases obtained of the capillary occupied by the mobile phase only. Sensiby surface silylation which were used in other packed-column SFC tivities for polycyclic aromatic hydrocarbons using this destudies (16). The polymer necessary to make the bonded polytector arrangement were on the same order of magnitude as siloxane was synthesized by mixing 2.5 g of dichlorodiphenylsile those commonly experienced in capillary gas chromatography and 1.5 g of trichloromethylsilane in a round-bottom flask which using a flame ionization detector. Practical working sample was partially immersed in an ice bath. Approximately 10 mL of loads were between 10 and 100 ng per component. As in all dry ethanol was slowly added and the mixture was refluxed for 7 h. Since €IC1 gas was produced in this step, the reaction was fluorometric detection systems, the solute quantum efficiencies carried out under EL hood. After the mixture was refluxed, 0.36 and the detector wavelengths chosen were major factors af-
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Table I. Contribution of Sample Loop Volume to Peak Width %
WI/W,a
sample loop volume, M L
0.30 mmb
0.20 mmb
0.y mm
0.5 0.2
8
22 9
123 49
3
a The ratio of the peak width due to sample loop volume to peak width due to column volume given in terms of percent. Column diameter.
fecting solute sensitivity (19). In order to investigate the feasibility of introducing the sample into the capillary column at ambient pressure and/or low temperature followed by rapid adjustment of these parameters to their initial operating values before solute migration occurs, we used an on-column injector. This method of sample introduction is desirable from the standpoint that a syringe could be used to transfer the sample to the column, and the sample could possibly be initially concentrated in a narrow band at the head of the column, much like what is observed in gas chromatography. The on-column injector was evaluated by using 0.30 mm i.d. capillary columns. Two different injection modes were investigated. One approach involved injecting the sample which was dissolved in methylene chloride with the oven at 50 "C, subsequently raising the oven temperature to 210 "C, and then ramping the pressure at various rates to 32 atm. The second approach involved injecting at the desired operating temperature (210 "C) with the sample in 1,2,4-trichlorobenzene, followed by a pressure rise to the desired operating pressure. The best results were obtained when injecting at the desired operating temperature and ramping the pressure at approximately 1.5 atm/min up to the desired operating value. Faster pressure ramping caused too much band broadening for the more volatile sample components. Even at the rate of 1.5 atm/min, the resolution of the low molecular weight components was quite poor. Since only small effects were observed for the resolution between coronene and ovalene for different initial pressure rise rates, sample concentration at the head of the column appeared feasible, although the overall performance of the injector was not as favorable as other sample introduction systems studied. Sample introduction using sample loops (valves) was evaluated with the chromatographic column at 32 atm and 210 "C in all cases. Trichlorobenzene was employed as the sample solvent since its boiling point is higher than 210 "C, allowing atmospheric loading of the sample into the valve. For heated valve sampling in which the valve was also heated to 210 "C, the 0.5-pL valve was employed when using 0.30 mm i.d. columns. For smaller diameter columns, the 0.20-pL valve was used. The reason for this is apparent when the contribution of the sample loop volume to band broadening is calculated. Assuming a %fold increase in sample volume upon injection, and calculating the ratio of the peak width due to the sample loop to that due to the column, the data in Table I were obtained. These figures were calculated for 20 m long columns, assuming a plate height equal to the column diameter. It was found that when the valve was turned to the injection position and left there for the entire run, an exponential decay was observed in all of the chromatographic peaks (20). This was especially noticeable for the less retained components. If the valve was left in the injection position for only a few seconds and then returned to the sample loading position, this problem was not observed. On evaluation of this system with coronene ( k = 0.33), typical plate heights were found to be 5 and 3 mm for 0.30 and 0.20 mm i.d. columns,
I
3
1
Linear V e l o c i l y
4
(cm/sec)
Flgure 2. Van Deemter plots for naphthalene at 20 atm and 210 OC with n-pentane as the mobile phase.
