Large-Volume Injection in Capillary Supercritical Fluid Chromatography Robert M. Campbell,' Hernan J. Cortes, and L. Shayne Green The Dow Chemical Company, Analytical Sciences, 1897A Building, Midland, Michigan 48667
A system was developed to Introduce large sample volumes (100 pL or more) Into a caplllary wpercrltlcal fluld chromatograph (SFC). Caplllary columns of 50-pm Internal dlameter (1.d.) were used wlthout a notlceable decrease In chromatographlc eff lclency and resolutlon. The sample Introductionsystem condstedof a serles of valves whlch were operated to effect sampllng Into an Inlet column, followed by a solvent elhnlnatlon step. A SF extraction step was then used to refocusthe analytes at the head of the capillary SFC column. Chromatography of the refocused analytes was accomplbhed wlth pressure programmlng of the COz moblle phase. Narrowbandswefe obtalned In the flnal SFC analytlcal step for a variety of analytes lncludlng alkanes, synthetlc trlglycerldes, polymer addltlves, and polyglycols In solvents such as methyhe chkrkle,chkrofonn, acetonltrlle, and water. A peak wlth greater than a ten to one signal to nolse railo was obtalned for a 100-pL Injectlon of a 10 parts per bllllon (ppb) solution of trlbehenln(C22:O) uslng flame lonlzatlondetectlon (FID). Trlton X-100 In water was analyzed at the 3 ppm level wing a 100-pL InJectlon. Injectlon reproduclbllltyat 1 ppm for 10-pL Injectlons of Irganox 1076 polymer addltlve was 0.16% relatlve, based on the raw peak areas. The largevokmoInJectbnsystemdescrlbedalkwedsensttlvltyIncreacres of 1000-fold, coupllng wlth llquld chromatography, Injectlonof a large varlety of solvents, Including water, and a 10-told Improvement In reproduclblllty.
INTRODUCTION Capillary supercritical fluid chromatography (SFC) has been used by the analytical community to solve problems, conduct research, and monitor industrial production processes. Many problems which would have been difficult or impossible to approach with conventional chromatographic techniques are readily amenable to capillary SFC. The combination of advantages of high chromatographic efficiency, predictable elution of increasing molecular weight, high inertness, and sensitive, universal detection for organic compounds has made SFC a valuable tool in industry, academia, and government. Compound classes such as polyglycols, polymer additives, waxes, lipids, reactive intermediates including epoxies, peroxides, and isocyanates are frequently easier and better analyzed by capillary SFC with FID than by any other method.' Columns of 50-pm i.d. are needed in capillary SFC to provide high chromatographic efficiency.l With these narrow diameters, even small amounts of injected solvent can flood the column, spreading the sample bands and resulting in broadened,distorted peaks. Consequently,injectionis usually done in a split mode with injected quantities in the nanoliter
* To whom correspondence should be addressed.
(1)Lee. M. L.: Markides. K. E. Anulvtical Sunercritical Fluid Chromatography and Extraction; ChromaLgraphy Conferences, Inc.: Provo, UT, 1990.
