Determination of eleven priority EPA phenolics at ng L−1 levels by on-line solid-phase extraction and liquid chromatography with UV and electrochemical detection

Summary The eleven priority, EPA phenolic pollutants were determined by liquid chromatography followed by two detectors in series; UV and electrochemical. Three different adsorbents, Envi-Carb (a carbon black) and two functionalized polymeric resins, Bond Elut PPL and another synthesized in our laboratory with an ocarboxybenzoyl moiety, were compared for solid-phase extraction (SPE) to detect lower concentrations of the eleven phenolics in natural waters. Higher recoveries were obtained using the functionalized polymeric adsorbents compared with Envi-Carb. When real samples were analysed, the synthetic adsorbent gave lower interference than Bond Elut PPL and phenol was determined at low levels with no humic and fulvic acid inter-ference when Na₂SO₃ was added. The linearity range for most compounds in tap water was 0.05–20 μg L⁻¹ and the limits of detection were <35 ng L⁻¹. Repeatability and reproducibility between days for real samples spiked at 0.1 μg L⁻¹, expressed as relative standard deviation, were <8% and 10%, respectively.     

HPLC determination of catabolic amines in silage using their dansyl derivatives and an electrochemical detector

Summary The use of the electrochemical detector (EC) in the high performance liquid chromatography (HPLC) of dansyl derivatives of biogenic amines is reported. Isocratic and gradient elution patterns of synthetic mixtures of putrescine, cadaverine, 1,6-diaminohexane, tryptamine, histamine, tyramine, spermine and spermidine on a reversed phase column are shown. Hydrodynamic voltammograms of standard compounds are presented. The detection limits of the EC are compared with those of ultraviolet and spectrofluorimetric detectors. A chromatographic profile of amines from the proteolytic catabolism of maize silage byClostridia is shown.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

Identification and quantitation of phenolics from green beans by high-performance liquid chromatography

Summary High-performance liquid chromatography with diode-array detection has been used for the separation and quantitation of phenolics from fresh and processed green beans. Whole beans, pods and seeds of green beans were studied separately. Chromatographic profiles from pods were more complex than those found in seeds. Flavonol glycosides were confined to quercetin and kaempferol classes and they were typical of external parts of the fruit. Flavan-3-ols monomers ((+) catechin and (−) epicatechin) as well as procyanidins structures were identified in pods and seeds. Chromatographic profiles from processed beans revealed a polyphenolic composition similar to those found in pod fresh green beans. The chromatographic method was carefully validated in regard to precision and accuracy. High reproducibility of peak area (RSD<3%) and calibration slopes (RSD<4%) was obtained. Recoveries between 94–104% revealed good accuracy for the overall method. Application to quantitative determination in a representative number of samples allowed good knowledge of the phenolic composition of green beans (Phaseolus vulgaris v.vulgaris).     

HPLC of porphyrinogens with electrochemical detection

Summary A reversed-phase system is described for the separation of coproporphyrinogen I, II, III, IV isomers, deethylisocoproporphyrinogen and isocoproporphyrinogen. The porphyrinogens are detected electrochemically with high sensitivity. The relative retention of the prophyrinogens is mainly governed by the arrangement of the ethyl and methyl substituents around the macrocycle, although steric effect may also be an important parameter.     

Fast HPLC with non-porous packing materials in the pharmaceutical industry

Summary This paper demonstrates the possibilities in transfering HPLC methods from porous to non-porous stationary phases. The procedures are transferred from 125 or 250 mm columns packed with porous stationary phases to 33 mm columns packed with non-porous particles. It is demonstrated that fast HPLC using non-porous columns reduces analysis times by a factor four to eight. Precision is comparable to HPLC with porous stationary phases. The costs for HPLC with porous and with nonporous packing materials are similar. The implementation of these fast HPLC columns is easy because standard equipment can be used.     

Optimisation of post-column reaction detector for HPLC of explosives

Summary Improvements in selectivity and sensitivity in the analysis of common explosives, like nitrate esters, nitramines and nitroaromatic compounds can be achieved by post-column derivatisation in a two step reaction detector. The first step in derivatisation is the photolysis of the analytes with UV at 254 nm. The photo reactor consists of a crocheted 20 m Tefzel capillary, which is coiled around a low pressure mercury lamp. In second step the nitrite ion generated is subsequently detected by a colourimetric reaction. The azo dye formed can be selectively detected at 540 nm.Addition of alkali after chromatographic separation to prevent oxidation of initially formed nitrite to nitrate during photolysis leads to a complex multistage arrangement. However, the contribution to peak broadening by the reactor is negligible and it is possible to detect 25–50 ppb of nitramines and 30–100 ppb of nitrate esters. Another advantage of the method is the selective detection of nitro compounds, even in complex matrices.The trace analysis of explosives is of growing interest in forensic science as well as in environmental analysis. It has been shown [1] that explosives can easily be extracted from soil and debris by the use of supercritical carbon dioxide. The separation and determination of explosives by gas chromatography is hindered by their thermal instability. In HPLC only the nitro aromatic explosives can be detected with sufficient sensitivity. Other types of explosives like the esters of nitric acid or nitramines do not absorb sufficiently in the UV region for sensitive detection. It has been shown [2] that explosives are liable to photochemical decomposition in the UV region, resulting in nitrate and nitrite, which have been detected after separation by ion-pair chromatography with electrochemical detection. A more sensitive and selective detection of nitrite has been possible in flow injection analysis [3]. Here a modified Griess reaction has been used. In a first step nitrite ions are used to form the diazonium salt with sulfanilamide which is coupled in a second step with N-[naphthyl-(1)]-ethylene diamine (NED) to form a redviolet azo dye with an absorption maximum at 540 nm. The advantage of this method is selective detection in the visible region, where hardly any other organic components are detected, which might be present in a crude environmental sample.In this paper the transfer of the Griess reaction to post-column derivatisation in RP chromatography of explosives will be described, and the optimisation of trace analysis of these solutes will be discussed.     

