and light gathered. This cell has a conductivity cell constant of 1.6 compared to the cell constant of 0.3 noted for the first cell. This means t h a t for a given salt concentration, the resistance of one cell is 5.3 times larger than the other, and thus the heating time constant is slower by the same amount. Center sections should be chosen with this in mind. T h e obvious limitations of the cell are that solvents that attack Lucite cannot be used and that wavelengths in the UV can not be reached with plastic windows.
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ACKNOWLEDGMENT The authors thank Paolo Priarone for the cell construction^
LITERATURE CITED (1) R M Reich and J R Sutter, Anal Chem, 49, 1081 (1977).
RECEIVED for review May 31, 1977. Accepted September 8, 1977.
Low Volume Reference Electrode for Electrochemical Detection and Fraction Collection in High Performance Liquid Chromatography Curt R. Freed* and Paul A. Asmus University of Colorado Medical Center, Division of Clinical Pharmacology: C23 7, 4200 East Ninth A venue, Denver, Colorado 80262
High performance liquid chromatography with electrochemical detection (LCEC) has been shown to provide selective and sensitive analysis of easily oxidized organic compounds and was recently reviewed ( I ) . The utility of this method for the analysis of catecholamines has been demonstrated ( 2 , 3 ) . We have used modifications of this technology to study brain catecholamine metabolism and have needed to measure both the concentration of trace metabolites and specific activity of a tritium label on dopamine and norepinephrine. Using a commercially available electrochemical detection system (Bio-analytical Systems, West Lafayette, Ind.), we could successfully measure the concentration of catecholamines in rat brain tissue. Because of the large volume of the reference electrode compartment in the standard electrochemical system, however, we could not collect fractions for radioactivity measurements without mixing of' adjacent chromatographic bands. We have developed a modification of the commercial detector which allows both monitoring of the chromatogram and collection of fractions. A simple low volume silver tube with a silver chloride coating is placed in the outflow stream of the electrochemical detection block and serves as a silver-silver chloride reference electrode. Current through the mobile phase is maintained by placing a short length of stainless steel tubing (auxiliary electrode) in the flowing stream. Fractions are collected directly from the steel tubing. Our present chromatographic system employs an ion-exchange column with acetate/citrate mobile phase and a mobile phase flow rate of 1 mL/min with peak widths of 3.5 and 5.0 mL for norepinephrine and dopamine, respectively. Since the total volume of the reference and auxiliary electrodes is 30 kL, no mixing of chromatographic peaks occurs. The signal-to-noise ratio and sensitivity are the same as the commercial electrode. Because the silver chloride coat of the reference electrode is in the outflow stream, the coating must be reapplied every few days by the electrolytic technique noted below. I t should be noted that the arrangment prescribed here will only be useful for trace analysis work where the current is small (typically nanoamperes) and no significant ohmic losses occur.
EXPERIMENTAL A silver tube was formed from 1 cm X 2 cm X 0.005 inch silver
Reference
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Carbon Paste E l e c t rode
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Reference
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Carbon- Pasle E lecirode
Auxiliary
A
B.
Flgure 1. (A) Standard electrochemical detector system (8)Modified low dead volume reference electrode system with silver-silver chloride tube as reference electrode and stainless steel auxiliary electrode
foil (MC & B, Norwood, Ohio) by rolling around a 1-mm 0.d. paper clip. The overlap was filled by silver soldering and the resulting tube cleaned with brief exposure to 6 N HN03 followed by 0.1 N HC1. Silver chloride was formed on ihe surface by connecting the silver tube t o the positive terminal of a 9-V battery. A platinum electrode was attached to the negative terminal and both electrodes were immersed and current was passed in 0.1 N HC1 for 30 s. Figure 1 shows a schematic diagram comparing the two electrode systems. The silversilver chloride electrode is connected to the outlet of the carbon paste electrode with a short piece of Teflon tubing and fittings. The stainless steel auxiliary electrode is connected to the reference electrode with a short piece of polyethylene tubing. Electrical connections are conveniently made with alligator clips.
LITERATURE CITED (1) P. T. Klsslnger, Anal. Chem., 49, 447A (1977). (2) C. Refshauge, P. T. Klsslnger, R. Drelllng, L. Blank, R. Freeman, and R . Adams, Life Sci., 14, 311 (1974). (3) R. Keller, A. Oke, I. Mefford, and R. N. Adams, L/fe Sci., 19, 995 (1976).
RECEIVED for review July 11, 1977. .4ccepted September 9, 1977. ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
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