2823
Anal. Chem. 1985,57,2823-2829
pressure (250 bar) and at subcritical temperature (20 'C). Influence of the Nature of the Modifier. The variation of the resolution with the nature of the mobile phase is shown in Figure 7a. We must emphasize that the addition of water to the modifier increases resolution while reducing the separation time. The lower the modifier polarity, the greater is this effect. Thus, with 2-propanol, the addition of water leads to an increase of about 50% in resolution, while the separation time, as clearly illustrated in Figure 7b, is about 2 times lower.
CONCLUSION Carbon dioxide super- and subcritical fluid chromatography, in conjunction with polar modifiers, permits the improvement of both column performance and resolution in the separation of enantiomers on chiral stationary phases. By choice of proper conditions and suitable modifiers, higher resolutions per unit of time than in classical liquid chromatography are obtained. Selectivity mainly depends on the nature of the polar modifier and on temperature, as in liquid chromatography, whereas the mobile phase density has no influence. Moreover, the retention mechanism of phosphine oxides seems to be similar in SFC and in LC. As a consequence, super- and subcritical fluid chromatography on a Pirkle phase can probably resolve all the racemics already separated in liquid chromatography in a shorter separation time. ACKNOWLEDGMENT We wish to thank Patrick Pescher for helpful discussions.
Registry No. COz, 124-38-9;MeOH, 67-56-1; EtOH, 64-17-5; 2-PrOH, 67-63-0; H20, 7732-18-5; methyl(4-methylnaphthy1)phenylphosphine oxide, 98687-96-8; methyl(2-methyoxynaphthy1)phenylphosphine oxide, 98687-97-9; methyl(1phenanthry1)phenylphosphine oxide, 98687-98-0; methyl(1naphthy1)phenylphosphine oxide, 37775-99-8;methyl(2-methylnaphthy1)phenylphosphine oxide, 98687-99-1. LITERATURE CITED Pirkle, W. H.; House, D. W.; Finn, J. M. J . Chromafogr. 1980, 192, 143. Pirkle, W. H., Flnn, J. M., Hamper, B. C., Schreiner, J., Pribish, J. R., Eliel, E. L., Otsuka, S., Eds. "Asymetric Reactions and Processes in chemistry"; American chemical Society: Washington DC, 1982; Am. Chem. Soc. Symp. Ser. No. 185, p 245. Pirkle, W. H.; Hyun, M. H.; Bank, B. J . Chromafogr. 1984, 316, 585. Pescher, P.; Tambute, A.; Oliveros, L.; Caude, M.; Rosset, R. N o w . J . Chim., 1985, 9 , 821. Mourier, P.; Sassiat, P.; Caude, M.; Rosset, R. Analusis 1984, 12, 229. Mourier, P.; Caude, M.; Rosset, R. Analusis 1985, 13, 299. &re, D. R.; Board, R.; McManigill, D. Anal. Chem. 1982, 5 4 , 736. Snyder, L. R. J . Chromatogr. Sci. 1978, 16, 233. Zief, M.; Crane, L. J.; Horvath, J. J . Li9. Chromafogr. 1984, 7 , 709. Thomas, J. P.; Brun, A.; Bounine, J. P. J . Chromafogr. 1979, 172, 107. Souteyrand, C.; Thlbert, M.; Caude, M.; Rosset, R. J . Chromatogr. 1983, 262, 1. Feist, R.; Schneider, G. M. Sep. Scl. Techno/. 1982, 17, 261. Lauer, H. H.;McManigili, D.;Board, R. Anal. Chem. 1983, 55, 1370.
RECEIVED for review April 29,1985. Accepted August 5,1985. This study was supported by the Soci6t6 Nationale ElfAquitaine (Production), Etablissement de Boussens.
Investigation of Retention and Selectivity Effects Using Various Mobile Phases in Capillary Supercritical Fluid Chromatography Bob W. Wright,* Henry T. Kalinoski, and Richard D. Smith
Chemical Methods and Kinetics Section, Battelle Northwest Laboratories, Richland, Washington 99352
The influence of varlous mobile phases on the retentlon and selectlvlty of a polarity-test mixture In caplllary supercrltlcal fluld chromatography (SFC) was Investlgated. Capaclty ratlos and selectlvltles were obtalned under equlvalent chromatographic operating condltlons using supercrltlcal carbon dloxide, nitrous oxlde, and ethane moblle phases and a relatively nonspeclflc 5 % phenyl poly(methylphenylsl1oxane) statlonary phase. A 2.5% (w/w) mlxture of methanol In carbon dloxlde was also evaluated. I n addition, chromatographic efflclency was compared at slmllar k' for each fluld. Carbon dloxlde and nitrous oxlde exhlblted similar solvatlng power with only Srlght dlfferences In selectlvlty. Ethane, belng less polar, displayed the poorest solvating power for the polar solutes and offered greater dlfferences In selectlvlty. The greatest dlfferences In selectlvlty were observed at lower temperatures and higher denslties. The carbon dloxldemethanol fluld mixture displayed dlfferent selectlvlty than pure carbon dloxlde, but not to the same extent as reported for packed column SFC.
