ANALYTICAL CHEMISTRY, VOL. 50,
F W
n n
U 3
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-0 9
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Figure 5. Cyclic voltammogram of solution of Figure 4 at stationary mercury drop electrode at a scan rate of 100 m V / s
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of the redox couple (Cp,Cr in our case), V is the volume of the solution, p is a constant dependent on the cell geometry and stirring rate, and i and F have their usual significance (9). A plot of log i vs. t for the chromocene oxidation gave a straight line with a value of p = 2.8 x (calculated from the slope) and a value of n = 1.18 (calculated from t h e intercept). T h e linearity of the log i vs. t relationship is again consistent with a n uncomplicated electron transfer in the oxidation. This was further proved by reversal coulometry. In this experiment, after electrolysis at 0 V for time t , t h e potential is set sufficiently negative to reduce any CpzCrf which may be present in solution. A potential of -1.0 V was employed. If the initial oxidation product is stable and not subject to follow-up reactions, t h e number of coulombs consumed in the oxidation and reductions steps, Qox and QM, respectively, are related by QRed -
-
--1
I
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1 - exp(-pt)
where t is the same for both electrolyses (1000 s in the present case). A value of 0.92 was predicted by Equation 2, using the value of p given above, and a value of 0.95 f .05 was measured by integration of the current-time data. A polarogram of the solution after the initial oxidation showed the 2 reduction waves of Cp2Crt very clearly (Figure 6). Cp&r can also be reduced. A diffusion-controlled wave with a half-wave potential of about -2.3 7\ involves formation of Cp@, followed by a reaction to give another electroactive product(s). Details of t h e reduction will be published in a subsequent paper concerned with metallocene reductions.
xc
-2 0
-0.5 VOLTS
YS
ACKNOWLEDGMENT
SCE
Flgure 6. DC polarogram of Cp,Cr+ solution produced by electrolysis of Cp,Cr at 0 V at mercury pool electrode
plicated by preceding or following reactions was obtained by our observation t h a t t h e anodic current function (ipa/t,' ' z , where iPl is the anodic peak current and u is the scan rate) was independent of scan rate over the range u = 50 to 500 mV/s. T h e long-term stability of t h e chromocene cation was investigated by bulk oxidation of t h e neutral complex a t t h e mercury pool anode, a t 0 V. For an uncomplicated anodic electron-transfer reaction, t h e current-time relationship is given by Equation 1,
We thank Roy Clark for his glassblowing efforts, and we gratefully acknowledge the support of t h e National Science Foundation in this work.
LITERATURE CITED (1) A. J. Bard, Pure Appl. Chem., 25, 379 (1971). (2) J. L. Mills, R. Nelson, S. G. Shore, and L.. B. Anderson, Anal. Chem., 43, 157 (1971). (3) W. E. Geiger, Jr., T. E. Mines, and F. C. Senftleber, Inorg. Chem., 14, 2141 (1975). (4) J. E. Harrar and I . Shain, Anal. Chem., 38, 1148 (1966). (5) J. Newman and J. E. Harrar, J . Electrochem. SOC.,120, 1041 (1973). (6) E. 0. Fischer and W. Hafner, 2. Naturforsch., 6 .8, 444 (1953); G. Wilkinson, J . Am. Chem. Soc., 7 8 , 209 (1954). (7) For leading references, see W. E. Geiger, Jr. J . Am. Chem. Soc., 9 6 , 2632 (1974). (8) E. 0. Fischer and K . Ulm, Chem. Ber., 9 5 , 692 (1962). (9) A. J. Bard and K. S.V. Santhananrn, Necfroanal. Chem., 4, 215 (1970); see especially pp 237-239.
Pt log i = -+ log nFVC, P (1) 2.3 where t is the electrolysis time, n is the number of electrons
RECEIWDfor review November 21, 1977. Accepted February
transferred, CR,is the bulk concentration of the reduced form
6 : 1978.
Liquid Sample Loop Geometry for Trace Analysis by High Performance Liquid Chromatography with Large Sample Volumes James M. Zehner" and Richard A. Simonaitis Stored-Product Insects Research and Development Laboratory, Science and Education Administration, USDA, Savannah, Georgia 3 1403
High performance liquid chromatography (HPLC) has many advantages compared with gas chromatography. However, in the case of trace analyses, the H P L C detectors sometimes lack sensitivity. This disadvantage can be overThis paper not subject to U S Copyright
come by injecting large-volume samples (1mL or larger). But when t h a t is done, it is necessary to dissolve the sample in a solvent which causes the components t u accumulate a t the front of the column and be eluted by rapidly changing the Published 1978 by the American Chemical
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ANALYTICAL CHEMISTRY, VOL. 50, NO. 7, JUNE 1978
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b
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Figure 1. Profiles of chromatograms caused by sample loops when 0.05% acetone was used in 60% methanol/water v/v on ODS "Permaphase" column, at 254 nm, 0.32 AUFS. (A) 1 mL, 'I8-in. (3.18-mm)0.d. loop; (B) 2 mL, '/8-in. (3.18-mm)0.d. loop; (C) 4 mL, (3.18-mm)0.d. loop; (D) 1 mL, 'Ilsin. (1.59-mm)0.d. loop: (E) 2 rn, 1/16-in. (1.59-mm)0.d. loop; (F) 4 m, 'I1& (1.59-mm)0.d. loop
composition of t h e mobile phase or by gradient elution. Fixed volume sample valves are sometimes used to inject large sample volumes. T h e technique involves loading the large volumes into a loop of tubing and then injecting them onto t h e column as part of t h e stream of mobile phase. T h e large loop (2 mL) furnished commercially for this purpose consists of an '/s-in. (3.8-mm) 0.d. stainless steel (S.S.) tubing with 1/16-in.(1.59-mm) 0.d. S.S. tubing soldered onto each end in order to fit the 1/16-in. (1.59-mm) low-volume fittings on the valve. When this loop is used, some mixing of solutions occurs a t the point where there is a change in tubing diameter. If there is a difference in absorbance between sample solvent and mobile phase, this mixing prevents a sharp return to baseline. This mixing also prevents the accumulation of the components in a narrow band a t the beginning of the column and causes the early eluting components to elute as short broad peaks. If the large (volume) loop had a constant internal diameter, perhaps this problem could be eliminated. We therefore compared loops with constant and variable internal diameters.