HPLC with electrochemical detection of metal-flavonoid-complexes isolated from food

Summary The electrochemical and chromatographic behaviour of flavonoid standards and of flavonoids extracted from food (green tea, black tea and onions) is investigated with respect to metalbinding properties. It is shown that metals such as iron, copper or aluminium are complexed by flavonoids, preferrably by those having an aromatic o-dihydroxy structure. This is confirmed by cyclic voltammetry on HPLC fractions (stopped flow) and by AAS measurement of metals. As the complexing sites of flavonoids are closely related to electrochemical properties, this is used for an indirect detection of metal species at low oxidation potentials. For iron species in particular a sensitive and selective detection is possible. For copper reductive detection can also be used.     

Determination of 2-mercaptobenzothiazole in river water by HPLC

Summary A method is proposed for the determination of 2-mercaptobenzothiazole in river waters using multi-electrode electrochemical detection HPLC. 2-mercaptobenzothiazole determination is unsatisfactory by gas chromatography as it degrades readily on the column. Multielectrode electrochemical detection HPLC combines sensitivity and the ability to screen out other electrochemically active species. The development work leading up to the proposed method is discussed. The method has a limit of detection of 0.798gl^–1 2-mercaptobenzothiazole and a total standard deviation of 2.06gl^–1 2-mercaptobenzothiazole at a concentration of 7.97gl^–1 2-mercaptobenzothiazole in river water.     

Full automation of serotonin determination by column-switching and HPLC

Summary A simple, fast, fully automated method for plasma serotonin determination is described. Full automation is obtained by coupling two devices: a sample processing station and a solid-phase autosampler. The sample processing station dilutes the plasma sample and is then connected, on-stream, with the solid-phase autosampler. It firstly fills a loop with all the solvents necessary for the sample clean-up, then, inverting the flow, pumps these solvents through the silica-bonded cation-exchange disposable extraction cartridge positioned on the autosampler. For the elution, the cartridge is switched on-stream with the HPLC analytical column and serotonin is eluted by the HPLC mobile-phase. The HPLC separation is performed by ion-pairing reversed-phase liquid chromatography. The column effluent is completely reduced by an electrochemical reactor and serotonin is detected in an oxidation-mode by a dual-cell electrochemical detector. The plasma sample is 50 l, the plasma sensitivity is 40 ng/l, the retention time is 6 min and the recovery is 95%. The repeatibility, the normal ranges for platelet-poor and for platelet-rich plasma have been established and correlation with manual HPLC calculated.Presented at the 17th International Symposium on Chromatography, September 25–30, 1988, Vienna, Austria.     

A specific sensitive HPLC method for determination of plasma dopamine

Summary We describe here a sensitive, selective and rapid method to quantitate plasma catecholamines, especially dopamine, using high-performance liquid chromatography with electrochemical detection. This method requires a 10-minute run time and has a threshold for detection of 2 picograms, (10pg/ml).A number of commonly employed mobile phases for catecholamine analysis have been tested and have failed to detect dopamine in biological samples. Neither acetonitrile (3–7%) or methanol, (5–8%) in the mobile phase has produced consistently interpretable data either due to inability to detect or interference from co-eluting substances. Optimal detection was achieved with a mobile phase containing sodium acetate (6.8g), citric acid (5.9g), EDTA (48mg), di-n-butylamine (270l), Na-1-octane sulfate (850mg), methanol (100 ml) (amounts refer to 1 liter aqueous solution) (pH 4.3). The mobile phase was passed through a Waters 5 resolve C₁₈ column using a Waters 590 pump and m460 electrochemical detector and 740 data module, Flow rate was 0.9ml/min. Using this method, normal values in human and swine left ventricular myocardium and human and swine plasma have been established for norepinephrine, epinephrine, and dopamine.     

HPLC Analysis of complex mixtures of triglycerides using gradient elutions and an ultraviolet detector

Summary A method of reserved-phase HPLC analysis for mixtures of triglycerides (TG's) that provides good resolution at acceptable analysis times for high-ECN TG's has been developed. An elution gradient of methyltert-butyl-ether (MTBE) in acetonitrile (ACN) was used with an ultraviolet detector operated at 215nm. The effect of the proportion of MTBE in the mobile phase, gradient time, temperature and sample solvent on TG retention and resolution was studied. Linear relationships were derived between the logarithm of the capacity factor (log k'), and the logarithm of selectivity (log α) and the above-mentioned chromatographic factors. The conditions selected were: an elution gradient of from 23 to 30% MTBE, an elution gradient time of 25 minutes, and a temperature of 30°C, which provided good resolution of soybean oil TG's in less than 30 minutes.     