Interest in supercritical fluid chromatography (SFC) is expanding. Supercritical fluids possess favorable properties
which render them attractive as mobile phases for chromatography. The density of a supercritical fluid approaches that of a liquid (and thus has similar solvating characteristics), but solute diffusivity remains higher and viscosity is lower ( I ) , which allows higher chromatographic efficiencies per unit time to be achieved relative to liquid mobile phases (2). Consequently, the potential advantages of both liquid and gas chromatography are combined. Furthermore, the solvating power of a supercritical fluid mobile phase is continuously variable and controllable as a function of pressure (or density). This property allows pressure-programming techniques to be used in SFC in an analogous fashion to temperature programming in gas chromatography or gradient elution in liquid chromatography if the pressure drop across the chromatographic column is minimal (3). Thus, pressure programming ranges in SFC are less restrictive for capillary column operation. In addition, several fluids have low critical temperatures that provide favorable conditions for the analysis of thermally labile compounds. As in liquid chromatography, there are a number of fluids available in SFC for mobile phases that offer a range of polarity and selectivity. However, current SFC practice and technology have been limited to relatively few fluids. Undoubtedly, the most popular fluid is carbon dioxide, which has been used extensively in both packed column (4-7) and
0003-2700/85/0357-2823$01.50/0 0 1985 American Chemical Society
2824
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
Table I. Critical Parameters of Fluids fluid COB NZO C2Hs C02/MeOH (97.502.5, w/w)
crit temp, "C 31.3
36.5 32.4 -35
crit press, atm 72.9 71.3 48.3 76
-
capillary column (8-12) applications. The widespread use of carbon dioxide can be accounted for by its mild critical parameters, low cost, chemical inertness, and compatibility with flame ionization (6,8) and ultraviolet absorption (4) detection. Pentane has also been used in a number of SFC applications (23, 141, but its more general applicability is limited by its higher critical temperature (197 "C) and incompatibility with flame ionization detection. Nitrous oxide (8))ammonia (5)) and mixtures of carbon dioxide and low concentrations of polar modifiers (15-1 7) have also seen limited applications. Several other fluids have also been used in earlier work (I). The objectives of this study were to quantitatively compare the selectivity and chromatographic performance of a limited number of mobile phases under equivalent capillary SFC conditions. Selectivity was measured in terms of relative retention (a)and a modified Kovats retention index system. Mass spectrometry was used for detection to provide high sensitivity and a wide range of fluid compatibility. Equivalent chromatographic operating conditions were achieved by operating each fluid a t the same reduced pressure (P, = P / P J and temperature (T, = T/T,). To minimize any differences in solute migration due to volatility effects, fluids were chosen with similar, low critical temperatures. The fluids used in this investigation were carbon dioxide, nitrous oxide, ethane, and a 2.5% (w/w) methanol in carbon dioxide mixture. Since exact critical parameters are not yet well-defined for carbon dioxide-methanol mixtures, only a semiquantitative comparison for this mobile phase system was possible. Capacity ratio and selectivity data were calculated for the various test probes of a polarity mixture for each fluid a t two different reduced temperatures and in some cases at different pressures. In addition, chromatographic efficiency was compared for each fluid at the two different temperatures.
