Anal. Chem. 1984, 56, 2647-2653
tert-butylphenol will have to be made using more transparent solvents in this region of the spectrum. CONCLUSIONS Through this study several conclusions can be drawn. First, it is apparent that although maximum infrared signal a t a particular frequency can be obtained in p-HPLC-FTIR experiments using the infrared scan taken at the chromatographic peak maximum, the maximum signal to noise is observed by integrating across the peak to f 1 . 3 7 ~from the peak maximum. Second, the FTIR has again been shown to be a concentration-dependent detector for chromatography. Although the early treatment of this matter for GC-FTIR implied that the FTIR has some characteristics of a mass-sensitive detector (13),we have shown that for the p-HPLC-FTIR experiment, this is not the case. It is much more beneficial, consequently, to optically modify the infrared beam to fit a suitable chromatographic cell for the p-HPLC-FTIR experiment than it is to try to force-fit the chromatographic system into the existing spectrometric system. Since the concentration profile of a p-HPLC separation is more closely followed as the detector cell volume decreases, the p-HPLC-FTIR experiment requires a very-small-volume flow cell. Finally, a multitude of improvements have resulted in enhanced detection limits for p-HPLC-FTIR. These improvements include the use of a cell with a longer effective path length, more sensitive MCT detector, and better data handling methodology. The total of these improvements probably does not enhance the detection limits to the extent observed. It appears that the extended dynamic range expected from a cell with nonuniform path length is the missing contribution to the observed sensitivity enhancement. ACKNOWLEDGMENT Special thanks to Don Sting of Spectra-Tech, Inc., for the use of the Barnes Model 600 Beam Condenser. The microbore electronics module, supplied by Louis R. Palmer of IBM
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Instruments, Inc., and the microbore column end fittings, supplied by Gene Desotelle of EM Science, are greatly appreciated. Registry No. 2,6-Di-tert-butylphenol, 128-39-2;o-methoxybiphenyl, 86-26-0;2-tert-butylphenol, 88-18-6;2-sec-butylphenol, 89-72-5; cyclohexyl acetate, 622-45-7. LITERATURE CITED (1) Vidrine, D. W.; Mattson, D. R. Appl. Spectrosc. 1979, 32,502. (2) Kuehl, D. T.; Griffiths, P. R. J. ch'Om8tOgr. Sci. 1979, 17,471-476. (3) Vidrine, D. W. I n "Fourier Transform Infrared Spectroscopy"; Ferraro, J. R., Basile, L. J., Eds.; Academic Press: New York, 1979; Voi. 2, pp 129-164. (4) Vldrine, D. W.J. Chromatogr. Sci. 1979, 17,477-482. (5) Brown, R. S.; Hausler, D. W.; Taylor, L. T.; Carter, R. C. Anal. Chem. 1981, 53, 197-201. (6) Combellas, C.; Bayart, H.; Jasse, B.; Caude, M.;Rosset, R. J . Chromatogr. 1983, 259,211-225. (7) Johnson, C. C.; Taylor, L. T. Anal. Chem. 1983, 55, 436-441. (8) Brown, R. S.; Taylor, L. T. Anal. Chem. 1983, 55, 1492-1497. (9) Brown, R. S.;Amateis, P. G.; Taylor, L. T. Chromatographia 1984, 18, 396-400. (10) Amatels, P. G.; Taylor, L. T. Anal. Chem. 1984, 56, 966-971. (11) Jinno, K.; Fujimoto, C. Chromatographia 1983, 17,259-261. (12) "CRC Standard Mathernatlcal Tables", 26th ed.; CRC Press: Boca Raton, FL, 1981. (13) Grlfflths, P. R. I n "Fourier Transform Infrared Spectroscopy"; Ferraro, J. R., Basile, L. J., Eds.; Academic Press: New York, 1978; Vol. 1, pp 143- 168. (14) deHaseth, J. A,; Isenhour, T. L. Anal. Chem. 1977, 49, 1977-1981. (15) Bowermaster, J.; McNair, H. M. J. Chromatogr. 1983, 279,431-438. (16) Sebes, B., Spectra-Tech Inc., private communication, 1984. (17) Baker, D. R. LC 1984, 2, 38-41. (18) Amateis, P. G. Ph.D Dlssertatlon, Virginia Polytechnic Institute and State Unlversity, Biacksburg, VA, 1984. (19) Hirschfeld, T. Anal. Chem. 1978, 50, 1225-1226. (20) Dasgupta, P. K. Anal. Chem. 1984, 56, 1401-1403.