Determination of phenol in raw and potable waters by HPLC multi-electrode electrochemical detection

Summary A method is proposed for the determination of phenol in raw and potable waters using multi-electrode electrochemical detection HPLC. The coulometric efficiency of the electrochemical cell together with an ability to ‘screen out’ other electrochemically active species precludes the need for trace enrichment concentration techniques. The development work leading up to the proposed method is discussed with reference to selection of solvent pH and electrode potentials. The method has a limit of detection of 0.034μgl⁻¹ phenol and a total standard deviation of 0.083μgl⁻¹ phenol at a phenol concentration of 1.048μgl⁻¹ phenol in river water.     

HPLC determination of morphine with amperometric detection at low potentials under basic pH conditions

Summary A simple, sensitive, accurate and reliable method for morphine determination in biological samples has been developed. It uses reverse-phase HPLC on a polymeric column with an eluent (0.05 mol/L dibasic sodium phosphate: acetonitrile, 8515) at pH 9.5, allowing both the suppression of ionization of morphine amine and the promotion of oxidation of the phenolic group. Amperometric detection at mild oxidizing potential (350 mV) proves very selective, and, therefore, only a simple and rapid one-step liquid-liquid sample preparation is required. Under these conditions clean chromatograms are obtained even with complex biological matrices such as cadaveric blood, urine and hair.Minimum detectable amount of morphine is about 200 pg injected. A calibration line with a correlation coefficient of 0.99996 has been produced over the range 7.5–250 ng/mL. Precision results comparable to other HPLC methods.A preliminary report was presented at the 25th T.I.A.F.T. Meeting 1988, Groningen (NL), June 27–30 1988.     

Mesoporous materials for the retention and separation of haloacetic acids

Summary Mesoporous silica-based materials have been synthesized and studied for applications in the environmental field. Haloacetic acids have been chosen as test compounds since they are byproducts of disinfection processes of water for human consumption. For this purpose mesoporous materials have been synthesized in acid, basic or neutral conditions. The effect of composition and synthesis conditions of mesoporous materials on their retention properties have been studied. The retention mechanism has also been elucidated in term of electrostatic interactions and as a function of the residual surfactant content in the materials. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001     

Optimization of a reaction detector for analysis at the femtomol level of carbamate insecticides by HPLC

Summary For the determination of trace amounts of carbamate insecticides in vegetables the combination of liquid chromatography with post column chemical derivatization (chemical reaction detector) is used. In a two step reaction detector the carbamates are firstly saponified in 0.01 M sodium hydroxide at 80°C with a reaction time of 30sec. To this mixture OPA reagent is added to detect the methylamine generated in the saponification. Optimization strategies for this reaction are demonstrated. At a temperature of 80 °C a reaction time of 80 sec is sufficient for quantitative transformation of the methylamine. The peak dispersion can be reduced and hence the detection limit improved by decreasing the diameter of the tubes and by diminishing dilution through the addition of reagent. A 1∶24 ratio of reagent to column (reactor) effluent is possible with cyclone-type mixers. The quantitation of carbamates in different vegetables is demonstrated. The detection limit is 20ppb at a signal to noise ratio of 10∶1. Thesis B. Lillig, Saarbrücken, 1984     

Increased sample throughput in HPLC using sample switching

Summary Column switching methods are described which demonstrate an alternative approach to the reduction of analytical time in HPLC. Although most methods use a combination of techniques, it has been possible to group them into major categories involving column cleanup, sample cleanup, multicolumn methods and boxcar chromatography. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Determination of free formaldehyde in the presence of donators in cosmetics by HPLC and post-column derivation

Summary The determination of free formaldehyde in the presence of its donators in cosmetic samples by a combination of reversed phase chromatography and post column reaction detection is described. The free formaldehyde is separated on a RP column with water as eluent from interfering formaldehyde-containing compounds and consecutively determined by the lutidine method in a reaction detector with knitted open tubes. With detection in the visible (420nm) the minimum detectable quantity is 40 ppb, with fluorimetric detection 15 ppb. The sample clean-up procedure for cosmetic products ranging from mascara to shampoo is by extracting the formaldehyde with water of pH 3, where the decomposition rate of the donators is minimal. Smaller amounts of free formaldehyde are always found compared to the standard lutidine method.     

Highly sensitive measurement of mepindolol in biological fluids using HPLC and electrochemical detection

Summary Mepindolol, a β-blocker agent, has an indolic structure which can undergo oxidation. A high-performance liquid chromatographic technique has been used to measure mepindolol in biological fluids using pindolol as the internal standard. The chromatography has been combined with electrochemical detection (coulometric detector). This method allows the determination of very low amounts of mepindolol with good precision and accuracy, the limit of quantification being 0.6 ng · ml⁻¹.     

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.