FemtoGram

Question: "For some reason, I do not seem to be getting the level of detection (LOD) reported in your applications note. What should I do?"

A selection of cell types are available for use with ESA electrochemical detectors. The approaches to optimization of response and noise reduction differ for amperometric cells (such as the model 5041) or coulometric cells (such as the model 5014B).

Dr. Bruce's Answer: Sensitivity issues can plague any HPLC assay, especially one that requires ultra-trace level performance, such as neurochemical assays using the electrochemical detector. The overall limit of detection of the system is dependant on the signal-to-noise ratio (S/N). This metric can be quantified for a given chromatogram by measuring the peak response (signal) and baseline deviations (noise) of a chromatographic trace taken for a standard at lower concentrations.

ESA's Coulochem® III detector can be used with either amperometric or coulometric cells, and how you maximize sensitivity depends on which technology you are using. For amperometric cells, the signal is flow-rate dependant. To achieve greater peak response, it's best to choose an amperometric cell with a 1.0, 1.5, or 2 mm ID HPLC column at flow rates from 0.05 – 0.25 ml/min. This will offer enhanced signals over more-conventional 3 or 4.6 mm ID columns at higher flow rates. However, column selection is not a factor when using coulometric cells, which are flow-rate independent (they operate with 100 % electrochemical efficiency). With both cell types, the best way to achieve better LODs is to identify the sources of noise and then minimize the noise effects.

The first two – detector potentiostat and flow cell – are very rarely, if ever, the problem: noise from the Coulochem III detector is extremely low since we engineer the electronics to provide less than 750 fA when using a test dummy load. In addition, we evaluate every cell we produce for noise on a fully functional HPLC system. All cells must pass stringent quality-control parameters. For example, our 5014B Microdialysis cell cannot leave the factory with cell noise above 75 pA using the same HPLC conditions used to measure biogenic amines and their acid metabolites. So, with the first two noise sources eliminated, most of the noise observed on a typical LCEC system comes from either mobile phase contamination or the HPLC system components.

The majority of mobile phases prepared for reversed-phase chromatography use highly aqueous buffers. The source of the water and chemical ingredients can play a major role in the amount of noise observed at the detector, and this can be readily measured as the amount of background current seen at the electrochemical cell. The use of clean water for the mobile phase alone will often minimize the noise at the cell and improve sensitivity. One can readily observe a decrease in detector background currents when a superior water source is employed in mobile-phase preparation. Sometimes, the water must be further polished over a C18 cartridge to remove trace levels of organics before it provides the lowest level of noise. Also, the grade of chemicals used in preparing the mobile phase can play a significant role in its final quality. Remember, when the electrochemical detector is approaching low-femtomole sensitivity (ppt), as it can with the Coulochem III, the level of contaminants in many buffer salts can exceed the ppm range. Some of these contaminants are electroactive (iron, nickel, heavy metals, etc.) and this can result in higher background currents at the detector. Make sure you purchase reagent buffers that have high purity and low levels of metal contaminants – this will decrease the noise and offer better LODs. ESA has prepared a technical note that discusses many of these issues in further detail. It also recommends some chemical brands that offer provide results when they are employed in mobile phase preparation.

Inherent HPLC noise is best reduced by selection of the proper pump. Often the noise can be further reduced by the inclusion of pulse dampers, which come standard with all ESA pumps.

Perhaps one of the most significant sources of noise comes from the choice of HPLC equipment used before the column and electrochemical detector. HPLC "front ends" that use metal fittings and steel components can be very problematic, since corrosion sites can cause a significant increase in background currents (noise) and even chemically interact with some of the compounds that are being analyzed. These interactions can facilitate auto-oxidation of the electroactive compounds found in the sample matrix, significantly raising the apparent limit of detection for the most-easily oxidized compounds. HPLC systems with significant amounts of metal can often be several times less sensitive than those systems made with non-metal components. ESA has designed the HPLC "front end" to be compatible with electrochemical detection by employing PEEK components wherever possible. This allows a new system to reach femtogram sensitivity almost immediately after installation.

Pump pulsations can also be another serious source of noise at the detector. Since the mobile phase used with EC detectors must have an electrolyte, any pump pulsations that cause flow disturbances will be observed as changes in the background current. These minor pump noise fluctuations are often observed as regular frequency saw-toothed patterns in the baseline. Some reduction in this pattern can be observed if a pulse damper is placed after the pump, but the best way to eradicate this issue isto use an HPLC pump with a very small stroke volume. The piston chamber of the ESA 584 pump is only 10 μl in volume, which essentially removes any noise caused by pump pulsations.

The final issue affecting sensitivity of the electrochemical detector is related to the fact that we are often dealing with chemicals which are very easily oxidized. Compounds such as dopamine or ascorbic acid can degrade quickly (auto-oxidize) when at low concentrations. Analyte-stability can become a serious problem during routine analysis and often prevents us from being able to store samples for even a few hours before analysis without a careful study of antioxidant additives. It becomes important to acidify the sample pH to provide a reducing environment. This can be accomplished with small additions of perchloric acid or other suitable preservatives to the sample during collection and storage. Often the best strategy is to analyze the sample while it is fresh to minimize or avoid these issues.

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