Unified high-pressure gas and supercritical fluid chromatography with

mobile phase was switched directly from high-pressure helium to supercritical ... stationary phase, the sample Injection, sample zone focusing, and In...
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Anal. Chem. lQQl, 63,926-930

Unified High-pressure Gas and Supercritical Fluid Chromatography with Microbore Packed Columns Yan Liu* and Frank J. Yangl Dionex Corporation, 1228 Titan Way, Sunnyvale, California 94088-3606

Unified gas chromatography (GC) and supercritical fluid chromatography (SFC) separation using the same mkrobore packed cdumn, restrictor, and detector was achieved. The mobile phase was switched directly from high-pressure heHun to supercritlcai Cot during the sample run. Several Important aspects of the unified GC and SFC technique such as the compatlbllity between the GC and SFC mobile phase, the transltlon process from the GC mode to the SFC mode, the statbnary phase, the sample Injection, sample zone focusing, and Instrument requirements were Investigated. Appllcatlons of unified microbore packed column GC and SFC for the separation of volatile and nonvolatile components in complex samples were demonstrated.

INTRODUCTION As early as 1965,Giddings pointed out that the perceived divergence between GC and liquid chromatography (LC) is arbitrary, artificial, and counterproductive (I). The rapid growth of microcolumn chromatography in the 1980s has resulted in many common features such as the use of a similar column or the same column, injector, detector, and other system components in the practice of capillary GC, microcolumn SFC, and microcolumn LC (2).The concept of unified microcolumn chromatography proposes that all three modes of chromatography may be carried out sequentially in the same column in one sample run using one instrument (2-4). Different chromatographic modes in one sample run may be obtained by selecting different mobile phases (e.g., using helium for GC, COz for SFC, and acetonitrile/water for LC) or by using pressure and temperature programming to change the physical state of a mobile phase from a gas in GC to a supercritical fluid for SFC to a liquid for LC. By virtue of combining all three chromatographic modes in one sample run, unified microcolumn chromatography promises to be an ideal separation technique to obtain an unprecedented amount of information about different classes of components in a complex sample, such as volatile ones from the GC mode and nonvolatile ones from the SFC and LC modes, in a single analysis. Several researchers have recently reported some preliminary results of carrying out GC, SFC, and LC modes of separation sequentially in a single column in one sample run. Ishii et al. (4,5)reported studies in which the selected GC, SFC, and LC separation conditions in a packed capillary column or a open tubular column within a sample run were achieved by changing the physical state of a mobile phase such as diethyl ether and methanol through temperature and pressure programming. It was shown that PAH components in a mixture were separated under the GC mode and styrene oligomers were separated under the SFC mode. Gemmel et al. (6,7)also

* Corresponding author.

'Present address: FFFractionation, Inc., 1270 W. 2320 South, Suite D, Salt Lake City, UT 84119. 0003-2700/91/0363-0926$02.50/0

reported similar studies with conventional packed columns. The approach of changing the physical state of a mobile phase to achieve unified chromatographic separation has limitations. The use of a single chemical as the mobile phase is likely not the optimum but is a severe compromise for each chromatographic mode to obtain optimal separation results that are possible with the microcolumn. The other approach is to use different but optimal mobile phases for each stage of the unified chromatographic run such as helium for GC, COPfor SFC, and acetonitrile/water for LC. This approach will require a much more complex instrument to direct much different mobile phases to the same column within a sample run, but it will certainly provide much more suitable conditions for each separation mode. Pentoney and co-workers (8)demonstrated the separation of volatile components by GC with helium and nonvolatile components by SFC with C02on the same open tubular capillary column with one sample injection. In that study, the transition from the GC to the SFC mode was not continuous; the column was transferred from a GC instrument to a SFC instrument to carry out combined GC and SFC separation on the same column because normal GC (1-3atm) and SFC (80-400atm) have different flow restriction requirements. More recently, Davies and Yang (9)also made their attempt to achieve unified GC and SFC separation on a single open tubular capillary column. High-pressure helium (160 atm) was used as the mobile phase in the GC mode such that the same restrictor could be used for both GC and SFC separations. They demonstrated continuous unified GC and SFC separation for complex samples such as a gasoline/crude oil mixture on the same column with one injection in one instrument. Packed capillary or microbore columns with proper stationary phases have the most potential to meet the requirements as universal columns that can be used in GC, SFC, and LC modes for unified chromatography (2).Our previous study showed that microbore columns packed with highly efficient microparticulate HPLC stationary phases could be used successfully to provide rapid and efficient separation of volatile compounds in the high-pressure GC mode (10).This paper reports the study of unified GC and SFC using microbore packed columns. The results demonstrate that unified GC and SFC separations on the microbore packed column can be achieved by using the same restrictor and flame ionization detector in one instrument with the mobile phase switched directly from high-pressure helium in the GC mode to supercritical COz in the SFC mode. Important considerations in mobile phases, column packing materials, and instrumental requirements for unified high-pressure GC and SFC on microbore packed columns are discussed. Application examples of unified high-pressure GC and SFC for separation of volatile and nonvolatile components in complex samples are presented. EXPERIMENTAL SECTION The instrumentationused for the unified high-pressure GC and SFC study is depicted in Figure 1. It consisted of a Lee Scientific Model 622 SFC system with a flame ionization detector (FID) (Salt Lake City, UT). An automated Rheodyne 7520 injection valve with a 0.5-pL internal loop (Cotati, CA) was used for the 0 1991 American Chemical Society