range.lt2 Such small injected volumes limit the sensitivity of the technique to 10-20 parta per million (ppm) using conventional split or timed-split injection for organic compounds detected by flame ionization (FID). To overcome this sensitivitylimitation, a variety of injection techniques for SFC have been investigated in order to allow larger volumes to be introduced into the SFC. These techniques include direct injection using an uncoated inlet,zJ solvent,venting,3-5 solvent dilution? density gradient focusing,' solvent back-flush? and combinations of these.8 SFC injection methods have been recently reviewed.9 Due to the small injection volumes and split injection mode used in SFC, analytical precision values obtained are seldom better than 345% relative, for raw peak areas.2 Although better precision can be obtained with internal standard methods, the precision obtained in SFC is a significant limitation of the technique. The development of a large volume injection system for capillary SFC also has utility in the field of multidimensional separations.10 Systems coupling microcolumn liquid chromatography (LC) to capillary gas chromatography (GC)11-13 and to conventional LC14J5 have been demonstrated for a variety of applications. However, a need remains for the capability to separate analytes of interest from a complex matrix in a multidimensional approach where the second dimension is SFC. Other workers have investigated LCSFC"3-'8 and the application of the principles demonstrated in this paper to the on-line couplingof LC to SFC were recently reported.19 Several on-line systems have been reported in which samples were SF extracted and the extractables deposited at (2)Richter, B. E.; Knowles, D. E.; Anderson, M. R.; Porter, N. L.; Campbell, E. R.; Later, D. W. HRC & CC, J.High Resolut. Chromatogr. Chromatogr. Commun. 1988,11,29. (3)Berg, B. E.;Greibrokk,T. HRCL CC, J.HighResolut. Chromatogr. Chromatogr. Commum. 1989,12,322. (4)Ashraf,S.;Bartle, K. D.; Clifford, A. A.; Davies, I. L.; Moulder, R. Chromatographia 1990,30,618. (5)Koski, 1.J.; Markides, K. E.; Lee, M. L. J. Microcolumn Sep. 1991, 3,521-529. (6)Hirata, Y.;Kadota, Y.; Hondo, T. J.Microcolumn Sep. 1991,3,17. (7)Lee, M. L.; Xu, B.; Huang, E. C.; Djordjevic, N. M.; Chang, H. C.; Markides, K. E. J. Microcolumn Sep. 1989,1, 7. (8)Liu, Z.;Farnsworth, P. B.; Lee, M. L. J.Microcolumn Sep. 1991, 3,435. (9)Koski, I. J.; Lee, M. L. J. Microcolumn Sep. 1991,3,481-90. (10)Cortes, H. J. In Multidimensional Chromatography; Cortes, H. J., Ed.; Marcel Dekker: New York, 1990; pp 251-300. (11)Cortes, H. J.; Olberding, E. L.; Wetters, J. H.Anal. Chim. Acta 1990,236,173. (12)Cortes, H. J.; Pfeiffer, C. D.; Richter, B. E. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1985,8,469-474. (13)Cortes, H. J.;Green, L. S.; Campbell, R. M. Anal. Chem. 1991,63, 2719-2724. (14)Cortes, H. J.;Bormett, G. E.; Graham, J. D. J.Microcolumn Sep. 1992,4,51. (15)Takeuchi, T.; Assai,U.; Haraguchi, H.; Ishii, D. J. Chromatogr. 1990,499,549. (16)Lurie, I. S.LC-GC 1988,6,1066. (17)Moulder, R.; Bartle, K. D.; Clifford, A. A. Anulyst, 1991,116, 1293-1298. (18)Hirata, Y. J. Microcolumn Sep. 1990,2,214. (19)Cortes, H. J.; Campbell, R. M.; Himes, R. P.; Pfeiffer, C. D. J. Microcolumn Sep. 1992,4,239-44.
0003-270Q/92/0364-2852$03.00/0 0 1992 American Chemical Society
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Figure 1. Schematlc diagram of the largevolume Injection CSFC system: (1) SFC pump; (2) tee; (3)10-port switchlng valve; (4) 4-port switchlng valve; (5) 10-port switching valve; (6) 8-port switchlng valve; (7) capillary Inlet column;(8) SFC Interface; (9) Caplilaty SFC column; (10) frit restrlctor; (11) GC oven; (12) FID; (13) vent; (14) vent; (15) sample loop.
the head of the SFC ~0lumn.2*23 These systems required manually opening the extraction vessels between analyses and were not amenable to automated analysis. Since these systems were designed primarily for solid samples, they did not readily accommodate analyses of liquid samples. In this report we describe a system which allows introduction of large liquid volumes into capillary SFC which is accomplished through solvent elimination followed by refocusing of the analytes by pressurizing the inlet and depositing the analytes in a narrow band at the head of the SFC column. The large volume injection SFC system described herein yielded high sensitivity and improved reproducibility.
