Flow-cells electrochemical detection

Another critical part of flow methods is the detector (electrochemical flow cell). An electrochemical detector uses the electrochemical properties of analytes for determination in the flowing stream. Electrochemical detection is usually performed by controlling the potential of the working electrode at a fixed value and monitoring the current as a function... 

Electrochemical Detectors Another common group of HPLC detectors are those based on electrochemical measurements such as amperometry, voltammetry, coulometry, and conductivity. Figure 12.29b, for example, shows an amperometric flow cell. Effluent from the column passes over the working electrode, which is held at a potential favorable for oxidizing or reducing the analytes. The potential is held constant relative to a downstream reference electrode, and the current flowing between the working and auxiliary electrodes is measured. Detection limits for amperometric electrochemical detection are 10 pg-1 ng of injected analyte. 

MWNTs favored the detection of insecticide from 1.5 to 80 nM with a detection limit of InM at an inhibition of 10% (Fig. 2.7). Bucur et al. [58] employed two kinds of AChE, wild type Drosophila melanogaster and a mutant E69W, for the pesticide detection using flow injection analysis. Mutant AChE showed lower detection limit (1 X 10-7 M) than the wild type (1 X 10 6 M) for omethoate. An amperometric FIA biosensor was reported by immobilizing OPH on aminopropyl control pore glass beads [27], The amperometric response of the biosensor was linear up to 120 and 140 pM for paraoxon and methyl-parathion, respectively, with a detection limit of 20 nM (for both the pesticides). Neufeld et al. [59] reported a sensitive, rapid, small, and inexpensive amperometric microflow injection electrochemical biosensor for the identification and quantification of dimethyl 2,2 -dichlorovinyl phosphate (DDVP) on the spot. The electrochemical cell was made up of a screen-printed electrode covered with an enzymatic membrane and combined with a flow cell and computer-controlled potentiostat. Potassium hexacyanoferrate (III) was used as mediator to generate very sharp, rapid, and reproducible electric signals. Other reports on pesticide biosensors could be found in review [17],... 

We have already briefly described a popular application of amperometry in Chapter 13. This was the electrochemical detector used in HPLC methods. In this application, the eluting mobile phase flows across the working electrode embedded in the wall of the detector flow cell. With a constant potential applied to the electrode (one sufficient to cause oxidation or reduction of mixture components), a current is detected when a mixture component elutes. This current translates into the chromatography peak... 

When electrochemical detection systems are used, it is essential that the mobile phases used are electrically conductive but only result in a low background current at the voltage selected. Cell volumes are of the order of 1 /d and the control of flow rate, pH and temperature is critical for reliable detection. 

Fluorescence detection, because of the limited number of molecules that fluoresce under specific excitation and emission wavelengths, is a reasonable alternative if the analyte fluoresces. Likewise, amperometric detection can provide greater selectivity and very good sensitivity if the analyte is readily electrochemically oxidized or reduced. Brunt (37) recently reviewed a wide variety of electrochemical detectors for HPLC. Bulk-property detectors (i.e., conductometric and capacitance detectors) and solute-property detectors (i.e., amperometric, coulo-metric, polarographic, and potentiometric detectors) were discussed. Many flow-cell designs were diagrammed, and commercial systems were discussed. 

Pulse polarographic studies have been described using a microcell of 0.5 mL capacity, which analyzed two 1,4-benzodiazepines, with the lowest detection limit reported to date being 10-20 ng/mL of blood [199]. Detailed construction of the cell and electrode assembly was also described (shown in Fig. 26.16). Further miniaturization of this type of three-electrode cell is not practical hence further increases in sensitivity will have to rely on electrochemical detector flow cells of microliter capacity such as those used in conjunction with liquid chromatography (see Chap. 27). 

Murakami, Y., Takeuchi, T., Yokoyama, K., Tomiya, E., Karube, I., Integration of enzyme-immobilized column with electrochemical flow cell using micromachining techniques fora glucose detection system. Anal. Chem. 1993, 65, 2731-2735. 

