Reversed-phase Paper Chromatography Research Articles

Determination of retinyl palmitate in homogenates and subcellular fractions of rat liver by liquid chromatography

It is widely acknowledged that, after crossing the intestinal barrier and circulating through the lymphatic vessels, vitamin A is stored principally in the liver which contains about 90% of the body’s total reserves of that vitamin [1,2]. Retinol is stored in large quantities in liver as fatty acyl esters and is released from the esters when it is required for its action. In the past, to study these fatty-acyl esters (RFAE), the separation was achieved either by reversed phase paper chromatography, or by thin layers of silica gel [3,4] and was quantitated by fluorometry. De Ruyter and De Leenheer [5] developed a reversed phase high-performance liquid chromatographic (HPLC) method for the simultaneous determination of retinol and retinyl esters and later modified the method to separate retinyl oleate from palmitate [6]. Alvarez et al have synthesized retinyl esters and analyzed these compounds by high-pressure liquid chromatography (HPLC) [7]. Little is known about the requirements, metabolism and subcellular distribution of fat-soluble vitamins in retinol deficiency. Plasma levels of this vitamin do not really give an indication of vitamin stores unless the stores are nearing depletion or approaching toxic levels [8]. In physiology or pathology, the determination of retinol in liver homogenates and subcellular fractions constitutes an essential element of assessment of vitamin A status [9]. Several procedures for quantitating vitamins and more specifically retinol essentially in the serum [5,6,10,11] have been previously published. In liver tissue, few results have been reported. Futterman et al [3] reported the presence of at least eight RFAE in rat liver; nevertheless, the results indicate that retinyl palmitate is the major fatty acyl ester (79%) [3,12,13]. Therefore it is of interest to develop a technique for subcellular distribution study of first the reserve form to determine the repartition in healthy subjects and also during vitamin A deficiency.

Separation of some transition metals by reversed-phase paper chromatography

A reversed-phase paper chromatographic method has been developed for the mutual separation of transition metals such as Fe(III), Cu(II), Pb(II), Ni(II), Ag(I) and Au(III). The separations were performed on Whatman No. 1 filter-paper with liquid ion exchangers such as trioctylamine, triisooctylamine or Aliquat 336 as stationary phases and sodium acetate, sodium malonate or sodium succinate as mobile phases.

The estimation of N-nitrosamines in tropical regions by reversed-phase paper and thin-layer chromatography

A simple and convenient procedure based on reversed-phase paper and thin-layer chromatography is reported for the detection and estimation of N-nitrosamines in food products, particularly cured meat. The method can easily be applied in tropical regions when plenty of sunshine is available. It is particularly useful in food-testing laboratories where sophisticated analytical instruments such as the gas chromatograph, mass spectrometer, etc., are not available. By this procedure, it is possible to detect up to 10-μg and to estimate up to 75-μg (reversed phase) and 50-μg (thin layer) amounts of N-nitrosamines.

Separation of -apocarotenals and related compounds by reversed-phase paper and thin-layer chromatography.

Tlie sclxuntion and clraractcrization of vitamins Al and An nnd related compoundsby reversed-pllasc paper cliromatogrnpl~y as well as ly thin-lqxr chromategraphy have hen rcportccl carlicrl * $. Thin-lnycr chromatography has also been used for the separatinn and charncterizatio11 of carotenoids from natural sourccs3~ ‘1. I-Iowcver, 130tr.rc,1~1~ofib scrvccl that carotenoid misturcs cannot be separated on a sin& aclsorhnt with ;1 sin& solvent. The scparntion and clctermi1wtion of carotenoid alclclydes from plants, microorganisms and animnl tissues have lxxn carriecl out by nicans of thin-layer clirf.~li~ato~apI~~U. Apocarotcnals awl apocarotcnoic acid have been detected in ornnges by the same technique’*

Chromatographic separation of vanadium, tungsten and molybdenum with a liquid anion-exchanger

In acidic solution only molybdenum(VI), tungsten(VI), vanadium(V), niobium(V) and tantalum(V) form stable, anionic complexes with dilute hydrogen peroxide. This fact has been used in developing an analytical method of separating molybdenum(VI), tungsten(VI) and vanadium(V) from other metal ions and from each other. Preliminary investigations using reversed-phase paper chromatography and solvent extraction led to a reversed-phase column Chromatographic separation technique. These metal-peroxy anions are retained by a column containing a liquid anion-exchanger (General Mills Aliquat 336) in a solid support. Then molybdenum(VI), tungsten(VI) and vanadium(V) are selectively eluted with aqueous solutions containing dilute hydrogen peroxide and varying concentrations of sulphuric acid.