EXPERIMENTAL Equipment. Two large loops (approximately 1 mL and 4 mL in volume) were constructed of '/*-in. (3.18-mm) o.d., 1.9-mm i.d. S.S. tubing with 1/16-in.(1.59-mm) o.d., 0.8-mm i.d. S.S. tubing soldered to the ends in the same manner as the manufacturer's 2-mL loop. In addition to the manufacturer's 1 mL constant 1/16-in.0.d. loop, coils were cut from 1/16-in.(1.59-mm) 0.d. x 0.05-in. (0.127-mm)i.d. S.S. tubing of 2 m and 4 m in length which corresponds to approximately 2.5- and 5-mL volume. A DuPont 820 liquid chromatograph equipped with an Altech 3000 psig rated sample valve, UV photometer at 254 nm, and 1 m X 2.1 mm i.d. S.S. column packed with ODS "Permaphase" (octadecylsilane permanently bonded to Zipax) was used. The chromatographic conditions were: mobile phase, 60/40 methanol-water (v/v); column temp., 60 "C; column messure, 450 psig; flow rate, 0.4 mL/min. Reagents. A solution of 0.05% acetone in water (v/v) and a solution containing 0.07 g dibenzyl (Eastman) (DB), 0.003 g 4,4'-dichlorobiphenyl (Aldrich (DCBP)) and 0.07 g pentachlorobenzene (Aldrich (PCB)) dissolved in methanol t o make 100 mL were prepared. Procedure. Profiles of 0.05% acetone solution were obtained as they were displaced from the sample loops. These profiles are shown in Figure 1. For comparison, 5 pL of the mixture of dibenzyl (DB) (0.07%), dichlorobiphenyl (DCBP) (0.003%),and pentachlorobenzene (PCB) in methanol were chromatographed by using the conditions listed. This solution was then diluted to make a final solution containing 30% methanol and 7070 water.
MINUTES
Figure 2. Chromatograms of mixtures of dibenzyl (a),4,4'-dichlorobiphenyl (b), and pentachlorobenzene (c). Chromatogram I, 5-WL sample; Chromatogram 11, 2 m, '/16-in, (1.59-mm) 0.d. loop; Chromatogram 111, 2 mL, 'I8-in. (3.18-mm) 0.d. loop
Table I. Comparison of Peak Height Ratios with Loop Geometry
Sample size 5 NL
1 mL, 1 mL, 2 mL,
2 m, 4 mL, 4 m,
-inch variable dia. loop -inch constant dia. loop -inch variable dia. loop -inch constant dia. loop -inch variable dia. loop -inch constant dia. loop
Ratio, DW PCB
Ratio, DCBP/ PCB
0.927 0.866 0.91 3 0.787
1.151 1.134 1.201 1.192 1.153 1.044 1.193
0.921
0.628 0.853
The final dilution for the 1-mL loops was approximately 240:l; that for the 2-mL and 2-m loops was 400:l; and that for 4-mL and 4-m loops was 800:l. The ratios of the DB and DCBP peak heights to PCB peak height were calculated from chromatograms obtained from all six sample loops. Sample chromatograms of the 5-pL sample, 2 m x '/&n. (1.59-mm) 0.d. loop and the '/& (3.18-mm) 0.d. 2-mL loop are shown in Figure 2.
DISCUSSION The profiles of Figure 1show that the response returns more sharply to baseline with the 1/16-in. (1.59-mm) diameter loops with uniform internal diameter than with corresponding variable diameter loops. This slow baseline response is also illustrated in Figure 2 where the DB peak elutes on the upward slope of the baseline caused by mixing of the 30% methanol and t h e 60% methanol mobile phase. The ratios of peak heights of D B to PCB and PCBP to PCB obtained from duplicate analyses are reported in Table I. Precision between duplicates was within approximately I170. There was a small effect of the various loops on the ratios of DCBP and P C B except for the 4-mL large diameter loop, which caused a ca. 107" decrease. However, the ratio of t h e DB to P C B peak heights decreased in the large diameter sample loops because of partial elution of the DB peak caused by mixing of mobile phase with sample before all the samples reached the column and was spread over a wider band. T h e 1/16-in.(1.59-mrn) 0.d. loops maintained a nearly constant ratio of DB to PCB, and there was only a small decrease (approx. 8 7 ~for ) the 4-m loop. Therefore, a longer constant-diameter loop is preferred over the shorter, variable-diameter loops.
RECEIVED for review December 1,1977. Accepted February 21, 1978. Mention of a commercial or proprietary product in this paper does not constitute an endorsement of this product by the U.S.Department of Agriculture.
