LCEC

Another dynamic measurement is the LCEC technique which can be thought of, simpHsticaHy, as EIA using a chromatographic column positioned between the sample injection port and the detector. Bioanalytical systems (BAS) of West Lafayette, Indiana, specializes in instmmentation for LCEC. Their catalogs come with extensive bibhographies covering a variety of appHcations. 

Because of this lack of resolving power, much electroanalytical research is aimed at providing increased selectivity. This can be accomplished in two ways. First, electrochemistry can be combined with another technique which provides the selectivity. Examples of this approach are liquid chromatography with electrochemical detection (LCEC) and electrochemical enzyme immunoassay (EEIA). The other approach is to modify the electrochemical reaction at the electrode to enhance selectivity. This... 

Liquid chromatography/electrochemistry (LCEC) has become recognized as a powerful tool for the trace determination of easily oxidizable and reducible compounds. This is because detection of as little as 0.1 pmol of material is readily accomplished with relatively simple and inexpensive equipment. Initial interest in LCEC was generated by the determination of several aromatic matabolites of tyrosine in the central nervous system. However, the application of LCEC into other areas of biochemistry has begun at a growing pace. A bibliography of LCEC applications is available... 

LCEC is a special case of hydrodynamic chronoamperometry (measuring current as a function of time at a fixed electrode potential in a flowing or stirred solution). In order to fully understand the operation of electrochemical detectors, it is necessary to also appreciate hydrodynamic voltammetry. Hydrodynamic voltammetry, from which amperometry is derived, is a steady-state technique in which the electrode potential is scanned while the solution is stirred and the current is plotted as a function of the potential. Idealized hydrodynamic voltammograms (HDVs) for the case of electrolyte solution (mobile phase) alone and with an oxidizable species added are shown in Fig. 9. The HDV of a compound begins at a potential where the compound is not electroactive and therefore no faradaic current occurs, goes through a region... 

With the multitude of transducer possibilities in terms of electrode material, electrode number, and cell design, it becomes important to be able to evaluate the performance of an LCEC system in some consistent and meaningful maimer. Two frequently confused and misused terms for evaluation of LCEC systems are sensitivity and detection limit . Sensitivity refers to the ratio of output signal to input analyte amount generally expressed for LCEC as peak current per injected equivalents (nA/neq or nA/nmol). It can also be useful to define the sensitivity in terms of peak area per injected equivalents (coulombs/neq) so that the detector conversion efficiency is obvious. Sensitivity thus refers to the slope of the calibration curve. 

Sensitivity by itself is not sufficient to completely evaluate an LCEC system for analytical purposes. The minimum detectable quantity (detection limit) is of more practical importance. The detection limit takes into consideration the amount of baseline noise as well as the response to the analyte. The detection limit is then defined as the quantity of analyte which gives a signal-to-noise ratio of three (a S/N of 3 is the generally accepted criterion although other values have been used). For a complete description of an LCEC application, both the sensitivity and detection limit, along with the S/N criteria used, should be provided. 

LCEC has become a widely used analytical technique for biomedical analysis. Several reports appear each month deairibing new LCEC analyses or reporting results obtain-with LCEC. While space does not permit a complete review of all bitwhemical applications, this section will consider applications of LCEC to general classes of compounds to provide an overview of the uses of this technique. For a thorough survey of the literature, a bibliography of LCEC applications is available... 

Most phenolic compounds are readily oxidized at carbon electrodes. The oxidation potentials vary widely depending upon the number of ring hydroxyl groups and their positions on the ring. Many compounds of biomedical and industrial interest are phenolic and LCEC based trace determinations are quite popular. 

The first and still most common LCEC application is determination of the catecholamines in biological samples. The number of papers describing methods for catecholamine determination for certain circumstances are far too numerous to cite, however, several good reviews are available The second major use of LCEC is in the... 

Phenols also constitute a major source of xenobiotic exposure to the body in the form of drugs and environmental pollutants. Oxidative metabolism of these compounds can lead to physiological damage, therefore the metabolism of these compounds is of great interest. LCEC has been a powerful tool for investigating the metabolism of aromatic compounds by the cytochrome P-450 system LCEC... 

Phenolic acids and polyphenols are natural plant constituents which impart flavor and textural components to beverages made from these plants. In order to better understand the role of these easily oxidized compounds in the flavor and stability of beverages, it is necessary to determine them at the low concentrations they occur. LCEC has been shown to be quite effective at these trace determinations... 

Many pharmaceuticals and environmental pollutants are aromatic amines. Like phenols, this class of compound is generally oxidizable at carbon electrodes. LCEC has been used to study the metabolism of aromatic amines of both environmental and pharmaceutical origin lcec has also been used for the trace determination... 

Many compounds of biomedical interest, both of endogenous and exogenous origin, are heterocyclic in structure. Many of these compounds are electroactive at potentials useful for LCEC analysis. Methods for the determination of both ascorbic acid and uric acid were developed in the early days of LCEC. The important enzyme... 