EXPERIMENTAL SECTION Reagents and Chemicals. The SFC system, including injection steps, was operated with SFE/SFC (FID and ECD) grade COz (Scott Specialty Gases, Plumsteadville, PA) which was further purified by passing it througha charcoal(SKC,Eighty Four, PA) trap, 30-cm X 9-mm i.d., followed by an alumina (BioRad, Richmond, CA) trap, 80-cm X 9-mm i.d., which was heated to 75 "C during pump filling. All solvents were HPLC grade or better (Fisher,Pittsburgh, PA). Methylenechloridewas pesticide grade (Burdick and Jackson, Muskegon, MI). Synthetic triglycerides and Triton X-100 were obtained from Sigma Chemical Co., St. Louis, MO. Irganox 1076,1330,and 1010were obtained from Ciba-Geigy, Basel, Switzerland. Instrumentation. The system used is similar to that previously reported.19 The CSFC injection system consisted of a series of switching valves mounted on a Model 5890 gas chromatograph (Hewlett-Packard, Avondale, PA). A schematic diagram of the system is shown in Figure 1. The carrier fluid, COz, was supplied with a syringe pump, 1 (Model 100D, Isco, Lincoln, NE). Valve VI (Model NlOWT, Valco, Houston, TX) allowed selection of either helium purge gas or liquid COz and was connected to valve V2 with l/az-in.-o.d.stainless steel tubing. The helium purge gas was supplied at 40 psi, and at this pressure its volumetric flow rate through the inlet column was approximately 2 mL/min. Valve V2 (Model NlOWT, Valco, Houston, TX) was equipped with a stainless steel sample loop, 15,of either 10or l00pL and was connected to valveV3 (ModelC8WE, Valco, Houston, TX) with the sample inlet column, 7. Valve V3, the inlet column, interface, and analytical column were mounted inside the oven. Valves V1, V2, and V4 were located outside the oven. Fill and waste lines were installed in the appropriate porta of valve V2. The inlet column was either a 15-m X 0.15-mm4.d. (20) Jackson, W.P.;Markides, K. E.; Lee, M. L. HRC & CC, J. High Resolut. Chromatogr. Chromatogr. Commun. 1986,9, 213. (21) Anderson, M. R.;Swanson, J. T.;Porter, N. L.; Richter, B. E. J. Chromatogr. Sci. 1989, 27, 371. (22) Xie, L. Q.; Markides, M. L.; Lee, M. L. J.Chromatogr. Sci. 1989, 27, 365. (23) Koski, 1: J.; Jansson, B. A,; Markides, K. E.; Lee, M. L. J. Pharmaceut. Bzomed. Anal. 1991,9, 281-290.
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fused-silica tube (Polymicro Technologies, Phoenix, AZ) or a 6-m- X 0.28-mm4.d. Silcosteel tube (Restek, Bellefonte, PA) or a 5-m- X 0.18-mm4.d. fused-silica column coated with a 0.2-pm film of methylsilicone(Rh-1,Restek, Bellefonte,PA). The outlet of this tube was connectedto valve V3which was in turn connected to the SFC column, interface, 8, via a linear restrictor, 30-cm X 20-pm i.d. X 90-pm 0.d. (Polymicro Technologies,Phoenix, AZ). A vent tube (20-cm X 250-pm i.d. fused silica, Polymicro) was connected to the port adjacent to the inlet column and a plug was installed in the port adjacent the restrictor. The linear restrictor was inserted through a glass-lined tee (Model SUT/16/005/005, Scientific Glass Engineering, Inc., Austin, TX) and into the interface tube, 20-cm X 100-pm i.d. (deactivated, Dionex/Lee Scientific, Salt Lake City, UT). The third leg of the tee was connected to valve V4 (Model C4W, Valco, Houston, TX) with a stainless steel tube 20-cm X 0.02-in. (508-pm) i.d. The outlet end of the interface tube was connected to the SFC analytical column, 9, 10-m X 50-pm i.d. (SB-PHENYL-5, Dionex/Lee Scientific, Salt Lake City, UT) with a stainless steel buttconnector (MVSU004,SGE, Austin, TX). The analytical column was equipped with a frit restrictor which was affixed with a pressed-fit connector. The outlet of the analytical column restrictor was installed in the flame ionization detector (FID). Carbon dioxide was supplied to valve V4 via a 40-cm X 0.01-in.(254-pm) i.d. stainless steel tube. A short fused-silica tube was installed in valve V4 as vent and the other port was plugged. Data were collected and processed with a computer-based chromatography data system (Access*Chrom, PE Nelson, Cupertino, CA). Procedure. A sample was injected on the system and analyzed as follows. The loop of valve V2 was filled with sample with a syringe. With valve V1 positioned to select helium, valve V4 positioned to supply COz to the analytical column, and valve V3 in the vent position, valve V2 was switched to put the loop in the helium flow stream. The system was held in this position for a period of time, usually 5 min, sufficient to allow the solvent to evaporate. The oven was held at 100 OC. Valves V4, V3, and V1 were then switched rapidly in succession, in that order. This supplied COz to the inlet column, vented the interface and analytical column, and diverted the outlet of the inlet column from waste to the restrictor and interface. The COz pressure was ramped at 53 atm/minute from 80 to 400 atm and held for a period of time (usually 5 min) in order to transfer the analytes from the inlet column to the interface. The COz was decompressed through the linear restrictor and vented as a gas while the analytes were deposited in the interface. The pump pressure was then decreased to 80 atm, and valves V4, V1, and V3 were switched. This supplied COz to the interface and analytical column and vented the inlet column. The pressure was then ramped at 12.8 atm/min to 400 atm and held for 6 min. The analytes were eluted from the analytical columnand detected by the FID.