The separation of chlorinated derivatives of hydroquinone using thin-layer and paper chromatography

Thin-layer and paper chromatographic procedures are described for the separation and identification of six chlorinated derivatives of hydroquinone. Adsorption thin-layer chromatography (silica gel) or reversed-phase paper chromatography (olive oil—stationary phase and diluted acetic acid—mobile phase) did not enable satisfactory separation of dichloro-derivatives. These compounds have been clearly separated on kieselguhr thin layers and paper impregnated with formamide using three solvent systems.

Sulphoxides as solvating reagents for the separation of metal ions

The solvent extraction properties of the following sulphoxides have been evaluated: di-n-octyl sulphoxide (DOSO), bis(n-octylsulphinyl) methane (BOSM), bis(n-octylsulphinyl)ethane (BOSE) and p-tolyl sulphoxide (PTSO). By use of reversed-phase paper chromatography as a qualitative surveying technique, the interactions of these sulphoxides with some fifty metal ions were investigated in several acid-ligand systems. All sulphoxides were studied in 1–10 M hydrochloric and nitric adds; DOSO and BOSM were also studied in perchloric acid and ammonium thiocyanate-perchloric acid mixtures. Observations are made concerning sulphoxide-metal interactions and the existence of several useful analytical separation systems is pointed out. The synthesis and characterization of BOSM and BOSE are described.

Elevated temperature reversed-phase paper chromatography of derivatives of selected drugs

Drug derivatives prepared by injection of a mixture of drug and reagent into a gas chromatograph can be collected and subjected to elevated temperature reversed-phase paper chromatography. This procedure provides additional parameters which have been found useful in identifying drugs in samples of blood, urine and liver.

The behaviour of hydrophylic substances in reversed-phase chromatography a contribution to the mechanism of reversed-phase paper chromatography

The chromatographic bahviour of both lipophylic and hydrophylic benzeneazo-2-naphtol derivatives was studied using untreated papers and papers impregnated with nonpolar or weakly polar organic stationary liquids and the same mobile phase. It was observed that on reversed-phase chromatograms dyes containing sulpho groups behaved as if the papers did not contain the organic stationary phase, whereas the lipophylic dyes followed normal reversed phase partition mechanism. Thus, two stationary phases must be considered to be present in the reversed-phase chromatograms: the organic liquid and the cellulose—water complex. The choice of the stationary phase involved in the separation process is dependent on the character of the compound chromatographed. In some case both stationary phases can be involved.

Inorganic chromatography on cellulose acetate pre-treated paper impregnated with 2-thenoyltrifluoroacetone

The R F values of inorganic ions on paper pre-treated with 0.25–2.0 % cellulose acetate and impregnated with various concentrations of 2-thenoyltrifluoroacetone (TTA) was studied by using acetate buffer as a developing agent. 1% cellulose acetate pre-treated paper impregnated with o.2 M TTA was found suitable for reversed phase paper chromatography. The R F values of inorganic ions were examined on the impregnated paper by using various pH's of the developing agent (0.1 M acetate buffer). Separations of PbCu, CoCu, ZnCu and FeCoNi were performed in this system.

Reversed phase paper chromatography of p-nitrophenyl methylphosphonates and sulfur analog

On account of differences in lipophilic character it is possible to separate p-nitrophenyl methylphosphonates and their sulfur analogs by reversed phase paper chromatography. Whatman No. 1 filter paper was impregnated with silicone oil 550; the upper layer of a chloroform-ethanol-water (5:5:3, v/v) system served as mobile phase. The relation R M = log (1/ R F —1) holds for homologous series of O-alkyl p-nitrophenyl methylphosphonates (I) and S-alkyl p-nitrophenyl methylphosphonothiolates (II) up to the n-pentyl homologues. Using a combination of Gibbs' and Dragendorff reagents it was possible to distinguish the phosphonates, phosphonothiolates and phosphonothionates.