1 1
1
Linear
Veloclfy
3
4
(crnfsec)
Figure 3. Van Deemter plots for pyrene at 29 atm and 210 "C with n-pentane as the mobile phase.
respectively. Less retained compounds gave smaller plate heights: anthracene (k = 0.02) gave plate heights of 0.34 and 0.20 mm for 0.30 and 0.20 mm i.d. columns, respectively. Room-temperature splitting using a 0.20-pL valve as the sample introduction device also gave a plate height of 5 mm for coronene on a 0.30 mm i.d. column. Naphthalene gave plate heights of 0.40 and 0.25 mm on 0.30 and 0.20 mm i.d. columns, respectively. Split ratios of 1:l and 2:l were used with these columns. The splitter was kept open continuously throughout the runs. Although both valve sampling systems gave about the same efficiencies for retained components at near optimum mobile phase velocities, differences could readily be seen at higher velocities. Using the split system, the observed plate height only doubled in going from 1to 4 cm/s, while plate heights increased five to six times for the same increases without splitting. On this basis the use of the split system is preferable since analysis times can be shortened with less loss in resolution. The large increase in plate height at increased velocities with the valve sampling system was probably due to a solvent effect resulting from the difference in sample solvent and mobile phase. This was minimized in the split system because of the dilution of the sample solvent with the mobile phase. By use of the room-temperature split sampling system, a comparison between the 0.30,0.20, and 0.10 mm i.d. columns was made by constructing van Deemter plots (Figures 2 and 3) for naphthalene and pyrene. As expected from theory, columns of smaller diameter yield lower plate heights. The shift to lower optimum velocities for higher molecular weight solutes can also be seen by comparing the plots for naph-
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BSSUre
I
10 T l e
T-
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I
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210-L-
Figure 4. Supercritical fluid chromatogram of a carbon black extract on a 60 m X 0.13 mm i.d. capillary column using n-pentane as the mobile phase.
thalene with those for pyrene. This is expected since the diffusion coefficient is smaller for higher molecular weight solutes. The Golay equation (21) predicts that the minimum plate height for a capillary column should be 0.289 times the column diameter. In this work, minimum plate heights equal to, or slightly greater than, the column diameters were obtained. This is approximately 31/2times greater than that predicted. A significant portion of this can be attributed to band broadening due to sample introduction, and, to some extent, less than ideal column efficiency. Further work is being done to overcome these problems. The rapid increase in plate heights for retained compounds corresponds well with that predicted by the Golay equation. On the basis of u minimum plate height of 0.10 mm for unretained compounds on the 0.10 mm i.d. column, the minimum plate height predicted for pyrene should be 0.30 mm, which was observed. The increase in plate height using the 0.20 mm and 0.30 mm i d . columns was somewhat higher, but not significantly higher than that based on theory. The analysis of asphalt and other similar samples using packed-column SFC has previously been reported (22). Figure 4 demonst,rates the application of capillary SFC to the separation of high-molecular-weight components in a carbon black extract which has been previously analyzed by conventional high-perfomance LC (23, capillary high-performance LC (a), and capillary gas chromatography (24). Since (a) the mobile phase linear velocity used in this application was approximately 10 times greater than the optimum, (b) the stationary phase was slightly soluble in the mobile phase and, hence, slowly stripped from the column, and (c) direct valve injection without splitting was used, somewhat less than the optimum efficiency and resolution was obtained. On the other hand, polycyclic aromatic compounds containing up to 11rings were separated, illustrating the potential application of this technique t o the analysis of high-molecular-weight compounds. Figure 5 shows a chromatogram of a coal tar sample obtained on a 20 m X 0.11 mm i.d. bonded-phase capillary column with split injection using supercritical n-pentane as the mobile phase. The initial mobile phase velocity was set at 4 cm/s. No changes in the restriction were made during the run to inaintajln this linear velocity. This column produced lo4 plates/m for a compound with k = 0.02 which approaches the efficiencies that are presently obtainable in capillary gas
Figure 5. Supercritical fluld chromatogram of a coal tar sample on a 20 m X 0.1 1 mm i.d. capillary column using n-pentane as the mobile phase.
