Anal. Chem. 2000, 72, 3620-3626
Modifier Effects on Column Efficiency in Packed-Column Supercritical Fluid Chromatography Wei Zou and John G. Dorsey*
Department of Chemistry, Florida State University, Tallahassee, Florida 32306-4390 Thomas L. Chester
Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, Ohio 45253
We investigate the effects on column efficiency of methanol, acetonitrile, ethanol, and 1-propanol used as modifiers in packed-column SFC. C-18, phenyl, and cyano columns were used with both nonpolar and polar solutes. For highly retained nonpolar solutes, addition of modifier significantly increased apparent column efficiency, especially for the C-18 column. For polar solutes, the presence of modifier dramatically improved retention and efficiency with an apparent efficiency dependence on modifier type and amount. Temperature and pressure effects on efficiency were also studied. In recent years there has been a remarkable rise in research interest and applications of packed column supercritical fluid chromatography (pSFC).1,2 In most cases of pSFC separation, polar modifiers are added to supercritical carbon dioxide to enhance the solubility of polar solutes in the mobile phase. The effects of modifiers on SFC retention have been reported in detail.3-10 Early systematic studies showed that adding modifiers reduces solute retention both by enhancing the solvating strength of the mobile phase and by deactivating the residual silanol groups on the stationary phase.3-5 Recent work includes studies on the effects of various modifiers and additives in SFC mobile phases from the point view of linear solvation energy relationships.6,7 However, unlike retention studies, effects of modifiers on column efficiency (1) Anton, K.; Berger, C. Supercritical Fluid Chromatography with Packed Columns; Chromatographic Science Series, Vol. 75; Marcel Dekker: New York, 1998. (2) Chester, T. L.; Pinkston, J. D.; Raynie, D. E. Anal. Chem. 1998, 70, 301R319R. (3) Randall, L. G. In Ultrahigh-Resolution Chromatography; Ahuja, S., Ed.; ACS Symposium Series No. 250; American Chemical Society: Washington, DC, 1984; pp 135-169. (4) Blilie, A. L.; Greibrokk, T. Anal. Chem. 1985, 57, 2239-2242. (5) Berger, T. A.; Deye, J. F. Anal. Chem. 1990, 62, 1181-1185. (6) Cantrell, G. O.; Stringham, R. W.; Blackwell, J. A.; Weckwerth, J. D.; Carr, P. W. Anal. Chem. 1996, 68, 3645-3650. (7) Blackwell, J. A.; Stringham, R. W.; Weckwerth, J. D. Anal. Chem. 1997, 69, 409-415. (8) Schmitz, F. P.; Leyendecker, D.; Leyendecker, D. J. Chromatogr. 1987, 389, 245-250. (9) Janssen, J. G.; Schoenmakers, P. J.; Cramers, C. A. J. High Resolut. Chromatogr. 1989, 12, 645-651. (10) Berger, T. A. J. Chromatogr., A 1997, 785, 3-33.
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have not been studied extensively or systematically, although there are several reports of peak shape and efficiency improvement with addition of modifiers, especially for polar solute separations.6,8-10 The symmetry and efficiency improvements with modified eluent were generally attributed to higher uniformity of the stationary phase caused by deactivation of residual silanol groups. There are few reports of how different types and amounts of modifiers affect column efficiency. A detailed study on modifier effects will not only contribute to a better understanding of mass transfer in modified pSFC but also will aid in the selection of modifiers in practical SFC separation. It has been shown in reversed-phase LC studies that chromatographic efficiency and selectivity are significantly related to the modifier or additive choice. Dorsey et al. found that addition of a small percentage of 1-propanol can dramatically improve peak shape and increase column efficiency in micellar chromatography.11 In a thorough study of secondary chemical equilibria in reversed-phase LC (RPLC), Foley and May also demonstrated the 1-propanol advantage in efficiency.12 The efficiency improvement was attributed to better wetting of the stationary phase by 1-propanol compared to other modifiers. This 1-propanol advantage has also been applied to practical LC separations to reduce the reequilibration time following gradient