EXPERIMENTAL SECTION The polarity-test mixture consisted of the following compounds (Chem. Service, West Chester, PA) at approximately 5 mg/mL in methylene chloride: n-decane, acetophenone, N-ethylaniline, naphthalene, 1-decanol, p-chlorophenol, and n-pentadecane. The mobile phases were 99.99% pure anaerobic carbon dioxide (Airco, Vancouver, WA), 99.99% ultrapure nitrous oxide (Matheson, Newark, CA), and 99.0% high-purity ethane (Oxarc, Pasco, WA). The carbon dioxide and nitrous oxide were further dried and purified by passing them through an activated charcoal and alumina adsorbent trap. The ethane was dried by passing it through alumina only. The binary fluid mixture of 2.5% (w/w) methanol in carbon dioxide was prepared by charging the highpressure syringe pump with the appropriate volume of dry methanol (Burdick and Jackson) and then filling the remaining volume of the pump with purified carbon dioxide and allowing the mixture to equilibrate. The instrumentation used for the capillary supercritical fluid chromatography-mass spectrometryhas been described previously (18-20). The SFC apparatus utilized a modified Varian 8500 syringe pump under microcomputer control to generate high pressure and pulse-free supplies of mobile phase. The accuracy and stability of the pressure control was kO.1 atm. A modified Hewlett-Packard 5790 gas chromatograph oven provided constant temperature conditions to k0.1 "C. The chromatographic column was prepared from an 18 X 50 clm i.d. length of fused silica tubing (Spectran Corp., Sturbridge, MA). The column was deactivated
crit density, g cm-3 0.46 0.45 0.20
-
Hildebrand solubility parameter (6) liq pr = 0.72 pr = 0.39 10.8 10.7
2.9
2.9
8.7
2.2
-
-
1.6 1.5 1.3 -
by using a poly(methylhydrosi1oxane)treatment (21) and coated with a 0.25-clm film of 5% phenyl poly(methylphenylsi1oxane) stationary phase (SE-54) that was rendered nonextractable by extensive cross-linking with azo-t-butane (22). Sample introduction was accomplished with a 0.06-clL Valco C14W injection valve operated at ambient temperatures and with a flow splitter adjusted to allow approximately a 1:30 flow into the chromatographic column. Consequently,approximately 10 ng of each solute component was placed on the column during an injection. Mobile phase decompression and regulation of the linear velocity were achieved by connecting the terminal end of the chromatographic column to a short length of small diameter (5 wm) fused silica tubing. The SFC effluent was injected axially into the chemical ionization (CI) source of a quadrapole mass spectrometer (20). Methane was used as the mass spectrometer chemical ionization reagent. The mass spectrometer was scanned from 92 to 220 amu at approximately 2 s/scan. The chromatographicoperating conditions were chosen so that the test solutes eluted with k'values between 0.5 and 5. A reduced pressure of 1.27 and reduced temperatures of 1.06 and 1.23 were selected for the quantitative studies. Pressure-programmed conditions were also used in selected situations. The void or hold-up times (to)used in k' and selectivity calculations were obtained from the elution time of butane, which was verified to be completely unretained at the various chromatographic conditions employed. Selectivitywas described in terms of the relative retention of adjacent peaks (avalues) and by modified Kovats retention indexes (23). The modified retention index system utilized n-decane and n-pentadecane as bracketing standards that allowed a retention index (RI) for each of the other five components to be calculated according to the following equation: log tk(x) - log tk(decane) RI = (1) log tk(pentadecane) - log tk(decane) where tk(x) is the adjusted retention time of the component of interest. In contrast to Kovats retention indexes (23), the magnitude of the RI is not related to carbon number since nonconsecutive paraffins were used as the bracketing standards and since only an arbitrary multiplier was utilized. However, the RI provides a direct measure of selectivity for the polar components relative to the alkanes.
RESULTS AND DISCUSSION Comparison of SFC Mobile Phases. An area of interest in supercritical fluid chromatography is the use of mobile phases with varying solvent properties. The choice of mobile phases in this study was restricted to those with similar and mild critical temperatures so that any differences in solute migration due to volatility would be minimized. The critical parameters of the fluids used in this investigation are summarized in Table I. The Hildebrand solubility parameters (24) are also included to provide a qualitative indication of the solvent strength for each of the fluids at the given reduced density. Values for the solubility parameter (6) were estimated from the relationship given by Giddings 6f =
81[Pf/P11
where 6f and 61 are the solubility parameters for the fluid and liquid, respectively, and pf and p1are the densities of the fluid and liquid, respectively. It should be noted that the calculated solubility parameters for the conditions used in this study are considerably smaller than for the normal liquid solvents (24).
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
Table 11. Chromatographic Operating Conditions
INAP
4
2825
temp (Tr), reduced parameters: press (PJ, density (p,) P, = T, = pr = T, = pr = 1.27,
1.06,
atm
"C
0.72," g mL-'
1.23,
fluid
"C
0.39," g mL-'
COZ N20 CzHG
92.2 90.3 61.1
50 55 51
0.33 0.33 0.14
100 108 103
0.18 0.17 0.08
=Ata reduced pressure of 1.27. lool
1 NAP
B
I
I
iI
CP
I
I
mdecanea
IM-ll+ 141
acetophenoneb
look
'
C
I
j:)
1
N-ethylanilineb
IIM,+i;+
naphthaleneb
I';,'d +
p-chlorophenola
o
'"1 0
l
loo!-
,
~
'
II '
'
1
'
"
'
I
n-pentadecsnea
'
1
m z
Figure 2. Methane chemical ionization mass spectra obtained during the chromatographic analyses of the polarity mixture used in this study. Spectra obtained with (a) carbon dioxide as the mobile phase and (b) nitrous oxide as the mobile phase.
