Anal. Chem. 1985, 57, 625-628
625
Selective Determination of Trace Level Chromium(II I)by Ion-Pair Reversed-Phase High-Performance Liquid Chromatography Based on Color Formation with 4-(2-Pyridylazo)resorcinol in the Presence of Triethanolamine Hitoshi Hoshino* and Takao Yotsuyanagi
Department of Applied Chemistry, Tohoku University, Aoba, Aramaki, Sendai 980,Japan
The color reaction of chromium( 11) Ion with 4-(2-pyridyiaro)resorclnol Is quantitatively accompilshed wlthin 30 mln in bolling triethanolamlne buffer solutlon at pH 8.5-8.9 to produce the red chelate having the molar absorptlon coefficient of 8.21 X lo4 dm3 mol-’ cm-I at 525 nm. On the basis of this finding, a rapid and highly seiectlve ion-pair reversedphase partltlon liquid chromatography/photometric detection system has been developed for the determination of trace chromlum at the ng cm-’ level. A detection limlt of chromium is down to 0.5 ng cm-3 at 0.005 absorbance unit full-scale range (525 mn), being free from the interferences by 100-200 ng cm-’ each of common cations such as AI(III), V(V), Mn(II), Fe(III), Co(II), NI(II), Cu(II), Zn(II), and Cd(I1) ions.
Trace determination of chromium(II1) ion is now increasingly important in biological, clinical, and evironmental analyses. The photometric method for trace chromium seems to be limited actually to the well-known 1,5-diphepylcarbohydrazide method (1,2), in which chromium(II1) ion is converted to the hexavalent form before the color development. Several workers have reported the color reaction of highly sensitive reagent, 4-(2-pyridylazo)resorcinol(PAR, HZL) with chromium(II1) ion and have suggested the analytical potentially (3-5). However, the methods have suffered from the lengthy and incomplete color development even under heating conditions. Certain auxiliary ligands which accelerate the color formation, thus, have been desired. We have now found that the formation of a Cr(II1)-PAR chelate is greatly enhanced by triethanolamine (TEA) in slightly alkaline solution (pH 8.5-8.9, boiling for 30 min) to produce an anionic chelate which has the larger molar absorption coefficient (E) value, 6.21 X lo4 dm3 mol-l cm-I a t 525 nm than any chelate reported previously (4,5). This E value is also 1.3 times larger than that of the 1,5-diphenylcarbohydrazidecomplex (E = 4.17 X lo4) reported by Allen (6). Nonetheless, the major problems which arose in application of the PAR method to chromium assay of biological or environmental materials were the provision of sufficient sensitivity and selectivity to detect the ng concentration in various matrix compositions. We developed successfully an ion-pair reversed-phase high-performance liquid chromatography (IPHPLC) system for Fe(III)-, Co(111)-, and Ni(I1)-PAR chelates (7,8), which is an alternative approach to conventional photometry for solving the sensitivity and the selectivity problems. The highly inert Cr(II1)-PAR chelate is separated effectively by IPHPLC using a bolaform electrolyte (diquaternary ammonium salt) as an ion-pairing reagent in the mobile phase. Hence, the combination of Cr(II1)-PAR chelate (enhanced by TEA) and the IPHPLC separation will provide sufficient selectivity and
sensitivity for the chromium determination within an acceptable analysis time. Some reports have appeared about the practical use of the HPLC method in chromium determination (9-14); however, they seem to be unsatisfactory in some respects, Le., incomplete peak resolution, low sensitivity, or requirement of a more sophisticated detection system. The method proposed in this work is applicable to a sample containing chromium in the ng cm-3 range and 100-200 ng cm-3 each of Fe(III), Co(II), Ni(II), Mn(II), Cu(II), V(V), Zn(II), Cd(II), and Al(II1) ions as the matrices. EXPERIMENTAL SECTION Apparatus. A Horiba M-5 pH meter and a Hitachi Model 124 double-beam recording spectrophotometer were used. The HPLC setup used consisted of a TWINCLE pump unit, a UVIDEC 100-111spectrophotometric detector with an 8 pL flow cell of 1 cm light path length, and a VL 611 loop injector (100 mm3)from Japan Spectroscopic Co., Ltd. (Hachioji, Tokyo, Japan). The detector settings of 0.01 and 0.005 absorbance unit full-scale (aufs) were used for 10 mV recorder output. A column used was a Hiber column packed with LiChrosorb RP-18 (ODs type 10 pm, 4 mm bore X 250 mm in length) from Cica-Merck (Tokyo Japan). Reagents. A standard chromium(II1)solution (0.01 mol dm-3) was prepared from guaranteed reagent grade KCr(SO4),.12Hz0, and was standardized by back titration with standard EDTA and zinc chloride solutions. Other metal ion solutions were prepared from the chlorides of nitrates and were standardized by accepted EDTA titrations. A triethanolamine solution (2.5 mol dm-3) was prepared from “for masking grade” TEA obtained from Dojindo Laboratories) (Kumamoto, Japan), and pH of the solution was adjusted to 8.7 with hydrochloric acid. A solution of PAR, 2.5 X mol dm-3 was prepared and standardized as reported previously (15). A bolaform electrolyte, N,N,N,N’,N’,N’-hexamethyl-l,4-butanediammaniumdibromide (BoBr,) was synthesized from trimethylamine and 1,Cdibromobutaneby refluxing in ethanol for 1.5 h (16).
