Reversed-phase Ion-pair Partition Chromatography Research Articles

Determination of serotonin and 5-hydroxyindoleacetic acid in urine by reversed-phase ion-pair partition chromatography with fluorimetric detection

The determination of serotonin and 5-hydroxyindoleacetic acid in urine is described. The compounds are separated by means of reversed-phase chromatography using tri- n-butylphosphate as the stationary phase and a mobile phase containing perchlorate as an ion-pairing agent. Detection is performed fluorimetrically. The compounds can be assayed directly, using two mobile phases with different pH values. Alternatively, both compounds can be determined with the same mobile phase after urine extraction on a small XAD-2 column for clean-up of serotonin. Two different approaches were evaluated for this XAD-2 extraction, viz.: (i) desorption of serotonin with a methanol-rich solvent after a washing step with an aqueous solvent; (ii) direct desorption of serotonin in the washing step using a solvent with a small percentage of methanol. Only the latter approach proved to be successful. The linearity of the method was investigated by standard additions to urine. The method was applied to the determination of both compounds in urine. The stability of the compounds in standard solutions and in urine was investigated under various storage conditions.

Determination of catecholamines in urine (and plasma) by liquid chromatograhy after on-line sample pretreatment on small alumina or dihydroxyborylsilica columns

The possibilities of the on-line pre-concentration of catecholamines on small columns packed with aluminium oxide (10 μm) or dihydroxyborylsilica (5 μm) prior to high-performance liquid chromatography (HPLC) were investigated. For both pre-concentration materials the influence of the pH of the mobile phase on the retention characteristics was studied for the catecholamines and some of their derivatives lacking the catechol and/or the amine function. The recovery of the catecholamines and the loading capacity on some pre-columns was then investigated. The compatibility of the pre-concentration system with several HPLC systems, viz., reversed-phase ion-pair, ion-exchange and reversed-phase ion-pair partition chromatography, was studied. Combined with amperometric or fluorimetric detection, the method was applied to the determination of free catecholamines in urine, using dihydroxybenzylamine or epinine as internal standards. An example of the determination of catecholamines in plasma containing 100 pg/ml of each catecholamine is also shown.

Separation and determination of stable metallo—cyanide complexes in metallurgical plant solutions and effluents by reversed-phase ion-pair chromatography

The advance of liquid chromatography made feasible the identification of the different metallo—cyanide complexes. A technique for their simultaneous separation and determination, involving reversed-phase ion-pair partition chromatography with UV detection, is proposed. The mobile phase contains 2.5 m M tetrabutylammonium hydrogen sulphate as the pairing reagent and methanol as the organic solvent. A C 18 Novapak cartridge with 4 μm packing is used as the stationary phase. The procedure is simple and rapid: separation and measurement are accomplished in 30 min. The method is accurate: the relative mean error is less than 1% for all complexes except Cu(CN) − 2 (2.72%). The precision is good, with relative standard deviations from 1.14% to 2.83% for the individual complexes.

Selective retention of catecholamines and their derivatives reversed-phase ion-pair partition chromatography

Some possibilities are described to improve the separation between catechoamines and their corresponding metanephrines in reversed-phase ion-pair partition chromatography. The retention of amines can be influenced by the perchlorate concentration of the mobile phase, while the retention of acidic metalolites is governed by the pH of the mobile phase. Nevertheless, a separation between the catecholamines and their corresponding metanephrines cannot be achieved by optimizing these mobile phase parameters. A decrease in the temperature of the chromatographic system leads to an improvement in the separation betweeen dopamine and 3-methoxytyramine; however, adrenaline and noradrenaline are not separated from metanephrine and normetanephrine, respectively, and the chromatographic efficiency decreases. Addition of boric acid to the mobile phase results in a selective decrease in the retention of compounds with a catechol function, the magnitude of this effect depending on the mobile phase pH. Applying this “boric acid effect” it is possible to obtain a complete and rapid (within about 9 min) of the catecholamines, their corresponding metanephrines, the acidic metabolites homovanillic acid and 3,4-dihydroxyphenylacetic acid and the neutral metabolite 3-methoxy-4-hydroxyphenyl-ethylene glycol.

