Post-column Derivatization Method Research Articles

Determination of aflatoxins in corn and peanuts using high performance liquid chromatography.

The various high performance liquid chromatographic techniques for the detection of aflatoxins in foodstuffs are examined. The need for resolution, selectivity, and sensitivity is emphasized and examples from the literature are used to illustrate this need. Examples include both normal and reversed-phase techniques. Sample cleanup, detection methods, chromatographic conditions, and pre- and post-column derivatization methods are discussed.

Fluorometric determination of biguanides in serum by high-performance liquid chromatography with reagent-containing mobile phase

A simple post-column derivatization method for the fluorometric determination of biguanides (buformin and phenformin) in serum by high-performance liquid chromatography is described. The serum was treated with 4% perchloric acid to precipitate proteins, and the supernatant was directly injected into the column. Synthesized 9,10-phenanthrenequinonesulphonate (PSQ) was used as a fluorogenic reagent and added to the mobile phase. Biguanides were separated within 10 min on a Radial-Pak μBondapak C 18 cartridge (10 μm, 10 cm × 8 mm I.D.) by reversed-phase ion-pair chromatography. They were then allowed to react with PQS in an alkaline stream and detected fluorometrically. This method was applied to the analysis of serum from patients with diabetes mellitus.

Fluorometric determination of streptomycin in serum by high-performance liquid chromatography using mobile phase containing fluorogenic reagent

A new postcolumn derivatization method for the fluorometric determination of streptomycin in serum by high-performance liquid chromatography is described. The serum was treated with 3.5% perchloric acid to precipitate proteins and the supernatant was directly injected into the chromatograph. Streptomycin was separated by reversed-phase, ion-pair chromatography with a mobile phase containing ninhydrin as a fluorogenic reagent, octanesulfonate, and 1,2-ethanedisulfonate as counterions, and was detected by fluorescence using continuous-flow, postcolumn derivatization in an alkaline stream with ninhydrin in the mobile phase. This method is sensitive to 1.0 μg/ml using only 100 μl of serum. Comparison with a fluorescence polarization immunoassay gave a good correlation coefficient of 0.976.

Fluorimetric determination of amino acids by high-performance liquid chromatography using a hollow-fibre membrane reactor

A high-performance liquid chromatographic method has been developed for the determination of primary amino acids. The method involves separation of primary amino acids on a C 18 column using sodium heptanesulphonate as an ion-pairing agent, post-column derivatization with o-phthalaldehyde and 2-mercaptoethanol introduced into the main flow stream using a sulphonated hollow-fibre membrane reactor immersed in their solutions and fluorimetric detection of derivatives (λ ex = 340 nm, λ em = 450 nm). A higher or similar sensitivity was obtained compared with the conventional post-column derivatization method. The detection limits were 0.2–2.3 pmol at a signal-to-noise ratio of 3. For amounts of 48–110 pmol, the precision was of the order of 1.1–4.0% (relative standard deviation, n = 20).

O-Phthalaldehyde—N-acetyl- L-cysteine as a chiral derivatization reagent for liquid chromatographic optical resolution of amino acid ernantiomers and its application to conventional amino acid analysis

A useful fluorescence derivatization reagent, o-phthalaldehyde-N-acetyl- l-cysteine, has been developed for the optical resolution of enantiomeric amino acids and for conventional amino acid analysis. Amino acids rapidly reacted with o-phthalaldehyde in the presence of N-acetyl- L-cysteine to give intensely fluorescent products, which were diastereoisomers. When this reaction was used in the precolumn derivatization of amino acid enantiomers, their diastereomeric derivatives were efficiently resolved on a reversed-phase column. Simultaneous analysis of common protein amino acid enantiomers was achieved by gradient elution within 70 min. In addition, the reagent was applied to the post-column derivatization of protein amino acids, combined with hypochlorite oxidation for the detection of amino acids, such as proline. Seventeen protein amino acids were sufficiently separated in 17 min by ion-pair chromatography with sodium dodecyl sulphate as a counter-ion, and they were determined at the same level of sensitivity by the above post-column fluorimetric detection system. Applications of both the precolumn and the post-column derivatization methods with the new reagent to hydrolysed protein samples are also described.