RESULTS AND DISCUSSION This injection system was designed to handle volumes ranging from 1pL to hundreds of microliters. Volumes less than 1 p L can be injected using direct injection with an uncoated inlet. As described in the Experimental Section, the sample was introduced into acapillary inlet where, through the action of helium gas and heat, the solvent was eliminated. A capillary column provides a more efficient separation of analyte from solvent than simple evaporation in a vial under a nitrogen gas stream. The use of this type of inlet system allows the application of all the solvent removal and analyte retention mechanisms which have been used in large-volume injection in GClO to injection in SFC. During the solvent elimination step, the solutes are spread over a wide band in the inlet column due to flooding of the column by the large amount of solvent. The solutes must be refocused into a narrow band prior to chromatography on the narrow-bore CSFC column. This was accomplished by fully exploiting the properties of COZ as a supercritical fluid. The analytes deposited on the walls of the inlet were dissolved in
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Flgure 2. LVI/CSFC chromatogram of synthetic trlglycerldes: (10) trldecanoin (C1O:O): (17)triheptadecanoln(Cl7:0):(22)tribehenin(C22: 0). Conditions: Injection volume, 10 pLL; concentration, 1 pg/mL: solvent, chloroform. Inlet column, 15-m X 150-pm 1.d. fused silica; oven temperature, 100 O C ; inlet He purge time, 5 mln; inlet pressure program, 80-400 atm at 53 atm/mln, 6min hold. Conditions for analytical column: 10-m X 50-pm i.d., 5 % phenylmethyislilcone,0.25pm film thickness; oven temperature, 100 O C : pressure program, 80400 atm at 12.8 atm/mln, 6 m l n final hold: detectlon, FID.
supercritical C02, transported through a restrictor, and deposited in the interface. The C02 decompressed through the restrictor and changed phase from a high-density, supercritical fluid to a low-density gas. Since the analytes were not soluble in the low-density, gaseous C02, they deposited in a narrow band at the outlet of the restrictor. It is important that the dead volume in the interface be minimized in order to prevent band broadening prior to the SFC analytical step which was accomplished using a 100pm4.d. interface tube with a 90-pm-0.d. restrictor tube. In early experiments, a large background was seen in the chromatograms.19 This problem was alleviated by using a better grade of C02, careful cleaning of the system componenta, and the use of heated traps during pump filling, as described in the Experimental Section. It was important to use a low pump filling rate (1-2 mL/min) to obtain clean C02 in this system. The heated trap caused the C02 to expand to a low density at this pressure, and the impurities were then no longer soluble in the fluid and deposited on the packed bed. If the filling rate was too high, the heater capacity was exceeded and impure liquid COZ flowed into the pump, resulting in high backgrounds. A typical chromatogram obtained using the described system is shown in Figure 2. The chromatogram represents a 10-pL injection of synthetic triglycerides, tridecanoin (ClO:O), triheptadecanoin (C17:0), and tribehenin (C22:O) dissolved in chloroforma t the 1p g / d level (ppm). Although actual column efficiencies could not be measured due to pressure programming, peak widths obtained were equivalent to those obtained with conventional split or timed-split injection. Another measure of the injection performance is resolution of closely related isomers. A chromatogram of pentaerythritol tetrastearate (PETS) polymer additive is shown in Figure 3. Resolution of the PETS isomers in this 10-pL injection of a 5 ppm solution was as good as that obtained using conventional split injection under similar chromatographic conditions.