To obtain equivalent chromatographic conditions, identical reduced pressures and temperatures were used for all fluids (Table 11). Total ion chromatograms of the polarity mixture obtained with carbon dioxide, nitrous oxide, and ethane at a reduced temperature of 1.06 and a reduced pressure of 1.27 are shown in Figure 1and illustrate the changes in selectivity which can be obtained. (See Table I11 for peak identification.) The solvent peaks are not shown since data storage was not initiated until after elution of the solvent front. The polar components exhibited little peak tailing, indicating that the chromatographic column was well deactivated and assuring that solute retention was being minimally influenced by adsorption. In addition to providing chromatographic profiles, good chemical ionization mass spectra were also obtained during the SFC-MS analyses. Typical spectra obtained from both supercritical carbon dioxide and nitrous oxide are shown in Figure 2. Since methane was used as the chemical ionization reagent, (M + 1)+ions predominate for the non-alkane components and (M - 1)+ ions for the alkanes. For a quantitative comparison of the solvent properties for the various fluids (P, = 1.27 and T,= 1.06), capacity ratios (k?, relative retentions of adjacent peaks (a),and the retention
2828
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
Table 111. Capacity Ratio and Selectivity Data (P,= 1.27 and T,= 1.06)
co2
polarity mixture compd n-C10 acetophenone (AP N-ethylaniline (EA) naphthalene (NAP) 1-decanol (DEC) p-chlorophenol (CP)
n-c,,
k'
0.53 * 0.01 0.79 i. 0.02 1.02 t 0.02 1.43 f 0.02 1.55 i: 0.01 2.00 * 0.01 3.23 * 0.02
-
2.16 3.59 5.46 5.93 T.33
1.49 1*29 1.40
k'
0.60 0.95
* 0.01
158 1:16
0.01 1.10 i: 0.02
i::
1.40 i: 0.02 1.62 i: 0.02 2.05 i: 0.04 2.66 * 0.06
11.7 min Did not elute between the alkane standards.
1.30
to = 11.0
C,H,
RI
CY
f
to =
a
-
N2O RI
CY
3.13 4.09 5.71 6.68 8.24
-
k'
0.58 i: 0.03 1.19 * 0.03 1.31 f 0.03 1.58 i: 0.04 2.07 t 0.05 3.44 i: 0.13 2.83 * 0.10
min
RI
cy
2.05
1*31
0.82
-
4.52 5.17 6.32 8.04 11.22a
-
to = 10.5 min
Table IV. Capacity Radio and Selectivity Data (Pr = 1.27 and Tr = 1.23) polarity mixture compd
CO, k'
01
N2 0
RI
-
n-C10 acetophenone N-eth ylaniline naphthalene 1-decanol p-chlorophenol
2.72 4.07 5.92 6.70 6.62
-
n-c,,
to =
7.2 min
indexes (RI)of the polarity mixture components are tabulated in Table 111. Multiple chromatographic runs were made at each set of conditions to evaluate the stability and reproducibility of the chromatography. In general, relative standard deviations of less than 3% were obtained. No changes in the physical configuration of the chromatographic system were made when switching from one fluid to another. Consequently, the hold-up times were slightly different due to changes in the mobile phase linear velocities (2.6-2.9 cm/s) as viscosity changed from fluid to fluid. As expected, carbon dioxide and nitrous oxide displayed very similar retention properties (solvent power) and selectivities for the polarity-test mixture components. Interestingly, the carbon dioxide eluted the polar compounds at slightly lower k'values and lower RI's than the nitrous oxide, while the nonpolar pentadecane was eluted at a significantly lower k'with the nitrous oxide, The differences in elution behavior for the ethane mobile phase were more marked. With the exception of pentadecane, the polarity mixture components were all eluted at higher k' values indicating lower solvent power. The selectivity was also different as evidenced by the higher RI values and the reversal in elution order of the chlorophenol and pentadecane (with the more polar chlorophenol showing greater retention). Even though chlorophenol eluted after pentadecane and a rigorous RI cannot be calculated, the apparent value is listed in Table III. As defined in eq 1,valid RI values range between 0.00 and 10.00. Comparison with Higher Temperature Separations. At higher temperatures differences in selectivity become less significant as solute vapor pressure increases and fluid phase solubility decreases. Capacity ratio and selectivity data obtained at a reduced temperature of 1.23 for the polarity mixture components are listed in Table IV. Variation of selectivity as a function of fluid was less pronounced than noted at the lower temperature (Table 111). These observations can be attributed to the reduced solvating power of the fluid and greater solute volatility, which renders the nature of the fluid phase somewhat less important in determining retention. The higher temperature also reduced the density of the mobile phases (see Table 11) and, in general, increased