3] Determination of thiamin and its phosphate esters by high-performance liquid chromatography

This chapter describes high-performance liquid chromatography (HPLC) of thiamin and its phosphate esters, which is considered the most sensitive method to determine these compounds at the subpicomole level within lo-20 min. This method is applicable to the analysis of thiamin and its phosphates not only in animal tissues but also in body fluids, foods, and pharmaceutical preparations. This method is also useful in the study of intermediary metabolism of thiamin. In the chromatographic mode, two systems, straight-phase HPLC and reversed-phase HPLC, are used. In the former system, thiamin, thiamin monophosphate (ThMP), thiamin diphosphate (ThDP), and thiamin triphosphate (ThTP) are eluted from a column in this order. In the latter system, the elution order of thiamin phosphates is the reverse. When detection of thiamin phosphates is carried out fluorometrically, two different procedures are used—namely, pre- and postcolumn derivatization methods. The mobile phase used in the precolumn derivatization method of HPLC for thiamin and its phosphates should contain a buffer solution of pH above 8, as thiochrome fluoresces at pH values above 8. All phosphate bonds remain intact when thiamin phosphates are converted to thiochrome phosphates by alkaline oxidation.

Determination of or-Keto and Hydroxy Acids by High Performance Liquid Chromatography Using Ferric Perchlorate as a Detection Reagent

A simple and selective method for the determination of α-keto and hydroxy acids was developed. These acids were detected spectrophotometrically by use of ferric perchlorate. Their simultaneous determination was performed by high performance liquid chromatography employing the post column derivatization method. Under our conditions, pyruvic acid, oxalacetic acid, α-ketoglutaric acid, lactic acid, tartaric acid, citric acid, malic acid, β-hydroxybutyric acid and oxalic acid were determined in the range of 2-100nmol at the sample size of 10μl. This method was also applied to the determination of pyruvic acid and lactic acid in human whole blood.

A rapid and sensitive method for the determination of hypusine in proteins and its distribution and developmental changes

A simple and sensitive method for determining hypusine in proteins was developed. A greater part of amino acids in the acid hydrolysate of proteins was separated from hypusine by treatment with an ion-exchange resin. The sample containing partially purified hypusine was then analyzed by high-performance liquid chromatography using the post-column derivatization method with o-phthalaldehyde. The recovery rate of hypusine through the overall procedure was more than 95%. Using this method, the distribution and developmental changes of hypusine in proteins were determined. The amino acid was found in proteins of all examined organs of rat. Its concentration was 5–40 nmol/g protein. The subcellular distribution in rat liver was also determined. About 60% of total amount of hypusine was present in the proteins of cytoplasmic and microsomal fractions and its relative concentration was high in the proteins of microsome and lysosome and low in mitochondria. In developing rat, the concentration of hypusine in the brain proteins was relatively high during the first 2 or 3 weeks of postnatal life and then decreased until adulthood. Its concentration in the liver proteins was highest at birth and then decreased continuously to the adult level.

High performance liquid chromatographic determination of sodium sulfate in anionic surfactants

AbstractSodium sulfate is separated from anionic surfactants on a separation column packed with strong anion exchange resin, and is detected by a post column derivatization method based on a chemical reaction between sulfate and ferric ion. A calibration curve shows a linear relationship in the range of 10\s‐300 mg/100 mL for sodium sulfate. The lower limit of determination is 2 mg/100 mL. Anionic surfactants such as sodium alkylbenzene sulfonate and sodium alkyl sulfate do not interfere with the determination. A personal computer was connected to the chromatographic system for data processing and control of an automatic sampler. This personal computer controlled liquid chromatographic system provides good recovery, reproducibility, agreement with conventional methods and full automatic determination.

Novel post-column derivatization method for the fluorimetric determination of norepinephrine and epinephrine

A novel method is described in which catecholamines are converted into fluorescent products by heating in alkaline borate buffer. The method was applied to the determination of norepinephrine and epinephrine after separation by high-performance liquid chromatography using a pellicular, strong cation exchanger. The new system is simpler than the system based on the trihydroxyindole reaction. It is suitable for the measurement of catecholamines in the range 0.25–20 ng. The assay of catecholamines in human urine is also described.