Anal. Chem. 1995, 67, 3057-3063
Quantitative Open-Tubular Supercritical Fluid Chromatography Using Direct Injection onto a Retention Gap T. L. Chester* and D. P. lnnis Miami Valley Laboratories, The Procter & Gamble Company, P.O. Box 538707, Cincinnati, Ohio 45253-8707
Injection in open-tubular supercritical fluid chromatography is usually accomplished using a dynamic-flowsplitting or time-splitting technique. These techniques limit the effective injectionvolumes to low-nanoliterlevels. Sample volumes can be increased significantly without broadening the detected peaks, and injection precision improved by using direct injection and a retention gap. The required instrument modifications are simple and very inexpensive. The initial oven temperature and pressure must be set with respect to the phase behavior of the binary mixture formed by the mobile phase and the sample solvent. Relative standard deviations of peak areas and peak heights (comparingthe same peak among repeated injections) are in the range of 0.6- 1.8%for wellbehaved solutes using injection volumes of 0.1 and 0.5 PL.
Open-tubular supercritical fluid chromatography (OT-SFC) is performed in capillary tubes with an inside diameter (id.) of typically only 50 pm. The potential for solute band broadening by sample-flooding processes is enormous in tubes of this diameter when sample volumes approaching just 1pL are injected. Dynamic flowsplitting injection' and time-s~litting~-~ injection techniques were developed for OT-SFC to limit the initial solute band spreading simply by reducing the injection volume to a few tens of nanoliters. Recent improvements in timesplitting injection were aimed at further reducing the valve-switching time to allow the injection of even smaller volume^.^,^ However, detection limits, expressed as concentration in the injected sample solution, worsen as effective injection volumes are reduced. This problem seriously limits the usefulness of these injection techniques. A second problem regards the accuracy and precision of analyses performed with splitting injection in SFC. Reports of the precision of absolute peak areas (that is, comparing the same peak on repetitive injections of a test solution) vary among workers. Several reports set the percent relative standard devia(1) Peaden, P. A; Fjeldsted, J. C.; Lee, M. L.; Springston, S. R.; Novotny, M. Anal. Chem. 1982,54, 1090-1093. (2) Moore, J. C. J Polym. Sci. 1962,A2, 835-847. (3) Harvey, M. C.; Steams, S. D. J Chromatogr. Sci. 1983,21, 473-477. (4) Harvey, M. C.; Steams, S. D.Anal. Chem. 1984,56. 837-839. (5) Richter, B. E. The Pittsburgh Conference and Exposition on Analytical Chemistry and Applied Spectroscopy, March 10-14,1986, Atlantic City, NJ, Paper 514. (6) Richter, B. E.; Knowles, D. E.; Andersen, M. R.; Porter, N. L.; Campbell, E. R.; Later, D. W. I. High Resolut. Chromatogr. Chromatogr. Commun. 1988, 11, 29-32. (7) Harvey, M. C.; Robinson, R. E.; Harvey, M. C.; Steams, S. D. J. Chromatogr. Sci. 1994,32, 190-194. 0003-2700/95/0367-3057$9.00/0 0 1995 American Chemical Society
tion (RSD) in OT-SFC in the range of about 2-6% for flow and about 2-8% for time ~ p l i t t i n g . ~ , ~Even * ~ J if~ J ~ the (absolute) peak area precision is excellent, any form of splitting injection is subject to systematic errors when the mass-transfer properties of the various sample and standard solutions are not identical, causing variations in the split ratios among the various sample components. In flow-splitting injection, the split ratio will change (from the volumetric ratio measured with mobile phase) any time the vent flow resistance is disturbed, for example, by the temporary accumulation of sample components in the splitter vent tube. In timesplitting injection, the effective split ratio depends on the volumetric flow rate of the sample in the injection valve and the time the valve loop is in the mobile phase flow The accuracy and precision of timesplitting injection is expected to be excellent in liquid chromatography (LC), where the mobile phase and sample are virtually incompressible and the mobile phase is delivered under flow-controlled conditions. However, in OT-SFC the pump is operated under pressure rather than flow control. Thus, viscosity differences among the test solutions can result in differences in flow resistance, volumetric flow rate through the valve, and effective injection volume of time-splitting injections. Schomburg et al. reported a reduction in absolute solute peak areas exceeding a factor of 8 in OT-SFC when switching the sample solvent from pentane (with a viscosity of 0.22 mPa s at 25 OCI3 ) to 2-propanol (2.04 mPa s at 25 0C13),9 Sampledependent systematic errors in the effective injection volumes of splitting-injection techniques are often diflicult to detect in practice. The use of internal standards, or other techniques using relative peak areas within one chromatogram, has often been considered necessary in OT-SFC with splitting injection to correct for split ratio uncertainty. The precision of relative peak areas has been reported in the range of 0.05% to about 5% RSD with flow ~ p l i t t i n g , ~ fand - ~ ~about J ~ 1-7% RSD for time ~plitting.~,~.~,ll Unfortunately, it is not always possible to use an appropriate internal standard in all analyses. (8) Kohler. J.; Rose, A; Schomburg, G.1. High Resolut. Chromatogr. Chromatogr. Commun. 1988,11. 191-197. (9) Schomburg, G.; Behlau, H.;HPusig, U.; Hoening, B.; Roeder, W. J. High Resolut. Chromatogr. 1989,12, 142-148. (10) Wang, 2.; Fingas, M. J. Chromafogy. 1993,641, 125-136. (11) Munder, A; Chesler, S. N. J. High Resolut. Chromatogr. 1989,12, 669-
674. (12) Jakota, N. IC; Stewart, J. T. J. Chromatogr. 1992,604, 255-260. (13) CRC Handbook of Chemistry and Physics, 73rd ed.; Lide, D. R. Ed.; CRC Press: Boca Raton, FL. 1992; Chapter 6. (14) Wang, 2.; Fingas, M. J. Chromatogr. Sci. 1993,31, 509-518.