Analysis of β-carbolines by reversed-phase ion-pair partition chromatography with fluorometric detection

A high-performance liquid chromatographic system was developed for the analysis of ten selected β-carbolines. It consists of tri- n-butyl phosphate as liquid stationary phase and buffered mixtures of water and methanol with perchlorate as the mobile phases. The separation was optimized by means of mobile phase parameters such as the perchlorate concentration, the methanol content and the pH. The relationship between these parameters and the retention of the β-carbolines was determined and compared with theory as derived from a model of the retention mechanism. The potential of spectrofluorometric and UV spectroscopic detection with regard to the detection limits, linear dynamic range and selectivity were investigated.

Reversed-phase ion-pair partition chromatography of iron, cobalt and nickel chelates with 4-hydroxy-3-(2-pyridylazo)naphthalene-1-sulphonic acid

The reversed-phase ion-pair partition chromatography of 4-hydroxy-3-(2-pyridylazo0naphthalene-1-sulphonic acid (α-PAN-4S) and its metal chelates was studied. Cobalt(III), iron(I) and nickel(II) chelates and excess of α-PAN-4S were separated on the column and eluted with acetonitrile—water (37–:60–63, v/v) containing 0.025 M tetrabuylammonium bromide, 0.005 M 3-(N-morpholino)propanesulphonic acid—sodium hydroxide buffer (pH 7) and 10 −4 M Na 2(EDTA). Cobalt, iron and nickel were simultaneously determined without interferences from other metals. The effects of the elunt composition and pH on the retention of all species were examined. The method was applied to the determination of cobalt, iron and nickel in a glass sample.

Analysis of serotonin and derivatives by reversed-phase ion-pair partition chromatography with fluorometric and electrochemical detection

A flexible and efficient system is described for reversed-phase ion-pair partition-chromatographic analysis of serotonin, its precursors tryptophan and 5-hydroxytryptophan, its main metabolite 5-hydroxyindoleacetic acid, and tryptamine. The chromatographic system consists of tri- n-butylphosphate as stationary phase and buffered water-methanol mixtures, containing perchlorate, as mobile phases. Retention can be selectively influenced by means of the pH, the perchlorate concentration, and the methanol content of the mobile phase, as well as the temperature of the phase system. The compounds of interest can be separated within 10 min and no interference from catecholamines and derivatives was observed. Compared with electrochemical detection, fluorometric detection yielded more favourable detection limits and was more selective when supernatants of brain tissue homogenates were directly injected. Both detection systems showed inadequate selectivity if urine samples were directly injected, but 5-hydroxyindoleacetic acid could readily be assayed.

Gradient Elution of Biogenic Amines and Derivatives in Reversed Phase Ion-Pair Partition Chromatography with Electrochemical and Fluorometric Detection

Abstract The compatibility of gradient elution and reversed phase ion-pair partition systems combined with electrochemical and fluorometric detection has been investigated. The phase system consisting of buffers with perchlorate counter-ions as mobile phases and tri-n-butylphosphate as stationary phase allows the use of pH and counter-ion gradients. It appeared that (i) use of gradients is time saving and favourable with respect to detection limits and (ii) dual electrode detection may offer a solution to the problem of gradient-induced baseline shifting in electrochemical Electrochemical detectors are rather sensitive towards changes in the mobile phase. Applying the differential coulometric detection principle the gradient induced baseline shift is reduced and consequently the quantification of a number of compounds is facilitated, although noise levels are slightly increased. Future research will be devoted to noise investigations in order to minimize detection limits.