RECEIVED for review June 4,1984. Accepted August 13,1984. The IBM LC/9533 was a donation to VPI & SU from IBM Instruments, Inc., through their University Gifts program. The financial support of the Department of Energy through Grant DE-FG22-81PC40799 and the Commonwealth of Virginia is appreciated.
Postcolumn Fluorescence Detection of Nitrite, Nitrate, Thiosulfate, and Iodide Anions in High-Performance Liquid Chromatography ~
Sun Haing Lee Department of Chemistry, Kyungpook National University, Taegu, Korea Larry R. Field*
Department of Chemistry, Southern Methodist University, Dallas, Texas 75275 A postcolumn fluorescence detection system is introduced for the detection of oxidizable anlons in anlon-exchange chromatography. The anlons (nltrke, thiosulfate, and lodlde) are analyzed by thls system using thelr reaction wlth Ce( I V ) to produce the fluorescent specles Ce( I1 I ) in a postcolumnpacked bed reactor. I n addltion, the nltrate and nitrite anlons are determined slmultaneously by uslng a postcolumn copperked reductor to reduce nitrate to nltrlte prior to Its oxldatlon wHh Ce( IV). The detection limit of thls method Is at the low ppb level with a llnear dynamlc range covering 2-3 orders of magnltude.
A new analytical method for the determination of nitrite,
nitrate, thiosulfate, and iodide ions using a cerium fluorescence detector in conjunction with anion-exchange chromatography is described. Analysis of the nitrite and nitrate ions is emphasized since these ions have been shown to cause adverse health effects in man. Even though nitrate analyses have been performed routinely for many years and a large number of chemical methods are available for this analysis, problems still remain in measuring this ion accurately, especially a t low levels. Manual methods for analyzing nitrate concentrations below about 0.1 mg NO3- as nitrogen/L are tedious and exhibit poor reproducibility, and matrix interferences may occur with each of these methods (1-16). Chromatographic separation of sample components is often a solution to the matrix problem. Sievers et al. (17, 18) and Tanner et al. (19) have developed methods for the analysis of nitrate and nitrite
0003-2700/84/0356-2647$01.50/00 1984 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984 / I I R
?PP
n
Figure 1. Flow diagram of packed bed reactor for determination of nitrite, thiosulfate, and iodide. R, solvent reservoir; P, LC pump; I, Injector; T, mixing tee; PP, peristaltic pump; BR, packed bed reactor; FD, fluorescence detector; RC, recorder; BC, back-pressure coil: W, waste bottle.
employing gas chromatography coupled with electron capture detection (GC-ECD). These GC-ECD techniques are sensitive, but severe interferences are sometimes present in the nitration reaction used in their method, and it is not possible to analyze both nitrate and nitrite anions simulmeously with this procedure. Gerritse (20) described a rapid method for the simultaneous determination of nitrite and nitrate by HPLC using UV detection at 210 nm. This method is sensitive, but it is limited by background fluctuation in the f a r - W light source and by the spectral purity of the mobile phase. Ion chromatography (IC) is a very versatile technique and satisfies an analytical need for a simple, rapid, and reliable method for the determination of common ions in either simple or complicated matrices (21,22).However, IC techniques do have several limitations (23),the most cumbersome of which is the frequent requirement of a suppressor column to remove background electrolytes. In addition, the time required to regenerate this suppressor column significantly increases both the time factor and complexity of the analytical procedure. The purpose of this paper is .topresent a sensitive detection system for the determination of some oxidizable anions (viz., nitrite, thiosulfate, and iodide) and a reducible anion (nitrate) using a postcolumn cerium reaction fluorescence detector in HPLC. This technqiue does not use a suppressor column and provides a highly sensitive method for the simultaneous determination of nitrite and nitrate. Nitrite, thiosulfate, and iodide are determined fluorometrically from the Ce(II1) ion produced from a simple oxidation reaction of these analyte anions with Ce(1V) in a postcolumn reactor. Nitrate is detected by reducing it in a postcolumn copperized cadmium reductor to nitrite and then oxidizing the nitrite formed by its reaction with Ce(IV) to generate the Ce(III) ion, which is subsequently measured by fluorescence detection. Excellent separation of these ions is achieved on the anion-exchange column. EXPERIMENTAL SECTION Materials and Reagents. Reagent grade sodium nitrite (Baker, Phillipsburg, NJ), potassium nitrate (Baker), potassium iodide (Baker), and potassium iodate (MCB, Nonvood, OH) were used as standards without further purification. Standard solutions of 10-1-10-2 M were prepared and serially diluted to the desired concentrations. All the solutions were prepared with deionized distilled water. Cerium(1V) sulfate (reagent grade, G. Frederick Smith Chemical M) were prepared as Co., Columbus, OH) solutions (1 X follows: 200 mg of sodium bismuthate was added to the cerium(1V) solution in 1L of 1N sulfuric acid and heated to boiling for several minutes. The boiled reagent solution was cooled in
Figure 2. Flow diagram of packed bed reactor with a Cu-Cd reductor for determination of nitrite and nitrate. RD, Cu-Cd reductor. Other terms are the same as In Figure 1.