chromatography. This is also equivalent to generating 40 plates/s, which is similar to what is presently achieved by high-performance LC. Furthermore, the time of analysis is approximately 120 min, which is far superior to analysis times presently achievable in capillary high-performance LC. In summary, experiments using the instrumentation dlescribed here have shown that a split injection system or a system allowing the use of the same sample solvent as t.he mobile phase is the most desirable. Sample concentration at the head of the column as often done in gas chromatography is feasible, but better results have been obtained with sample loop valving methods. On-column fluorometric detection provides sufficient sensitivity and minimal band broadening. Furthermore, the system described here has allowed the achievement of results close to those predicted by capillary column theory. LITERATURE CITED (1) Klesper, E.; Corwin, A. H.; Turner, D. A. J. Org. Chem. 1982, 17, 700-701. (2) Jentoft, R. E.; Gouw, T. H. J. Chromafogr. Sci. 1970, 8 , 136-1412. (3) Conaway, J. E.; Graham, J. A.; Rogers, L. B. J . Chromafogr. Sci. 1978, 16, 102-110. (4) Novotny, M.; Springston, S.R.; Peaden, P. A,; Fjeldsted, J. C.; Lee, M. L. Anal. Chem. 1981, 5 3 , 407 A-414 A. (5) Springston, S. R.; Novotny, M. Chromatographia 1981, 14, 679-684. (6) Novotny, M.; Bertsch, W.; Zlatkis, A. J. Chromafogr. 1971, 6 1 , 17-28. (7) Graham, J. A.; Rogers, L. B. J. Chromafogr. Scl. 1980, 18, 75-84, (8) Sie, S. T.; Van Beersum, W.; Rljnders, G. W. A. Sep. Scl. 1988, 1 , 459-490. (9) Jentoft, R. E.; Gouw, T. H. Anal. Chem. 1972, 44, 681-686. (10) Klesper, E.; Hartmann, W. Eur. Polym. J . 1978, 74, 77-87. (11) Karayannls, N. M.; Corwin. A. H.; Baker, E. W.; Klesper, E.; Walter, J. A. Anal. Chem. 1988, 40, 1736-1739. (12) Gouw, T. H.; Jentoft, R. E. J . Chromafogr. 1972, 6 8 , 303-323. (13) Asche, W. Chromadographia 1978, 7 1 , 411-412. (14) Van Lenten, F. J.; Rothman, L. D. Anal. Chem. 1978, 4 8 , 1430- 1432. (15) Blomberg, L.; Wannman, T. J . Chromafogr. 1979, 768, 81-88. (16) Jentoft, R. E.; GOUW,T. H. Anal. Chem. 1978, 48, 2195-2200. (17) Yang, F. J. CRC CC J. High Resolut. Chromafogr. Chromafogr. Cornmun. 1981, 4 , 83-85. (18) Jorgenson, J. W.; Lukacs, K. D. Anal. Chem. 1981, 53, 1298-1302. (19) Das, B. S.; Thomas, G. H. Anal. Chem. 1978, 5 0 , 967-973. (20) Coq, B.; Cretier, G.; Rocca, J. L.; Porthault, M. J. Chromafogr. Sci. 1981, 70, 1-12. (21) Golay, M. J. E. "Gas Chromatography 1958"; Desty, D. H., Ed.; Academic Press: New York, 1958; pp 36-55. (22) Gouw, T. H.; Jentoft, R. E. Chromatogr. Sci. (Chromatogr.Pet. Anal.) 1979, I f , 313-327. (23) Peaden. P. A.: Lee. M. L.: Hirata. Y.: Novotnv. M. Anal. Chem. 1980. 52, 2268-2271. (24) Hirata, Y.; Novotny, M.; Peaden, P. A,; Lee, M. L. Anal. Chim. Acta 1981, 127, 55-81.
RECEIVED for review November 24,1981. Accepted February 8, 1982. This work was supported by a grant from the Utah
Energy Consortium DE-AT03-77-AD72002,from the United States Department of Energy.