elution in RPLC.13 It is worthwhile to examine modifier effects on separation efficiency in pSFC, since pSFC also uses conventional LC columns and modifiers. EXPERIMENTAL SECTION (1) Chemicals. Test solutes included naphthalene and anthracene obtained from Sigma (St. Louis, MO), pyrene, chrysene, and dibutyl phthalate (DBP) from Aldrich (Milwaukee, WI), and phenol from Fisher Scientific (Fair Lawn, NJ). Modifiers tested were HPLC grade methanol, acetonitrile, and 1-propanol from Fisher Scientific and ethanol (absolute) from CAPER Alcohol & Chemical Co. (Shelbyville, KY). SFC grade carbon dioxide was the main mobile-phase component and was obtained from Air Products (Allentown, PA). (11) Dorsey, J. G.; DeEchegaray, M. T.; Landy, J. S. Anal. Chem. 1983, 55, 924-928. (12) Foley, J. P.; May, W. E. Anal. Chem. 1987, 59, 110-115. (13) Cole, L. A.; Dorsey, J. G. Anal. Chem. 1990, 62, 16-21. 10.1021/ac991417u CCC: $19.00
© 2000 American Chemical Society Published on Web 06/23/2000
(2) Chromatographic System. A Hewlett-Packard G1205A SFC system with two pumps was used in this study. Pump A was used for neat CO2, and pump B, for neat modifier. The amount of modifier added into the carbon dioxide mobile phase was expressed in volume. Sample injection was performed using a Rheodyne 7410 injection valve fitted with a 0.2 µL sample loop. Detection was at 254 nm with a HP diode array detector (DAD). Pressure was controlled downstream from the column outlet and the DAD. Three commercial HPLC packed columns were used: Supelcosil LC-18-DB (250 × 4.6 mm, 5 µm); DuPont Zorbax Phenyl (150 × 4.6 mm, 5 µm); Beckman Ultrasphere Cyano (250 × 4.6 mm, 5 µm). The system was equilibrated for at least 30 min for each change in chromatographic conditions. Each solute was injected at least in triplicate. The void time was determined by the baseline deflection of methanol for the C-18 column and the refractive index change peak of hexane for the phenyl and cyano columns. (3) Calculation of Efficiency and Resolution. The FoleyDorsey equation for efficiency calculation was used:14,15
N)
41.7(tR/W0.1)2 (B/A + 1.25)
(1)
Here tR is the retention time of solute, W0.1 is the peak width at 10% height, and B/A is the asymmetry factor of the peak. Resolution was calculated as
RS )
∆tR 0.5(Wb1 + Wb2)
(2)
where ∆tR is the difference of retention time of the pair of solutes and Wb1 and Wb2 are the base widths of the two adjacent peaks. RESULTS AND DISCUSSION (1) Retention. Addition of organic modifier considerably decreased retention of solutes on all three columns, especially for polar solutes which are very strongly retained with pure CO2 as the mobile phase. The change of retention factor, k′, with the modifier concentration in the mobile phase has the same trend for our solutes as has been previously shown.3-5,9 The four modifiers used here show similar effects on retention. Except for phenol, the decrease of k′ with acetonitrile as modifier is much less than that with alcoholic modifiers. The retention factors of dibutyl phthalate (DBP) and phenol with different modifier concentration on phenyl and cyano columns are shown, respectively, in Figure 1. It should be noted that, on the phenyl column, the elution order of DBP and phenol with acetonitrile as modifier was reversed compared to other modifiers. With acetonitrile used as modifier, phenol eluted later than DBP, while, with other modifiers, phenol was eluted earlier. The reversed was not found on the cyano column, where phenol eluted later than DBP with all four modifiers. The decrease of solute retention with addition of modifier is believed to be due to the increase in solvent strength of the mobile phase and to the deactivation of the stationary phase. Although (14) Foley, J. P. Dorsey, J. G. Anal. Chem. 1983, 55, 730-737. (15) Bidlingmeyer, B. A.; Warren, F. V. Anal. Chem. 1984, 56, 1583A-1596A.
Figure 1. Plots of modifier effects on retention, log k′, for DBP and phenol at 80 °C and 210 bar, 2.0 mL/min: (A) cyano column; (B) phenyl column. Solute: filled symbols with solid lines are for DBP; open symbols with dotted lines are for phenol. Modifier: 9 (0) ) methanol; ( ()) ) 1-propanol; 2 (4) ) acetonitrile.
