Nonionic micellar liquid chromatography coupled to immobilized enzyme reactors

Immobilized enzyme reactors are used as post-column reactors to modify the detectability of analytes. An immobilized amino acid oxidase reactor was prepared and coupled to an immobilized peroxidase reactor to detect low level of amino acids by fluorescence of the homovanilic dimer produced. A cholesterol oxidase reactor was prepared to detect cholesterol and metabolites by 241 nm UV absorbance of the enone produced. The preparation of the porous glass beads with the immobilized enzymes is described. Micellar liquid chromatography is used with non-ionic micellar phases to separate the amino acids or cholesterol derivatives. It is demonstrated that the non ionic Brij 35 micellar phases are very gentle for the enzyme activity allowing the reactor activity to remain at a higher level and for a much longer time than with hydro-organic classical chromatographic mobile phases or aqueous buffers. The coupling of nonionic micellar phases with enzymatic detection gave limits of detection of 32 pmol (4.8 ng injected) of methionine and 50 pmol (19 ng injected) of 20alpha-hydroxy cholesterol. The immobilized enzyme reactors could be used continuously for a week without losing their activity. It is shown that the low efficiency obtained with micellar liquid chromatography is compensated by the possibility offered by the technique to easily adjust selectivity.     

Detector for organophosphorus compounds in liquid chromatography based on the cholinesterase inhibition reaction

A sensitive method for the post-column reaction detection of organophosphorus compounds is described. The method relies on cholinesterase and is particularly suitable for the analysis of potent inhibitors such as sarin, soman and tabun. The compounds are separated by reversed-phase chromatography with methanol-water as the mobile phase in a linear gradient system. The reactor used for the detection comprises conventional autoanalyzer equipment with air segmentation of the reactor stream. The detection limits are 10 pg for sarin and soman and 60 pg for tabun. A quantitation method is presented, based on the linear correlation between the residual enzyme activity and the inhibitor concentration. The repeatability is +/- 1%. As a test of the system, the model compounds were detected against a background of urban air.     

High-performance liquid chromatographic assay of L-alpha-glycerophosphorylcholine using a two-step enzymic conversion.

A high-performance liquid chromatographic method using an enzymic reactor for determination of L-alpha-glycerophosphorylcholine in pharmaceutical forms is described. The procedure includes incubation of L-alpha-glycerophosphorylcholine with glycerophosphorylcholine phosphodiesterase (EC 3.1.4.2), giving choline and glycerophosphate, and subsequent chromatography of choline with a post-column enzymic reactor and electrochemical detection. The results obtained show a close linearity of the whole assay from 2 to 150 nmol/ml L-alpha-glycerophosphorylcholine, the sensitivity being 2 pmol per 20 microliters of injected sample. The precision of the method in the analysis of L-alpha-glycerophosphorylcholine in pharmaceutical forms, ampoules and capsules, was 1.34 and 1.21%, respectively.     

Enzymatic production and analysis of d-xylulose with a recirculating flow system and post-column liquid chromatographic detection using a co-immobilized enzyme reaction and a chemically modified electrode

A pure d-xylulose and standard was produced by isomerization of d-xylose in a recirculating flow system incorporating an enzyme reactor containing immobilized xylose isomerase. The d-xylulose formed was purified chromatographically. A selective chromatographic detection system was used in the post-column mode. It consisted of a co-immobilized enzyme reactor (CIMER) with xylose isomerase, mutarotase and glucose dehydrogenase on-line with a chemically modified electrode for selective elctrochemical oxidation of NADH. The pure standard was compared with commercially available d-xylulose, which was confirmed to contain impurities of d-glucose and d-xylose.     

Determination of acetylcholine and choline in the femtomole range by means of HPLC,a post-column enzyme reactor,and electrochemical detection

Summary The measurement of choline and acetylcholine by means of HPLC, a post-column enzyme reactor, and electrochemical detection has been simplified and optimised. The use of a cation exchanger and enzyme reactor fitted in a cartridge holder appeared to result in reproducible, sensitive, and selective measurement of endogenous choline and acetylcholine with a lower detection limit of 50 fmole.     

Post-column derivatization in liquid chromatography using immobilized enzyme reactors and amperometric detection

The advantages to be gained by conducting enzyme assays of carbohydrates in liquid chromatography are discussed. The enzymes are contained in immobilized enzyme reactors and used in the post-column mode. The product formed in the reactor is selectively detected amperometrically at a chemically modified electrode mounted in a flow-through detector. Selected examples are given to illustrate the advantages obtained.     

Flow-rate dependence of post-column reaction chromatographic detectors and optimization of reactor length for slow chemical reactions

In high-performance liquid chromatography, use of any post-column reactor invariably involves a compromise between the conditions needed to obtain complete reaction and avoidance of excessive dispersion by band broadening in the reactor. Flow rate and the reactor geometry interact to establish the final chromatographic performance. Based on the flow-rate dependence of the peak area and peak height, post-column detectors constitute a distinct class of detectors which differ from mass-flow and concentrations-sensitive detectors such as the flame ionization and absorbance detector, respectively. The concept of reactor length optimization is developed for first-order chemical reactions in a post-column detector. The findings are applicable to both chromatographic and flow-injection systems.     