All other reagents and solvents used were of guaranteed reagent grade. Procedure. A sample solution (40 cm3or less) containing 0.020 to 2.5 pg of chromium(II1) ion is pipetted into a 100-cm3beaker to which 2 cm3each of PAR and TEA solutions are added. The mixture is boiled down cautiously to 30 cm3 for 30 min. After cooling, the solution is transferred into a 50-cm3volumetric flask and diluted to the mark. An aliquot of the solution (ca. 1cm3) is injected into the HPLC system through a 100-mm3loop. The Cr(II1)-PAR chelate eluted is detected at 525 nm. As the mobile phase, an aqueous methanol (40 wt % ) solution containing 9.8 X loy3mol kg-’ BoBr,, 2 X loy3mo1 kg-’ Tris-HC1 buffer (pH 7.5) and mol kg-’ disodium EDTA was used in a flow rate of 1.0 cm3 m i d .
0003-2700/85/0357-0625.$01.50/00 1985 American Chemical Society
626
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985 I
0
5%
0 450
-7
550
.
8
7
1
600
x Figure 1. Absorption spectra of Cr(II1)-PAR chelate and PAR at pH 8.7: (1)Cr(II1)-PAR, corrected for the spectrum of excess PAR, (2) PAR; C, = 1.03 X mol dm-3, C, = 1.0 X mol dm-3, boiling for 30 min in 0.054 mol drns TEA solution.
I I
06;
2
0
I
I
I
I
1
I
I
1
3
5
7
9
11
Figure 3. Molar ratio plots for Cr(II1)-PAR chelate at pH 8.7 in the presence of TEA after 2 h of boiling: Ccr = 1.02 X mol dm-3; full circles, , C = 0.05 mol dm3; open circles, CEA = 0.1 mol dm-3. i 0 8 t
o
0
30
60
o
90
120
a
111
Flgure 2. Absorbance-pH curves of the Cr(II1)-PAR system: (1) Cr(II1l-PAR-TEA system, absorbances corrected for those of excess PAR at 525 nm, C, = 1.02 X mol dm-3, CL = 1.06 X mol dm-3, C,,, = 0.05 mol dm-3, after 2 h of boiling; (2)prepared Cr(111)-PAR chelate used, at 525 nm, Ccr = 9.94 X lo-' mol dm-3.
