HPLC-ECD (Electrochemical Detection) Fundamentals

Electrochemical (EC) detection (ECD) coupled with HPLC is a powerful tool for the detection of neurotransmitters, environmental assessment, and the detection of phenol compounds from food samples. Various neurotransmitters are detectable. Such as norepinephrine, dopamine, serotonin, glutamate, GABA, acetylcholine, as well as others. The sample matrix is widespread. Including but not limited to microdialysis samples, tissue, cell lysate, cell culture medium, blood plasma, and more.

Principles

Using a separation column and mobile phase on an HPLC is how you separate target compounds (analytes). After separation, the compounds present within the mobile phase enter the electrochemical detector (ECD). These are oxidized or reduced. Upon oxidation, free electrons are being released to the counter electrode. With the compound reduction, electrons are provided from the counter electrode to the analyte. The ECD detects this electrical current which linearly correlates to the analyte concentration loaded into the HPLC. Read on to learn how to use this principle for practical applications. 

Advantage of Electrochemical Detection

Electrochemical detection (ECD) is very sensitive and capable of detecting in the femtomole/L (10-15 M) range. Depending on the analyte and sample matrix. Choosing the appropriate applied voltage for the oxidation/reduction potential and material of the working electrode, a more chemical-specific detecting condition can be obtained. For example, thiols are detected by a gold working electrode. Where hydrogen peroxide is relatively specific to the platinum electrode. A high sensitivity concomitant detector with good specificity is the tandem mass spectrometer (LC/MS/MS). But the cost is prohibitive. HPLC-ECD has the perfect balance of price and performance. For routine measurements of biological or environmental samples. Such as catecholamines, acetylcholine, glutamate, glycine, GABA, and others. 

Amperometric and Coulometric Detection

Two types of electrochemical detectors use electrolysis; amperometry and coulometry. Eicom carries both types of electrolysis cells. Coulometry uses a procedure that electrolyzes compounds 100% using a porous electrode structure and a larger surface area when an adequate voltage is applied. Amperometry uses a smooth electrode surface and only partially electrolyzes the compounds. This method has less noise and is more sensitive when compared to coulometry. Please read on to learn how recent technology applies these two different methodologies to maximize results. 

More About Sensitivity

The signal-to-noise ratio defines sensitivity, not solely by signal intensity. Electronics enable signal amplification but this usually accompanies noise interference as well. Removing the noise at the detector level is required for reliable analysis. The amperometric cell has a lower noise level and results in higher sensitivity. The amperometric detector cell has a solid non-porous working electrode with a smooth surface, which lowers the noise level. Eicom employs the low noise amperometric cell for its detectors. This enables a dramatically lower limit of detection compared with the coulometric cells. When Eicom began the production of the ECD in 1986, we only produced the most innovative amperometric detector cells to realize excellent sensitivity. In recent years we also began offering coulometric detectors per our customers’ request. 

Applied Potential

To change the redox status of an analyte, the appropriate potential must be applied. The ECD applies this potential as a voltage and controls it with a third electrode called a reference electrode. The applied potential needs to be set higher than the redox potential of the compound but also needs to be kept to a minimum. If the potential is set higher than required, other compounds hidden or having higher potentials can be detected. This results in a loss of selectivity. Even if the potential is low enough for the target compound and the peak includes other compounds that also have a lower redox potential, the other compounds can also be detected and not distinguished from the target compound on the detector. How do we resolve this issue? See below. 

Specificity is made by Separation and Detection

As explained in the Applied Potential section, losing specificity at the ECD can be a problem. This can be prevented by choosing the appropriate separating conditions before the detector and so removing any peak that may be included at the lower potential. Thus, choosing the appropriate separating conditions specific to the sample is as important as choosing the appropriately applied potential. This is the only way to prevent a loss in selectivity. Theoretically, filtrating out the lower potential compounds by applying a lower potential before the final detector would ease this selectivity problem. When you need robust sensitivity, this procedure is not practical. The electric current upstream from the lower potential compounds flows to the other detector(s) located downstream. This is a complicated system and can also become an obstacle in attaining high sensitivity. In the next section, we will describe the latest and most practical technology of ECD using two cells. 

A Combination of Amperometry and Coulometry

Coulometric cells can completely electrolyze compounds if the applied voltage is high enough. Another advantage of coulometry is a less distribution of flow in the cell. This results in most of the analytes in the coulometric cell reaching the amperometric detector linearly. Thus, the combination of the coulometric cell (characterized as a less distribution of flow and 100% electrolysis cell), and the amperometric cell (higher sensitivity) work excellently to detect certain compounds which are more difficult to oxidize than to reduce. In preparatory electrolyzing, the analytes are electrochemically changed and easily detected in the second cell. For example, 3-nitrotyrosine (3-NT) is reduced easily but has a high oxidative potential. After the separation column, the coulometric cell works to apply a reduced potential and convert the 3-NT to its reduced form. At the amperometric detector located after the coulometric cell, the reduced form of 3-NT can be oxidized with high sensitivity using a lower potential than the oxidative potential of the 3-NT. 

HPLC-ECD Method Optimization

Did you think the detection method for 3-nitrotyrosine sounded a bit complicated? This is normal. As common as HPLC-ECD is, there are still many important variables involved in achieving the optimal analytical conditions. This specific information requires years of training and experience. Eicom can provide it to you efficiently in a short time. Some factors influencing detection are as follows, but are not limited to column types, mobile phase pH, type of buffer solution, ion pairing level, methanol concentration, temperature, and detector setting. Finding the best conditions for both the HPLC and ECD may be more difficult and time-consuming than you expect. Yet, if you use Eicom’s HPLC-ECD methods, the time optimizing analytical methods will be reduced.

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