k'
cy
0.54 0.96 1.18 1.61 1.90 1.88 3.90
1.78 1.23 1.36 1.18
0.99 2.07 to =
7.4 min
C2H6
RI
2.90 3.92 5.52 6.35 6.21
-
k'
0.54 1.14 1.39 1.88 2.39 2.52 4.46
cv
2.11 1.22 1.35 1.27 1.05 1.17 to =
RI
3.5 7 4.49 5.93 7.05 7.31
-
6.3 rnin
retention. It is also interesting that the fluid viscosities at this temperature were such that the linear velocity was greater for carbon dioxide than for nitrous oxide rather than the inverse as was the case at the lower temperature. At the higher reduced temperatures nitrous oxide eluted the polarity-test mixture components at lower k'values than carbon dioxide (in contrast to higher k'values at the lower temperatures). The differences may be (at least) partly attributed to the slighly higher temperature for nitrous oxide. The RI values were also lower for nitrous oxide, suggesting greater selectivity. The ethane still displayed the highest k' values, indicating lower solvent power. The selectivities for each of the fluids was also altered at the higher temperatures. For instance, decanol was eluted just after the chlorophenol (rather than before) at the higher temperature with both carbon dioxide and nitrous oxide. (These compounds nearly coeluted and would have been indistinguishable if mass spectrometric detection had not been employed.) This reversal in elution order can probably be attributed to a more gaschromatographic-like retention mechanism at the higher temperature. This is reasonable since gas chromatographic retention studies with the same stationary phase at both 50 "C and 100 "C showed the same eluficn order, as was observed for the carbon dioxide and nitrous oxide mobile phases at the higher reduced temperatures. The most notable change in selectivity, however, was with the ethane mobile phase in which elution of chlorophenol was shifted prior to pentadecane. The k'value was actually less than at the lower temperature. The acetophenone also eluted at a slightly lower k'value in the higher temperature ethane. Again, it is likely that these more polar solutes, which would have lower solubility in the ethane mobile phase, were eluted earlier at the higher temperature due to an increase in vapor pressure. In general, shorter analysis times for a wide range of compounds can best be achieved under pressure-ramped conditions and elution order often resembles isobaric separations, but changes in elution order are possible (25). A t the higher temperature (T, = 1.23), the ethane mobile phase provided similar elution profiles under both isobaric and pressureramped conditions. An example of a separation of the po-
ANALYTICAL CHEMISTRY, VOL. 57,NO. 14, DECEMBER 1985
2827
Table V. Comparative Capacity Ratio and Retention Index Data at Various Pressures CzH8(T,= 1.06)
COZ (TI = 1.06)
polarity mixture compd n-clo
acetophenone N-ethylaniline naphthalene 1-decanol
p-chlorophenol n-CH
PI = 1.57 (76 atm)
P, = 1.27 (61.1 atm)
P, = 1.37 (100 atm)
PI = 1.27 (92.2 atm) k'
RI
12'
RI
k'
RI
k'
RI
0.53 0.79 1.02 1.43 1.55 2.00 3.23
2.16 3.59 5.46 5.93 7.33 -
0.31 0.37 0.44 0.55 0.48 0.67 0.72
2.08 4.11 6.74 5.07 9.06 -
0.58 1.19 1.31 1.58 2.07 3.44 2.83
4.52 5.17 6.32 8.04 11.220 -
0.13 0.26 0.26 0.29 0.28 0.64 0.28
11.51' 11.51' 13.04' 12.6S0 26.40° -
ODid not elute between the alkane standards.
Table VI. Comparative Capacity Ratio and Relative Retention ( a ) Data for the CO,/MeOH (2.5% w/w) Fluid System 119 O C , 95 atm 108 'C, 93 atm 108 'C, 114 atm polarity mixture compd k' 01 k' 01 k' 01 0.61 0.67 n-C 10 0.36 1.64 1.66 2.03 1.00 1.11 acetop henone 0.73 i 0.03 1.29 1.29 1.22 1.29 1.43 N-ethylaniline 0.89 i 0.03 1.39 1.43 1.37 1.79 2.04 naphthalene 1.22 i 0.03 1.12 1.12 1.19 2.00 2.28 1-decanol 1.45 i 0.04 0.95 1.01 1.20 1.90 2.31 p-chlorophenol 1.74i 0.04 to = 21.8 min to = 16.5 rnin to = 14.1min larity-test mixture obtained with a pressure ramp of 2.5 atm/min, starting at 50 atm, is shown in Figure 3. The pressure-ramped separation was completed in approximately 20 min compared