Development of post-column enzymic reactors with immobilized alcohol oxidase for use in the high-performance liquid chromatographic assay of alcohols with electrochemical detection

The development of a very sensitive, direct injection high-performance liquid chromatographic method, using a post-column reactor with immobilized alcohol oxidase, was undertaken with the aim of determining methanol and ethanol levels in microlitre volumes of biological samples. After reversed-phase chromatography to separate methanol and ethanol, the analytes were enzymically converted into the respective aldehydes with formation of stoichiometric amounts of hydrogen peroxide, which could be measured via electrochemical oxidation at a platinum electrode. Some problems were encountered in the development of solid-phase enzymic reactors, using a delicate enzyme, that is prone to lose activity, such as alcohol oxidase. Owing to the slightly alkaline pH required for the optimum activity of alcohol oxidase, polymeric columns seemed to be preferable for the chromatography. HEMA copolymer was chosen as the stationary phase, but the methanol and ethanol peaks eluted close together and posed severe problems of limiting post-column band spreading. Reactors based on coarse supports for enzyme immobilization gave unacceptable band spreading, causing the methanol and ethanol peaks to overlap. On the other hand high-performance liquid chromatographic packings maintained the efficiency of the chromatographic separation, quite independently of the reactor volume. Polymeric supports proved superior to silicas in maintaining the enzyme activity. However, relevant changes in the enzyme substrate specificity were observed after immobilization.     

The high-performance liquid chromatography and detection of phospholipids and triglycerides: Part 2. Comparison of an ultraviolet absorption and post-column reactor detector

A liquid chromatographic ultraviolet absorption detector (at 195 nm) and a novel postcolumn reactor detector are compared for use in the detection of triglyceride and phospholipid molecular species. The detection limit for the u.v. detector depends on the degree of unsaturation of the lipid sample (3 × 10^-6–2 × 10^-8 M in the detector cell for 0–3 double bonds per acyl group). The post-column reactor detector is responsive to equivalents of lipid and has detection limits of 5 × 10^-7 M for triglycerides and 2 × 10^-6 M for phospholipids. The log u.v./post-column reactor detector response ratios are linearly related to the log of the degree of unsaturation of the lipid, indicating the usefulness of both detectors for quantifying triglycerides and phospholipids.     

Novel quartz flow-cell as a post-column photochemical reactor for high-performance liquid chromatography

The construction of a new post-column photochemical reactor with quartz flow cells in series for high-performance liquid chromatography (HPLC) is described. The performance of the new reactor was compared with a conventional open tubular PTFE coil reactor. The sensitivity, accuracy and precision obtained with both reactors are comparable. The new reactor has the obvious advantages of smaller cell volume as well as inertness and resistance to not only light and heat produced by the UV lamp, but also to organic solvents in the mobile phases, which results in greatly improved durability, reduced peak broadening and shorter chromatographic run times. Application of the new reactor to the fluorescence detection of DU-6859a, a new fluoroquinolone antimicrobial agent, in human serum is reported.     

An enzyme reactor electrode for urea determinations.

An enzyme reactor electrode system for the determination of urea is described. A buffer is pumped through an enzyme reactor (0.4 ml) containing urease immobilized with glutaraldehyde to glass. The effluent is mixed with sodium hydroxide pumped through a second channel and fed through an ammonia gas electrode. Samples are introduced via a third flow channel and mixed with the buffer. The conversion of urea to ammonia is quantitative for sample concentrations of less than 0.03 M for a flow rate of 40 ml h^-1. The reactor electrode shows a Nernstian slope of 57 mV/decade for 5·10^-5–3·10^-2 M urea. The response is independent of variations in the flow rate, enzyme activity or temperature of the reactor.     

Specific detection of acetyl-coenzyme A by reversed-phase ion-pair high-performance liquid chromatography with an immobilized enzyme reactor

A selective chromatographic detection system for the determination of acetyl-coenzyme A (CoA) is reported. The short-chain acyl-CoA thioesters were separated by reversed-phase ion-pair high-performance liquid chromatography (HPLC), and then acetyl-CoA was selectively detected on-line with an immobilized enzyme reactor (IMER) as a post-column reactor. Thio-CoA liberated enzymatically from acetyl-CoA was determined spectrophotometrically after reaction with Ellman's reagent in the reagent stream. The IMER with phosphotransacetylase had a substrate specificity sufficient to determine acetyl-CoA and was active and stable in the mobile phase containing methanol and the ion-pair reagent. The calibration graph was linear between 0.2 and 10 nmol, with a detection limit of 0.05 nmol. This HPLC system with detection by IMER allows the selective identification and determination of acetyl-CoA in a mixture of acetoacetyl-CoA and 3-hydroxy-3-methylglutaryl-CoA, which are difficult to separate with ion-pair HPLC.     