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Figure 3. LVI/CSFC chromatogram of pentaemritoi tetrastearate (PETS). Conditions: InJection volume, 10 pL; concentration,5 pg/mL; solvent,chloroform; Inlet column, 6m X 0.29-mm 1.d. Slkosteel; Inlet Dressure program final time, 8 mln. Other conditlons are as in Fbwe 2. 43102 3
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Figure 4. LVI/CSFC chromatogram of Irganox 1076 (l), 1330 (2), and 1010 (3). Conditions: InJectlon volume, 10 pL; concentration, 1 pg/mL except Irganox 1010 which was 2 pg/mL; solvent,chloroform. Other conditlons are as in Flgure 2.
The injection system was also applicable to compounds of intermediate polarity. Figure 4 shows the SF chromatogram of a 10-pL injection of hindered phenol polymer additives. Irganox 1076 and 1330 were injected a t a concentration of 1 ppm, while Irganox 1010 was at the 2 ppm level. Resolution and peak shape were equivalent to those obtained with conventional injection typically restricted to much higher concentration levels (100-200 ppm). The capability to analyze dilute solutions of polymer additives is important since many of the additives are found in polymers at trace levels. A 10-pLinjection of Triton X-100 polyglycol a t the 30 ppm level in chloroformis presented in Figure 5. This was injected at a level which is much lower than the percent levels which are usually necessaryin split injection. Although the pressure
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Flgurr 5. LVIlCSFC Chromatogram of Triton X-100. Conditions: injection volume, 10pL; concentration,30 pglmL; solvent,chloroform; inlet column, 6 m X 0.28-mm i.d. Siicosteei; inlet pressure program final time. 10 min. Other conditions are as in Figure 2.
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Figuro 7. LVIlCSFC chromatogram of synthetic triglycerides. Conditions: concentration, 0.01 pglmL (10 ppb). Other conditions are as in Figure 6. 49888
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Flgurr 6. LVI/CSFC chromatogram of synthetic triglycerides: tridecanoin (10);triheptadecanoln (17); Mbehenin (22). Conditions: injection volume, 100 pL; concentratbn,0.1 pglmL; solvent,methylene chloride; inlet He purge time, 20 min; inlet pressure program final time, 10 min. Other condklons are as in Figure 2.
program was not optimized here for resolution of all the isomers, peak shapes are not significantly worse in width or symmetry than what is seen in conventional split injection SFC. Further optimizationof the pressure program and other column/inlet conditions will likely result in improved resolution for this material. This demonstrates the applicability of the large-volume injection system to polyglycols. Injection volumes much larger than 10pL were also possible with this system, making trace analysis a reality for capillary SFC. Figure 6 shows a 100-pL injection of the triglycerides in methylene chloride at the 0.1 ppm level. Figure 7 shows the same triglycerides injected a t the 10 parts per billion (ppb) level, again with 100 p L injected. The signal to noise level was greater than 10 for tribehenin, suggesting that
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Flgurr 8. LVIlCSFC chromatogram of Irganox 1076. Conditions: injection volume. 10 pL; concentration, 1 pg/mL; solvent, water with 0.2% (w:w)acet!cacid;inlet column, 5m X 0.18"i.d. methylslicone capllary,0.2-pm film; inlet He purge time, 10min; inlet pressure program final time, 18 min. Other conditions are as in Figure 2.
