What is electrochemical detection?
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 neurotransmitters, catecholamines, aminoglycosides, carbohydrates, thiols and phenols. 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 with the same instrument.
HPLC with an electrochemical detector.
How does electrochemical detection work?
In amperometric electrochemical detection the electrical current is measured resulting from oxidation or reduction reactions (see video). 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 detectable signal for data acquisition.
What is HPLC?
HPLC or High Performance Liquid Chromatography is an analytical technique to identify and quantify substances in a solution. It can be applied to measure catecholamines in urine for clinical purposes, pesticides in surface water for environmental analysis, or carbohydrates in food and beverages, to name a few.
An HPLC system consists of one or more pumps, an autosampler, a detector and data acquisition software. At the heart of the HPLC system is the chromatographic column where separation of substances in a mixture takes place. The pump(s) supply a carrier solution, called mobile phase through the system. The samples to be analyzed are introduced in the flow path by an autosampler.
The separation principle in HPLC is based on partitioning of a substance in stationary phase (column filled with particles) and mobile phase (solvent flowing through). In a well functioning HPLC system the time spent in the column is unique for each substance in the mixture. This ‘retention time’ identifies the substance. The detector continuously measures the mobile phase that flows through the detection cell. Substances eluting from the column result in a deflection of the signal called a ‘peak’. The signal is analyzed in a chromatogram by data acquisition software on a PC that is connected to the detector.
Schematic HPLC configuration, injection valve is part of an autosampler.
What is a chromatogram?
A HPLC detector is connected to a computer where the data is collected and analyzed in data acquisition software. The resulting chromatogram 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.
By analyzing standards of different concentration, a linear calibration plot can be constructed. The concentration of a substance in a sample is found by entering the signal value in the linearity equation of the calibration plot.
Care must be taken that the peak measured in the sample chromatogram is not co-eluting with other substances with the same retention time. This, and other checks, are part of an assay validation.
A chromatogram with calibration standards of several neurotransmitters.