Ion chromatographic determination of transition metals in irradiated nuclear reactor surveillance samples

The determination of transition metal ions in radioactive (+/-25 microCi/g) low-alloy steels (nuclear reactor surveillance samples) by ion chromatography (IC) is described. The analysis has been done directly without prior separation of the iron matrix. The eluted metal ions have been detected with a UV-visible spectrophotometric detector after post-column complexation with 4-(2-pyridylazo)resorcinol. The results are in a good agreement with the certified values for the standard reference material used. The method was applied to nuclear reactor surveillance samples for the determination of Cu, Mn, Co and Ni.     

Enzymatic Assay by Flow Injection Analysis with Detection by Chemiluminescence: Determination of Glucose,Creatinine, Free Cholesterol and Lactic Acid Using an Integrated Fia Microconduit

Abstract

A miniaturized flow injection system for the determination of D-glucose, L-lactic acid, creatinine and free cholesterol is described. All substrates are degraded enzymatically by means of oxidases which, along with ancillary coenzymes (creatinine assay), are immobilized on controlled porosity glass and incorporated into small PVC column reactors. The hydrogen peroxide generated by the individual oxidases is determined by chemiluminescence with an alkaline reagent containing luminol and hexacyanofer rate (III). The injection valve, flow channels, enzyme reactor and light detector are integrated into a FIA microconduit. The detection limits were 0.03 mg glucose/dl, 0.03 mg lactate/dl, 0.3 mM creatinine and 0.5 mg cholesterol/dl. The enzyme reactors all showed little change in activity over a 3 months period of operation and were found fully compatible with serum samples.     

Determination of glycerol, 1,2-propanediol and triglycerides by high-performance liquid chromatography and a post-column reactor containing immobilized glycerol dehydrogenase

A fluorimetri method is described for the determination of glycerol, 1,2-propanediol and triglycerides in serum by high-performance liquid chromatography with an on-line post-column reactor containing immobilized glycerol dehydrogenase. Before separation, triglycerides are cleaved with lipase and esterase. The polyhydric alcohols are separated from each other on a Finepak SIL C₁₈ (10 μm) column with water as eluent. The NADHI produced from the enzymatic reaction is monitored by fluorimetry. Calibration curves are linear between 0.01 mM and 1.0 mM for glycerol or 2.0 mM for 1,2-propanediol. The method gave satisfactory results for control sera.     

Computer-assisted optimization of HPLC with post-column reaction for the determination of amino-acids

Computer-assisted optimization of a high-performance liquid chromatograph and associated post-column reactor is reported for the determination of six amino-acids. First six, then seven, experimental variables were considered. Non-standard experimental conditions were found which gave significantly improved colour development in the ninhydrin reaction. The composite modified simplex method for experimental optimization and a novel system-response function facilitated rapid and simultaneous improvement of separation, sensitivity and analysis time. The approach described is directly applicable to many similar systems.     

Use of enzymatic reactors in the high performance liquid chromatographic determination of ethanol and methanol with electrochemical detection

Despite numerous applications both in industry and in analytical chemistry, immobilized enzyme technology has encountered only limited success in modern liquid chromatography. The strict requirements of enzymatic reactors for high performance liquid chromatography (HPLC) (i.e., mechanical resistance, limited void volumes, high contact surface, high binding capacity, stable enzyme-to-matrix bond without loss of enzyme activity) are not yet fully met by commercially available products. In our laboratory, the development of post-column reactors with immobilized alcohol oxidase on-line with an HPLC/EC system has been undertaken. Three home-made reactors were produced: in the first alcohol oxidase from Candida boidinii was quasi-immobilized onto an HPLC-sized anion exchange support, in the second the enzyme was bound to a silica-based support via Schiff's base formation, and in the third a covalent linkage to an epoxy-activated hydroxyalkyl methacrylate material was chosen. Chemical immobilization proved superior to the ionic binding. The importance of limiting the post-column band spreading due to the reactor void volume, by using HPLC-size supports, was confirmed.     

High-performance liquid chromatographic determination of histamine N-methyltransferase activity

A method for the determination of histamine N-methyltransferase (HMT) activity by high-performance liquid chromatography based on post-column derivatization with omicron-phthalaldehyde is described. The determination involves the separation of the substrate, histamine, from its product. N tau-methylhistamine, using a weak cation exchanger, followed by on-line derivatization of these imidazoleamines with omicron-phthalaldehyde and their detection and quantitation with a fluorimetric detector. This assay method is suitable for the measurement of HMT activity during enzyme purification.     

微透析液中葡萄糖的液相色谱—电化学测定

文介绍了一种灵敏测定大白鼠皮下微透析液中葡萄糖的液相色谱－电化学分析方法。本法用柱后葡萄糖氧化酶反应器将色谱分析柱中分离出的葡萄糖转化成过氧化氢，再用过氧化物酶电极检测生成的过氧化氢。     

Micro enzymatic assay coupled to capillary electrophoresis via liquid junction

Summary A system for capillary electrophoresis combined with enzymatic assay has been evaluated for the two enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Instrumentation included a post-column reactor coupled to the separation capillary by a liquid junction. A technique for generating a substrate solution flow into the reactor by utilizing two high voltage supplies is proposed. This method offers a high degree of freedom in optimizing the separation and enzymatic reaction conditions individually. Possibilities for improving the enzymatic assay sensitivity were also examined.