RESULTS AND DISCUSSION In ordinary exchange runs. very small amounts of resins were used, so that final C1- concentrations in solution up to 5X mequiv/L were reached. With the fastest resins, the exchange reaction was completed after about 200 s: hence. an mequiv/L s-' of chloride average buildup of about 2.5 X was t o be continuously monitored. With system response times experimentally ranging from about 3 to 30 s. depending mainly on Na2S04 concentration, conventional calibration techniques based on equilibrium potentials were not useful in this situation. Experimental potential vs. time plots were then referred to dynamic calibration curves prepared with C1- feed rate 52.5 X 10-j mequiv/L s-'. The ratio of electrode response time to C1- feed rate ranged accordingly between 1 x 10''and 1.2 x 10' L s*/mequiv. The results of some typical rate measurements made with this procedure are shown in Figure 1. where the degree of exchange (U= [Cl-],;[Cl-],) with time
over a quadruplicate set of runs is reported. As can be seen, good reproducibility is obtained, with a standard deviation lower than l t 6 7 ~ .Furthermore, by allowing the same limited time for potential measurements in the kinetic runs and in the related calibration curves, the precision of the method was assured (actual C1~concentrations. obtained by this technique, differed less than k770 from values calculated on the basis of weight and full exchange capacity of the resin). The proposed procedure is expected to work also in similar applications of continuous ion-selective potentiometry, provided that a sufficiently high ratio (110" L s'lmequiv) between electrode response time and primary ion feed rate is allowed.
LITERATURE CITED ( 1 ) 6 . Fleet. T. H Ryan, and M J D Brand, Anal Chem , 46, 12 (1974)
RECEIVED for review March 4, 1977. Accepted July 18, 1977.
Demountable Temperature Jump Cell John R. Sutter" Chemistry Department, Howard University, Washington, D.C. 20059
Richard M. Reich American Instrument Company, Silver Spring, Maryland 209 10
In genersl, Temperature Jump Cells are designed with permanently mounted cylindrical or conical quartz rods to guide monochromatic light through the solution onto the detector. This arrangement, however, presents certain limitations for any given cell. Depending on the reaction being investigated, one might need a cell with: long path length in order to use very dilute solutions or compensate for a low absorptivity; minimum heated volume to conserve a precious reactant or to compensate for a small reaction V-l: a geometry designed to study the fastest reactions (minimize to maintain the temperature after perturbation for the longest possible time in order to study a slower reaction. We have been experimenting with the cell design shown in Figure 1. The center, transparent, sample compartment is the important feature of the cell. This section is milled from Lucite and the optical surfaces are polished. It is sandwiched between the two Kel-F halves forming the body of the cell and holding the electrodes. The four bolts holding the cell together are not shown in Figure 1. The center section shown has a 1.0-cm by 1.0-cm hole and a thickness of 0.2 cm. The lower electrode mounts flush whereas the upper one is recessed 0.1 cm from the Lucite plate. This recess increases the heated volume to 0.3 mL. The cell has a 1.0-cm light path, and a conductivity cell constant. d / A , of 0.3 cm-'. Also of importance is the improvement in light gathering ability of this cell. The 1.0- by 0.2-cm cross-section yields a 0.2-cm area which is to be compared to a 0.3-cm diameter circular rod functioning as the cell window. The rod offers only 0.07 cm2 of window. This represents a maximum increase of 2.8 in available light, producing a corresponding increase in SIN. The Temperature-Jump apparatus used with the cell has 2378 * ANALYTICAL CHEMISTRY, VOL. 49, NO. 14, DECEMBER 1977
Figure 1. Temperature Jump Cell
been described previously ( I ) . The cell of Figure 1 was tested in the manner outlined there. A block diagram of the cell is also contained there. .4 heating time constant of 300 ns is easily obtained. The temperature of the heated solution will persist, with no noticable sag, for about 3 s after perturbation. After this time, the observed absorbance will drift slowly and exponentially (30-45 q ) back to the absorbance corresponding to that at the thermostat temperature. Various sample spacers have been tried, e.g. a. 0.5- by 0 5 c m hole with 0.3-cm thickness will give twice the jump height compared to the cell just described, at a sacrifice in path length
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
I
__
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Carbon Paste E l e c t rode
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I
Reference
I:
-2
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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