Anal. Chem. 1990, 62, 1554-1560
1554
Supercritical Fluid-Solid Chromatography Using a Carbonaceous Stationary Phase Tina M. Engel and Susan V. Olesik*
Department of Chemistry, The Ohio State Uniuersity, 120 West 18th Auenue, Columbus, Ohio 43210
Results from a prellmlnary evaluatlon of a porous glassy carbon (PGC) material for packed-column supercrltlcal fluid chromatography (SFC) are reported. The material Is characterlzed In terms of its retentlon characterlstlcs. Increasing temperature at constant pressure causes lnitlal increases In solute capaclty factors with subsequent slow decreases when sufficient temperatures (-145 "C) are reached. Varlatlon of temperature at constant carbon dloxide mobile phase densities yields enthalpies of Interaction for low molecular weight from -0.5 to -1.5 kcal/mol. PGC Is highly organlcs (C&,) retentive; use of a supercrltlcal fluid mobile phase that competes wlth solutes for adsorptlon onto the PGC allows slgnlflcant control of solute retention. PGC offers potential advantages not provided by other materials. PGC-SFC exhibits reverse-phase characterlstlcs slmllar to those found for PGC-HPLC, retention behavior previously unavailable In SFC. PGC Is highly stereospeclflc and will be useful in SFC applications requiring separations of Isomers and molecules with only slight structural differences.
INTRODUCTION In 1982, Gilbert, Knox, and Kaur (I) demonstrated the use of porous glassy carbon (PGC) as a stationary phase for use in packed column high-performance liquid chromatography (HPLC) and gas chromatography (GC). This PGC was extensively characterized by Knox et al. (2) Recently, PGC was successfully applied in HPLC analyses of aromatics (3)and basic pharmaceuticals (4, 5). PGC has many desirable characteristics as a stationary phase, including 3- to 10-wm spherical particles, 80% porosity, high surface area (150 m2/g), a spongelike structure resistant to shearing forces, strong hydrophobicity, and a uniform surface evidenced by the absence of active sites ( 2 ) . PGC is stable to acids, bases, and high pressure, allowing use of PGC with harsh mobile phase conditions. The chromatographic behavior of PGC as a stationary phase has been compared to that of reverse-phase materials such as ODS-silica ( 2 ) . The retention of solutes on PGC is independent of solute functional groups and determined primarily by the interactions of the delocalized A electrons of the graphitic carbon with the solute's electron cloud. Retention order is therefore strongly controlled by the polarizability of the molecule. One interesting application of the unique retention on PGC caused by electronic interactions is the separation of cations and anions of a radioactive pharmaceutical (6). Due to the molecular-level flat nature of the graphitic ribbons of PGC, the configuration of a molecule can also affect its retention. PGC-HPLC has been demonstrated to be a viable means of separating structural and configurational isomers (7-9). Supercritical fluids with polar modifiers can readily solvate many polar species. This attribute is commonly used in supercritical fluid extraction (SFE) processing ( I O ) . Presently, SFC is more limited than SFE in the scope of polar molecules that it can separate. Since retention on PGC is not affected by functional group polarity, we believe that its use in SFC 0003-2700/90/0362-1554$02.50/0
may expand the scope of applications of SFC. This paper describes initial studies demonstrating use of PGC as a stationary phase in SFC. Included are descriptions of observed retention mechanisms and column efficienciesand a discussion of possible extensions and applications of SFC on PGC. EXPERIMENTAL SECTION Column Preparation. An acetone slurry of PGC particles (Hypercarb PGC 99, manufactured by Shandon Scientific, Ltd., England, and provided by Keystone Scientific, Inc., Bellefonte, PA) was placed in a reservoir manufactured from 4-mm-i.d. X 10-cm-long stainless steel tubing. The slurry was pushed with 272 atm pressure into 318-wm-i.d. X 25-cm-long untreated fused silica tubing (Polymicro Technologies) equipped with a 15-pm4.d. x 20-cm-longfused silica linear restrictor. The PGC packing was held in the column with a microbore column end fitting (U-434, Upchurch Scientific, Inc., Oak Harbor, WA) equipped with a replaceable