the solvent strength of acetonitrile/CO2 binary mixtures is less than that of methanol or 1-propanol/CO2 mixtures,16 the change of retention for phenol caused by the different solvent strength should not be so significant; otherwise it should also be seen for DBP as the solute. As expected from its structure, retention of phenol should be much more affected by silanol groups on the stationary phase than DBP. Therefore, the greater retention of phenol and the reversed elution order of phenol and DBP with acetonitrile as modifier both imply poor deactivation of the stationary phase by acetonitrile, which is consistent with the fact that acetonitrile is not a good hydrogen bonder.17 This proposition is also supported by the effect of acetonitrile on column efficiency with comparison to other modifiers as is discussed later. The consistent retention order of phenol and DBP for acetonitrile and other modifiers can be explained by the fact that the bonded cyano column is much more polar than the phenyl column. The cyanobonded phase retains phenol much more than DBP so that the difference of the coverage of silanols between acetonitrile and other modifiers is not enough to change the elution order of phenol and DBP. However, the poor deactivation of cyano column by acetonitrile is still shown in the later efficiency study. The effects of temperature and pressure on solute retention with added modifier (shown in Figure 2,A,B, respectively) are (16) Berger, T. A. In Supercritical Fluid Chromatography with Packed Columns; Anton, K., Berger, C., Eds.; Chromatographic Science Series, Vol. 75; Marcel Dekker: New York, 1998; pp 19-58. (17) Johnson, B. P.; Khaledi, M. G.; Dorsey, J. G. Anal. Chem. 1986, 58, 23542365.
Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
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Figure 2. Plots of solute retention vs temperature (A) and pressure (B) on C-18 column, 2.0 mL/min: (A) CO2 with 5% methanol at 210 bar; (B) CO2 with 3% methanol at 40 °C. Solute: ( ) naphthalene; 9 ) anthracene; 2 ) pyrene; × ) chrysene.
similar to those with pure CO2 as the mobile phase. Under constant pressure, an increase in temperature first increased retention. After reaching a maximum, k′ values dropped at higher temperatures. While at constant temperature, retention of solutes decreases with an increase of pressure. The temperature effect can be explained as the results of two opposite effects with increase of temperature as has been pointed out in the literature with pure CO2 as the mobile phase:18 the decrease in mobilephase density and the increase in vapor pressure of the solute. The former decreases the solubility of the solute while the latter increases it. The pressure effect at constant temperature simply followed the density change. An increase in pressure decreased retention due to the increase in density of the mobile phase. According to approximate calculations and previous studies in our laboratory,19 the critical point of the modified mobile phase should fall in our temperature and pressure study range (40-200 °C, 90-300 bar) above a certain percentage of modifier. No observation of an abrupt change in retention in our temperature and pressure studies reflects that there is no discontinuity of mobile phase state from subcritical to supercritical condition. (2) Efficiency. It should be noted that, in packed-column SFC, a pressure drop along the column causes expansion of the mobile phase and weakening of its solvent strength, resulting in a new spatial gradient on the column with more retention as peaks near the outlet, the inverse of what occurs in gradient elution LC. Thus, (18) Leyendecker, D. In Supercritical Fluid Chromatography,; Smith, R. M., Ed.; The Royal Society of Chemistry: London, 1988; pp 53-101. (19) Ziegler, J. W.; Dorsey, J. G.; Chester, T. L.; Innis, D. P. Anal. Chem. 1995, 67, 456-461.