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sample injection. The volume of sample injected was controlled with the automated injection valve by setting the injection time to between 0.05 and 0.5 s. The sample injector was heated to 120 "C. A Rheodyne 7010 valve was used as the mobile-phase selection valve to direct the appropriate mobile phase to the microbore packed column in the unified GC and SFC run. The microbore packed column was connected to the FID via a stainless steel capillary tube (30 cm X 0.002 in., i.d.), which was crimped at the FID end with a pair of pliers. The restriction was adjusted by crimping so that the outlet gas flow rate from the restrictor was about 45 mL/min at ambient pressure with the column inlet pressure of 160 atm of helium. A open stainless steel capillary tubing (30 cm X 0.002 in. i.d.) was used in some high-pressure GC experiments. Helium and SFC grade COOsupplied by Scott Specialty Gases (Plumsteadville, PA) were used as the mobile phases. In a typical unified GC and SFC run, the sample was injected into the column with high-pressure helium delivered directly from the helium cylinder at a cylinder pressure of 160 atm. The high-pressure GC separation with helium as the mobile phase was then carried out with a temperature program. A t the end of the GC temperature program, the mobile-phase selection valve was switched so that the supercritical C02 delivered by the syringe pump was directed to the same column to complete the SFC separation with a pressure program. The same restrictor was used in both GC and SFC modes. In addition to high-pressure helium gas, a mixture of helium and COSgases with partial pressures of about 120 atm for helium and 40 atm for COPwas also used for the high-pressure GC separation in unified GC and SFC runs to study the effects of different GC mobile phases. The gas mixture was prepared by filling another Lee Scientific syringe pump with COz to 40 atm and then switching the pump inlet line to the 160-atm helium cylinder to continue to fill the pump with helium. The filling was stopped immediately when the pump pressure reached 160 atm. In some unified GC and SFC experiments, the C02/heliumgas mixture was used as the mobile phase in the GC mode and was delivered by the additional syringe pump at a constant total pressure of 160 atm. Two columns were used in the unified GC and SFC studies. One column (15 cm X 0.75 mm i.d.) was prepared by slurry packing with a highly cross-linked ethylvinylbenzeneand divinylbenzene (EVB-DVB) macroporous polymer. The polymer packing material was developed in this laboratory for packed column SFC applications (11). It has an average particle size of 5 pm, pore size of 60 A, and surface area of 300 m2/g. A Deltabond octylsilica (5pm particles) column of 10 cm X 1 mm i.d. (Keystone Scientific, Bellelfonte, PA) was also used. Experiments were conducted to obtain van Deemter plots for the EVB-DVB polymer column in both high-pressure GC and SFC modes. In the high-pressure GC mode, different mobile-phase flow rates were obtained by changing the mobile-phase pressure at the column inlet; the SFC syringe pump was used to deliver the mobile-phase gases with constant pressure control, ranging from 20 to 350 atm for helium gas and 15 to 60 atm for C02gas. In the SFC mode, column inlet pressure were kept at 120 atm and different mobile-phase flow rates were obtained by adjusting the crimping to the outlet tip of the stainless steel capillary restrictor. The samples used in the study included n-alkanes, n-alcohols, and n-free fatty acids, and a gasoline/crude oil mixture.

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Linear Velocity, cmls Figure 2. van Deemter plots for high-pressure GC and SFC on microbore packed column. High-pressure helium GC conditions: 170 OC, helium pressure varied from 70 to 350 atm, hexane as the test solute. CO, SFC conditions: 150 OC, 120 atm, tetradecane as the test solute, EVB-DVB polymer column.

