R. W. Frei

Preconcentration and LC analysis of chlorophenols, using a styrene-divinyl-benzene copolymeric sorbent and photochemical reaction detection

SummaryA specific HPLC method has been developed for the trace analysis of lower chlorinated aromatic compounds. The method consists of an on-line preconcentration and a post-column reaction step. On-line preconcentration of mono- and dichlorophenols from aqueous samples has been performed using PRP1, a divinylbenzene-styrene copolymeric sorbent as packing material for both pre-and analytical column. Enrichment factors of over 300 were obtained compared to regular (100 μl) loop injections, even for the highly polar monochlorophenols. After reversed-phase separation, post-column photochemical dechlorination is carried out directly in the eluent stream, using a photochemical reactor. Upon dechlorination, fluorescent products are formed, which can be detected selectively. The resulting fluorescence signal shows a linear response to the quantity of solute present over 2 to 3 orders of magnitude (correlation coefficients: 0.990–0.98). For the mono- and dichlorophenols, the detection limit of the photoconversion method is in the lower nanogram range. The method is especially suitable for the analysis of complex matrices such as effluent water samples or biological fluids containing traces of the polar chloroaromatic compounds together with large amounts of other constituents, which interfere when using more general methods of detection like UV absorption. The potential of this technique for automation has been demonstrated by using a microprocessor-controlled column switching unit.

Optimization of a peroxyoxalate chemiluminescence detection system for the liquid chromatographic determination of fluorescent compounds

SummaryA peroxyoxalate chemiluminescence detection system for liquid chromatography is described. The excitation of fluorophores is generated by the reaction of bis-(2,4,6-trichlorophenyl)oxalate or bis(2,4-dinitrophenyl)oxalate and hydrogen peroxide, which are added to the column effluent. The influence of the solvents and the concentrations of the reagents have been investigated. The influence of the flow cell volume on sensitivity and on band broadening have also been studied and a “chemical band narrowing effect” has been observed. Different types of apparatus have been compared for detection of the emitted light. The system has been used for the detection of the dansyl derivative of a drug with a secondary amine functional group in serum samples. The detection limits are in the 1–10pg range.

Reaction detectors in modern liquid chromatography

SummaryReaction detectors are considerably enhancing the application potential of HPLC particularly when dealing with trace analysis in complex matrices. In principle one distinguishes between segmented and non-segmented reactor system. The theory of band broadening of these designs and some criteria for the choice of the proper type are brieffly discussed. It seems that with a proper construction of the phase separators segmented systems have a wider range of applicability. Applications of reaction detectors in the areas of pharmaceutical and environmental analysis are discussed.

Photochemical reaction detection of phylloquinone and menaquinones. A comparison with chemical post-columns reduction for fluorescence detection

SummaryA new photochemical reaction method for the on-line fluorimetric detection of natural vitamin K homologs was developed. In addition to its high sensitivity, the method features an interesting selectivity for the determination of these compounds in a complex matrix such as human serum. The formation of different photolysis products as a function of reaction conditions, and the optimalisation of a segmented flow reaction system will be discussed. The results indicate that the method is 4 times more sensitive than UV detection, and at least as selective as fluorescence detection after post-column chemical reduction.

Miniaturization of solid-phase reactors for on-line post-column derivatization in narrow-bore liquid chromatography

SummaryThe use of solid-phase reactors for post-column derivatization in narrow-bore HPLC (1.0mm i.d. analytical columns) is evaluated. Two systems are described, viz. for the determination of N-methylcarbamate pesticides and for that of urea and ammonia. The solid-phase reactor is packed with a strong anion exchange resin and urease immobilized on silica, respectively, to effect the catalytic hydrolysis of the solutes eluting from the analytical column. In both systems, the hydrolysis product is reacted with o-phthalaldehyde followed by fluorescence monitoring.Analytical data are presented and band broadening from various parts of the reaction detector system is discussed. An on-line trace enrichment procedure via a micro precolumn is descried for the trace level determination of N-methylcaramates in surface water samples.

Gas chromatographic analysis of phenylurea herbicides following catalytic hydrolysis on silica gel

SummaryA method is described for the rapid catalytic hydrolysis of phenylurea herbicides on silica gel at elevated temperatures. After derivatisation of the anilines produced with heptafluorobutyric acid anhydride final analysis is done on a gas chromatograph equipped with an electroncapture detector. Detection limits are in the 1–5 picogram range. The method has successfully been applied to residue analysis of water samples at the 1 ppb level. The determination of free anilines present in water samples and the potential of various techniques to be used to discriminate between free anilines and parent herbicides are also discussed.

Immunoaffinity precolumn for selective sample pretreatment in column liquid chromatography: Immunoselective desorption

SummaryAn automated liquid chromatographic system is described using immunoaffinity precolumns for sample pretreatment. The system consists of a column-switching unit allowing preconcentration from a large volume of sample (e.g. 15 ml of urine) onto an immunoaffinity precolumn (containing polyclonal antibodies immobilized on Sepharose). After sorption, the analytes are desorbed by a mixture of two cross-reacting solutes, followed by reconcentration on a C-18-bonded silica precolumn, and then separation on a C-18-bonded silica analytical column. Using oestrogen steroids as model compounds and UV absorbance detection, the minimum detectable concentration is ca. 200 ng/l with a repeatability of 6–8%. The total analysis time is 45 min which allows the unattended analysis of 30 samples per day. The features of the sample pretreatment method, especially of the immunoselective desorption, are evaluated and the general applicability of the system is discussed.

Phosphorescence detection of polychloronaphthalenes and polychlorobiphenyls in liquid chromatography

Abstract Room temperature phosphorescence in liquid solutions (RTPL) is applied as a detection method in the chromatographic analysis of technical mixtures of polychloronaphthalenes (PCNs) and polychlorobiphenyls (PCBs). The identification of PCBs is strongly facilitated by combining UV detection and detection based on sensitized RTPL of biacetyl, since the compounds substituted at position 2 are not detected by the latter method. For PCNs, in addition to sensitized RTPL, quenched RTPL detection can be successfully applied. This method is based on the partial quenching of the direct RTPL of biacetyl by the analyte. Combination of UV detection with the two phosphorescence detection methods provides interesting possibilities for identification.

Sensitized room-temperature phosphorescence for detection in continuous flow and chromatographic systems

SummaryIt is shown that sensitized room-temperature phosphorescence in liquid solutions can be successfully applied as a detection method in continuous flow and chromatographic systems. The limits of detection for a series of halogenated naphthalenes and biphenyls obtained by plug injection are roughly the same as those in batch experiments — in the subnanogram region. Liquid chromatograms of mixtures of PCBs, PCNs and polychlorodibenzofurans are given.

Fluorescence enhancement for aflatoxins in HPLC by post-column split-flow iodine addition from a solid-phase iodine reservoir

SummaryA method for post-column derivatization of the highly carcinogenic aflatoxins with iodine has been developed. It involves splitting of the mobile phase used for the reversed phase HPLC separation. One part flows through the injection valve and the C18 analytical column to achieve the separation. The other part flows through a column packed with solid iodine. The iodine-containing solution is recombined with the flow coming from the analytical column. The derivatization reaction takes place in a knitted open tubular reactor maintained at 60 °C. Detection is done by fluorescence measurement. Due to the low solubility of iodine in the mobile phase, the iodine solid-phase column can be used for very long periods of time before refilling is necessary. The analytical system consists of only one pump and therefore gives the opportunity to carry out low-cost post-column reaction detection. The method yields reproducible results, a linear response over at least two orders of magnitude and detection limits of about 1 ppb, both for standard solutions and for peanut butter samples.