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the column efficiency, which is measured from the retention time and temporal width of peaks at the column outlet, cannot be interpreted in SFC the same as in HPLC and GC where retention of solute is independent of pressure drop. Here, all efficiencies reported are apparent efficiency since we have no way to estimate the focusing effect of the column pressure drop. The apparent efficiency will approach to true efficiency as the mobile phase becomes less and less compressible. Efficiencies reported are averages of three or more measurements, and the relative standard deviation never exceeded 8%. (a) Nonpolar Solutes. The dependence of apparent column efficiency on modifier type and amount for PAH solutes on the C-18 column is shown in Figure 3. Addition of modifiers significantly increased the apparent column efficiency for late eluting solutes, while there is little change for early eluting ones. For naphthalene, the plate number did not change much in the presence of modifier. It actually decreased a bit with large amounts of ethanol. Plate numbers for anthracene, pyrene, and chrysene increased with addition of modifier and were related to the amounts of modifier in the mobile phase. For anthracene, efficiency increased with the modifier concentration up to about 5%. However, efficiency for pyrene and chrysene continuously increased with modifier concentration. Improving symmetry of peaks does not contribute very much to the increase in apparent efficiency for solutes because tailing or fronting is not severe even with pure CO2 as the mobile phase. For PAH solutes, 1-propanol does not improve peak shape compared with other modifiers. In fact, there is no apparent difference in efficiency effects among the four modifiers used here except for very late eluting solutes pyrene and chrysene, where methanol shows a little more efficiency, while acetonitrile is slightly worse. The absence of a 1-propanol advantage in efficiency in SFC can be explained by the change of mobile phase polarity. Carbon dioxide, the main mobile phase component in SFC, is a nonpolar solvent.20 Therefore, the extremity of the nonpolar stationary phase and polar mobile phase, which is the case in RPLC, does not exist in SFC, so the better wetting effect of 1-propanol is not applicable in SFC. Both phenyl and cyano columns, which are more polar than C-18 columns, show similar behavior of apparent column efficiency in the presence of modifiers for nonpolar PAH solutes. The dependence of plate numbers on modifiers for the phenyl column is shown in Figure 4, and the trend is very similar for the cyano column (figures not shown). Addition of modifier has relatively little effect on apparent efficiencies for naphthalene, anthracene, and pyrene. Only the last eluted peak, chrysene, shows an increase in apparent efficiency when modifiers are added to the mobile phase. Unlike the C-18 column, the increase in plate count for chrysene is not dramatic and does not continue with increase of modifier amount. Again, the four modifiers exhibit little difference in efficiency for either phenyl or cyano columns. The early observation of an increase in separation efficiency in SFC with addition of modifier, especially for polar solutes, was attributed to coverage of “active sites”, the residual silanol groups, by the adsorption of modifiers.8-10 However, this cause is not (20) Johnston, K. P.; Harrison, K. L.; Clark, M. J.; Howdle, S. M.; Heitz, M. P.; Bright, F. V.; Carlier, C.; Randolph, T. W. Science 1996, 271, 624-628.
Figure 3. Plots of modifier effects on efficiency for nonpolar solutes on C-18 column at 80 °C and 210 bar, 2.0 mL/min. Solute: (A) naphthalene; (B) anthracene; (C); pyrene; (D) chrysene. Modifier: solid line with 9 ) methanol; broken line with b ) ethanol; dotted line with ( ) 1-propanol; dashed line with 2 ) acetonitrile.
Figure 4. Plots of modifier effects on efficiency for nonpolar solutes on phenyl column at 80 °C and 210 bar, 2.0 mL/min. Solute: (A) naphthalene; (B) anthracene; (C) pyrene; (D) chrysene. Modifier: solid line with 9 ) methanol; dotted line with ( ) 1-propanol; dashed line with 2 ) acetonitrile.
adequate to explain the following two features shown for PAH solutes in our study: first, the continuous increase of apparent efficiency with the increase of modifier concentration for late
eluting solutes on the C-18 column; second, the rather symmetric peaks even with pure CO2 as the mobile phase. Since it was suggested in other SFC studies and is demonstrated in our later Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