frit (C407X, Upchurch). When the tubing was completely filled with PGC, the high-pressure pump was turned off, and the column was allowed to gradually return to ambient pressure by venting through the restrictor. The fused silica column proved to be fragile at temperatures exceeding 100 "C; apparently, the temperature-dependent expansion and/or contraction rates of PGC and fused silica differ enough to shatter the fused silica housing. Because PGC is harder than fused silica, the fused silica is probably etched during heating, which causes weakening and eventual fracture of the containment material. Temperature variation studies were therefore conducted with a column prepared in glass-lined stainless steel tubing. No loss in efficiency due to fracturing of the glass liner was observed. This column was prepared as described earlier, except the slurry was packed into a 300-gm-i,d. X 30-cm-long piece of glass-lined stainless steel (Scientific Glass Engineering). Chromatographic System. An ISCO LC-2600 precision syringe pump or a pLC-500 micro flow pump (ISCO, Lincoln, NE) wm used to pressurize and deliver supercriticalfluid mobile phase. Samples were injected with a W-series high-pressure injection valve that was fitted with a 60-nL rotor (Valco Instruments, Houston, TX). The column was maintained in a Hewlett-Packard 5790 series gas chromatograph. The detector for most studies was a Spectroflow 757 UV detector operated at 210 nm. Detector dead volume was minimized by preparing a flow cell from 318wm-i.d. untreated fused silica tubing with the polyimide coating removed. Temperature variation studies were conducted with the standard HP 5790 series flame ionization detector (FID). Materials. Mobile phases included supercritical grade carbon dioxide (COz)(Scott Specialty Gases), nitrous oxide, ethane, and 5.15% methanol in COz (Matheson). Carbon dioxide modified with acetonitrile (spectrophotometric grade Mallinckrodt) or methanol (anhydrous spectrophotometric grade Mallinckrodt) containing citric acid (reagent grade Mallinckrodt) was prepared by mixing known volumes in a syringe pump. Much of the evaluated data were generated by using a standard methylene chloride solution containing 1mg/mL each of benzene, phenol, o-cresol, p-cresol, o-nitrotoluene, and 3,5-xylenol. Temperature-variation experiments were made using a standard carbon disulfide solution containing 1 mg/mL each of ethylbenzene, n-butylbenzene, n-decane, n-pentylbenzene, n-dodecane, and n-hexylbenzene. Standard materials were obtained from Aldrich Chemical Co. and were specified at 96% or greater purity. RESULTS AND DISCUSSION Mechanism of Retention in PGC-SFC. The initial purpose of this study was to demonstrate the applicability of 0 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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capillary column using supercritical COP mobile phase. Compounds are (S) solvent, (1) benzene, (2) phenol, (3) o-cresol, (4) p-cresol, (5) o-nitrotoluene, and (6) 3,5-xylenol.
the PGC stationary phase to packed column SFC. We s t a t e d with COP, the most common SFC mobile phase, and used typical temperature and pressure conditions. Figure 1shows a chromatogram of the test mixture obtained with supercritical C 0 2 mobile phase at 40 "C and 251 atm. Some of the test mix components, especially p-cresol and 3,5-xylenol, had tailing peaks. In addition, attempts to elute larger molecules such as naphthalene by increasing the mobile phase pressure or temperature were unsuccessful. However, the addition of modifiers such as methanol (Figure 2A) to COz drastically improved the peak shapes and lowered the retention. The effect of supercritical solvent on the resultant chromatography is described in detail later in this section. The principal controlling mechanism of adsorption on graphitic carbon is dispersive interactions between the solute and the stationary phase (11-13). Snyder (13) described adsorption on solids from liquids by the general equation log KO = log V,
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A and B were generated with the same column as in Figure 1 using supercritical COPmodified with 5% methanol and CO, modified with 5% methanol containing 0.18% citric acid mobile phases, respectively. Compound identifications are given in Figure 1.
