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Sensitivity and long-term stability of electrochemical detection in high-performance liquid chromatography.

Sensitivity and Long-Term Stability of Electrochemical Detection in High-Performance Liquid Chromatography

In high-performance liquid chromatography (HPLC), one of the most sensitive methods of measurement is electro chemical detection (ECD). Compounds down to the picogram level can be detected easily. As well as sensitivity, ECD has good selectivity for those compounds that can be oxidized or reduced at voltages between -1 and +1.4 V. Furthermore, electrochemical detectors are linear over a wide range and prices are relatively low.

Detection is based on electrochemical oxidation or reduction of compounds at the surface of a measurement electrode, placed in the effluent stream (Figure 1). Oxidation or reduction is activated by applying a constant voltage (potential) difference between the measurement (or working) electrode and the mobile phase. The resulting electron flow is amplified and converted into a signal that can be recognized as a peak in a chromatogram.

The thin-layer cell has a relatively small surface and only 5 to 10% of the sample can reach the active area of the electrode. The electrochemical yield (current) is limited by the diffusion rate of the analyte from the mobile phase to the surface area of the electrode.

In the wall-jet design, the surface area is even smaller and the yield is determined by the convection rate of the analyte to the electrode surface. Typically, the yield is also below 10%.

In the porous through-flow cell, the active area is much larger, about two orders of magnitude. So the electrolytic efficiency is 100% (coulometric detection). But it is incorrect to assume that this cell design gives better sensitivity. Increasing the surface area increases the background noise at least as much as the signal height. Both the wall-jet as well as the thin-layer design result in lower detection limits. On the other hand, the long-term stability of porous through-flow flow cells is increased, because it takes longer to contaminate the larger surface area with sample and reaction products. But once it is contaminated, the electrode cannot be repolished any more, it has to be replaced. Another disadvantage is that the large cell volume of porous through-flow cells causes band broadening when using microbore columns. Figure 2 shows the schematics of the three most frequently used flow cell designs: thin-layer, wall-jet and through-flow cells.

One goal of our work was to combine the high sensitivity typically achieved with the wall-jet and thin-layer designs, with long-term stability.


For the experimental work we used an HP 1050 Isocratic Pump, and HP 1050 Automatic Liquid Sampler and an HP 1049A Programmable Electrochemical Detector.

Cell Design

The HP 1049A is an amperometric detector with a thin-layer cell design. Three working electrodes are available: glassy carbon, gold and platinum. The working electrode is a small disc with a diameter of 8mm and a thickness of 3mm. It is sealed with a spacer. The cell volume is 0.5[mu]. This design ensures that air bubbles, when generated by the electrochemical reaction, are flushed out of the cell area immediately.

The working electrode of the HP 1049A is not embedded in an isolator as is normally the case and it has a bigger working area than the usual built-in electrodes. This feature has several advantages. It is easy to exchange the electrode when it is necessary to use different materials. The electrode is effectively sealed so that current creepage is prevented. The glassy carbon electrode can be cleaned in chromic sulfuric acid or, if the contamination is irreversible, it can be disposed of.

Reference Electrode

As well as the working electrode, the reference electrode is important because if affects the stability of the results. Reference electrodes are used to maintain a stable potential on the working electrode. A conventional reference electrode consists of a silver wire coated with silver chloride in a saturated potassium chloride solution. A ceramic or porous glass frit acts as an ion bridge to the mobile phase. This design is often a source of an unstable baseline, if molecules from the mobile phase diffuse into the reference cell.

These problems can be eliminated by using a so-called solid state reference electrode. This consists of a silver wire coated with silver chloride which is in direct contact with the mobile phase. Chloride ions are added to the mobile phase in a concentration of about 1mmol. This ensures that the concentration of the chloride ions is constant over a long period. This type of reference electrode can be used whenever the addition of chloride ions does not interfere with other chromatographic parameters.

Optimizing Stability by

Electrochemical Cleaning

Between two runs

Typically, commercial electrochemical detectors operate by applying a single fixed potential to the working electrode. During the electrochemical process, many compounds may form reaction products which can deposit on the working electrode and foul the electrode surface. The effects include reduced response, increased noise, and extensive drift.

The standard method of restoring a fouled electrode is mechanical polishing; this wastes operators' time and decreases instrument uptime. In many cases, the electrode can be cleaned electrochemically by applying a second, or even a third, potential between two injections. Changing the potential manually also wastes operators' time. In addition, the period of time during which a certain potential is applied to the electrode cannot be controlled accurately.

With the pretreat mode built into the HP 1049A, it is possible to program the detector so that two selected potentials are applied for a specified duration between two analyses. When the potential cycle is complete, the detector sends a "ready message" to the auto-injector to perform the next injection after a programmable time buffer, or after a user-definable threshold of current drift caused by equilibration of the baseline. The duration of the pulses depends on the elctrode material; for glassy carbon it is close to one second, for platinum or gold, the millisecond range is adequate.