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discussion for polar solutes that a certain amount of modifier is enough to fully cover the residual silanol groups on the stationary phase, the observation of a continuous increase in plate number for the C-18 column with the modifier concentration changing from 0 to 20% implies that the deactivation of residual silanols on the stationary phase is not the only contribution for the increase of apparent column efficiency for late eluting nonpolar solutes. The second feature also indicates that the effect from silanol groups on retention of PAH solutes is not as important as it for polar solutes. Therefore, there should be other causes for the increase of apparent column efficiency for nonpolar solutes with addition of modifier besides the deactivation of silanols. Improvement of mass transfer in the presence of modifier should be either related to changes of properties in the mobile phase or in stationary phase. The mobile phase contribution to efficiency improvement is not straightforward from the point view of enhancement in solvent strength of the modified mobile phase because it has been reported that the higher solvent strength with modifier is believed to cause “clustering” of the modifier molecules around solutes,21 which should not improve mass transfer from the mobile phase to the stationary phase, and the diffusivity of solutes in the mobile phase changes very little with addition of modifier.22 If contributions of the mobile phase are ruled out, the increase of efficiency with modifier is more likely caused by the improved uniformity of the stationary phase due to adsorption of the mobile phase components. The uniformity discussed here should not only include the coverage of the residual silanol groups on the stationary phase by the modifier but also the composition change of the stationary phase. The composition of the stationary phase will change with increasing modifier concentration in the mobile phase due to the adsorption of modifier as well as CO2.23,24 The small effect for early eluting solutes with all three columns is consistent with our attribution of efficiency improvement to modification of the stationary phase because the interaction between early eluting solutes and the stationary phase is much less than for late eluting solutes. The effects of modifier on resolution of PAH solutes are shown in Figure 5. For the C-18 column, addition of small amounts of modifier increases resolution until the percentage of modifier reaches about 5. Further increases in modifier amount decreases resolution. For phenyl and cyano columns, the presence of modifier decreased the resolution continuously with increase of modifier concentration. The increase of resolution with small amounts of modifier on the C-18 column is caused by the efficiency improvement although retention and selectivity both decreased with addition of modifier. For phenyl and cyano columns, the decrease of resolution is simply due to decreased retention and the negligible change of efficiency with modifier. These results imply that it is beneficial to add small amounts of modifier into the mobile phase for separation of nonpolar solutes on C-18 columns because of improved efficiency and resolution with shorter retention time. For phenyl and cyano columns, addition of modifier is not as helpful from the view of separation efficiency. (b) Polar Solutes. Because DBP can be eluted from both phenyl and cyano columns with pure CO2 as the mobile phase, it (21) Kim, S.; Johnston, K. P. AIChE J. 1987, 33, 1603-1611. (22) Smith, S. A.; Shenai, V.; Matthews, M. A. J. Supercrit. Fluid 1990, 3, 175. (23) Strubinger, J. R.; Song, H.; Parcher, J. F. Anal. Chem. 1991, 63, 98-103. (24) Strubinger, J. R.; Song, H.; Parcher, J. F. Anal. Chem. 1991, 63, 104-108.
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Figure 5. Plots of modifier effects on resolution for PAH solutes at 80 °C and 210 bar, 2.0 mL/min. Column: (A) C-18; (B) phenyl; (C) cyano. Key: ( for anthracene/naphthalene; 9 for pyrene/anthracene; 2 for chrysene/pyrene.
was chosen as the polar solute to study the effects of addition of modifier. Phenol was used as a probe of silanol activity on the stationary phase as in other studies.25 although it cannot be eluted with pure CO2. The modifier effects on apparent column efficiency for DBP on the phenyl and cyano columns are shown in Figure 6. Both phenyl and cyano columns show similar behavior. Addition of modifier into the mobile phase dramatically increased the plate number for DBP as solute. The very broad and tailing peak with pure CO2 as the mobile phase was significantly improved with plate counts increasing to 9000 for the phenyl column and 20 000 for the cyano column. Around 1-2% percent of modifier is enough to reach maximum efficiency for both columns. The severely tailing and broad peak was quickly improved to a sharp and symmetric shape when the modifier amount was above 2%. Further increase of modifier amount in the mobile phase shows no effect on efficiency. There is no apparent difference among (25) De Weerdt, M.; Dewaele, C.; Verzele, M.; Sandra, P. J. High Resolut. Chromatogr. 1990, 13, 40-46.
Figure 6. Plots of modifier effects on efficiency and asymmetry factor for dibutyl phthalate (DBP) at 80 °C and 210 bar, 2.0 mL/min. (A) phenyl column; (B) cyano column. Filled symbols with solid line for efficiency; open symbols with dot line for asymmetry factor. Modifier: 9 (0))methanol; ( ()))1-propanol; 2 (4))acetonitrile.
Figure 7. Plots of modifier effects on efficiency and asymmetry factor for phenol at 80 °C and 210 bar, 2.0 mL/min: (A) phenyl column; (B) cyano column. Filled symbols with solid lines represent efficiency; open symbols with dotted lines represent the asymmetry factor. Modifier: 9 (0) ) methanol; ( ()) ) 1-propanol; 2 (4) ) acetonitrile.
