Anal. Chem. 1997, 69, 623-627
Subcritical Water Chromatography with Flame Ionization Detection David J. Miller* and Steven B. Hawthorne
Energy & Environmental Research Center, University of North Dakota, Grand Forks, North Dakota 58202-9018
A chromatographic method has been developed that allows subcritical (hot/liquid) water to be used as a mobile phase for packed-column reverse-phase LC with solute detection by flame ionization detection. Detection limits (S/N > 3:1 for butanol) of 1 ng were achieved with water flows of 20 and 50 µL/min and 5 ng with flows ranging from 100 to 200 µL/min. Quantitative determinations of ethanol in alcoholic beverages are in excellent agreement with label values and replicate injections have RSDs of ∼2%. Increasing the column temperature lowers the dielectric constant (polarity) of water, decreases the elution time, and improves the peak shape for compounds ranging from alcohols to hydroxy-substituted benzenes to amino acids. Temperature programming up to 175 °C can be used to improve separations and decrease analysis times. Two important issues for liquid chromatography (LC) are the need for a universal and sensitive detector [such as the flame ionization detection (FID) used in gas chromatography] and the need to reduce organic solvent usage. The use of water as an LC carrier is potentially attractive since water has no significant FID response, and its use generates no organic solvent waste. While the most common modes of detection used with water as an LC carrier are spectrographic (UV or fluorescence), there have been several reports of thermionic and flame emission detectors having been adapted for use with liquid water.1-5 A few early attempts have also been made to adapt an FID for use with water. Rudenko6 and Nonaka7 used steam as a mobile phase for packedcolumn gas chromatography with FID and a flow rate of steam of 5-20 mL/min (corresponding to ∼4-16 µL/min liquid water). Nitrogen was added to the FID hydrogen in order to maximize both the sensitivity and signal-to-noise ratio.7 Krejci et al.8 used a modified FID with liquid water as the mobile phase in capillary column liquid chromatography. The capillary columns used had inner diameters of less than 15 µm and the mobile phase flow rate was extremely low (10-5 µL/s). An obvious problem with coupling LC to FID is the high detector response caused by any organic solvent. Since reverse(1) McGuffin, V. L.; Novotny, M. Anal. Chem. 1981, 53, 946-951. (2) McGuffin, V. L.; Novotny, M. Anal. Chem. 1983, 55, 2296-2302. (3) Gluckman, J. C.; Novotny, M. J. Chromatogr. 1984, 314, 103-110. (4) Kientz, C. E.; Verweij, A.; de Jong, G. J.; Brinkman, U. A. Th. J. Chromatogr. 1992, 626, 59-69. (5) Kientz, C. E.; Verweij, A.; de Jong, G. J.; Brinkman, U. A. Th. J. Chromatogr. 1992, 626, 71-80. (6) Rudenko, B. A.; Baydarovtseva, A.; Kuzovkin, V. A.; Kucherov, V. F. J. Chromatogr. 1975, 104, 271-275. (7) Nonaka, A. Anal. Chem. 1972, 44, 271-276. (8) Krejci, M.; Tesarik, K.; Rusek, M.; Pajurek, J. J. Chromatogr. 1981, 218, 167-178. S0003-2700(96)00729-9 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Schematic diagram of the water LC-FID system. Components are described in the text.
phase separations are performed with solvent programming, it would appear that coupling to FID detector would have little utility. However, increasing temperature (with enough pressure to maintain the liquid state) can be used to dramatically lower the polarity of water,9 and thus temperature programming with pure water may be a useful method to mimic solvent programming. While this approach has not been exploited for LC, subcritical water extractions at temperatures up to 250 °C have recently been used to extract moderately polar (e.g., phenols) and nonpolar (e.g., PAHs and PCBs) organics from contaminated soils and sludges.10,11 The present study describes the use of a standard FID detector for reverse-phase LC with water flow rates typical of packed columns (up to 200 µL/min liquid water). The ability of temperature programming up to 175 °C with pure water as the eluant phase to decrease reverse-phase retention times and improve peak shapes is demonstrated with a variety of alcohols, hydroxysubstituted benzenes, and amino acids. EXPERIMENTAL SECTION Figure 1 shows a schematic diagram of the components of the water LC-FID system contained in the oven of a Hewlett-Packard Model 5890 gas chromatograph (Hewlett-Packard, Wilmington, DE). An Isco Model 100D syringe pump (Isco, Lincoln, NE) operating in the constant-flow mode was used to provide the water mobile phase. The injection system consists of a Valco HPLC injector (Model CI4UW.5) fitted with a 0.5-µL sample loop. A 15cm × 2-mm i.d. HPLC column (5 µm PRP-1, P/N 79366, Hamilton (9) Haar, L.; Gallagher, J. S.; Kell, G. S. National Bureau of Standards/National Research Council Steam Tables; Hemisphere Publishing Corp.; Bristol, PA, 1984. (10) Hawthorne, S. B.; Yang, Y.; Miller, D. J. Anal. Chem. 1994, 66, 29122920. (11) Yang, Y.; Bowadt, S.; Hawthorne, S. B.; Miller, D. J. Anal. Chem. 1995, 67, 4571-4576.