an ice bath to below 5 OC. The cold solution was transferred t o a brown bottle reservoir which was connected to a peristaltic pump via silicon tubing (1.52 mm i.d., Rainin Instrument Co., Inc.). This solution was mixed with the column effluent in a simple tee fitting (see Figure 1). The reagent solutions were prepared daily to enhance reproducibility. A packed anion-exchangecolumn (4.6 mm i.d. X 25 cm, Vydac 302 IC, Hesperia, CA) was used for the separation of nitrite, nitrate, thiosulfate, and iodide ions. The packed bed reactor was made from a Teflon tube (6 mm i.d. X 20 cm) which was filled with DMCS-treated glass beads (100/120 mesh, Alltech Associates). Bed packing was accomplished by the tap-fill method (24). HPLC Teflon frits (Alltech) with an average pore size of 5 wm were used to retain the glass beads in the reactor column. The cadmium reductor column was made from a piece of Pyrex glass tubing (4 mm i.d. X 10 cm) and washed sequentially with dilute hydrochloric acid, distilled water, methanol, and ether. The cleaned glass tubing was dried and then packed, as above, with cadmium particles (Alfa, 1OQ-250 mesh, Danven, MA) which were sieved to a uniform size range of 100-200 mesh. The cadmium was physically separated from the porous stainless-steel frits (5 pm pore diameter) by a porous Teflon frit that was used to retain the packing at high pressure. The Willis method (25)for preparation of copperized cadmium columns was modified in the following manner. First, the copperized cadmium (Cu-Cd) reductor was prepared by pumping a minimum of 10 mL of each of the following solutions through the column in the order listed (i) deionized/distilled water, (ii) EDTA buffer solution (pH 7.0), (iii) copper(I1) sulfate solution, and (iv) deionized/distilled water. Second, the reductor was connected to the anion-exchange column as is illustrated in Figure 2. The EDTA buffer solution was prepared by dissolving 38 g of the disodium tetrahydrate salt of EDTA in ca. 700 mL of distilled water. The pH was adjusted with 2 M potassium hydroxide to 7.0, and the solution was diluted to 1L. The copper(I1) solution was prepared by dissolving 38 g of EDTA and 12.5 g of copper(I1) sulfate pentahydrate in ca. 700 mL of distilled water. The pH was adjusted to 7.0 with 2 M potassium hydroxide, and the solution was diluted to l L. This solution contains a 2:l EDTAcopper(1I) ratio. The mobile-phase solutions were prepared by dissolving suitable amounts of succinic acid (SA) or potassium acid phthalate (KHP) and sodium sulfate in 700 mL of deionized/distilled water. When a pH meter was used, the pH of the solution was adjusted to 7 with sodium borate and then the solution was diluted to 1 L. All other reagents used were Baker reagent grade or better and were used without further purification. Apparatus. Two Waters Model 6000A HPLC (Waters Associates, Milford, MA) pumps were used in this work. One pump was used to provide mobile-phase flow through the column while the other pump was used to introduce the postcolumn reagent which was used to increase the pH of the effluent so that efficient reduction of nitrate to nitrite could take place. A peristaltic pump (Harvard Apparatus) with dual pumping channels was used to
ANALYTICAL CHEMISTRY, VOL. 56, NO. 14, DECEMBER 1984
introduce the cerium(1V) solutions. The back-pressure limit of the peristaltic pump was ca. 70 psi. Injections were accomplished by means of a liquid sampling valve (Rheodyne, Berkeley, CA) with sample loop volumes of 20 and 100 pL. The packed bed reador was connected to the effluent side of the anion-exchange column after the Teflon “tee” (see Figure 1). The effluent from the packed bed reactor passed directly to the fluorescence detector, and the resulting fluorescence signals were recorded by a Linear Instruments chart recorder. A piece of Teflon tubing (0.8 mm X 6 m; see Figure 1)was attached to the output of the fluorescence flow cell to maintain back pressure on the cell and to avoid high