RESULTS AND DISCUSSION To achieve unified microbore packed column GC and SFC with mobile-phase switching directly from helium for GC to supercritical C02 for SFC, the GC separation needs to be carried out a t a pressure similar to that of the SFC mobile phase (typically 80-400 atm) so that the same column, restrictor, and FID can be used for both GC and SFC modes. The conventional low-pressure GC (typically 1-3 atm) is clearly not compatible due to the much different operation pressure. In unified microbore packed column GC and SFC, there are several important aspects to be carefully studied such as the compatibility between the GC and SFC mobile phases, the transition process from the GC mode to the SFC mode, the stationary phase, the sample injection, initial sample zone focusing, the detector interfacing, etc. If the same column and restrictor are used in both GC and SFC modes, it is necessary for the same flow restrictor that provides a desirable mobile-phase linear velocity in the GC separation to yield a mobile-phase linear velocity that is at or near the optimal linear velocity in SFC separation; otherwise, the advantages of the unified GC and SFC technique are likely to be severely compromised. To study the mobile-phase linear velocity compatibility between high-pressure GC and SFC, van Deemter plots of high-pressure GC and SFC were obtained with the ED-DVB polymer column as shown in Figure 2. The optimal linear velocity (uopJof supercritical COSon the column was found to be 0.5 cm/s at a constant column inlet pressure of 120 atm using tetradecane as the test solute, which is typical in packed column SFC (12). Two high-pressure GC van Deemter plots were obtained when using hexane as the test solute with two different flow restrictors by varying the helium gas pressure at the column inlet from 100 to 350 atm. uoptwas found to be about 0.6 cm/s (corresponding to the column inlet pressure of 260 atm of helium) with restrictor A, and uoptwas found to be about 0.7 cm/s (160 atm of helium) with restrictor B, which had a smaller flow restriction than restrictor A. Figure 3 shows the helium and COBGC van Deemter plots for the same EVB-DVB column obtained without using a flow restrictor at the column outlet; the mobile-phase pressure at the column inlet was changed from 30 to 90 atm for helium and 15 to 60 atm for C02. Much higher optimal linear velocities were obtained with lower column inlet mobile-phase pressures; uoptwas 2.8 cm/s (70 atm of helium) with helium as the mobile phase and uoptwas 1.4 cm/s (35 atm of COP) with C02 as the mobile phase. The above data indicate that the flow restrictor determines the column inlet mobile-phase pressure that produces the optimal linear velocity for a given column in high-pressure

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GC. The optimal mobile-phase linear velocity decreases with the increase of mobile-phase pressure in high-pressure GC. This is due to the fact that the diffusion coefficient of the solute in the mobile phase is inversely proportional to the mobile-phase preasure. Thus,the mobile-phase linear velocity decreases as the mobile-phase pressure increases for the same contribution of the resistances to mass transfers to the column plate height. The results in Figures 2 and 3 clearly show that the optimal linear velocity of helium in the GC mode can be shifted to a lower value by carrying out the GC separation with high-pressure helium so that the optimal linear velocity of helium in the GC mode can be very close to that of supercritical C02 in the SFC mode under the proper experimental conditions. The results also indicate that both GC and SFC mobile phases can be operated at or near their optimal linear velocities using the same column and restrictor in the unified GC and SFC separation. It should be pointed out that it is important to choose a restrictor with the proper amount of flow restriction. For microbore packed columns used in this study, the experimental results show that the restriction of a flow restrictor should be adjusted such that the optimal linear velocity in the GC mode is obtained with a helium column inlet pressure at about 160 atm. A restrictor with a flow restriction that is too high would require using helium at inconveniently high pressures. It would force supercritical COz to be operated at an undesirably low linear velocity and also limit the low end of the SFC pressure operation range. Figures 2 and 3 also show that the reduced plate high of the EVB-DVB column in both GC and SFC modes was on the order of 10-20, which is higher than typical. This is possibly due to the fact that the retention of solutes on the macroporous EVB-DVB polymer stationary phase is governed by the surface adsorption-desorption mechanism, and mass transfer is relatively slow under the experimental conditions as observed in our previous study (11). The unified GC and SFC separation of a synthetic mixture of n-alkanes on the octylsilica column is shown in Figure 4. The separation of volatile components such as Cl-Cll n-alkanes was obtained in the high-pressure GC mode using 160 atm of helium as the mobile phase, with temperature programming from 40 to 150 OC. The mobile phase was then switched directly from high-pressure helium to supercritical COz at 28 min from sample injection. The separation of less volatile and nonvolatile components such as C12-C30 evencarbon-numbered n-alkanes was achieved in the SFC mode with pressure programming from 140 to 400 atm. Neither high-pressure GC nor SFC alone could be used t o separate all volatile and nonvolatile compounds over such a wide boiling point range in a single analysis. On the other hand, the