Identification of the thiazide diuretic drugs.

Abstract A method for detecting and identifying ten thiazide diuretic drugs in tablets, gastric washings and urine by ultraviolet spectrophotometry and paper chromatography is described. The thiazides can be detected by absorption maxima in the region 264 to 294mμ at concentrations of 5 μg/ml or lower. Identification is by ascending high temperature reverse phase paper chromatography using tributyrin treated paper and developing for 20 min at 90° with a phosphate buffer (pH 7614). Further differentiation is obtained with the solvent system amyl alcohol—0 880 ammonia (9:1) on Whatman No. 1 paper. The thiazides are located as absorbing or fluorescent spots in ultraviolet light (2534 Å) and these are confirmed by the stable red colour given by an alkaline sodium 1,2-naphthaquinone-4-sulphonate spray reagent. Many of these diuretics are co-extracted with barbiturates and may interfere with barbiturate determinations.

Studies on Coenzyme Q: THE BIOSYNTHESIS OF COENZYME Q9 IN RAT TISSUE SLICES

Abstract Unequivocal proof that individual rat tissues carry out the biosynthesis of coenzyme Q9 (CoQ9) from naturally occurring small molecules has been provided by the demonstration of incorporation of radioactivity from acetate-1-14C, DL-mevalonate-2-14C, uniformly labeled L-phenylalanine-14C, and uniformly labeled tyrosine-14C into CoQ9 in rat liver, kidney, heart, and intestine slices. The rates of synthesis of CoQ9 and cholesterol in various tissues were variable, although the rate of the over-all synthesis of cholesterol exceeded that of CoQ9 by a ratio of 350:1 in rat liver slices and 15:1 in rat kidney slices. The incorporation of mevalonate-2-14C into both cholesterol and CoQ9 obeys saturation kinetics as measured by the ratio of specific activity of the product to precursor. The apparent Km for cholesterol biosynthesis was 1.0 mM, whereas that for CoQ biosynthesis averaged 0.2 mM. Although the incorporation of label from mevalonate-2-14C into cholesterol was linear with time, a lag of about ½ hour was observed before significant incorporation of label into CoQ9 occurred. This lag in labeling of CoQ9 was attributed to the accumulation of intermediates at a slow point in the reaction sequence. The incorporation of mevalonate-2-14C into CoQ9 in rat tissue slices was independent of the carbon dioxide tension in the reaction medium but was dependent upon the partial pressure of oxygen. Neither the addition of pyruvate as an exogenous energy source nor the additional rat serum stimulated the rate of biosynthesis in liver slices. A radioactive fraction moving faster than CoQ9 on reverse phase paper chromatography from the crude CoQ fraction isolated from alumina has been identified as a mixture of radioactive isoprenoid alcohols. Tyrosine was approximately 3 times as effective as phenylalanine in labeling CoQ9 in vitro and approximately twice as effective as acetate. Incorporation of label into CoQ9 from the aromatic amino acids was little affected by addition of 5 mM nonradioactive mevalonate, whereas that from acetate-1-14C was inhibited 95% by means of isotope dilution. These findings support the dual origin of the side chain and aromatic portion of CoQ9 and indicate the feasibility of studying its biosynthesis in vitro.

Formation and enterohepatic circulation of metabolites ofc retinol and retinoic acid in bile duct-cannulated rats

Four hours after intraportal injection of retinoic acid-(14)C into bile duct-cannulated rats, less than 10% of the radioactivity was recovered in the liver, intestine, and kidneys. Within 6 hr, 40% of the radioactivity had appeared in bile. When suspensions of retinol-(14)C or retinal were similarly injected, 25-35% of the dose was excreted in bile within 24 hr and equivalent amounts were deposited in the liver as retinol ester. The isolated perfused liver also produced these bile metabolites and is probably the major site of their formation in vivo. The intestine may metabolize retinoic acid, however, since some metabolites were found in the intestinal wall and lumen, even in bile duct-cannulated rats. The bile metabolites of retinol-(14)C and retinoic acid-(14)C undergo extensive enterohepatic circulation. The bile radioactivity was not volatilized on boiling at acid pH, was not present in digitonin-precipitated sterols, and did not migrate with bile salts on reversed-phase paper chromatography. Anion-exchange chromatography resolved the metabolites of bile into three fractions containing nonionic compounds, acidic substances like retinoic acid, and more polar acidic derivatives.