to approximately 34 min for the isobaric separation. A faster ramp rate or a higher isobaric pressure would also have decreased the analysis times. Variation of Reduced Pressure. Capacity ratio and retention index data for carbon dioxide and ethane at two different pressures are given in Table V. For carbon dioxide, increasing the reduced pressure from 1.27 to 1.37 (and the density from 0.33 g/mL to 0.40 g/mL) increased the solvent power of the fluid, as evidenced by a significant decrease in retention (up to a factor of 4 for some components). In addition, the selectivity was also altered, as is evident by the reversal in elution order for decanol and naphthalene. In terms of RI, the data show that at the higher pressure decanol and acetophenone were eluted earlier (explaining why decanol switched elution order) while the other polar components were eluted later. Ethane at a reduced pressure of 1.57 displayed significantly decreased retention of the polar components with the acetophenone and ethylaniline coeluting and the naphthalene and decanol essentially coeluting. However, relative to the pentadecane, the chlorophenol was actually retained longer at the higher pressure (a= 2.29 compared to 0.82). In fact, all of the polar components were eluted at significantly higher RI values at the higher pressure, which is not surprising for a nonpolar supercritical fluid solvent such as ethane. This again emphasizes the enhanced importance of the solvating power of a fluid as density is increased. Comparison with a Carbon Dioxide-Methanol Fluid Mixture. The use of small amounts of fluid modifiers (e.g., methanol) has been shown to significantly alter retention in previous studies with packed columns (1517). The present studies show relatively minor changes and suggest the previous observations resulted from modification of the stationary phase (16),possibly by solvent adsorption on active column sites. A typical chromatogram illustrating the separation of the polarity mixture using a carbon dioxidemethanol mixture (2.5% w/w) is shown in Figure 4. The restrictor on the chromatographic column was different than that used for the previously discussed fluids, and the linear velocity was slower
I
I
67 5
77 5
I 87 5 Prerrvie
/Blrn/
Figure 3. Capillary SFC-MS total ion chromatogram of a polarity mixture using ethane as a mobile phase at 103 O C and a pressure ramp of 2.5 atmlmin, starting 5 min after injection at 50 atm. See Table 111 for peak identifications.
than for the chromatograms shown in Figures 1 and 3 (1.4 cm/s compared to -2.7 cm/s). Consequently, the overall analysis time was longer but elution times were similar. At these operating conditions (119 "C and 95 atm), the decanol eluted after the p-chlorophenol while both eluted close to naphthalene. Capacity ratio and relative retention data for the binary fluid mixture at various operating conditions are listed in Table VI. Since the exact critical point data for the carbon dioxide-methanol mixture have not been well established, it was not possible to assure reduced parameters equivalent to those used for the other fluids. Preliminary experiments indicate that the critical parameters for the 2.5% methanol mixture approximate those listed in Table I. Visual observation of the fluid mixture in a high-pressure cell was used to ensure that all operating conditions were above the critical point and that a single phase system existed. The 108 "C and 93 atm conditions were chosen to approximate a reduced
2828
ANALYTICAL CHEMISTRY, VOL. 57, NO. 14, DECEMBER 1985
Table VII. Column Efficiencies for Various Mobile Phases at Different Temperaturesn
fluid
capacity ratio (k')
linear velocity,
theoretical plates/m,
retention time,
plates/min,
cm s-*
n/m
min
n/min
1.43 1.40 1.55
2.6 2.7 2.9
3980 3760 5090
28.2 26.1 26.8
2540 2580 3420
1.95 1.88
4.2 4.8
4670 5220
21.3 18.2
3950 5180
2.04
1.8
7900
50.14
2840
T, = 1.06 COZ NZO CZH,
T,= 1.23 COZ CZH6
108 O C , 93 atm
C02/MeOH (97.5/2.5 w/w) a
Naphthalene was used as the test solute.
C 0 2 - MeOH (2 5 % ) 95 atm. 11 9 O C
I
I
40
50
1
I
8a
70
T me ( mnules
Flgure 4. Capillary SFC-MS total ion chromatogram of a polarity mixture using a 2.5% (w/w) methane in carbon dioxide binary fluid as a mobile phase at 119 O C and 95 atm. See Table I11 for peak identifications.