While most amino acids are not electroactive at analytically usable potentials at carbon electrodes, much work is currently directed at general methods of LCEC amino acid detection by electrode surface modification or derivatization of the amino acid. Kok et al. have directly detected amino acids at a copper electrode. Several derivatization methods for amino acids have also been reported 227.228)... 

The pentapeptides, met- and leu-enkephalin, have been detected in rat striatum tissue by LCEC at a glassy carbon electrode These peptides can be detected directly... 

Thiols are easily oxidized to disulfides in solution, but this reaction occurs only very slowly at most electrode surfaces. However, use can be made of the unique reaction between thiols and mercury to detect these compounds at very favorable potentials. The thiol and mercury form a stable complex which is easily oxidized, in a formal sense it is mercury and not the thiol which is actually oxidized in these reactions. For the LCEC determination of thiols a Au/Hg amalgam electrode is used Using a series dual-electrode both thiols and disulfides can be determined in a single chromatographic experiment... 

Many drugs are electroactive, and as such, have been determined using LCEC. Space does not permit a discussion of the relevance of electrochemical detection to each class of drug. Table 4 lists several compounds of pharmaceutical interest (by therapeutic type and electroactive functionality) which have been determined by LCEC. 

The majority of LCEC applications have used oxidative detection. This is likely because of the perceived difficulties encountered with reductive detection. In particular, dissolved oxygen and trace metal ions must be removed to prevent high background currents. These problems are not difficult to overcome and more applications of reductive detection should appear as this is more generally realized. 

Aromatic nitro and nitroso compounds are easily reduced at carbon and mercury electrodes. Other nitro compounds such as nitrate esters, nitramines, and nitrosamines are also typically easily reduced. The complete reduction of a nitro compound consists of three two-electron steps (nitro-nitroso-hydroxylamine-amine). Since most organic oxidations are only two-electron processes, higher sensitivity is typically found for nitro compounds. Several LCEC based determination of nitro compounds have been reported... 

Several heterocycles of biomedical interest are reducible. Among these the K vitamins and the pterins have been determined by LCEC. Some heterocyclic... 

Enzyme linked electrochemical techniques can be carried out in two basic manners. In the first approach the enzyme is immobilized at the electrode. A second approach is to use a hydrodynamic technique, such as flow injection analysis (FIAEC) or liquid chromatography (LCEC), with the enzyme reaction being either off-line or on-line in a reactor prior to the amperometric detector. Hydrodynamic techniques provide a convenient and efficient method for transporting and mixing the substrate and enzyme, subsequent transport of product to the electrode, and rapid sample turnaround. The kinetics of the enzyme system can also be readily studied using hydrodynamic techniques. Immobilizing the enzyme at the electrode provides a simple system which is amenable to in vivo analysis. 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

Lequea et al. used the activity of tyrosine apodecarboxylase to determine the concentration of the enzyme cofactor pyridoxal 5 -phosphate (vitamin B6). The inactive apoenzyme is converted to the active enzyme by pyridoxal 5 -phosphate. By keeping the cofactor the limiting reagent in the reaction by adding excess apoenzyme and substrate, the enzyme activity is a direct measure of cofactor concentration. The enzymatic reaction was followed by detecting tyramine formation by LCEC. The authors used this method to determine vitamin B6 concentrations in plasma samples. 

A sandwich electrochemical enzyme immunoassay has been described for IgG Alkaline phosphatase was again used as the enzyme label with the conversion of phenyl phosphate to phenol being determined electrochemically by LCEC. A detection limit of 10 pg/mL was reported. 

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

LCEC Liquid chromatography with L2ToFMS Laser-desorption laser-... 

Whatever the future of electroanalysis really will be, it is worth considering what Faulkner recently said190 at a meeting on "Electrochemistry Faces Reality , where he mentioned more explicitly the significant impact of LCEC, liquid chromatography electrochemistry (see p. 346) and cybernetic instrumentation. 

LCeo, of wastewater, 25 881. See also Lethal concentration (LC50) lcec technique, 9 587-588 LCP resins, prices for, 20 861 LD50 data, 25 228. See also Lethal dose (LDE0)... 

The use of polarographic assays for the determination of drugs in blood is the most demanding on the detection limitations of the technique. Differential pulse polarography, stripping voltammetry, and LCEC are the only electrochemical methods currently available for routine determination of drugs below 1.0 ng/mL of blood. 

Liquid chromatography with electrochemical detection (LCEC) is in widespread use for the trace determination of easily oxidizable and reducible organic compounds. Detection limits at the 0.1-pmol level have been achieved for a number of oxidizable compounds. Due to problems with dissolved oxygen and electrode stability, the practical limit of detection for easily reducible substances is currently about 10-fold less favorable. As with all detectors, such statements of the minimum detectable quantity must be considered only with the proverbial grain of salt. Detector performance varies widely with the analyte and the chromatographic conditions. For example, the use of 100- m-diameter flow systems can bring attomole detection limits within reach, but today this is not a practical reality. 

---