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mixture. Conditions: 160 atm of helium as the mobile phase with temperature program from 40 to 150 O C at 10 OC/mIn in the GC mode, mobile phase switching at 28 min, CO, pressure program from 140 to 400 atm at 6 atmlmin in the SFC mode, Deltabond octylsilica column. advantages of the unified GC and SFC technique is clearly evidenced by the complete resolution of all volatile and nonvolatile compounds with one sample injection using the same column, restrictor, and FID in one instrument in a single sample run. In unified GC and SFC, the sample is injected into the column with high-pressure helium in the GC mode, and there is a transition of the mobile phase from helium gas to supercritical COP during the sample run. Sample injection, sample zone focusing, and transition from GC and SFC modes are very important aspects in unified GC and SFC. Figure 5 shows the unified GC and SFC chromatogram of the same C1-Cm n-alkane mixture when the temperature of the sample injector was at 30 "C. Good separation of C1-Cll n-alkanes in the GC mode was obtained, as expected. However, severely split peaks of C14and C16and the C12peak to a small degree were observed, and the separation of C18-C30 even-carbonnumbered n-alkanes became normal again in the SFC mode of the sample run. When the temperature of sample injector was maintained 120 OC, the split peak phenomena did not occur under otherwise identical experimental condition as shown in Figure 4. In conventional GC, the sample injector is usually operated a t elevated temperature in order to vaporize the sample injected and to ensure the proper sample zone transfer to the column. On the other hand, the sample injector is normally operated at room temperature in SFC; the problem of proper sample zone transfer from the injector to the column is usually much less critical due to the solvating power of the supercritical COP. In unified GC and SFC, the sample is injected with high-pressure helium in GC mode. When the sample injector is operated at low temperature, the sample zone injected is likely not focused, especially for less volatile compounds. The more volatile solutes injected are transferred onto the stationary phase, and some portion of less volatile solutes may be stuck along the inner walls of the injector and column inlet tubing. This problem may cause band broadening for the solutes eluted in the GC mode. For solutes that can only be eluted in the SFC mode, the problem is negligible

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since the sample zone is likely to be refocused during SFC separation. For solutes such as C12, C14, and c16 in the nalkane sample eluted during or closely after the mobile phase is switched from helium to supercritical COz, the proper transfer and focusing of sample zone is most critical. In the GC mode, some portion of the solute may already be migrating through the column and the other portion may still be stuck on the inner walls of the injector and column inlet due to the low injector temperature. When the mobile phase is switched to supercritical COz, the later portion of the solute is eluted from the injector and column inlet tubing. However, it may not be able to catch up and refocus with the portion of the solute that is already being eluted through the column. The split peak of the solute thus results. The results indicate that the sample injector should be heated to ensure proper sample zone transfer and focusing. The experimental results show that heating the sample injector to 120 "C is adequate for most samples tested in this study such as alkanes, alcohols, and free fatty acids over a wide boiling point range. The effects of different GC mobile phases in unified GC and SFC separation were also studied. Gaseous COPinteracts with stationary phase and solute molecules even a t very low densities and is a much stronger mobile phase compared to helium (IO). This is because COz molecule posssesses in-

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duced-dipole moments while helium is an inert atomic gas. To study the effect of gaseous COPas a mobile phase in the GC mode in unified GC and SFC, a gas mixture containing COPa t a partial pressure of 40 atm and helium at a partial pressure of 120 atm was prepared. This COP/He gas mixture was used as the mobile phase in the GC mode so that COP would remain at the gaseous state in the mobile phase in the GC mode at a total pressure compatible for the unified GC and SFC operation. Figure 6 shows the unified GC and SFC separation of C1-CI8 alcohols using 160 atm of helium as the mobile phase in the GC mode. Figure 7 shows the separation of the same sample using the COz/He gas mixture as the mobile phase in the GC mode under otherwise identical experimental conditions. The GC mode was changed to the SFC mode 19 min after the sample injection in both separations. The results show that the retention of volatile alcohols eluted