temperature of 1.23 and a reduced pressure of 1.27. The data for the carbon dioxidemethanol mixture indicate that simliar retention was obtained with both the binary fluid mixture and pure carbon dioxide (Table IV). Selectivity differences were most significant for the polar components. The relatively minor impact of the 2.5% methanol modifier is reasonable if only fluid properties impact retention (i.e., the stationary phase is not being modified by the fluid), since the solubility parameter for the fluid mixture is only increased by about 10% (assuming that the contributions to the solubility parameter are additive and directly proportional to composition). As expected, increasing the pressure from 93 to 114 atm (at 108 "C) reduced the retention of the mixture components since the solubility parameter was increased by approximately 30%. No major changes (e.g., peak reversals) in selectivity occurred. Increasing the temperature to 119 OC (at 95 atm) also decreased the retention for the mixture components. This is reasonable since only a small decrease in density ( < 5 % ) results, which would be expected to be counteracted by the increase in migration rate due to volatility. The main difference in selectivity at the higher temperature was the elution of chlorophenol prior to decanol rather than after. This is consistent with the temperature effect observed for all the fluids as can be seen by comparison of Tables I11 and IV. Fluid Effects oh ChromatographicEfficiency. Due to differences in solute diffusivity, different chromatographic efficiencies would be expected for the various supercritical fluid mobile phases. Increased diffusivity as temperature is
increased and density decreased should increase efficiency. Measurements of column efficiency for the various fluids at different temperatures are listed in Table VII. Naphthalene was used for the test solute since, in most cases, it eluted at a similar k'under the various test conditions. The mobile phase linear velocities were all significantly higher than predicted for optimum efficiency (26,27),and thus, the efficiencies are less than obtainable. At constant reduced temperature (TI= 1.06) and identical k', the carbon dioxide and nitrous oxide generated essentially equivalent efficiencies (described as theoretical plates per meter). This would be expected since the solvent properties and the diffusivities are nearly identical. However, a higher efficiency was obtained with ethane due to the greater diffusivity in this fluid. Increasing the reduced temperatures of the fluids, which increased diffusivity, also increased the efficiencies. Furthermore, since the mobile phase linear velocities were higher at the increased reduced temperatures, the analyses were faster and greater efficiencies per unit time were also generated. Although the k' values are not identical and the linear velocities increased, the trend is still obvious. The higher efficiency for the carbon dioxide-methanol mixture can be accounted for, in part, by the lower linear velocity. These results indicate that operation at higher temperatures allows increased chromatographic efficiency to be obtained. Operation at higher temperatures (TI = 1.15-1.35) is generally preferred when sufficient compound thermal stability exists even though the fluid density is lower.
CONCLUSIONS This work demonstrates the successful application of supercritical fluid chromatography-mass spectrometry using a variety of fluid systems, including a binary mixture, as mobile phases and the flexibility afforded by mass spectrometric detection. The various fluids provided slightly different solvent strengths and selectivities,with the greatest differences evident for ethane, the least polar of the fluids. In general, variations in selectivity for the different fluids were less pronounced at higher temperatures, inferring that the nature of the fluid was becoming less important in determining retention behavior and that volatility and a gas chromatographic mechanism were becoming more important. The binary fluid mixture of 2.5% (w/w) methanol in carbon dioxide displayed slightly different selectivity than pure carbon dioxide but did not show the dramatic changes reported for packed column SFC. This suggests that in many instances fluid modifiers are affecting the stationary phase rather than grossly changing the fluid's solvating power. In addition, increased solute diffusivity in the mobile phase obtained from either increasing the temperature or using a different fluid demonstrated that higher chromatographic efficiencies could be obtained. Additional studies are required to explore other fluid systems
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Anal, Chem. 1985,57,2829-2836
and to fully understand the influence of solvent modifiers in capillary SFC.
ACKNOWLEDGMENT We thank C. R. Yonker and H. R. Udseth for helpful discussions and D. F. Couch for preparation of the manuscript. Registry No. Carbon dioxide, 124-38-9; nitrous oxide, 10024-97-2;ethane, 74-84-0; methanol, 67-56-1.
LITERATURE CITED Gouw, T. H.; Jentoff, R. E. Adv. Chromatogr. (NY) 1975, 73,1-40. Giddings, J. C. Anal. Chem. 1984, 36, 1890-1892. Peaden, P. A,; Lee, M. L. J . Chromatogr. W83, 259, 1-18. Gere, D. R.; Board, R. D.; McManigill, D. Anal. Chem. 1982, 54, 736-740. (5) Lauer, H. H.; McManlglll, D.; Board, R. D. Anal. Chem. 1983, 55, 1370- 1375. (8) Rawdon, M. G. Anal. Chem. 1984, 56, 831-832. (7) Takeuchl, T.; Ishll, D.; Sslto, M.; Hlbl, K. J . Chromatogr. 1084, 295, 323-33 1. ( 8 ) FJeldsted,J. C.; Kong, R. C.; Lee, M. L. J . Chromatogr. 1983, 779, 449-455. (9) FjeMsted, J. C.; Lee, M. L. Anal. Chem. 1984, 56, 6lQA-628A. (IO) Chester, T. L. J . Chromatogr. 1984, 299, 424-431. (11) Wright, B. W.; Udseth, H. R.; Smith, R. D.; Haziett, R. N. J . Chromatogr. 1884, 3 7 4 , 253-262. (12) Smith, R. D.; Kaiinoski, H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 56, 2476-2480. (13) Peaden, P. A.; Fjeldsted, J. C.; Lee, M. L.; Sprlngston, S. R.; Novotny, M. Anal. Chem. 1982, 54, 1090-1093. (I) (2) (3) (4)