Analytical Chemistry, Vol. 69, No. 4, February 15, 1997 623
Co., Reno, NV) was connected to the injector with a 10-cm length of 1/16-in. × 0.3-mm i.d. glass-lined stainless steel tubing (Scientific Instrument Services, Ringoes, NJ). The outlet of the HPLC column was connected to the FID maintained at 400 °C using an Isco stainless steel supercritical fluid extraction restrictor with an inside diameter of 57 µm (i.e., the restrictor is rated at 1.5 mL/ min by the manufacturer for SFE using CO2). This restrictor was required to maintain pressure in the column so that water remained liquid at temperatures of >100 °C. At 40 °C, this resulted in a pressure (measured at the pump) of ∼20 bar with a water flow of 20 µL/min and ∼65 bar at 200 µL/min). No modifications were made to the standard HP FID. Fisher HPLC-grade water (purged with nitrogen to remove dissolved oxygen) was used for all separations. All chemical standards used in this study had a purity of >99%. Standards were prepared in water at concentrations of typically 0.1-1 mg/mL. Determinations of ethanol concentration in beverages was based on direct injection of a 1:10-1:100 dilution in water. Quantitations were based on three-point standard curves (0.1-1% ethanol in water) with 0.5% methanol as an internal standard. RESULTS AND DISCUSSION FID Optimization at Different Water Flow Rates. In order to determine the useful range of water flow rates that could be used and still maintain acceptable FID sensitivity, the ratio of hydrogen to air was varied at several mobile-phase flow rates. For these determinations, the HPLC column was removed and the inlet of the stainless steel restrictor connected directly to the injector (oven temperature held at 40 °C). Several dilutions of tert-butyl alcohol were prepared to test the sensitivity of the FID. Initial experiments were conducted with a water flow rate of 20 µL/min and hydrogen and air flow rates of 40 and 240 mL/ min, respectively (the normal FID gas flows for capillary GC). It was found that varying the air flow rate had little effect on the detection limit of the FID as long as the air flow rate was ∼100 mL/min greater than the hydrogen flow rate. However, the position of the transfer restrictor in the FID had a large effect on the signal-to-noise (S/N) ratio of the FID. It was very difficult to keep the FID lit if the transfer restrictor was placed just below the tip of the jet (as in capillary GC). Positioning the restrictor 1 or 2 cm below the tip improved the S/N ratio with the best results achieved at 3 cm below the tip (Figure 1). This position provided maximum sensitivity with minimum noise and was used in all subsequent experiments. Table 1 shows the results of FID optimization with the restrictor outlet placed 3 cm below the tip of the FID jet. When relatively slow water flow rates (e.g., 20 or 50 µL/min) are used, normal FID gas flow rates produce detection limits of 1-2 ng, which is only ∼1 order of magnitude poorer than the same FID used in the normal GC mode. For example, when a water flow rate of 50 µL/min is used, a detection limit of 1 ng was achieved with a hydrogen to air flow ratio of 80/240 mL/min. At higher water flows (100 µL/min and greater), an increasing amount of hydrogen was needed to keep the flame lit and to achieve acceptable sensitivity. In general, the hydrogen flow needed only to be increased enough to keep the flame lit. With a water flow rate of 100 µL/min, the hydrogen flow rate had to be increased to 150 mL/min (nearly double that for 50 µL/min, Table 1) to obtain a detection limit of 5 ng. Further increases in the water flow necessitated increasing the amount of air to the FID as well 624 Analytical Chemistry, Vol. 69, No. 4, February 15, 1997
Table 1. Effect of Water Flow Rate and Flame Ionization Detector Gas Flows on Detection Limits flow, µL/min
H2, mL/min
air, mL/min
detection limit, ng (S/N > 3:1)
20
20 40 80 80 120 150 150 215 215 300 300
240 240 240 240 240 240 330 240 330 330 430
1 2 10 1 2 5 5 10 5 5 5
50 100 150 200
as the amount of hydrogen. At water flow rates of >200 mL/ min, a stable flame and FID signal could not be achieved. However, a water flow of 200 µL/min, hydrogen flow of 300 mL/ min, and air flow of 430 mL/min yielded a reasonable detection limit (5 ng) and no problems in flame stability. These conditions were used for all subsequent studies. Effect of Temperature on the Elution of Alcohols. For an HPLC system using water as a mobile phase and an FID detector, the addition of organic cosolvents to improve separation and elute less polar solutes was not an option. However, previous work with water as an extraction fluid has demonstrated that increasing the water temperature (under enough pressure to maintain the liquid state) dramatically increases the solubility of organic solutes.10,11 This is due to the fact that the polarity (i.e., the dielectric constant) of water decreases with increasing temperature. For example, increasing the temperature of water (with enough pressure to maintain the liquid state) from ambient to 200 °C decreases from 80 to 35, similar to the dielectric constant of methanol.12 Therefore, it was hoped that solvent polarity programming could be achieved with pure water by temperature programming. The Hamilton PRP-1 column was chosen for its good thermal stability, since it was anticipated that a wide temperature range would be needed. To determine the effect of temperature on separation, a mixture of seven alcohols (1 mg/mL each of methanol, ethanol, n- and isopropyl alcohol, n-, sec-, and tert-butyl alcohol) in water was injected with the column oven at several temperatures. Figure 2 shows the separations achieved at 40, 80, and 140 °C and the use of a temperature program (all at 200 µL/min of water). Performing the separation at 40 °C (Figure 2, top) results in acceptable peak shapes for methanol and ethanol but as the size of the alcohol increases, the peaks broaden and begin to tail. In addition, n-butyl alcohol did not elute even after 50 min at 40 °C. Increasing the temperature of the separation to 80 °C has a dramatic effect on the peak shape of all seven of the components (Figure 2, second from top). The methanol, ethanol, and two propanol peaks are reasonably narrow, and the three late-eluting butanols are much improved. n-Butyl alcohol (which did not elute at 40 °C) elutes in