(1)
where K O is the equilibrium distribution coefficient for the adsorption-desorption interaction of the solute with the stationary phase, V , is the adsorbent surface volume, and AE is the net dimensionless free energy of adsorption. For a solute molecule to be adsorbed, it must first displace solvent (mobile phase) molecules. Accordingly, the net free energy of adsorption is more completely described in the equation log
B
+ a d ( s d - AB€&+ a(So- As€") (2)
where a d and a are terms that describe the surface activity toward dispersive and specific (H-bond, dipole-dipole,induced dipole-dipole) interactions, respectively, S d and So are the standard dispersive and selective adsorption energies of the solute in a standard chromatographic system, respectively, A, is the area required by the solute when it is adsorbed on the solid adsorbent surface, and 6d and eo are the dispersive and selective solvent strength parameters, respectively. The second and third terms on the right portion of eq 2 describe the net adsorption energy caused by dispersive interactions in the interchange of solvent molecules with solute molecules, and the fourth and fifth terms describe the interaction energy caused by selective interactions involved in the interchange. If dispersive interactions predominantly control the adsorption on carbon, then the fourth and fifth terms in eq 2 are negligible. Equation 2 then simplifies to
For a specific column of adsorbent, V , is constant throughout a chromatographic analysis. In addition when molecules such as those used in this study are adsorbed, then
A, varies minimally between solutes (14). Accordingly, a linear relationship between the dispersive energies of the analytes, s d , and the log of the distribution coefficient should exist if the adsorption retention mechanism on the PGC stationary phase with supercritical fluid solvent is predominantly controlled by dispersive interactions with the carbon. In general, dispersive forces associated with functional groups in a molecule are additive. Values of Sdcan therefore be estimated by adding the dispersive adsorption energies associated with various functional groups of the solute. By use of tables of group adsorption energies for the interaction with carbon (13),the values for each model compound were calculated. Figure 3 shows the s d values plotted against the log of the capacity factors observed from the two test mixtures using COPmobile phase. Retention of the test mixture analytes shows a linear relationship with their dispersive energies. As shown in Figure 3, o-nitrotoluene has different retentive behavior than the phenols in the test mixture. The retention of o-nitrotoluene is less than that predicted on the sole basis of dispersive interactions of the compound with the PGC. The assumption was made in the derivation of eq 3 that specific interactions do not contribute to retention on the PGC and solute-solvent interactions in the mobile phase negligibly affect the overall retention behavior. Nitro groups are facile electron acceptors. Accordingly, the formation of charge transfer complexes with electron donor molecules is well documented (15-1 7). The decreased retention of o-nitrotoluene with addition of methanol or acetonitrile modifier is not surprising since preferential solvation of polar groups by such modifiers is also well documented (18). However, the lower than expected retention of o-nitrotoluenein pure carbon
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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Flgure 3. Dependence of log k' on standard dispersive adsorption energy: (0)supercritical CO, mobile phase at 40 "C and 189 atm with points representing compounds (lower left to upper right) phenol, ocresol, p -cresol, o-nitrotoluene, 3,5-xylenol, and o-nitrophenol; (A) supercritical CO, mobile phase containing 5 % methanol modifier at 40 " C and 189 atm with points representing compounds (lower left to upper rlght) phenol, ocresol, p-cresol, onkrotoluene,3,5-xylenol,and o-nitrophenol; (e)supercritical COP mobile phase at 45 " C and 238 atm with points representing compounds (left to right) ethylbenzene, n-butylbenzene, n-decane, n-pentylbenzene, n-hexylbenzene, and n-dodecane.
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As previously discussed, retention in adsorption chromatography on carbon is believed to be mainly due to dispersion interactions between the solute and the carbon surface. Larger molecules with increasing polarizabilities are preferentially adsorbed. Another useful characteristic of PGC is the molecular-level flat surface of the material which leads to preferential adsorption of planar molecules and a definite selectivity for isomers. Separations of molecules with only slight structural differences are frequently observed. For example, the separation of antibiotic and steroid stereoisomers using PGC-HPLC was recently reported (7). By use of supercritical COz mobile phase at 40 "C and 189 atm, k'values of 2.4, 3.2, and 3.5 were observed for o-cresol, rn-cresol, and p-cresol, respectively. The order of selectivity for the cresol isomers is the same as found in reverse-phase HPLC; however reverse-phase HPLC cannot discriminate between the ortho and meta cresol isomers (7). Bassler and Hartwick have also demonstrated the separation of all three cresol isomers by using PGC-HPLC (7). However, the order of retention was reversed from that found in PGC-SFC. The effects of supercritical solvents on retention were considered further. Acetonitrile and methanol modifiers were added to the C 0 2 to determine the effect on compound peak shapes and