(14) Fjeldsted, J. C.; Jackson, W. P.; Peaden, P. A.; Lee, M. L. J . Chromatogr. Sci. 1983, 27, 222-225. (15) Randall, L. 0. "Ultrahigh Resolution Chromatography"; Ahuja, S., Ed.; American Chemical Society: Washington, DC, 1984; ACS Symp. Ser. No. 250, Chapter 11. (18) Wright, B. W.; Smith, R. D., submitted for publication In J . Chromatogr (17) Levy, J. M.; Ritchey, W. M. "Proceeding of the Sixth International Symposium on Capillary Chromatography"; Huethig: Heidelberg, 1985; pp 925-943. (18) Smith, R. D.; Udseth, H. R. Anal. Chem. 1983, 55, 2266-2272. (19) Wright, B. W.; Udseth, H. R.; Smith, R. D.; Hazlett, R. N. J . Chromatogr. 1984. 374, 253-282. (20) Smith, R. D.; Kalinoski, H. T.; Udseth, H. R.; Wright, B. W. Anal. Chem. 1984, 56, 2476-2480. (21) Woolley, C. L.; Kong, R. C.; Richter, B. E.; Lee, M. L. HRC CC, J . Hlgh Resolut Chromatogr Chromatogr Commun W84, 7 , 329-332. (22) Wright, B. W.; Peaden, P. A.; Lee, M. L.; Stark, T . J. J , Chromatogr. 1982, 248, 17-34. (23) Ettre, L. S. Chromatographla 1973, 6 , 489-495. (24) Giddings, J. C.; Meyers, M. N.; McLaren, L.; Keller, R. A. Science 1988,-162, 67-73. (25) Smith, R. D.; Chapman, E. 0.; Wright, B. W. Anal. Chem. 1985, 57, 2829-2836. -- -- - - - -. (26) Fields, S. M.; Kong, R. C.; Lee, M. L.; Peaden, P. A. HRC CC, J . Hlgh Resolut. Chromatogr, Chromatogr, Commun, 1984, 7 , 423-428. (27) Fields, S. M.; Kong, R. C.; Fjeidsted, J. C.; Lee, M. L.; Peaden, P. A. HRC CC , J . Hlgh Resolut. Chromatogr Chromatogr . Commun 1984, 7 . 312-318.
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RECEIVED for review June 7, 1985. Accepted July 26, 1985. This work was supported by the U S . Army Research Office under Contract DAAG29-83-K-0172.
Pressure Programming in Supercritical Fluid Chromatography Richard D. Smith,* Elaine G. Chapman, and Bob W. Wright
Chemical Methods and Kinetics Section, Pacific Northwest Laboratory, Battelle Memorial Institute, Richland, Washington 99352
A theoretlcal and experlmental study of pressure programmlng in capillary supercritlcal fluid chromatography (SFC) Is reported. For the case of llnear density programming, reiationshlps are derlved between retention time, programmlng rate, and peak width uslng the capaclty ratlo-density dependence obtained from Isobaric studles. Llnear velocity in SFC Is shown to depend on both pressure and programmlng rate due to fluid compressibility. Numerical calcuiatlons are shown to accurately predlct retention tlmes under rapid pressure programming and account for changes in llnear velocity. Similarly, peak width data can be approximated by numerical methods using the capacity ratio at elutlon and the often substantlal contribution due to peak compression. These results are used to explore the reiatlve quality of separations obtained under pressure programming, and it is shown that a significant improvement In separatlon quality per unit time Is feasible at Increased programmlng rates. Further practical lmpllcatlons of these results relevant to column evaluation, injector-splitter design, and determlnation of optimum separatlon parameters are discussed.
Supercritical fluid chromatography (SFC) is attracting increased attention for the analysis of high molecular weight and thermally labile compounds (1-5). The use of open tubular capillary columns for SFC has been demonstrated to provide high efficiencies for the analysis of complex mixtures
and, more recently, fast separations using very rapid pressure programming techniques (6, 7). The control of pressure (and, hence, density) allows the solvating power of the supercritical fluid to be easily manipulated in a manner somewhat analogous to temperature programming in GC and gradient elution in HPLC. However, pressure-programmed SFC has the advantage that limitations upon the gradient rate are not significant and pressure increases are essentially instantaneous along the column (6). The practicality of pressure programming is further enhanced by the use of capillary columns where pressure drop along the column is typically negligible. In this report a detailed investigation of pressure programming in capillary SFC from theoretical and experimental viewpoints is described. Simple relationships are derived for prediction of retention times, and separation quality for the case of linear density programming and numerical methods are described for more complex situations. Rapid pressure programming and its unique aspects (e.g., significant peak compression and large column velocity gradients) and their implications are described. It is demonstrated that significant improvements in the quality of the separation per unit time can be obtained by using these techniques. Finally, numerical calculations are compared to experimental rapid pressureprogrammed SFC separations, and the practical implications of these developments are discussed.
EXPERIMENTAL SECTION The instrumentation used for pressure-programmed capillary SFC has been described previously (6,8-10). The system consisted
0003-2700/85/0357-2829$01.50/0 0 1985 American Chemical Society