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Flgure 9. Unified GC and SFC separation of a synthetic alcohol and free fatty acid mixture. Conditions: 160 atm helium as the mobile phase with temperature program from 30 to 170 OC at 20 OC/min in the GC mode, mobile phase switching at 15 min, CO, pressure program from 130 to 350 atm at 5 atm/min in the SFC mode, EVB-DVB polymer column.

in the GC mode was reduced noticeably when the C02/He gas mixture was used as the mobile phase compared to helium. For example, the retention time of 2-propanol was 10.6 min with the C02/He gas mixture as the mobile phase and it was 13.8 min with helium. The most significant difference between the two separations was that 1-butanol was eluted at 16.9 min in the GC mode with the COz/helium mixture as the mobile phase while the same solute was eluted at 22.2 min in the SFC mode when helium was used as the mobile phase in the GC mode. This kind of retention time and selectivity modification in the GC mode resulted from using different mobile phases and would be useful in some separations in unified microbore packed column GC and SFC. The unified high-pressure GC and SFC technique was applied for analysis of several complex samples. Figure 8 shows the unified GC and SFC separation of a mixture of an unleaded gasoline and a crude oil on the octylsilica microbore packed column. The volatile components in the sample were eluted in the GC mode with a temperature program, and the nonvolatile componenb from the crude oil fraction were eluted in the SFC mode with a pressure program. The microbore column packed with the highly inert EVB-DVB polymer stationary phase was found to be very useful in the separation of polar volatile and nonvolatile compounds in unified GC and SFC. Figure 9 shows the separation of a synthetic mixture of alcohols and free fatty acids. Several volatile alcohols and acetic acid in the sample were separated in the GC mode. A very good separation of C3-CB n-free fatty acids was obtained in the SFC mode with supercritical COz as the mobile phase. In summary, the results of this study show that the unified GC and SFC can be carried out on the same microbore packed column using a conventional SFC system with minor hardware modifications for mobile-phase switching. The results also indicate that unified microbore packed column GC and SFC

is a very powerful technique for separation of volatile and nonvolatile compounds in complex samples since GC and SFC alone may not provide complete separation of all components over a wide range of polarity and volatility. The development of suitable stationary phases that can be used in the GC, SFC, and LC modes is one of the key aspects in the development of unified microcolumn chromatography. The ultimate unification of GC, SFC, and LC separation on the same microcolumn in one instrument would undoubtedly provide an extremely valuable separation tool for probing complex samples. LITERATURE CITED (1) Giddings, J. C. Dynamics of Chromatography; Marcel Dekker: New York, 1965. (2) Yang, F. J. I n Microbore Column Chromatography: A U n l M Approach to Chromatography; Yang, F. J., Ed.; Marcel Dekker: New YOrk, 1989; pp 1-36. (3) Bartle, K. D.; Davies, 1.; Raynor. M. W.; Clifford, A. A.; Kithinji, J. P. J . Microcol. Sep. 1989, 1 , 63-70. (4) Ishii, D.; Takeuchi, T. J . Chromtogr. Sei. 1989, 27, 71-74. (5) Ishii, D.; Niwa, T.; Ohta, K.; Takeuchi, T. Hi?C&CC, J . High Resolut. Chromatogr. Chromatogr. Commun. 1988, 1 1 , 800-801. (6) Gemmel, E.; Schmitz, F. P.; Kiesper, E. M C & CC, J . Hbh Resdut. Chromatcgr. Chromatogr. Commun. 1988, 1 1 . 901-903. (7) Gemmei, B.; Schmitz. F. P.; Klesper, E. J . Chromatogr. 1988, 455, 17-21. (8) Pentoney, S. L., Jr.; Giorgetti, A.; Griffiths, P. R. J . Chromtogr. Sei. 1987, 25. 93-98. (9) Davies, I.; Yang, F. J. Unified Open Tubular Column Gas and Supercritical Fluid Chromatography. Unpublished results. (10) Liu, Y.; Yang, F. J. High Pressure Microbore Packed Column Gas Chromatography Using Common Liquid Chromatography Stationary Phases. Unpublished results. (11) Liu, Y.; Yang. F. J.; Pohi, C. J . Mlcrocol. Sep. 1990, 2 , 245-254. (12) Ashraf-Khorassani, M.; Shah, S.; Taylor, L. T. Anal. Chem. 1990, 62, 1173-1 176.

RECEIVED for review November 12, 1990. Accepted January 24, 1991.