retention. Specifically, COz containing 5% or 10% acetonitrile, 5% methanol, or 5% methanol containing 0.18% citric acid was evaluated. Previous work has shown that low percentages of citric acid added to methanol modifiers in COz significantly improves peak shapes in bonded phase SFC (19). Example chromatograms obtained by using 5% methanol and 5% methanol/0.18% citric acid modifiers at 40 "C and 189 atm are shown in parts A and B of Figure 2, respectively. The variation of the analyte capacity factor, k', with the percentage of acetonitrile or methanol modifier is shown in Figure 4. In general, addition of acetonitrile or methanol modifier decreases the capacity factors of analytes and eliminates tailing of the para-substituted phenols chromatographic peaks. The addition of a very small amount (0.18%) of citric acid to the 5% methanol modifier also results in significant capacity factor reductions, especially in the case of o-nitrotoluene. The addition of acetonitrile, methanol, or methanol/citric acid to the
0
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Figure 5. Effect of alternative supercritical fluid mobile phases on compound capacity factors. Data were generated by using the same column and conditions as in Figures 2 and 4; compound Mentiflcatlons are given in Figure 4. COz mobile phase also allows elution of naphthalene, but not condensed aromatics with more than two rings a t 40 "C and pressures less than 251 atm. However, Figure 5 shows that supercritical nitrous oxide or supercritical ethane causes increasingly dramatic reductions of the capacity factors. With supercritical ethane as the mobile phase, larger aromatic compounds were readily eluted, such as naphthalene ( k ' = 1.2), biphenyl (k' = 1.3), o-terphenyl (k' = 1.3), 1,2,3,4-tetraphenylbenzene ( k ' = 7 . 5 ) , acenaphthylene (k' = 7.9), and fluorene (k' = 14.5). The purpose of introducing a modifier or using a different supercritical fluid is to adjust the capacity factors of molecules. In packed-column SFC using silicon-based stationary phases the primary effect of modifiers is to cover silanol active sites
ANALYTICAL CHEMISTRY,
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AH fkca//mo/) Figure 6. Product of dispersive solvent strength parameter and molecular surface area (A2) vs heats of adsorption on carbon (kcal/mol). on the stationary phase (20-22). However, PGC does not have active sites; therefore the polarity of a molecule has little bearing on its retention on carbon ( 2 ) . More importantly, dispersive interactions control the adsorption of both the solute and the solvent on carbon. Colin and Guiochon proposed an eluotropic series for liquid mobile phases interacting with carbon stationary phases (23). The solvent strength, €d, was determined from theory based on the Hildebrand solubility parameter or from the proportionality of the interfacial tension of a liquid and its dispersive interaction (23). This solvent strength parameter described well the solvent effect in HPLC. As expected, the solvent strength was found to increase with increasing molecular size and aromaticity. We initiated the development of a similar eluotropic series for supercritical fluids. One method of determining solvent strength was to use the Hildebrand solubility parameter, 6, for supercritical fluids as described by Giddings (24) and determine a relationship between 6 and the elution strength of the solvent. The Hildebrand solubility parameters for supercritical ethane, nitrous oxide, and C02 for the conditions described above are 6.9 H, 7.5 H, and 7.7 H, respectively. If these parameters are good measures of the solvent strength of supercritical ethane, nitrous oxide, and COPas they interact with the carbon, then the COP would have the strongest affinity for PGC and ethane the least. However, the reverse affinity was observed. Clearly the solubility parameter derived by Giddings does not solely measure dispersive solvent strength of supercritical fluids. Another means of determining the dispersive solvent strength of a supercritical fluid was considered. Gas-solid chromatography (GSC) has been used extensively to measure the heats of adsorption on graphitic carbon (25). The heats of adsorption determined by GC describe well the adsorption of molecules at the monolayer level. Previous work has shown that adsorption of liquids on graphitic carbon does not involve multilayer adsorption (26). Minimal multilayer adsorption of solute is expected for temperatures above the critical point of solvents as well (26). Therefore a linear relationship would be expected between the GSC-measured heats of adsorption, AH,and the product of the solvent strength parameter and the molecular surface area of the solvent, td X A. Figure 6 shows a plot of this product plotted versus the GSC heats of adsorption for the compounds studied by Colin and Guiochon (25,27). By use of the equation of the line in Figure 6, the molecular surface areas (12, 14) and heats of adsorption on carbon (25, 27) for COP, nitrous oxide, and ethane, the approximate solubility parameters were determined to be -0.0017, -0.0012, and -0.0011, respectively. These values properly describe order of solvent strength of the supercritical fluids used in this study (ethane > nitrous oxide > COz). This method is a means of providing approximate measures of solvent strength of supercritical fluids while providing di-
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Flgure 7. Van't Hoff plot collected at (A) 0.70 g/cm3density and (e) 0.55 g/cm3 density: (A)ethylbenzene; ( 0 )n-decane; (V)n-butyl-
benzene; ( * ) n-pentylbenzene; (W) ndodecane; and benzene.