Analytical Chemistv, Vol. 67, No. 77, September 1, 1995 3057
Mobile phase supply
UncOaled inlet tube -0.25 m (or more) at rwm temperature ...
Safely shield nd shown)
oven w 1 1
...and2.5m (ormore) inlet
tube in the oven functioning
8s B retention gap
Figure 1. Schematic representation of the injector. Although the retention gap and column are shown separately in the figure, in practice they are wound together on the same column suppolt.
Efforts to increase the effectiveinjection volume and to avoid splitting have led to a variety of alternative injection techniques for OT-SFC including delayed ~plitting,’~ solvent venting,’6 solvent bacM~sh,’~ dilution chamber methods,l7-I9pressure focusingwith solvent venting,”’ and solid-phase Among these techniques, the pressurefocusingholvent-venijngtechnique has achieved the most impressive results, with RSDs of 0.16-0.63% and injection volumes up to 100pLm Unfortunately, users cannot add this or any of these more sophisticated techniques to commercial OT-SFC instruments without considerable effort and expense. Additional components are required, commercial SFC instrumentsmay not provide enough control capability to antomate these components, the modifications required are not supported by the instrument manufacturers, and in the case of pressure focusingholvent venting, a US. patent has been issued disclosing this technique.2I Therefore,the need existsfor yet another simple, effective, inexpensive, and readily available injection technique providing larger injection volumes and better quantitativeperformance than the flow- and timesplitting techniques. Direct injection using a retention gap” (Fkgre l), when combined with howledge and use of the phase behavior of the binary mixture formed by the mobile phase and the sample solvent, provides significantly larger sample volumes than flowand timesplitting injection. Direct injection, in principle, avoids the potential systematic errors of the splitting techniques by transferring the entire contents of the injection loop and is (15)Lee. M.L:Xu,B.: Huang. E.C.: Diordjevic,N. M.;Chang. HC. K Markides, K E.:J Microcolumn Sep. 1989. 1.7-13. (16)Greibrokk. T.:Berg. B. E. Trends Anal. Chom., 1993. 12,303-308. (17)Hirata. Y.:Inomata. K 1. Microrolums Sen. 1989,1, 242-248. (18)Hiram. Y.;Koshiba. H.: Maeda, T. J. H ~ Resobt. h Ckmmalofl. 1990,13, 619-623. (19)Hiram. Y.;Kadota. Y.:Kondo, T.I. Mterocolvnrn Sep. 1991,3,17-25. (20) Campbell. R M.: Corks, H. J.: Green, L S.Anal. Ckem. 1992,64,28522857. (21)Corte, H.J.; Campbell. R M.:Himes. R P.: Pfeiffer, C. D. US.Patent 5 234 599, 1993. (22)Koski, I. J.; Lee. E. D.;Ostmvsky, 1.: Lee. M. L Anal. Chm. 1993,6.5. 1125-1129. (23)Cheter, T.L:Innis. D. P. J. Micmcoluni Srp. 1993. 5,261-273.
3058 Analytical Chemistw, Vol. 67,No. 17, September 1, 1995
completely compatablewith commercialSFC instruments. A few meters of fused-silica tubing and one union are the only new components required. No additional instrument control is neces-
w. Direct Injection-Retention Gap Technique in OT-SFC. Outside the oven, the mobile phase (Cod exists as a liquid. The short, room-temperature segment of inlet tubing is necessary to dampen the injection pressure pulse and its recoil (or echo) from the much more compressible supercritical mobile phase present in the oven. The pressure pulse occurs because actuation of the injection valve momentarilyblocks and then abruptly restores the mobilephase flow. The recoil, if not sufticiently dampened, can displace some of the sample solution into the mobilephase supply tube (on the pump side of the injection valve), its valve connector, and the valve port, causing unacceptable mass-transfer problems. Initial chromatographic conditionsare set so that either liquid mobile phase or liquid sample solution exists at all times in the room-temperature components. Note that even though a piece of the retention gap tubing is simply withdrawn from the oven to function as the inlet tube and pulse dampener, this roomtemperature segment does not function as a retention gap. The total volume of the room-temperature segment of inlet tubing is not much larger than the sample injection volume, so there is little opportunity for sample dilution by the mobile phase. Furthermore, sample solvents are chosen that are miscible with liquid COz so there is never a phase separation between the sample solution and mobile phase outside the oven. Since no evaporation, no phase separation of the liquid components, and little dilution by mobile phase are allowed to occur before the sample solution reaches the oven, sample components are predominantly transported to the oven in the original liquid sample solvent. The actual retention gap processes, i.e., phase separation of the sample solution and the mobile phase, separation of sample componentsfrom the sample solvent, and the subsequent refocusing of the solutes into narrow initial bands, occur in the oven. The liquid-/vapor-phase separation is accomplished by the (isobaric) change in temperature as the sample solution is
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