Anal. Chem. 1990, 62, 1173-1 176
1173
Column Efficiency Comparison with Supercritical Fluid Carbon Dioxide versus Methanol-Modified Carbon Dioxide as the Mobile Phase M. Ashraf-Khorassani Suprex Corporation, 125 William Pitt Way, Pittsburgh, Pennsylvania 15238
S. Shah and L. T. Taylor* Department of Chemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 -0212
The efflclency of packed columns containing either cyanopropyl or phenyl stationary phases has been determined with both a polar (carbazole) and a nonpolar (naphthalene) anawe. Supercrtlical Huld CO, and methanoknodlfled CO, were employed as mobile phases. Relatively high percentages of methand have Nttie effect on mlnLnum plate height regardless of the polarity match between analyie and stationary phase. High moMle phase linear velocitles, however, appear to have less effect on cdumn efficiency when polarity match is more slmllar between analyte and stationary phase.
INTRODUCTION Elution of polar compounds using supercritical fluid (SF) C02 or modified SF COPhas been an interest to many laboratories. While many workers have reported the use of both types of mobile phases for elution of polar compounds (1-5), there appears to be no fundamental comparison such as van Deemter plots between SF COPand modified SF COz for polar analytes eluted from packed columns. Several reports have been published regarding comparison of high-performance liquid chromatography (HPLC) and supercritical fluid chromatography (SFC) for elution of polynuclear aromatic hydrocarbons (6, 7) and nitrogen-containing compounds (8). Column efficiency was noted to be essentially the same at optimum linear velocity. Since the diffusivity of a SF is considerably higher than that of a liquid, the maximum efficiency was realized a t a much higher linear velocity in the SF case. Most fundamental studies on SFC efficiency involving both packed and capillary columns deal with nonpolar analytes and 100% SF COP. In order to address this deficiency, we have recently completed (9) a study of capillary and packed columns employing amide analytes which revealed that low SF density-low temperature conditions enhance column efficiency as long as the column is not overloaded. Direct injection (i.e. overload) in the capillary case surprisingly suggested that higher temperature-higher SF density enhanced efficiency. This research, therefore, describes a study on the efficiency of packed columns using either a polar or nonpolar stationary phase for the elution of both a polar and a nonpolar analyte using SF COPand methanol-modified SF CO,. Also, in this study the efficiency of columns using different percentages of methanol-modified COPwill be compared. The effect of high linear velocities of COPand methanol-modified COP on packed column efficiency is also a concern of this study. EXPERIMENTAL SECTION
AU chromatographicseparations were carried out under isobaric (3000 psi) and isothermal (80 "C) conditions employing a Hewlett-Packard 1084B liquid chromatographmodified for SFC. The 0003-2700/90/0362-1173$02.50/0
h e m velocity was varied by changing the flow rate of mobile phase while keeping the pressure at the end of the column fixed with a back pressure regulator. Modifier was varied by utilizing a Suprex micro LC pump. The dual pump system employed in this study is similar to that described previously (IO). Samples were injected into the chromatographicsystem with a Rheodyne 7129 injection valve. A variety of packed columns (Zorbax cyano (Du Pont), Deltabond cyano (KeystoneScientific),and phenyl (IBM)) were used. The dimensions of all columns were kept constant to ensure comparable results (25 cm X 4.6 mm i.d., 5 - ~ m particle size). Van Deemter plots were obtained for both naphthalene and carbazole using 100% GO2and methanol-modifiedC02as the mobile phase. A mixture of components varying in polarity (diphenylamine, N-phenyl-1-naphthylamine, 5-ethylindole, carbazole, a-phenylindole) was used to estimate resolution at different flow rates. The concentration of componentsin each mixture was in the range 500-700 ng/pL. Each mixture was dissolved in HPLC grade chloroform (Fisher Scientific, Raleigh, NC) and approximately 0.1 pL of each mixture was injected on to the chromatography columns. Ultraviolet detection at 254 nm was employed. To calculate column efficiency, simple triangulation was used employing the equation N = 16[tR/W12
where N is the plate number of the column, t R is the uncorrected retention time of the analyte, and W is the peak width at the base line. Height equivalent theoretical plate (HETP) was calculated by dividing the length of the column in micrometers by the column plate number. Each point in every plot represents three replicate injections.