(+)
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rection on which solvent systems will be the best for the separation of high molecular weight components by PGC-SFC. Clearly this series predicts that a supercritical solvent system such as supercritical C 0 2 modified with higher molecular weight alkanes such as octane and nonane should provide a higher solvent strength. No attempt was made to include the modified mobile phases in this approximate description of an SFC eluotropic series. A more detailed fundamental study of the important parameters controlling solvent strength in PGC-SFC is underway with encouraging results (28). Enthalpy of Interaction. The enthalpies of interaction of low molecular weight organic molecules with the PGC stationary phase using supercritical COPmobile phase were estimated by monitoring compound capacity factors while increasing the operating temperature under constant density conditions. Experiments were carried out using a density of 0.70 g/cm3 a t temperatures ranging from 45 to 90 "C and a density of 0.55 g/cm3 at temperatures ranging from 80 to 135 "C. The resulting van't Hoff plots obtained by using the two density conditions are shown in parts A and B of Figure 7 , respectively. The partial molar enthalpy of adsorption of the solute at infinite dilution was calculated from the slopes of the lines in Figure 7 and are listed in Table I. Enthalpies of adsorption ranged from -0.57 to -1.5 kcal/mol. The enthalpy of adsorption values are similar in magnitude to those found for liquidsolid chromatography (29). In addition, the enthalpies of adsorption are more positive a t higher mobile phase densities and more negative as the size of the test molecule is increased; these observations are consistent with previously reported trends in bonded-phase SFC (22).
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ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
Table I. Enthalpy of Interaction kcal/mol
compound
ethylbenzene
-0.57 -0.94 -0.83 -0.99 -1.2 -1.1
n-decane
n-butylbenzene n-pentylbenzene n-dodecane n-hexylbenzene
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Dependence of k'on Temperature. The effect of temperature on retention with PGC and supercritical C02 mobile phase was studied by holding the pressure constant at 238 atm, varying the temperature from 45 to 175 "C in 10 "C increments, and monitoring the capacity factors of the test analytes. Test analyte capacity factors observed a t different temperatures are shown in Figure 8. At temperatures below approximately 145 "C, capacity factors for all test analytes increased with increasing temperature. This is strong evidence that at these temperatures isobaric retention variation with temperature is controlled by solvation forces, not the adsorption process. The adsorption process is an exothermic reaction. Accordingly, if adsorption is controlling the variation of k 'with temperature, then k'will decrease with increasing temperature. At temperatures exceeding approximately 145 "C the capacity factors for the test analytes decreased, indicating that at higher temperatures isobaric retention with temperature is predominantly controlled by the adsorption process. Temperature-dependent retention behavior results from volatility and solvation contributions. At lower temperatures, the solvating power of the supercritical fluid predominates; as the temperature of the fluid increases, the density of the fluid decreases and analyte retention increases. At higher temperatures, the solvating power of the fluid does not compete as well with the adsorption reaction; therefore the analyte capacity factors begin to decrease with temperature increase. Sie and Rijnders (23)observed similar retention behavior in supercritical fluid-solid chromatography on alumina. Yonker et al. (30) and Chester et al. (31) observed a sharp rise in capacity factor at temperatures immediately past the critical temperature followed by a gradual decrease at even higher temperatures for capillary SFC with bonded phases such as OV-17 and BP-10. Both groups also indicated that significant reductions in capacity factors could be obtained by modest temperature increases (30,31). However, Figure 8 shows that for PGC-SFC a significant reduction in capacity factor is not expected under isobaric conditions unless the temperature is
