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ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
(Dean's Office), School of Public Health, University of North Carolina at Chapel Hill; and Specific Cooperative Research Agreement (12-14-7001-1221, Science and Education AdDepartment of Agriculture and Botany ministration, U.S. Department, North Carolina State University a t Raleigh.
Mention of trade or company name does not constitute a guarantee or warranty of the product by the [J.S. Department of Agriculture or the North Carolina State University and does not imply their approval to the exclusion of other products that may be suitable.
AIDS FOR ANALYTICAL CHEMISTS Removal of Excess Chelating Reagent Prior to Gas-Liquid Chromatography of Chelated Chromium E. L. Arnold" and 8 . L. Dold Clinical Sciences Division, USAF School of Aerospace Medicine, Brooks Air Force Base, Texas 78235
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Gas-liquid chromatography (GLC) of 3-diketone chelates has been shown t o be a very sensitive procedure for the '\ GLASS WOOL PLUG analysis of certain trace metals. While a number of 3-diketones form volatile metal chelates suitable for gas chromatographic analysis, t h e majority of practical applications ONE HOLE STOPPER of this technique involve the use of l,l.l,-trifluoro-2,4-penI - C H E L E X lOO(200 400 tanedione (H(tfa)) as the chelating reagent. This reagent has MESH) R E S I N been used by Taylor et al. ( 1 ) and Taylor and Arnold ( 2 ) to prepare beryllium chelates, by Hansen et al. ( 3 ) and Black 110 rnm TO W A T E R and S e v e r s ( 4 ) to prepare chromium chelates, and by Wolf ASPIRATOR e t al. ( 5 ) for both beryllium and chromium. Quantitative analyses of these metal chelates utilizing electron capture. 600 m l SUCTION FLAS mass spectrometric, and microwave emission detectors have FRiTTED GLASS been reported. While these detectors vary greatly in charE N D PLATE acteristics, they are all hampered by the presence of the large amounts of unreacted chelating reagent (H(tfa)) found in chelation reaction mixtures. T o remove this source of in16 x 125 mm TEST T U B E terference, researchers have relied on a base extraction procedure whereby the residual (H(tfa))is converted to (tfa-) and extracted into the aqueous phase prior to chromatography Figure 1. Chelex-100 resin column for removal of H(tfa) and suction of the organic phase which contains the metal chelates. apparatus for increasing and stabilizing hexane flow rate Because this technique suffers from irreproducible removal of H(tfa), possible degradation of metal chelates, and multiple transfers, we have investigated alternative means of H(tfa1 removal from samples to be analyzed for chromium content Crjtfa by GLC. We have found that the use of small glass columns filled with the ion-exchange resin, Chelex-100, is an effective means of accomplishing this, a t least in the case of chromium trifluoroacetylacetone chelates.
EXPERIMENTAL Glass columns of dimensions, 5 X 110 mm with a 4-mL top reservoir and fritted glass end plate. were fabricated in-house. They were prepared for use by slurrying approximately 1 g of Chelex-100 ion-exchange resin (200-400 mesh) (BioRad, Richmond, Calif.), in 0.1 N KOH, and filled by gravity flow. After filling, they were washed with an additional 20 mL of 0.1 K KOH and then aspirated from the bottom employing vacuum from a water aspirator. Hexane (J. T. Baker analyzed reagent grade) was aspirated through the columns until a flow of 0.5-1.0 mL/min was established. The hexane flow rate obtainable varied depending upon the batch number of Chelex-100 used. When resin manufactured prior to 1974 (batch 12670) was employed, a flow by gravity of 0.5-1.0 mL/min was possible. However, when recently manufactured resin was used (batch 159041, a vacuum assistance device was necessary to obtain this same flow rate (Figure 1). The use of coarser resin (50-100 mesh) t o obtain a higher flow is not recommended because of a significant reduction in efficiency of H(tfa) removal. Care should be taken to establish
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Figure 2. Chromatograms of (A) pure Cr(tfa),, (B) and (C) extracts of chelation reaction mixtures processed on Chelex-100 columns and (D), base washed reaction mixture extract. GLC column = 2.0 M X 2.0 mm i.d. glass, packed with 3% OV-1 on 80/100 mesh Supelcoport, column oven temperature = 135 OC, N, flow = 40 mL/min. "Ni electron capture detection
an adequate, constant flow of hexane prior to adding the sample.