Electrochemical detectors measure the current resulting from the application of a potential (voltage) across electrodes in a flow cell. They respond to substances that are either oxidizable or reducible and may be used for the detection of compounds such as catecholamines, carboxylic acids, sulfonic acids, phosphonic acids, alcohols, glycols, aldehydes, carbohydrates, amines, and many other sulfur-containing species and inorganic anions and cations. Potentiometric, amperometric, and conductivity detectors are all classified as electrochemical detectors. 

Karube and Yokoyama presented an overview on the developments in the biosensor technology [60]. The overview describes the use of micromachining fabrication techniques for the construction of detection units for FIA, electrochemical flow cells and chemiluminescence detectors. Acetylcholine microsensors using carbon fiber electrodes and glutamate microsensors for neuroscience were discussed. 

To summarize, the type 1 equipment, in which the spectroscopic detection is combined with an electrochemical flow cell, has the advantage that the experimental setup is relatively simple. The lower limit of the time window is ca. 5 s, with the major drawback that only relatively stable intermediates can be detected. On the other hand, this ensures that homogeneous conditions are always attained. If the lifetime of the intermediates is less than 1 s, they exist in a reaction layer within the diffusion... 

The most common electrode material used in LC-EC is carbon, either as solid glassy carbon disks in thin-layer cells, or as a high-surface-area porous matrix through which the mobile phase can flow. Gold electrodes are useful to support a mercury film and these are primarily used to determine thiols and disulfides, and also for carbohydrates using pulsed electrochemical detection... 

Many detectors have been developed for use with liquid chromatographs (Table 6-4). Examples include photometric, spectrophotometric, fiuorometric, and electrochemical detectors. A key and integral component of such detectors is the flow cell (Figure 6-18), through which passes the eluate from the chromatographic column. Dissolved analytes are then detected and an electronic signal generated. (Mass spectrometers, which also have been used as LC detectors, are discussed in Chapter 7.)... 

Electrochemical Detectors. In amperometric electrochemical detectors (see Chapter 4), an electroactive analyte enters the flow cell, where it is either oxidized or reduced at an electrode surface under a constant potential. Electroactive compounds of clinical interest conveniently analyzed by HPLC with electrochemical detection include the urhiary catecholamines (see Chapter 29). In addition, electrochemi-cally active tags (e.g., bromine) are added to compounds such as unsaturated fatty acids or prostaglandins. 

Murakami Y, Takeuchi T, Yokoyama K, Tamiya E, Karube I and Suda M 1993 Integration of enzyme-immobilized column with electrochemical flow cell using micromachining techniques for a glucose detection system Anal. Chem. 65 2731-5 Olson B, Stalbom B and Johansson G 1986 Determination of sucrose in the presence of glucose in a flow Injection system with immobilized multi-enzyme reactors Anal. Chim. Acta 179 203-8... 

Microscale fluidic systems use small volumes so sensitivity of detection can be a challenge. Any detector for chip-based LC needs to be small and ideally have low power consumption. It is generally a problem of interfacing. Flow cell geometry is also a big factor, e.g. a U cell instead of linear flow cell can give a ten-fold increase in sensitivity for absorbance measurements. Electrochemical detection is very common, mainly ampero-metric and potentiometric, and very amenable to detection on chip. Fluorescence is more sensitive than UV-Vis absorbance and chemiluminescence is sensitive down to a single molecule, similar to LIF. 

M. E. Herrera, Analytical and Neurochemical Studies with Flow Injection Analysis and Electrochemical Detection. Part I. Pulsed Flow Electrolysis with the Reticulated Vitreous Carbon Electrode. Part II. On-Line Determination of Secreted Catecholamines from Cultured Adrenal Cells. Diss. Abstr. Int. B, 46 (1986) 1894. 

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