RESULTS AND DISCUSSION The chromatographic parameters under investigation in this study were linear velocity, stationary phase, mobile phase modifier, and analyte polarity. First, we wished to study the efficiency of the same packed columns exhibiting polar and nonpolar stationary phases for elution of both a polar (carbazole) and a nonpolar (naphthalene) compound using relatively high flow rates of SF COP. Second, the efficiency of packed columns for elution of the same compounds using methanol-modified COzwas of interest. For this latter study, the percentage of methanol in COz was varied between 2% and 20%. Also, it was of interest to compare the efficiency of select columns under subcritical and supercritical conditions. In the first part of this study, columns with different stationary phases were employed with 100% COPusing both a nonpolar (naphthalene) and a polar (carbazole) analyte. Van Deemter plots for naphthalene using Deltabond cyano, Zorbax cyano, and IBM phenyl columns were similar (Figure 1A). The bonded phase phenyl and Zorbax cyano columns, however, showed a slightly lower HETP for naphthalene, which we believe is due to more extensive partitioning (and less adsorption) of the solute between the mobile and these eta@ 1990 American Chemical Society
ANALYTICAL CHEMISTRY, VOL, 02, NO. 11, JUNE 1, 1990
1174
(Deltabond cyano), 3.7 s (Zorbax cyano), and 3.1 s (IBM phenyl). Since peak widths were quite similar for naphthalene on each column, the triangulation method used for computing column plate number was believed to be satisfactory. Capacity factors for the three columns were 0.28, 0.41, and 0.48, respectively. Similar columns were used to generate van Deemter plots for carbazole analyte, Figure 1B. The plots for Deltabond cyano and Zorbax cyano were practically identical. The triangulation method was also used with carbazole as the analyte; although, thismethod is not preferred for asymmetric peaks. A comparison appears justified since both cyano columns yield similar peak widths. The strange behavior of the plot for carbazole on the phenyl column could be due to incompatibilityof the solute and stationary phase and/or an insufficiently deactivated column giving rise to adsorption of carbazole to silanol sites rather than partitioning with the stationary phase. Base peak width for carbazole with the phenyl column (K’ = 5.8) was quite large as compared with the two cyano columns (e.g. 34.0 s, phenyl; 11.7 s, K‘ = 4.3, Deltabond; 14.0 s, K’ = 5.0, Zorbax). An examination of Figure 1 reveals that the efficiency of each column for naphthalene decreased at the same rate as linear velocity increased above approximately 0.5 cm/s. However, for carbazole increasing linear velocity from 0.4 to 0.8 cm/s resulted in an insignificant lost in column efficiency for both Zorbax cyano and Deltabond cyano. Shah et al. (9) have observed similar behavior for elution of amides from a Deltabond cyano column at low density. With increasing density, however, the van Deemter plot in the study by Shah et al. exhibited a much steeper upward slope with increased linear velocity in spite of what was believed to be a good polarity match of analyte and stationary phase. The Deltabond cyano column was further studied by separating at test mixture of five components varying in polarity. Figure 2 shows the separation of this mixture at three different
80 70
l0o, t B , I
0
0.1
0.2
0.3
0.4
0.6
0.8
0.7
0.8
0.9
1
1.1
Linear Velocity (cm/sec) 100 Nlphmw* 100% coz
BO -L 80
’
--
70
so 60:: 40 --
30 -20
--
+
lo--A , 0 ,
tionary phases. The polymer-coated Deltabond cyano column was slightly less efficient. Even though the particle sizes were identical, the relative stationary phase loading for each column was not known, which may account for the slight differences in minimum HETP. Peak widths at the base for naphthalene on all three stationary phases with 100% C02 were 3.6 s
WL/MIN
5
c
*0 .1 .2 ,3
/
3
6
r7
&
1
o
i
2
5
2
4
1
0
.
1
.
2
A
TIME (mW Figure 2. SFC separation of basic compounds on Deltabond cyano column at different CO, flow rates: 80 O C ; 3000 psi back pressure; UV (254 nm) detection; (1) diphenylamine, (2) N-phenyl-I-naphthylamine, (3)B-ethylindole, (4) carbazole, (5) a-phenyllndole.