This paper not subject to U S . Copyright. Published 1978 by the American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 12, OCTOBER 1978
0 2 4 6 TIME [MINUTES] Figure 3. Chromatogram of the hexane extract from a H(tfa) treated urine specimen containing 0.01 pg/mL added chromium after Chelex-100 column processing. Chromium concentration in this sample calculated as 0.017 pg/mL. Chromatographic conditions are in Figure 2
Chromium chelates (Cr(tfa),) were prepared by a modification of the method of Wolf (6). Chromic chloride (CrC1,.6H20) solutions of varying concentrations were buffered to pH 4.7 by addition of 0.1 M acetic acid/sodium acetate buffer. One mL of this buffered solution was heated for 2 h with 0.2 mL redistilled H(tfa) (Pierce Chemical Co., Rockford, Ill.) in sealed ampules. After cooling, the chromium chelates were extracted with four 0.5-mL washes of n-hexane. Aliquots (0.5 mL) of the hexane extract were added to the Chelex-100 columns and eluted with an additional 3 mL of hexane. The purified sample consisted of the first 3.0 mL of column eluent after the 0.5-mL sample had been added. After collection. samples were concentrated to 0.5 mL under vacuum, then analyzed by GLC.
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broad, tailing peak typical of H(tfa) seen in chromatogram D above is entirely eliminated. While we have not attempted to identify the extraneous peaks seen in B and C , it appears likely that they result from contaminants in the reaction buffer or t h e H(tfa). It should also be noted that the peak height of the Cr(tfa), peak in chromatogram B is essentially the same as that in a standard (A) containing the same concentration of Cr(tfa), while that in chromatogram D is reduced. either due to loss of the chelate or difficulty in accurate measurement. We have used this procedure to remove unreacted H(tfa) from reaction mixtures ranging in chromium mncentration from 0.2 to 0.002 gg/mL without detectable loss of the metal chelate. While the normal concentration of H(tfa) in these reaction mixtures prior to Chelex-100 column processing is about 10% v/v, concentrations as higjh as 5 0 7 ~v j v have been successfully processed with no evidence of residual H(tfaj in the chromatograms. The use of this procedure is also applicable to the removal of H(tfa) from biological sample reaction mixtures. Figure 3 shows a chromatogram of the hexane extract of a buffered urine specimen to which 0.01 pg/mL Cr3+as the, chloride salt had been added. While a considerable increase in the size and number of extraneous peaks is noted, the sample can be seen t o be free of H(tfa) interference. Chelex columns used in this procedure can be washed with 15-20 mL of hexane after use, then reused for processing as many as five additional samples before discarding the resin. After the processing of a single sample, a yellow ring will appear at the top of the resin column which gradually moves down the column with additional samples. T h e movement of this ring to a position near the co1u:mn exit, signals the end of the useful life of the resin and indicates that replacement is required. By staggering additions of sample to the Chelex-100 columns, a technician can process samples as rapidly as they can be chromatographed (5-6 min/samplej.
RESULTS AND DISCUSSION
ACKNOWLEDGMENT
Figure 2 shows four chromatograms obtained under different conditions using 63Nielectron capture detection (Tracor model MT 220 gas chromatograph). Chromatogram A is that of a standard hexane solution containing 0.1 Fg Cr(tfa)3/mL (equivalent to 0.01 pg chromium/mL). B shows a chromatogram obtained from the hexane extract of a reaction mixture containing 0.02 pg chromium/mL equivalent to 0.1 gg Cr(tfa),/mL after processing on a Chelex-100 column. A reagent blank processed in the same manner as B is shown in C while D illustrates the results obtained when the hexane extract of B was shaken for 5 min with 1 N NaOH instead of column processing. While those extracts processed by the Chelex-100 column technique contain a number of extraneous peaks, the region of the chromatogram displaying the Cr(tfaj, is relatively free of interference even a t high EC detector sensitivity. T h e
The research reported in this paper was conducted by personnel of the Clinical Pathology Branch, Clinical Sciences Division?Aerospace Medical Division. AFSC. lrnited States Air Force, Brooks AFB. Texas.
LITERATURE CITED (1) M. L. Taylor, E. L. Arnold, and R. E. Sievers, Anal. Letf., 1, 735 (1968). (2) M. L. Taylor and E. L. Arnold, Anal. Chem., 43, 1328 (1971). (3) L. C. Hansen, W. G. Scribner. T. W. Gilbert. and R. E. Sievers. Anal. Chem., 43, 349 (1971). (4) M. S. Black and R. E. Sievers. Anal. Chem.. 48. 1872 11976) (5) W. R. Wolf, M. L. Taylor. E.M. Hughes, T. 0. Tiernan, and R. E. Sievers, Anal. Chem., 44, 616 (1972). (6) W. R. Wolf, J , Chromatogr., 134, 159 ('1977).
RECEIVED for review February 13, 1978. Accepted June 8. 1978.
Logarithmic Data Compression Strategy for Gas Chromatography D. L. Doerfler and I.
M. Campbell*
Department of Biological Sciences, University of Pittsburgh, Parran Hall, 730 DeSoto Street, Pittsburgh, Pennsylvanir? 7526 7
This Aid describes how the output of the electrometer of a gas chromatograph (GC) can be transmitted in logarithmic form to an on-line data system, and how the original signal 0003-2700/78/0350-1709$01 OO/O
value can be reconstituted following digitization A hardware device is used to take the logarithm; the antilog operation is accomplished in software in a subroutine added to a pre1978 American Chemical Society