Spectrophotometric determination of platinum (II) and palladium (II) with 4-(2-pyridylazo)-resorcinol by reversed phase ion-pair partition chromatography

白金(II)及びパラジウム(II)は,塩化物イオン共存下2-プロパノール-水混合溶媒中で4-(2-ピリジルアゾ)レソルシノール(PAR,H2par)と反応して,金属イオン:塩化物イオン:PAR;1:1:1の三元錯体を形成する.1価の陰イオン種[PtCl(par)-]及び[PdCl(par)-]は,テトラブチルアンモニウムイオンを対陽イオンとして,逆相イオン対分配高速液体クロマトグラフィーによる分離が可能である.これを利用して,PARによる白金(II)及びパラジウム(II)の同時定量法を確立した.本法により1×10-6moldm-3の白金(II)及び5×10-7moldm-3のパラジウム(II)が定量できた.1×10-5moldm-3の目的成分に対し,10-4moldm-3の鉄(II),コバルト(II),ニッケル(II),銅(II),亜鉛(II),アルミニウム(III),クロム(III)及びロジウム(III)の共存は許容された.

Reversed-phase ion-pair partition chromatography of bromo-anion complexes of platinum group metals (rhodium(III), palladium(II), and platinum(IV)).

A novel method for the separation of bromo-anion complexes of platinum group metals ( Rh, Pd and Pt ) by reversed-phase ion-pair partition chromatography has been developed. The excellent separation of RhBr52- PdBr42-and PtBr62-was accomplished on a Fine SIL C18 ( ODS ) packed column, where acetonitrile - phosphate buffer ( pH 7 ) mixture containing tetrabutylammonium bromide and sodium bromide was used as a mobile phase. These anion complexes can be detected at 310 nm, and the amount of PtBr62- as low as 1 × 10-11mol can be determined with the peak height method which gave a straight calibration curve. In this paper, the effects of ion-pair reagents, acetonitrile-water ratio, pH, temperature and light on the chromatogram are reported.

Determination of Citrinin in Grain by Reversed-phase Ion-Pair Partition Chromatography

Citrinin is a toxic metabolite produced by several Penicillium spp. and a few Aspergillus spp. Citrinin is nephrotoxic and has been implicated in disease outbreaks associated with moldy rice.A reversed-phase ion-pair high-performance liquid chromatographic method was developed for determining citrinin. Citrinin was extracted from a grain sample, then the extract was cleaned up by partitioning with solvents, and an aliquot was injected into a stainless steel column containing Partisil-10 ODS-3. The mobile phase was 47% ethanol/PIC-A (tetrabutylammonium phosphate 0.005M) and 53% water/PIC-A. Detection was achieved by ultraviolet absorbance measurement at 254nm. Retention time and peak hight were highly reproducible. The relationship between peak height and quantity injected was linear over a range of 1-103ng. The detection limit of the method is 20ng/g. Recovery of citrinin from a grain sample spiked at the level of 0.25μg/g was 66%. The method was applied to the analysis of commercial rice and barley samples. Citrinin was not detected in 10 samples examined.

Reversed-phase ion-pair partition chromatography of phenylacetic, mandelic and pyridinecarboxylic acid derivatives

Phenylacetic, mandelic and pyridinecarboxylic acids of physiological interest have been separated by ion-pair partition chromatography with 1-pentanol as stationary phase and tetrabutylammonium as counter ion in the aqueous mobile phase. The stability of the chromatographic system is very high and the support is spontaneously re-coated with stationary phase. Untreated plasma samples can be injected in relatively large volumes without serious loss of separating efficiency. A venting system was found efficiently to reduce disturbing early peaks on injection of biological samples.

Reversed-phase ion-pair partition chromatography of carboxylates and sulphonates

A reversed-phase partition chromatographic system for separation of organic anions as ion pairs with quaternary ammonium ions has been developed. Commercial, hydrophobized silica supports are used with 1-pentanol as stationary phase and aqueous solutions of tetrabutylammonium (TBA) as mobile phase. The separation of aromatic sulphonates and benzoic acid derivatives is demonstrated. The use of the TBA concentration of the mobile phase to regulate the capacity factor of the anions, as a means of improving separation by gradient elution and direct injection of large sample volumes, is demonstrated. The isolation of nicotinic acid from human serum samples is shown.