background noise resulting from pressure fluctuations and bubble formation in the cell. For the simultaneous analysis of nitrite and nitrate, a Cu-Cd reductor was placed between the column and the packed bed reactor (see Figure 2). The analytical column and the reactor were conditioned for at least l/z h with flowing mobile phase and reagents before use to increase the precision and reproducibility of the postcolumn reaction detector. Conditioning was found to be especially important for freshly prepared Cu-Cd reductors. Liquid Chromatography and Detection. A Perkin-Elmer (Norwalk, CT) fluorescence spectrophotometer Model 650-10s with an 18-pL horizontally illuminated microflow cell was used as the chromatographic detector. The fluorescence flow cell was aligned initially so that the signal-to-noise ratio of the Raman line of water was greater than 25. Pressure limitations of the peristaltic pump used restricted the mobile-phase flow rate between 1.0 and 1.5 mL/min. Flow rates for the peristaltic pump were generally maintained between 0.5 and 0.8 mL/min (typically 0.7 mL/min). The flow rate of the Ce(1V) sulfate solution was calibrated by measuring the effluent volume per unit time with a graduated cylinder. The sensitivity of the postcolumn cerium fluorescence detector was compared to a commercial ion conductivity (IC) detector (Model 213A, Vydac, Santa Clara, CAI. Drinking water samples were analyzed directly while seawater samples from Puget Sound were prepared for nitrate analysis by diluting 1:l (V/V) with distilled deionized water. A guard column was constructed and inserted between the injector and the anion-exchange column to protect the ion-exchange column from contamination when real-world samples were injected. The guard column (3.5 cm X 4 mm i.d.) was filled with silica gel (40-pm particle size, E. Merck, Darmstadt, Federal Republic of Germany).
RESULTS AND DISCUSSION Analysis of Nitrite, Thiosulfate, and Iodide. The Ce(IV) reagent solutions were stabilized by the addition of sulfuric acid and sodium bismuthate which was used to oxidize any of the cerium(II1) present in the Ce(1V) solutions. Trace amounts of Ce(II1) remaining and/or produced on standing would cause a background fluorescence signal which must be subtracted out electronically in order to maintain a reasonable base line. The addition of these reagents to the Ce(1V) solution also minimizes certain self-absorption effects as reported by Katz (26). Nitrite, thiosulfate, and iodide ions react with Ce(1V) to produce highly fluorescent Ce(II1) when mixed in the postcolumn bed reactor. Nitrite, thiosulfate, and iodide are oxidized with Ce(1V) according to the reaction.
+ H20 = NO, + 2Ce(III) + 3Hf 2S2032-+ 2Ce(IV) = S40G2-+ 2Ce(II1)
HNOz + Ce(1V) 21-
+ 2Ce(IV) = IZ+ 2Ce(III)
(1)
(2)
(3) The relative fluorescence response is stoichiometric for each of the three ions since no side reactions occur. The responses of each species differ, however, because of differences in the kinetics of each reaction and differences in the chromatographic properties of the individual ions. Therefore, in order to optimize the system the chromatographic conditions were held constant while the reagent and the experimental conditions were varied to determine the relative reaction kinetics of each ion.
1.o
2.0
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3.0 (MINI
REACTION TIMES
Figure 3. Changes in fluorescence response with time resulting from the reactions of nitrite, thiosulfate, and iodide with Ce(1V).
The reaction kinetics strongly influence the choice of a particular reactor design. The kinetics of the reactions described above were investigated by measuring the fluorescence intensity in an open system (no chromatographic column) in order to find a suitable reactor design. As seen in Figure 3, the reaction rate of nitrite with Ce(1V) is relatively slow (ca. 2 min to reaction plateau) while the reactions foi. thiosulfate and iodide are almost instantaneous (a maximum is approached in