RESULTS AND DISCUSSION Absorption Spectra and Effect of pH. Absorption spectra of Cr(II1)-PAR chelate (A, = 525 nm) and PAR in slightly alkaline solution are shown in Figure 1. The absorption-pH curve after the Cr(II1)-PAR-TEA system was boiled for 2 h is shown in Figure 2 (curve 1). The maximal absorbance was obtained in the pH range 8.5-8.9. The formation of Cr(II1)-PAR chelate was strongly facilitated in slightly alkaline solution as compared with that in the acetate buffer solution system (4). This indicates that the hydrolysis of Cr(II1) ion is favorably avoided by the formation of Cr(111)-TEA complex. Once the color was developed, the absorbance was constant over the pH range 6-11 (Figure 2, curve 2). In this case, the decrease in the absorbance below pH 6 is due to the protonation equilibria represented as (17)
The highly inert Cr(II1)-PAR chelate is not decomposed even at pH 2 at room temperature. The predominant species under the condition with the TEA buffer solution is the anionic one. The mobile phase pH should also be adjusted at 7 to 8. Molar Ratio Plots and the E Value. The molar ratio plots for the Cr(II1)-PAR chelate are given in Figure 3. In the concentrations of TEA, 0.05, 0.10, and 0.25 mol dm-3, the chelate was quantitatively formed when at least 4-fold molar excess of PAR (4 X mol dm-3) against Cr(II1) ion was added. The plots clearly show that the composition of the chelate is Cr:PAR = 1:2. The 6 value of the chelate is calculated from the plots to be 6.21 X lo4 dm3 mol-l cm-l which is similar to that of the Fe(II1)-PAR chelate, 6.11 X lo4 a t 495 nm (8). Rates of Color Development. Boiling time-absorbance curves for the Cr(II1)-Par-TEA system at various pH and TEA concentrations are shown in Figure 4. Ten cubic centimeter aliquots of the reaction mixture on boiling in a three-necked flask fitted with a dimroth condenser were pipetted at prescribed time intervals and were added to 10 cm3
Figure 4. Boiling tlme-absorbance curves for Cr(I 11)-PAR-TEA system. C,, = 2.03 X mol dm-3; CL = 2.12 X mol dm3; (1)Cm = 0.01 mol dm3, pH 8.22;(2)CEA = 0.04mol dm3, pH 5.88 (3)Cm = 0.1 mol dm-3, pH 9.25;(4)Cm = 0.04 mol dm3, pH 7.99. Absorbance values on the ordinate are half those of the reaction mixture.
of ice-cooled water. The absorbance values in Figure 4 are, thus, one-half of those for the reaction mixture. Consequently, when more than 0.04 mol dm-3 of TEA is added at pH above 7.5, the constant absorbance is reached within 30 min. On the basis of the results given in Figures 2, 3, and 4, boiling for 30 min with 1.0 X lo4 mol dm-3 of PAR and 0.10 mol dm4 of TEA at pH 8.7 is recommended for the color development. Reasonably, this is also the case for Cr(II1) ion at ultratrace concentration as pseudo-first-order conditions with respect to Cr(II1) ion may hold, i.e., the relatively high concentrations of PAR and TEA being used. Carbonate, nitrite, sulfite, and tartrate ions as well as urea have been known as an accelerator in the formation of Cr(111)-EDTA chelate (1419)but in the PAR system they failed to work, since the final absorbances were quite small under any pH conditions. Yamamoto and Takano (5) examined an acceleration effect of hydrogen peroxide on the Cr(II1)-PAR chelate formation in acetate buffer solution at pH 5.3. However, they reported the somewhat smaller E value, 5.10 X lo4 at 530 nm, as compared with that obtained in this work, 5.86 X lo4 (from the data in Figure 2, curve 2 at pH 5.3). TEA is an excellent auxiliary ligand for acceleration of the aqueous complexation of Cr(II1) ion with PAR. The Cr(111)-TEA complex intermediate may undergo the replacement reaction with PAR much faster than aquo or hydroxo Cr(II1) species do. A further role of TEA is to prevent hydrolytic olation of Cr(II1) ion, thus providing quantitative formation of the PAR chelate in slightly alkaline solution. Chromatography and Interference Studies. Typical chromatograms are shown in Figure 5. The IPHPLC separation of PAR chelates of Fe(III), Co(III), and Ni(I1) ions (7) and Pd(I1) and Pt(I1) ions (20)on an ODS column have been developed using tetrabutylammonium bromide (TBABr) as an ion-pairing reagent in aqueous methanol or aqueous tetrahydrofuran mobile phase. Under the conditions similar to those in ref 7 and 20, however, the satisfactory resolution of Cr(II1)-PAR chelate from the corresponding chelates of Fe(II1)
ANALYTICAL CHEMISTRY, VOL. 57, NO. 3, MARCH 1985
627
Table I. Reproducibility of the Chromium Determination l
i
u c
0 4 8 1 2 RcrL ? l o " I l P L
f
sample no. 1
none
2
Fe 1.77, Co 1.74, Ni 1.56, Cu 1.60, Mn 1.59, V 1.60, A1 1.60, Zn 1.58, Cd 1.92, Mg 3.2, Ca 3.2, Sr 3.2, Ba 3.2
3
Fe 3.54, Co 3.48, Ni 3.12, Cu 3.20, Mn 3.18, V 3.20, A1 3.20, Zn 3.16, Cd 3.84, Mg 6.4, Ca 6.4, Sr 6.4, Ba 6.4
4 5
Fe 5.11 Fe 12.2
.,I1
Figure 5. IPHPLC separation of Cr(II1)-PAR chelate: sample, Ccr = 1.23 X lo-' mol dm-3, CL = 1.0 X lo4 mol dms, ,C = 0.1 mol
dm-3; forelgn metal ions (A) none, (e) the same as sample no. 3 in Table I, (C) the same as sample no. 5 in Table I; column, Hiber column (LiChrosorb RP-18); mobile phase, 40.0 "fa% aqueous methanol containing 9.8 X lo3 mol kg-' BoBr,, 2 X 10 mol kg-' Tris-HCI, and mol kg-' EDTA; flow rate, 1 cm3 min-'.