During run

Pulse mode is similar to the pretreat mode and is used with the gold or platinum electrode. The difference from the pretreat mode is in the time interval. The pulse mode is used during the run in order to avoid contamination of the electrode with compounds that fould the electrode rapidly. In this case, the use of a single potential electrochemical detector is practically impossible. The fouling of the electrode is eliminated by using a pulsed waveform with one or more potentials capable of cleaning the electrode. The detector measures the current only during a short sampling interval, in contrast to a single potential mode where the current is continually measured. Because the potential is also pulsed during the elution of a compound, the duration of the pulse cycles must be much less than 1 second. Short pulse cycles are necessary in order to get a higher signal-to-noise ratio by averaging a larger number of sample measurements per time unit.

Optimizing Sensitivity

In order to get the best sensitivity, there are several rules that must be fulfilled for any part of the total chromatographic system. Table 1 gives an overview of parameters that influence the sensitivity of electrochemical detection The influence of these parameters are described in the literature[1] and in most of the operating manuals supplied with the instruments.

Table : Table 1 Overview of Parameters
Detector parameters parameters
detection potential flow rate
electrode material and area pump pulsations
cell geometry mobile phase
reference electrode stability (type, pH, purity)

temperature stability potentiostat/amplifier stability

We will only discuss here the most important of the detection parameters, ie., how to select the best potential for individual applications. When developing a new method, finding the optimal voltage is important in order to get the best sensitivity and/or selectivity. The potential must be high enough (positive or negative) to activate the electrolysis. On the other hand, if the potential selected is too high, the signal-to-noise ratio decreases because the baseline noise is too high. In addition, selectivity is lost if molecules other than the analyte are electrolyzed. Therefore, when developing a method for EC-detection it is important to find the correct potential easily.

Finding the best working potential for

single compounds

One way to find the best potential is to change the potential continuously and to record the current. When using an X-Y recorder or a data system and suitable software, you can obtain voltammograms. If voltammograms are required in order to find the best detection voltage for an HPLC analysis, they must be acquired under the flow conditions that are identical to the HPLC sample run. Therefore it is recommended that the standard should be added to the mobile phase when acquiring voltammograms.

Finding the best working potential for mixtures

The standard method of finding the optimal voltage for a single component is to acquire a voltammogram. A limitation of this method is that the optimal voltage can be found with solvent flow only. This means that the sample must be added to the mobile phase. This procedure is time consuming if the optimal voltage has to be found for a large number of compounds. In this case it might be easier to inject the standard mixture several times and to change the voltage automatically between each injection. The best signal-to-noise ratio for each individual compound, as well as for the mixture, can be found by overlaying the chromatograms.


Originally, electrochemical detection was developed in response to the need for a convenient method for determining tyrosine metabolites in tissue samples (brain and body fluids). The initial applications were directed toward measurements of catecholamines in small animal brain tissue. Meanwhile, the application range has been expanded to many other compounds in biomedical research and to other areas such as pharmaceuticals, phenols, thiols, pesticides, aromatic amines, industrial oxidants and more recently, to carbohydrates.


Catecholamines are aminoethyl derivatives of brenzcatechin. The most important examples are adrenaline and noradrenaline which have important functions as hormones and neurotransmitters. High performance liquid chromatography with electrochemical detection enables the direct analysis of catecholamines in tissue, serum, plasma and urine. The compounds are oxidized at a relatively low potential of 0.4 to 0.6 V; this ensures a high signal-to-noise ratio.


The traditional method for measuring carbohydrates in HPLC is refractive index (RI) detection. RI detectors lack sensitivity, and high selectivity is usually required for samples in complex matrices, e.g. food. Conventional electrochemical detectors with single potential capability cannot be used for carbohydrate analyses because the electrodes are rapidly deactivated by the reaction product. When using a pulsed amperometric detector, the potential is changed alternately to another value to remove the reaction product.


The determination of organic samples in water is fundamental to solving problems in environmental protection. Phenol derivatives are widely used as insecticides, antiseptics and disinfectants and are found in various environmental matrices such as waste water and soil. As environmental samples are usually contaminated with a large number of compounds, selective detection methods are preferred. Phenols and their derivatives can be analyzed by HPLC and detected with electrochemical detection using relatively high potentials: 1.0 to 1.3 V.

Figure 3 shows the analysis of river water highly contaminated with chlorophenols and other compounds. Two hundred injections were spread over one week and, as Figure 3 shows, there was no loss in response.


Electrochemical Detection in High Performance Liquid Chromatography - A Practical Primer. Hewlett-Packard Co., 1989. Pub. no. 12-5952-0034.

PHOTO : Figure 1 Principle of electrochemical detection

PHOTO : Figure 2 Different cell designs for electrochemical detectors

PHOTO : Figure 3 Analysis of chlorophenols in river water
COPYRIGHT 1990 Chemical Institute of Canada
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 1990 Gale, Cengage Learning. All rights reserved.

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Author:Gratzfeld-Huesgen, Angelika; Haecker, Wolfgang
Publication:Canadian Chemical News
Date:Mar 1, 1990
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