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Electrochemical detection for HPLC

Introduction

Electrochemical detection (ECD) for HPLC or uHPLC is an extremely selective and sensitive detection technique that is applied in a number of analyses such as the neurotransmitters dopamine, serotonin and noradrenalin. In combination with the proper electronics, ECD has an enormous linear dynamic range of more then 7 orders of magnitude. This means that concentrations can be measured as low as 10 pmole/L and as high as 100 µmol/L or more.

Fig. 1. HPLC with an electrochemical detector.

Detection principle

In amperometric electrochemical detection the electrical current is measured resulting from oxidation or reduction reactions (see video 1). A sample is introduced in HPLC and separated on the chromatographic column. The column is connected to an ECD cell, which is an electrochemical sensor where a reaction takes place at an electrode. Electrochemically active substances that elute from the column undergo an electrochemical reaction, electrons are transferred resulting in an electrical current. The electrodes are connected to an electronic circuitry with a powerful -low noise- amplifier that converts a pico- or nanoampere current in a signal in the range of ± 1 Volt which is commonly used in data acquisition.

Video 1. HPLC/ ECD explained in 2 min. (double click to expand). Full version is elsewhere on this website and on YouTube.

Chromatogram

The detector is connected to a computer where the data is collected and stored in data acquisition software. The resulting chromatogram (fig. 2) shows the detector response and is used for identification and quantification, the ultimate goal of analysis. Using calibration standards, the signal height (current, in nA) is related to concentration and the retention time identifies a substance.

Fig. 2. A chromatogram with calibration standards of several neurotransmitters.