and Co(II1) could not be obtained. By the use of a bolaform electrolyte, BoBr2, peak resolution was successfully achieved as shown in Figure 5. The bolaform cation affects the resolution differently than TBA" ion. TEA gave no effects on the separation of the PAR chelates under the conditions. The Co(II1)-PAR chelate is eluted faster than the Cr(II1) chelate, while the Fe(II1) and Ni(I1) chelates have an almost similar retention time to PAR (HL-) as shown in Figure 5B. The peaks of cobalt and chromium seem to badly overlap in chromatogram B, but they are well base-line resolved when the cobalt concentration is below lo4 mol dm-3. Under such the conditions, cobalt can be determined simultaneously with chromium. Actually, there are no interferences by the metal ions at their molar concentrations comparable to that of Cr(II1) ion. The main potential interferences are caused by iron, cobalt, and nickel ions in large amount, since they produce the very stable and inert PAR chelates whose peaks appear in the chromatogram. These can be circumvented by adding PAR in sufficient excess and by judicious adjustment of the mobile phase conditions. Under the conditions, Co(II) and Ni(1I) ions were tolerated up to the weight ratios against 6.4 ng cm-3 Cr(II1) ion of Co/Cr = 30 and Ni/Cr = 50, respectively. Ferric ion, which is most likely to be encountered in a real system, did not interfere individually at 1.22 X mol dmJ (680 ng ~ m - Fe/Cr ~, = 106) as shown in chromatogram C in Figure 5. Phosphate ion, added as KH2P04,at 4 X mol dm-3 had no influence on both the color development and the IPHPLC. Calibration Curve and Reproducibility. The peak height calibration at 0.005 and 0.01 aufs is linear over the concentraton range of Cr(II1) ion, (0.10-6.0) X lo-' mol dme3 (0.5-30 ng ~ m - ~ which ), is represented by the following equation: recorder response in absorbance unit = 9.84 X Cr (pmol dm-3) - 1.80 X The lower limit of detection is at a concentration which gives a signal twice the peak-to-peak base-line noise. The absolute on-column detection limit is down to 1.0 pmol of chromium. The sensitivity of the method is very high, comparable with flameless atomic absorption spectrophotometry (21), gas chromatography with electron capture detection (22, 23), neutron activation analysis (24),and a chemiluminescence method (25). As the blank (or contamination) value was approximately equal to the detection limit, extra care should always be taken to prevent contamination from laboratory dust, vessels, reagents, solvents, and solutions. The reproducibility of the method is shown in Table I, where an ac-
concns of foreign metal ions (IO4 mol d m 9
run peak height?b no. cm 1 2 3 4
3.00 3.10 2.95 3.00
5 6
2.90 2.95
7 8 9
2.95 3.00 2.95 2.98 h 0.04 av 1.90% RSD
aO.O1 aufs at 525 nm, 0.001 absorbance/2.5 cm. *Chromium mol dm-3 (6.40 ng ~ m - ~ ) . added 1.23 X
ceptable precision [relattive standard deviation 1.90% (n = 9)] is obtained for 6.4 ng cm-3 chromium in the presence of various metal ions. One HPLC run takes as little as 15 min or less and the overall analysis time for one sample with four calibration standards does not exceed 2 h. PAR is one of the most sensitive reagents for the photometric determination of chromium. The analytical sensitivity can be further increased by use of a highly sensitive photometric detector (0.005 aufs range is readily available). In addition, interference problems can be eliminated to a great extent by coupling with the IPHPLC separation system. The HPLC technique is now universally available in most chemical and clinical laboratories. An on-line preconcentration method for the Cr(II1)-PAR chelate in a manner similar to that described by Haring and Ballschmiter (26) seems highly promising for the determination of chromium in the picogram per cubic centimeter range. Since the use of TEA in the Cr(111)-PAR system as an accelerator has yielded a sufficient reduction in analysis time, the IPHPLC method, inherently sensitive and selective, has proved to be compatible with the practical applicability on a routine basis. In addition, when iron, cobalt, and nickel ions are to be determined in the presence of chromium ion in the same sample, the color development for those ions with PAR can be made at room temperature, followed by IPHPLC separation under the conditions reported previously (7, 8). The detection limits of those metal ions are also at the sub-nanogram-per-cubic-centimeter level. Registry No. PAR, 1141-59-9; TEA, 102-71-6; Cr, 7440-47-3.