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990
Table I. Resolution of Peak Pairs in Figure 2'
carbon dioxide flow rate, mL/min 2.0 2.5 3.0 3.5
4.0 4.5 5.0
100
Rzs
R34
R46
8.0 7.8 7.4 7.3 6.7 6.2 6.8
6.2 6.0 4.8 4.1 3.7 3.5 3.0
11 11 12 11 11 11 11
4.8 4.8 4.5 4.0 4.0 4.0 3.7
cwwn
cnsauca? D M m M CN
30 20
0
Key: 1, diphenylamine; 2, N-phenyl-1-naphthylamine;3, 5-
0.1
0.2
0.3
0.4
0.6
0.6
0.7
0.8
0.9
1
1.1
0.9
1
1.1
Linear Velocity (cm/8ec)
ethvlindole; 4. carbazole: 5, a-Dhenvlindole.
I:
PIab Helpht (urn)
90
R 12
1175
Plate Helght (Urn) 100
Plate Helght (urn)
,
100 1
90 80 70
70
60
60 40
30
30 --
20
20 --
lo--A 08
,
''16 0 ,
,
,
0
0.1
0.2
,
0.3
0.4
0.6
0.0
0.7
0.6
Linear Velocity (cm/sec) Figure 4. van Deemter plots for cyano and phenyl packed columns with analytes naphthalene and carbazoie, 2%-20% methandmodlfled
100 90
co*.
--
NlphlNWW 2% CH,ORRl%
i;
0tll.t"ml
Phenyl
40
30 20
--
--- B
lo O (
CN
1
I
co2
of 2% methanol to C02 changed drastically both the shape of the van Deemter plot and the chromatographic peak (peak width 34.0 s (K' = 5.8) for 100% COz,9.2 s (K'= 2.0) for 98% CO2/2% CH30H))for carbazole on the phenyl column (Figure 3A). This suggests that methanol modifies the phenyl stationary phase probably by hydrogen bonding to accessible silanol sites. This process leads to a less active column and greater partitioning of solute with the modified stationary phase rather than adsorption at accessible silanol sites. The optimum efficiency of the Deltabond cyano column for naphthalene using 2% methanol-modified COz was also approximately the same compared to 100% C02. In this case peak width was unaffected by the addition of 2% methanol to the mobile phase. At higher linear velocities the methanol modified column was perceptibly less efficient for naphthalene elution. Overall, column efficiency was more dependent on linear velocity in this case (Le. rising slope was steeper). For the phenyl column/naphthalene case, the addition of 2% methanol also had minimal effect on peak shape and on the optimum efficiency, but the dependency of efficiency on linear velocity was again more pronounced in the modified mobile phase situation, Figure 3B. It was of interest to study the effect of higher percentages of modifier on the efficiency of packed columns. Experiments were, therefore, conducted employing 2% to 20% methanol-modified COP Figure 4 shows the van Deemter plots for naphthalene and carbazole under these conditions. For naphthalene with the polar stationary phase, increasing percentage of methanol in COz gave rise to increased HETP at high linear velocities. This increase in HETP with linear velocity was more pronounced with increasing percentages of methanol. It is believed this increase in HETP is due to an increase in viscosity of the mobile phase and also to a decrease in partitioning of the nonpolar analyte between the increasingly more polar mobile phase and polar stationary phase. It is important, however, to point out that the minimum (Le. optimum linear velocity) HETP for naphthalene obtained by using different percentages of modifier remained constant (Le.
1176
ANALYTICAL CHEMISTRY, VOL. 62, NO. 11, JUNE 1, 1990 Plats Hdght (urn)
60
lop 0 ,
0
I
0.1
0.2
0.3
0.4
0.6
0.6
0.7
0.8
0.0
1
1.1
Linear Velocity (cm/sec) 100
NlphIkMn 2% CH,OnqSW
90 --
co?
DMlbona CN
-7 0 --
80
60
--
60
--
4 0 --
30 -20 --
I
lo+B 0 , , 0
0.1
0.2
0.3
0.4
0.6
0.6
0.7
0.8
0.9
1
1.1
Linear Velocity (cmhec) van Deemter plots for cyano packed column with analytes naphthalene and carbazole with subcritical and supercritical methanol-modified COP. Figure 5.