Reversed phase paper chromatography of metal ions with phenylbenzohydroxamic acid

The chromatographic behavior of some 35 different metal ions has been studied using paper impregnated with phenylbenzohydroxamic acid and 2-octanone. A number of selective separations can be accomplished using eluents varying in acidity from Ph 3 to 5 or 6 molar acid.

Reversed-phase paper chromatography of some cations with two nitrophenylthiphosphate derivatives as statinary phases

Reversed-phase paper chromatography of some cations with two nitrophenylthiphosphate derivatives as statinary phases

Reversed-phase paper chromatography of the rare earths, thorium and uranium using methylenebis(di-n-hexylphosphine oxide) as the stationary phase.

Reversed-phase partition chromatography was used to separate the rare earths, thorium and uranium on papers treated with methylenebis(di-n-hexylphosphine oxide), MHDPO. The separation factors between adjacent rare earths were found to increase with the acid concentration in both nitric and hydrochloric media with the larger separation factors in nitric acid. Opposite trends were observed in hydrochloric and nitric acid. In the former case the partition coefficient increased with acid concentration, but in the latter case it decreased. This was explained on the basis of the solvation energies of the cations and anions, the mass action effect and the competitive extraction of nitric and hydrochloric acid. Solvation number studies indicated a 1:1 adduct between MHDPO and the rare earth nitrates. Corresponding studies on the rare earth chrlorides gave anomalous results. It was postulated this was caused by non-equilibrium conditions.

FATTY ACIDS IN DROSOPHILA MELANOGASTER. I. THE QUALITATIVE ANALYSIS OF UNSATURATED FATTY ACIDS IN DROSOPHILA MELANOGASTER.

The pupal lipids of Drosophila melanogaster were mercurated and the unsaturated fatty acids were separated by reversed-phase paper chromatography. Comparative quantitative analyses were made by means of densitometry and by comparison of extinction coefficients of absorption. In all the strains tested, the following acids were commonly found: oleic, palmitoleic, myristoleic, lauroleic, linolenic, linoleic, and arachidonic acids.Within a particular strain, the content of oleic and palmitoleic acids varied with that of linoleic and linolenic acids, respectively. Based on the contents of these unsaturated fatty acids, the strains can be sorted into three groups: group A, the wild types (Tokyo, Oregon, Oregon-RS, Saikyo, and Canton-S); group B, vermilion (v), cinnabar (cn), scarlet (st), clot (cl), sepia (se), and brown (bw); and group C, white (w), along with the compounds v;bw, cn;bw, and bw;st, all of which represent different genetic blocks to eye pigment formation.

The application of thin-layer chromatographic techniques to the analysis of pesticide residues.

The introduction of paper chromatography by Consden et al. (1944) as an extension of the partition chromatographic technique of Martin and Synge (1941) was followed by a very rapid expansion of the use of these procedures. The advantages of paper chromatography in particular, e.g., small sample size, excellent resolution, and short times involved, over earlier separatory methods were so great that the accompanying disadvantages tended to be overlooked. While being admirably suitable for amino acids and hydrophilic compounds in general, many difficulties arose when lipophilic compounds were studied. These were only partially overcome by reversed-phase paper chromatography and although the classical adsorption chromatography introduced by Tswett (1906 a and b) worked very well for materials of this nature, no general analytical technique was available.

A METHOD FOR THE SEPARATION AND ESTIMATION OF NEUTRAL STEROIDS IN THE URINE OF NEWBORN INFANTS.

SUMMARY A method is described for the separation and estimation of neutral steroids in infant urine. The free steroids are obtained by a two-stage enzyme hydrolysis. Following preliminary chromatography, the extracts containing steroids of low and medium polarity are suitable for final resolution. The extract containing steroids of high polarity requires further purification by partition between ether-ethanol and aqueous ammonium sulphate, followed by reversed-phase paper chromatography. Final resolution of steroids in fractions of different polarity is achieved using Bush aqueous methanol systems. Losses incurred in the method are estimated by the use of 4-14C steroids.