LITERATURE CITED Sandell, E. B. "Colorimetric Determination of Traces of Metals 3rd. Ed"; Intersclence: New York, 1959; p 392. Bryson, W. G.; Goodail, C. M. Anal. Chlm. Acta 1981, 124, 391. Tataev, 0. A.; Abdulaev, R. R. Zh. Anal. Khim. 1970, 2 5 , 930. Yotsuyanagl, T.; Takeda, Y.; Yarnashita, R.; Aornura, K. Anal. Chlm. Acta 1973, 67, 297. Yamamoto, K.; Takano, Y. Bunseki Kagaku 1978, 2 7 , 460. Allen, T. L. Anal. Chem. 1958, 30, 447. Hoshino, H.; Yotsuyanagi, T.; Aornura, K. Bunseki Kagaku 1978, 27, 315. Hoshlno, H.; Yotsuyanagi, T. Talanta 1984, 3 1 , 525. Wlllet, J. D.; Knight, M. M. J . Chromatogr. 1982, 237, 99. Krull, I . S.; Bushee, D.; Savage, R. N.; Schielcher, R. G.; Smith, S. B., Jr. Anal. Lett. 1982, 1.5 ( A -3),267. Larochelle, J. H.; Johnson, D. C. Anal. Chem. 1978, 50, 240. Wenclawiak. B. Fresenlus' Z.Anal. Chem. 1982, 310, 144. Tande, T.; Petersen, J. E.; Torgrlmsen, T. Chromatographla 1980, 13, 607. Bond, A. M.; Wallace, 0. G. Anal. Chem. 1982, 54, 1706. Hoshlno, H.; Yotsuyanagl, T.; Aomura, K. Anal. Chlm. Acta 1976, 83, 317.
628
Anal. Chem. 1985, 57, 628-632
(16) Menger, F. M.; Wrenn, S. J . Phys. Chem. 1974, 78,1387. (17) Hoshlno, H.,Tohoku Unlverslty. unpublished results, 1982. (18) Phatak, G. M.; Bhat, T. R.; Shankar, J. J. Inofg. Nucl. Chem. 1970, 32, 1305. (19) Ohashi, K.; Suzukl, S.; Kubo, K.; Yamamoto, K. Bunseki Kagaku 1976, 25, 693. (20) Watanabe, E.; Nakashima. H.; Ebina, T.; Hoshlno, H.;Yotsuyanagl, T. Bunsekl Kagaku 1983, 32,469. (21) Surles, T.; Tuschal, J. R., Jr.; Collins, T. T. Env/ron. scj. Techno/. 1975, 9 , 1073.
(22) (23) (24) (25) (26)
Gosink, T. A. Anal. Chem. 1975, 47, 165. Lovett, R. J.; Lee, G. F. Environ. Scl. Techno/. 1976, 70, 67.
Salbu, B.; Steinnes, E.; Pappas, A. C. Anal. Chem. Ig75, 4 7 , 1011. Hoyt, S. D.; Ingle, J. D.,Jr. Anal. Chim. Acta 1976, 8 7 , 163. Haring, N.; Ballschmiter, K. Talanfa 1980, 27,873.
RCEIVED for review May 25, 1984. Accepted November 15, 1984.