the three modifiers used here. 1-Propanol is not better than other modifiers, and slightly more acetonitrile is needed to obtain the best efficiency compared to other modifiers. Phenol only eluted in the presence of >0.5% modifier in the mobile phase. Figure 7 shows its efficiency dependence on modifier for the cyano and phenyl columns. Increasing modifier amount significantly increased apparent efficiency for alcoholic modifiers (methanol and 1-propanol) but not for acetonitrile. Although phenol can be eluted from either column with acetonitrile as modifier, the apparent efficiency is extremely low with severely tailing and broad peaks. Even with up to 20% acetonitrile, the plate count is still below a few hundred, which is lower than the plate numbers at 0.5% methanol or 1-propanol. Asymmetry factor (B/A) changes show that the extremely low efficiency is consistent with peak tailing with acetonitrile as the modifier. For methanol and 1-propanol as modifiers, the poor peak shape is improved dramatically with increases of the modifier amount. It also should be noted that, for the phenyl column, around 5% alcoholic modifier is enough to obtain maximum efficiency and symmetry, while, for the cyano column, the optimum amount of modifier becomes 7%. For both columns, methanol seems a little better than 1-propanol in efficiency. The extremely low efficiency for phenol on both phenyl and cyano columns with acetonitrile as the modifier confirms our previous statement that acetonitrile is not a good silanol blocker. The poor coverage of silanols by acetonitrile can be further substantiated by the result that even with a large percentage of
acetonitrile (>15%), when the retention of phenol is equivalent to that with methanol or 1-propanol as modifier (see Figure 1), the apparent efficiency with acetonitrile is still extremely low with severe peak tailing. The little difference in retention and efficiency of DBP between acetonitrile and alcoholic modifier indicates that the effect of silanols is insignificant for DBP retention, where the improvement of efficiency is more likely the results of the composition change on the stationary phase due to the adsorption of the mobile phase components. Although the polarities of the bonded phase are different for phenyl and cyano columns, the lack of a 1-propanol advantage in efficiency again suggests little wetting effects in SFC. Since phenol is used as a probe for silanol activity, its efficiency and peak symmetry can be used as an indication of the extent of silanol coverage by adsorption of modifier. The results in Figure 7 show that 5% is the minimum amount of modifier required for complete silanol coverage for the phenyl column because above 5% the apparent efficiency and peak symmetry for phenol reach maximum. For the cyano column, 7% is the least required modifier amount. Because the polar solutes were mostly unretained on the C-18 column, the modifier effect on efficiency for DBP and phenol on the C-18 column was not studied. Resolution is not discussed for polar solutes because it becomes less illustrative and practical here due to the difference of properties between DBP and phenol. (c) Temperature and Pressure Effects. The dependence of the apparent column efficiency on pressure for pyrene on the C-18 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
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Figure 8. Plot of pressure effects on efficiency of pyrene as solute with and without modifier at 80 °C, 2.0 mL/min: circle symbols for the C-18 column; square symbols for the phenyl column. Mobile phase: filled symbols with solid lines for pure CO2; open symbols with dotted lines for CO2 + 5% methanol.
and phenyl columns is shown in Figure 8 (other solutes have similar results which are not shown). There is little effect of pressure on plate number for either column with or without modifier, although changes in pressure do change retention of the solute. It has been demonstrated that the adsorption of the mobile phase components changes very little with change of pressure when the chromatographic conditions are well above the critical point of the mobile phase,23,24 which is the likely case in our pressure study (150-300 bar). The result of our pressure effect study implies that pressure can be used to control separation without any sacrifice in efficiency in SFC. The effect of temperature on pyrene apparent efficiency with and without modifier in the mobile phase is shown in Figure 9. With pure CO2 as the mobile phase, the apparent efficiency for pyrene first decreases and then increases with increase in temperature. The valley profile for pure CO2 is the combined result of two effects caused by the increase of temperature: an increase of linear velocity of the mobile phase and increase of the diffusivity
3626 Analytical Chemistry, Vol. 72, No. 15, August 1, 2000
Figure 9. Plot of temperature effects on efficiency of pyrene as solute with and without modifier at 80 °C, 2.0 mL/min: circle symbols for the C-18 column; square symbols for the phenyl column. Mobile phase: filled symbols with solid lines for pure CO2; open symbols with dotted lines for CO2 + 5% methanol.
of solute in the mobile phase. Higher linear velocity decreases efficiency while higher diffusivity of solute increases plate number. However, this trend becomes obscure in the presence of modifier. With 5% methanol in the mobile phase, the apparent efficiency of pyrene does not change much on the C-18 column with increase of temperature, while, on the phenyl column, addition of modifier seems to rule out the diffusivity effect on efficiency, showing a continuous decrease of efficiency with increase in temperature. Other solutes show similar temperature effects on efficiency as pyrene with and without modifier. ACKNOWLEDGMENT J.G.D. is grateful to The Procter & Gamble Co. for continued support of our work.
Received for review December 10, 1999. Accepted March 27, 2000. AC991417U