increased to values greater than ca. 200 "C. Dependence of Plate Height on Mobile Phase Velocity. The variation of the plate height, H , as a function of linear velocity for the test mixture using supercritical C 0 2 at 40 "C and 189 atm was evaluated. The linear velocity was varied by changing the length of the linear restrictor. All plate height values were corrected for contributions due to band dispersion in the connecting tubing using the Golay equation for open tubes (32). The variation in the column plate height as a function of linear velocity is shown in Figure 9. The lowest observed plate height was approximately 0.05 mm, which corresponds to a reduced plate height of 7. No obvious minimum of the curve was observed. For each compound studied the plate height varied minimally over the entire range of linear velocities studied. This behavior is a well-documented attribute of packed column SFC using bonded polysiloxane stationary phases. However, when supercritical fluidsolid chromatography was performed using an uncoated silica phase, the increase in the plate height with velocity was large (33).Slow diffusion in the pores of the silica was believed to be the cause. PGC-SFC clearly does not suffer from this problem. Comparison of Performance w i t h Liquids a n d S u percritical Fluids. As previously mentioned, PGC has been used as a stationary phase for liquid chromatography. Comparison of the chromatographic behavior of this material with supercritical fluids as opposed to liquids is useful. The PGC material used for this study was provided with a test chromatogram generated by using a liquid mobile phase. The manufacturer used 3,5-xylenol to assess PGC performance, but 3,5-xylenol demonstrated poor peak shape when supercritical C 0 2 was used as the mobile phase. For this reason, further studies of plate height vs linear velocity were conducted with other supercritical fluid mobile phases that gave improved peak shape for 3,5-xylenol. Additional plate height vs linear velocity curves were constructed by using COSmodified with 5% methanol containing a small percentage (0.18%) of citric acid at 40 "C and 189 atm. Plate heights were corrected for extracolumn contributions to dispersivity as described earlier. Data for o-cresol and 3,5-xylenol collected by using these conditions are shown in Figure 10. A minimum plate height of 0.05 mm, or reduced plate height of 7 , was observed. When a HPLC test chromatogram provided with the PGC was used, a plate height of 0.05 mm was also determined for 3,5-xylenol when using 95:5 methanol/ water mobile phase. Therefore, using reduced plate height as the evaluation criteria, equivalent performance to the manufacturer's HPLC test conditions was achieved by using supercritical fluid mobile phase. Previous publications claimed reduced plate heights of 2 to 3 for basic pharmaceuticals ( 4 ,
ANALYTICAL CHEMISTRY, VOL. 62, NO. 15, AUGUST 1, 1990
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packings, 4 varies over the range of 500 to 1000 (34). Alternatively, well-packed capillaries have values of 1 W 3 0 0 (35) and therefore are more permeable to flow than conventional columns. The flow resistance parameter for PGC has not been reported. Additionally, we are not aware of having being reported for any packed-column SFC. We compared the flow resistance parameter of CI8-packed SFC columns to that of the PGC-packed capillary SFC column used in this study. From data reported by Gere et al. (36)a flow resistance value of 1260 was calculated for a 4.6-mm-i.d. X 15-cm-long column packed with 3-pm Spherisorb ODS and using supercritical COz (392-atm inlet pressure, 154-atm column pressure drop, and 33 "C) as the mobile phase. The flow resistance parameter for a 318-pm-i.d. x 25-cm-long capillary column packed with 7-pm PGC was 3210 to 3350 using C 0 2 modified with 5% methanol at 40 "C, column inlet pressures ranging from 158 to 251 atm, and column pressure drops ranging from 70 to 126 atm. The same approximate value of flow resistance was also found for PGC in packed-capillary HPLC (37). The flow resistance parameter of the CI8SFC column was in the approximate range of values expected for an analytical-scale column with spherical packing. However, the PGC column demonstrated higher 4 values than expected in SFC and HPLC (37). The permeability of irregularly shaped packings can be much lower than that of spherical packings due to the presence of fine particles that are created in the packing process (38). The PGC material was studied with high-resolution microscopy. The particles were generally spherical, but the particle size varied considerably from approximately 2 to 10 pm. This nonuniform particle size may have caused the low permeability of the column.