Separation Function for Measuring the Information in Complex Chromatograms D. R. Van Hare' and L. B. Rogers* Department of Chemistry, University of Georgia, Athens, Georgia 30602
The separatlon functlon for use In the optlmlratlon of complex separations relates the lnformatlon content of a chromatogram to the number and the extent of resolution between adjacent peaks. Each fully resolved peak Is arbitrarily assigned 2.0 btls of lnformatlon whlle partlally resolved peaks are asslgned 1 to 2 bits dependlng upon the depth of the valley between them. Palrs that glve no valley but are detectable using a second-derlvatlve method are asslgned 0.5 bit. No a prlorl lnfonnatlon Is needed so the separation functlon may be easlly Incorporated Into exlstlng optlmlratlon schemes.
Recently, the optimization of chromatographic separations has moved away from conventional methods in which a chromatographer uses experience and intuition to judge the quality of a complex separation. Systematic methods are now being used which provide a more objective optimization of separations and are amenable to computer control. Laub and Purnell (I) searched for conditions that gave the highest minimum a: value (ratio of capacity factors) for all pairs of adjacent peaks. Later Glajch et ai. (2) used resolution in a similar manner. Morgan and Deming (3)suggested the use of a peak separation factor (4) as a quality measure in their simplex optimization procedure. Later Watson and Carr (5) added a weighting factor for the time of analysis as well as a consideration of the actual peak separation compared to the desired peak separation. Wegscheider et al. (6) developed a response function that operates differently than those mentioned above, i.e., as a product rather than a summation, and it takes into account the noise level of the chromatogram. In addition, there have also been peak-counting algorithms. Spencer and Rogers (7) proposed a separation number using a procedure that obtained information from badly overlapped peaks, even before a valley appeared. Later, Berridge (8)introduced a response function in which the resolution of each pair of adjacent peaks was summed up to a maximum value of 2.0. In addition, the number of peaks was weighted as were the analysis times for the first and last peaks in the chromatogram. A new response function has been developed to overcome some of the limitations of the previous functions. For example, those that utilize the natural logarithm of a fraction will Present address: Savannah River Laboratory, E. I. du Pont de Nemours and Co., Inc., Aiken, SC 29801.
approach zero a t the optimum. However, one cannot determine whether a large number of peaks has been fairly well separated or a few have been poorly separated. In addition, peak crossing causes problems since the function is zero for totally overlapped pairs of peaks, causing false optima. The function of Wegscheider et al. (6),even though it operates as a product of the individual peak separations rather than the sum of natural logarithms, suffers from the same problems. Furthermore, for all of the above functions, the number of components being separated should be known in order to ensure proper operation of the function. Berridge's response function (8)does keep track of the number of peaks detected during each separation. One drawback of his function is the need for calculating the resolution of each adjacent pair of components. In practice, unsymmetrical and severly overlapped peaks may present problems. In contrast, the separation number of Spencer and Rogers (7) is essentially a peak-counting algorithm that can provide information about overlapped peaks and does not require the calculation of resolution. However, the information for an isolated peak depended upon its shape and retention time. Furthermore, the function lacked general applicability because it was limited to isothermal or isocratic separations. In the present study, a new response function has been tested which incorporates some aspects of the others. It operates as a peak-counting function that assigns a value between 0.5 and 2.0 for each component that is detected. It is applicable to the optimization of all chromatographic methods and requires no a priori information. Furthermore, it can easily be expanded to include additional weighting terms similar to those of Watson and Carr (5)and Berridge (8). Both simulated and real chromatograms have been examined in this study. EXPERIMENTAL SECTION Chemicals. All of the chemicals were reagent grade or better and were used without further purification. Ethanol (U.S. Industrial Chemicals Co., New York) was used as the solvent for the gas chromatographic test mixture. This mixture was composed of 2,3,3-trimethylbutane, 2-pentanol, 2-methylheptane, cycloheptane, n-octane (Aldrich Chemical Co., Inc., Milwaukee, WI), tetrahydrofuran, m-xylene (J.T. Baker Chemical Co., Phillipsburg, NJ), 2-pentanone, o-xylene (Eastman Kodak, Rochester, NY), and cyclohexane (Fisher Scientific Co., Norcross, GA). The compressed gases used to operate the gas chromatograph were all obtained from the same source (Selox, Inc., Gainesville, GA). They were first passed through a gas purifier (Alltech Associates, Norcross, GA) which contained Drierite and molecular
0003-2700/85/0357-0628$01.50/00 1985 American Chemical Society
