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 inverse liquid chromatography detector configuration for studying solid-liquid adsorption

Inverse liquid chromatography (ILC) is a physicochemical chromatographic technique for studying solid-liquid interactions and surface properties of unknown solid materials. However, such studies are dependent on the ability to self pack the sample powder into a LC column in a reliable and robust manner. In practise, this is often difficult and anomalous chromatograms can be encountered quite frequently. Results obtained with a novel LC system configuration utilising both pre and post-column UV-vis detectors for ILC studies are reported here which greatly assist in our attempts to interpret such chromatograms. The operational issues for using a two detector system are discussed including the use of both detectors in the linear concentration regime; with the choice of an optimal detector wavelength being the most critical factor. The two detector system was demonstrated to be capable of verifying reversible adsorption processes, as well as assisting in the interpretation of complex or problematic chromatograms. This paper demonstrates that the two detector ILC system can offer a number of practical benefits in the study of complex chromatographic phenomena.     

A post-column enzyme reactor for detection of oxidized cholesterols in h.p.l.c. separations

An enzyme reactor is used as a post-column detector after the h.p.l.e. separation of a mixture of cholesterol and some auto-oxidation products. The enzyme reactor contains cholesterol oxidase immobilized on controlled-pore glass. A method for purification of the enzyme is described. The properties and the solvent dependence of the reactor are discussed. A post-column system for dilution of the eluent with aqueous buffer is described.     

Analysis of trace components in gases by gas chromatography

Summary Relationships are derived describing how the detection limit of a chromatographic system depends on the minimum detectable limit of the detector and the chromatographic parameters such as column length, efficiency, carrier gas flow rate and the capacity factor. Performance data of detectors developed in the last 25 years at the Dalian Institute of Chemical Physics of the Chinese Academy of Sciences are given and a few selected application examples are listed. These include trace analysis by preconcentration and by direct analysis and the utilization of multidimensional gas chromatography with two columns, two detectors, a 12-port valve, and a catalytic conversion reactor.     

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.     

Determination of Thiamethoxam residues in honeybees by high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor

A method for the determination of Thiamethoxam in bee samples was set up by means of high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor (HPLC-h nu-ED). Analytical method was based on a rapid sample extraction procedure with acetone, followed by chromatographic separation into a C18-RP column isocratically operated by 60 mM phosphate buffer/acetonitrile (75/25) mobile phase at pH 2.7. A photochemical reactor was used as a tool to verify and eventually quantify the presence of Thiamethoxam in the samples by distinguishing it from interference contribution. Detection was performed with a potential of 880 mV after a photoactivation with a 254 nm light. The least detectable dose was 0.002 mg kg(-1). Recovery rates ranged between 59.88 and 71.62%.     

Fluorescence high performance liquid chromatographic determination of 3 alpha-hydroxysteroids in urine of 21-hydroxylase deficiency

A fluorescence high performance liquid chromatographic method using an immobilized 3 alpha-hydroxysteroid dehydrogenase column as a post-column enzymatic reactor was developed for the determination of corticosteroid metabolites in the urine of subjects with congenital adrenal hyperplasia. 3 alpha-Hydroxysteroids, such as pregnanetriol, pregnanediol and pregnanetriolone, in the eluate from mu-Bondapak phenyl column (300 x 3.9 mm I.D.) using 0.05% ammonium phosphate buffer (pH 7.1)-acetonitrile-methanol (100:55:15) as the mobile phase was mixed with NAD+ solution in the enzyme column at 30 degrees C to generate NADH, which was monitored by a fluorophotometric detector. Each steroid was measured at the 2.5 micrograms/dl at the highest sensitivity of the detector. The mean recoveries and reproducibilities were 91.5-108.2% with 0.9-6.5% (CV%).     

Characteristics and performance of gas chromatographic detectors with glass capillary columns

A comparison of the characteristics and Performance of the flame ionization, electron capture, and flame photometric detectors with capillary columns is described. Factors which may affect the limits of detection, linearity, chromatographic peak shape, and other detector performance characteristics are discussed and compared with the results of a model derived for the behavior of concentration and mass flow-rate dependent chromatographic detectors used with capillary GC systems. Examples are given of highly complex and labile mixtures such as pesticide residues and products from coal hydrogenation.     

Application of power functions to chromatographic data for the enhancement of signal to noise ratios and separation resolution

Chromatographic detection responses are recorded digitally. A peak is represented ideally by a Guassian distribution. Raising a Guassian distribution to the power ‘n’ increases the height of the peak to that power, but decreases the standard deviation by √n. Hence there is an increasing disparity in detection responses as the signal moves from low level noise, with a corresponding decrease in peak width. This increases the S/N ratio and increases peak to peak resolution. The ramifications of these factors are that poor resolution in complex chromatographic data can be improved, and low signal responses embedded at near noise levels can be enhanced. The application of this data treatment process is potentially very useful in 2D-HPLC where sample dilution occurs between dimension, reducing signal response, and in the application of post-reaction detection methods, where band broadening is increased by virtue of reaction coils. In this work power functions applied to chromatographic data are discussed in the context of (a) complex separation problems, (b) 2D-HPLC separations, and (c) post-column reaction detectors.     