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5); however, evaluation of provided chromatograms indicates that this calculation was done by using an unretained peak. The published plate heights for retained peaks were obviously much larger. Plate height vs linear velocity curves for o-cresol using COP and C02 modified with 5% methanol/citric acid were evaluated to determine if mobile phase composition has a significant effect on column efficiency. These data are shown in Figure 11. A slight improvement in efficiency is observed when neat supercritical C 0 2 is used as the mobile phase. Di Corcia and Liberti noted that the mass transfer coefficient in gas-solid chromatography is much smaller than in gas-liquid-solid chromatography because of slowed passage of the solute through the gas-liquid interface and retarded diffusion through the liquid phase (11). This slowed equilibrium is evidenced by lower efficiencies especially at higher mobile phase flow rates. The lower plate heights observed when using C02 mobile phase as compared to methanol-modified COPmay therefore indicate formation of a partial layer of methanol on the surface of the carbon. Flow Resistance. The flow resistance parameter, 4, is a unitless term that is often used to assess column performance. The value can be determined from experimentally measurable variables by using the equation
+
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(4)
where AP is the pressure drop across the column, q is the mobile phase viscosity, u is the linear velocity of the mobile phase, L is the column length, and d, is the particle diameter. This expression shows that the column permeability is inversely proportional to the flow resistance parameter. For analytical-scale and microbore columns of porous spherical
CONCLUSIONS In summary, preliminary results indicate that PGC is a viable stationary phase for SFC. Good efficiency, peak shapes, and selectivity were demonstrated. A major drawback of this material is its high affinity for large molecules. However, these studies indicate that through the appropriate choice of the supercritical fluid mobile phase a wider molecular weight range of molecules can be separated. Perhaps the three most interesting aspects of PGC are its structural stability, selectivity for structural isomers, and nonpolar nature. The use of PGC in SFC may provide distinct advantages in difficult analytical separations, allowing separations of molecules with only slight structural differences. In addition, PGC-SFC also provides reverse-phase HPLC-like retention mechanisms previously unavailable in SFC. In this article, we demonstrated the separation of moderately polar molecules. Since the polar functionalities have very little effect on the retention on carbonaceous stationary phases, this phase may also allow the extension of SFC applications to more polar compounds than previously possible. ACKNOWLEDGMENT The authors wish to thank Keystone Scientific, Inc., for providing a Hypercarb graphitized carbon HPLC column and bulk PGC for these studies. LITERATURE CITED (1) Gilbert, M. T.; Knox, J. H.: Kaur, 6. Chromtographia 1982, 76. 138-1 46. (2) Knox, J. H.; Kaur, B.; Millward, G. R. J . Chromatogr. 1988, 352, 3-25. (3) Ghauri, F. Y . K.:Simpson, C. F. Anal. Proc. London 1989, 26, 69-71. (4) Berridge, J. C. J . Chromtogr. 1988, 449, 317-321. (5) Mama, J. E.; Fell, A. F.: Clark, B. J. Anal. Proc. London 1989, 26, 71-73. (6) Emery, M. F.: Lim, C. K. J . Chromatogr. 1989, 479, 212-215. (7) Bassler, 6. J.; Hartwick, R. A. J . Chromatogr. Sci. 1989, 2 7 , 162-1 65. (8) Pawlak, Z.,; Kay, 0.: Clark, B. J. Anal. Roc. 1990, 2 7 , 16-18. (9) Fell, A.; Clark, 6.;Mamba, J. J . Chromatogr. 1988, 434, 337-384.
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(10) MCHugh, M. A.; Krukonis. V. J. Supercrifical Fluid Exfracfionh 7 C @ k S and Practice; Butterworth Publishers: Stoneham, MA, 1986; pp 181-198. (1 1) Di Corcia, A.; Liberti, A. In Advances in Chromatography;Giddings, J. C., Grushka, E., Cazes, J., Brown, P. R.. Eds.; Marcel Dekker, Inc.: New York, 1976; Vol. 14, pp 305-366. (12) Snyder, L. R. In Principles of Adsorption Chromatography;Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker, Inc.: New York. 1968. (13) Snyder, L. R. J . Chromafogr. 1968, 36, 455-475. (14) McClellan, A. L.; Harnsberger, H. F. J . Colloid Interface Sci. 1967, 2 3 , 577-599. (15) Bassler, B. J.; Kaliszan, R.; Hartwick, R. A. J . Chromafogr. 1989, $61, 139-147. (16) Smejkal, F.; Popl, M. 8hovB, A.; ZBzvorkovB, M. J . Chromafogr. 1980, 197, 147-153. (17) Mourey, T. H.; Siggia, S. Anal. Chem. 1979, 51, 763-767. (18) Kim, S.; Johnston, K. P. Ind. Eng. Chem. Res. 1967, 2 6 , 1206-1 2 13. (19) Giorgetti, A.; Pericles, N.; Widmer, H. M.; Anton, K.; Datwyler, P. J . ChrOmatOgr. SCI. 1989, 2 7 , 318-324. (20) Lee, M. L.: Markides. K. E. Science 1987, 235, 1342-1347. (21) Pekay, L. A.; Olesik, S. V. Anal. Chem. 1989, 61, 2616-2624. (22) Engelhardt. H.; Gross, A.; Mertens, R.; Peterson, M. J . Chromafogr. 1989, 477, 169-183. (23) Colin, H.: Guiochon, G.; Jandera, P. Chromatographia 1982, 75, 133- 139. (24) Giddings, J. C. Science 1968, 762, 67-73.