20-30 pm). The triangulation method was again used to compute column plate number. The base peak width for naphthalene remained fixed upon changing methanol from 2% to 20% (K' = 0.14-0.06). The behavior of van Deemter plots for carbazole with increasing amounta of modifier in C02 was different. Since carbazole is more polar than naphthalene, the addition of modifier (up to 10%) does not appear to affect column efficiency, Figure 4A. This apparent insensitivity to increasing amount of methanol may result from a combination of factors. Carbazole base peak width changes from 8.2 s (K' = 2.1) for 2% CH30H to 7.7 s (5% CH30H) to 5.3 s (K'= 1.5) for 10% CH30H. Therefore, the data in Figure 4 may reflect relative efficiency more so than absolute column efficiency. With 20% methanol-modified COz, HETP was increased slightly. The modifier in COz increases the polarity of the mobile phase which should cause the polar analyte to partition better between the polar mobile phase and polar stationary phase. The addition of polar modifier, however, increases the viscosity of the mobile phase which should decrease the efficiency of the column. In this case (Le. carbazole/Deltabond cyano) no decrease in column efficiency was observed a t optimum linear velocity. It was, however, observed that with increasing percentage of methanol in COz the pressure drop
acro9s the column became larger, which no doubt results from the greater viscosity of the mobile phase. Finally, the efficiency of a packed column was compared under sub- and supercritical conditions. Figure 5A shows the van Deemter plot for carbazole obtained a t 3000 psi and 30 "C (subcritical) and at 3000 psi and 80 "C (supercritical) with 2% methanol modified COz. Little difference in efficiency for the column regardless of linear velocity in either of these situations is apparent. This observation is surprising since the density and diffusivity at these two temperatures are expected to be different. For this naphthalene/cyano column case (Figure 5B), the optimum linear velocities and optimum column efficiencies correspond closely, although the decrease in column efficiency with increasing linear velocity is more pronounced under supercritical conditions. These results support the unified chromatographic principle espoused for gas-supercritical fluid-liquid chromatography (I1). In summary, relatively high percentages of methanol have little effect on minimum plate height regardless of the polarity match between analyte and stationary phase. High mobile phase linear velocities, however, appear to have less effect on column efficiency when polarity match is the same between analyte and stationary phase. We, therefore, conclude that packed column efficiency is essentially insensitive to changes in linear velocity, mobile phase density, temperature, modifier concentration, and mobile phase critical conditions when analyte-stationary phase polarity is well-matched. It can further be concluded that even when the polarity is not well-matched, these variables do not drastically affect the overall net packed column efficiency.
ACKNOWLEDGMENT Discussions of these results with Terry Berger and Jerry Deye were extremely helpful. The loan of a micro liquid chromatographic pump by Suprex Corp. is greatly appreciated. LITERATURE CITED Field, S. M.; Markiies, K. E.; Lee, M. L. J . Chromatogr. Scl. 1987, 25, 223. Yonker, C. R.; Smith, R. D. J . Chromatogr. 1986, 367, 25. Ashraf-Khorassani, M.; Taylor, L. T.; Berger, T. A.; Deye, J. F. J . Chromatogv. Sci. 1989, 27, 105. Levey, J. M.; Richey, W. M. J . Chrometogr. Sci. 1988. 24, 242. Wright, B. W.; Halinoski. H. T.; Smith, R. D. Anal. Chem. 1985, 57, 2823. Randall, L. G. I n Ultra tUgh ResduMon chrometogrephy; Ahuga, S., Ed.: ACS Symposium Series No. 2 5 0 American Chemlcal Soclety: Washington, DC, 1984; p 135. Fjeklsted. J. C.; Jackson, W.P.; Peaden, P. A.; Lee, M. L. J . Chromatogr. Sci. 1983, 27, 222. Ashraf-Khorassani, M.; Taylor, L. T. J . Chromatogr. Sci. 1988, 28, 331. Shah, S.; Taylor, L. T. Chromatographie,in press. Shah, S.; Taylor, L. T. J . Chromatogr.,in press. Martire, D. E.; Boehm, R. E. J . Phys. Chem. 1987, 97, 2433.
RECE~VED for review November 28,1989. Accepted February 23, 1990. The financial support provided by the Environmental Protection Agency is appreciated.