Applications of a new vapour-phase pyrolyser

The application of a device for the post-column vapour-phase pyrolysis of compounds after gas chromatographic separation is described. The system proposed permits a mixture to be separated, pyrolysis to be carried out and pyrograms to be obtained in the chromatograph with one flame ionization detector. The reproducibility of the retention time and the intensity of the pyrogram peaks were studied. Some examples of applications to the identification of unknown components in a single gas chromatographic analysis are given.     

Configuration and optimization of semiconductor gas sensors as gas chromatographic detectors

A tin oxide, gas-sensitive semiconductor sensor was configured as a gas chromatographic detector and its performance was optimized. Two sensor housings were compared but little difference was found in performance. The flow rate and temperature of the column and the internal heater voltage of the sensor affected both the sensitivity and peak shape. The temperature of the sensor surface was the most critical parameter. Optimal conditions for the gas chromatographic detection of a mixture of alkanes (C₁–C₅) and hydrogen were identified by using the simplex technique. The detection limit for hydrogen was improved by a factor of five to 20 ppb (v/v), illustrating the value of optimization and the excellent sensitivity of the detector. It is concluded that semiconductor gas sensors offer significant advantages as gas chromatographic detectors for the determination of reducing gases.     

Development of a selective post-column detector for phenols separated by high-performance liquid chromatography

An on-line post-column reactor based on air-segmented continuous flow is described for the determination of phenols. The reaction used is the coupling of diazotized sulfanilic acid with phenols to form highly colored azo dyes. The effect of experimental parameters on the detector response was inveestigated by both univariate and simplex approaches in order to establish optimum reaction conditions. The aqueous reaction system is compatible with common reverse-phase solvents. The detection limit for phneol with the derivatization detector (71 μg 1^?1 shows a 16-fold improvement over u.v. detection of the underivatized phenol. Imprecision, based on multiple injections of sample into the HPLC system and measurement of the peak heights, is ± 0.64% (RSD). The technique is applied to the determination of phenols added to river water and present in residual fuel oil samples.     

Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors

In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.     

Optical photothermal detection in HPLC

A mode-mismatched parallel dual-beam thermal lens spectrometer with a far-field single-channel detector system was used as a detector in HPLC. An expert estimation of the measurement results was applied to optimize the optical-scheme configuration of the spectrometer to achieve the longest linear calibration range and highest repeatability under chromatographic flow conditions. Chelates with 4-(2-pyridylazo)resorcinol were separated and determined with the limits of detection of n x 10(-8)- n x 10(-7) mol L(-1); the relative standard deviation of measurements was 46%. Xylenol Orange, 4-(2-thiazolylazo)resorcinol, and dithizone were studied as post-column reagents in thermal lens detection in ion chromatography. The limits of detection were n x 10(-8)- n x 10(-7) mol L(-1); the linear calibration ranges were about three orders; the relative standard deviation of measurements was 3-7%. A combined photothermal-refractometric detector for HPLC based on a polarization interferometer is proposed. Metal complexes as 4-(pyridylazo)resorcinol chelates (limits of detection of n x 10(-8)- n x 10(-7) mol L(-1)) and sugars (limits of detection of 10-20 ng L(-1)) were investigated as model substances. Obtained results were compared with results for traditional detectors, which show that photothermal detection has higher sensitivity than photometric and other absorption detectors.     

Determination of plasma tranexamic acid using cation-exchange high-performance liquid chromatography with fluorescence detection

A procedure is described for the determination of plasma tranexamic acid concentrations using cation exchange high-performance liquid chromatography with fluorescence detection following post-column derivatisation with omicron-phthalaldehyde. The chromatographic conditions were optimised with respect to detector performance and the method applied to measuring the plasma tranexamic acid levels of patients in a double-blind trial.     

Quantitative liquid chromatographic determination of cefatrizine in serum and urine by fluorescence detection after post-column derivatization

A fast, specific and sensitive high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. The drug is determined by the internal standard method, using cephradine as the internal standard. The separation is carried out on a reversed-phase column, filled with octadecylsilane chemically bonded microparticles. The eluent is a mixture of acetonitrile with 0.025 M sodium phosphate buffer (pH 7). Quantitation is effected by fluorescence detection of the fluorophores formed after post-column derivatization with fluorescamine in a packed-bed reactor. The chromatographic conditions and the conditions for the post-column derivatization are discussed. The method has been applied to serum and urine samples, which were analysed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by the determination of the cefatrizine content in spiked serum and urine samples.     

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.     

Trace analysis of heavy metal ions by ion chromatography

Summary An ion chromatographic separation technique for heavy metal ions is described. Using pressure-stable, silica-based, ion-exchange supports and standard HPLC equipment with post-column reaction detector high resolution is achieved as well as extremely high sensitivity in the parts per trillion (ppt)-range.     

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.     

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.