(25) Belyakova, L. D.; Kiselev, A. V.; Kovaleva, N. V . Russ. J . Phys. Chem. 1986, 40, 811-815. (26) deBoer, J. H. The Dynamical Character of Adsorption: 2nd ed.; Oxford University Press: London, 1968. (27) Avgul, N. N.; Kiselev, A. V. In Chemistry and Physics of Carbon; Walker, P. L., Jr., Ed.; Marcel Dekker: New York, 1970; Vol. 6. (28) Engel, T. M.; Olesik, S.V. Unpublished results. (29) Knox, J. H.; Vasveri, G. J . Chromafogr. 1973, 83, 181-194. (30) Yonker, C. R.; Wright, B. W.; Peterson, R. C.; Smith, R. D. J . Phys. Chem. 1985, 8 9 , 5526-5530. (31) Chester, T. L.; Innis, D. P. HRC& CC, J . HighResoiuf. Chromatogr. Chromatogr. Common. 1985, 8 , 561-566. (32) Sternberg, J. C. In Advances in Chromatography; Giddings, J. C., Keller, R. A., Eds.; Marcel Dekker, Inc.: New York, 1966: Vol. 2, pp 205-270. (33) Mourler, A.; Caude, M. H.; Rosset, R. H. Chromatographia 1982,2 3 , 21-25. (34) Knox, J. H.; Laird, G. R.; Raven, P. A. J . Chromafogr. Sci. 1980, 18, 453-461. (35) Knox, J. H. J . Chromatogr. Sci. 1980, 18, 453-461. (36) Gere, D. R.; Board, R.; McManigill. D. Anal. Chem. 1982, 5 4 , 736-740. (37) Cui, Y.; Olesik, S. V. Unpublished data. (38) Bristow, P. A . J . Chromafogr. 1978, 149, 13-28.
RECEIVEDfor review January 10,1990. Accepted May 3,1990.
Computer Simulation (Based on a Linear-Elution-Strength Approximation) as an Aid for Optimizing Separations by Programmed-Temperature Gas Chromatography D. E. Bautz, J. W. Dolan, W. D. Raddatz,' and L. R. Snyder* LC Resources, Inc., 3182C Old Tunnel Road, Lafayette, California 94549
If the dependence of retention on temperature is specified for the various components of a sample in isothermal gas chromatography (GC), it Is possible to predlct retention, bandwidth, and resoiutlon for programmed-temperatureGC separatlons as a function of experimental conditions. The use of a linear-elution-strength (LES) approxirnatlon for isothermal retention allows these predictions to be carried out more easily and convenlently, in turn facilitatlng rapid simulations with a personal computer. This approach to GC method development appears promising, especially if segmented-temperature programs are used. The LES approximation also provides added insight Into how different factors affect separation in programmed-temperature GC.
A rigorous treatment of programmed-temperature GC separation has existed for several years, as summarized by Harris and Habgood (1)and used by Dose (ref 2; see also other citations of ref 2 ) for the computer simulation of GC separation as a function of experimental conditions. Dose (3) further demonstrated that computer simulation can be a valuable tool in the systematic optimization of these separations, varying (for example) the initial temperature and programming rate. Several studies (3-10) suggest that band spacing (values of the separation factor a ) and resolution can often be varied by changing either (a) the separation temperature in isothermal GC or (b) the programming rate and/or starting Department of Mathematics, Linfield College, McMinnvilie,
97128.
OR
temperature in temperature-programmed GC. By analogy with liquid chromatographic separations based on gradient elution (11-14), this suggests the use of segmented-temperature programs as a potentially useful tool for maximizing overall sample resolution in GC separations. Our understanding of programmed-temperature GC is limited by the inherent complexity of the equations (1)that describe these separations. This has also prevented the wider use of computer simulation (as in refs 2 and 3) for GC method development. Thus, the application of computer simulation requires an initial knowledge of the dependence of (isothermal) sample retention on temperature. This information is most conveniently obtained (as described here) from two experimental runs using programmed-temperature GC, rather than a larger number of time-consuming isothermal runs-as has been required in previous attempts at the computer simulation of programmed-temperature GC. Furthermore, previous attempts at computer simulation (e.g., ref 2) have used numerical integration for the solution of the rigorous equations that describe separation as a function of experimental conditions. This approach results in calculation times of 10-30 s/chromatogram when using a personal computer (2, 15). These relatively slow computation times can be inconvenient when a large number of simulations are required to determine optimum final conditions (see ref 3 and the discussion of our Figure 6). In the present study we describe an approximate treatment of programmed-temperature GC that overcomes the various problems cited above. This so-called LES approximation is similar to the linear-solvent-strength (LSS) treatment for gradient elution in liquid chromatography (16,17); it allows a comparable simplification of the various relationships be-
0003-2700/90/0362-1560$02,50/0@ 1990 American Chemical Society