detection limits of less than 10 ppb are possible. Larger injection volumes may be feasible and it appears that sensitivity with the reported injection technique may only be limited by the background level of impurities in the sample. In addition to organic solvents, aqueous samples were also introduced into the system. Figure 8 shows the injection of 10 pL of Irganox 1076 in water a t the 1ppm level. A small amount (0.2 5% ) of acetic acid was added to the sample to prevent adsorption of the analyte onto the walls of the glass vial and injection syringe. As can be observed, peak shapes obtained were not affected by the type of solvent introduced. Figure 9 representa a chromatogram of a 100-pL injection of triglycerides in water a t the 0.1 ppm level, while Figure 10 represents a chromatogram of 100 pL of water containing Triton X-100polyglycol nonionic surfactant a t the 3 ppm
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Table I. Raw Areas and Precision Results for Irganox 1076 and Triheptadecanoin at 1 ppm and 10-rL Injections in Chloroform
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182 258 182 366 182 178 182 176 182 418 181 704 182 174 182 768
184 762 183 186 186 438 185 296 185 574 186 860 185 008 186 336
0.9864 0.9955 0.9772 0.9832 0.9830 0.9724 0.9847 0.9809
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Flgure 0, LVI/CSFC chromatogram of synthetic triglycerides. Conditions: injectbn volume, 1OOpL; Concentration,0.1 pg/mL; solvent, water with 0.2% acetic acid. Other conditions are as in Figure 8. 49528
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Figure 11. LVI/CSFC chromatogram of Irganox 1076 and triheptadecanoin. Conditions: injection volume, 10 pL; concentration, 1 pg/mL; solvent, chloroform;inlet column,6-m X 0.26-mm 1.d. SWcosteei; inlet pressure program final time, 10 min. Other conditions are as in
Figure 2. 17503 0
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Figure 10. LVIICSFC chromatogram of Triton X-100. Conditions:
injection volume, 100 pLL; concentration, 3 pg/mL, solvent, water. Other conditions are as in Figure 8.
level. The results obtained demonstrate the applicability of the technique for the analysis of trace level components in aqueous media. Samples such as process streams, waste streams,or blood plasma, for example,willlikely be candidates for analysis using the reported approach. A coated column was used as the inlet column for the aqueous injections. The separation mechanism employed in the inlet column during aqueous injections was different from that used in the organic solvent injections. In the organic solvent injections, the introduction temperature was above the boiling point of the solvent, and the solventwas evaporated in the uncoated inlet tube and exited as a vapor. The solutes were retained on the basis of differential volatility. In the aqueous case, the sample was forced through an inlet column coated with methyl silicone stationary phase with the temperature below the boiling point of water. The water exited the inlet column as a liquid, rather than a vapor as in
the organic solvent case. The organic analytes in the aqueous solution were trapped in the silicone stationary phase through a partitioning mechanism, analogousto open tubular reversedphase liquid chromatography. The separation between the water solvent and the organic solutes was based on water/ silicone partitioning coefficients rather than on volatility. One important benefit obtained using the system described is in injection precision. Conventional split injection techniques yield precision values in the range 3-5 % RSD for the raw peak areas. Using this large-volume injection, however, much better precision than this was obtained. Table I lists the results of a series of eight 10-pL injections of Irganox 1076 and triheptadecanoin at the 1ppm level. A representative chromatogram from this series is shown in Figure 11. An RSD value of 0.16 % was obtained for Irganox 1076, while triheptadecanoin yielded an RSD value of 0.6%. The results obtained represent a 10-fold increase in injection precision over conventional split and timed split techniques. It should be noted that the precision values reported here do not necessarily reflect a maximum level of precision possible with this system. As in any technique, the actual precision obtained will be affected by the type of analyte, the matrix, sample type, concentration, analysis conditions, etc. Further
ANALYTICAL CHEMISTRY, VOL. 64, NO. 22, NOVEMBER 15, 1992
work on the optimization of this system will likely yield even better precision values. The precision of the peak area ratios as would be used in internal standard methods was about the same as the raw area precision. Since the two compounds used in this case were of different compound classes and did not have closely related retention times, the relative precision obtained is not a good indicator of what will be possible with this injection system. The same set of valves, including valve V3 in the oven, was used over a period of months for hundreck of injections without leakage or failure. The oven temperature was typically at 100 OC, although some injections were made a t 80 or 120 OC. In this report we have described a large-volume injection system and have shown that it brings four important advances to CSFC injection technology. First, with analyses down to 10 ppb, sensitivity has been improved 1000-fold over conventional split techniques. Second, LC was coupled to CFSC
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for improved specificity and trace analysis of complex samples.1QThird, aqueous samples can be injected without prior extraction or prior removal of the water matrix. Fourth, with large-volume injections using a sample loop, a 10-fold improvement of injection precision was realized over conventional split techniques in CSFC. In addition, since the system is operated completely with switching valves, it can be easily automated. With this injection system, the major limitations which have heretofore been associated with CSFC injection were overcome.
ACKNOWLEDGMENT The authors wish to thank R. P. Himes and C. D. Pfeiffer for helpful discussions and their kindly assistance. RECEIVED for review June 1, 1992. Accepted September 1, 1992.
