Preparative isolation of monogalactosyl and digalactosyl diglycerides by thin-layer chromatography

Monogalactosyl and digalactosyl diglycerides were separated from leaf and Chlorella vulgaris lipid extracts by thin-layer chromatography with Silica Gel G as the stationary phase and acetone-acetic acid-water as the mobile phase. Phospholipids are completely removed and with two-dimensional development can themselves be fractionated.     

Separation of sterol acetates by column and thin-layer argentation chromatography

Column and thin-layer chromatographic systems employing silver nitrate-impregnated adsorbents are described for the separation of sterol acetates which differ in the number of double bonds in the steroid nucleus or side chain.     

Preparative scale separation of neutral lipids and phospholipids by centrifugally accelerated thin-layer chromatography

Mixtures of lipids and phospholipids were separated by centrifugally accelerated thin-layer chromatography on a preparative scale (300-500 mg lipid mixture per run). The isolated lipids and phospholipids were identified by 1H and 13C NMR spectroscopy and their fatty acid composition was determined by GLC and GLC-MS of their methyl esters.     

The behaviour of the pulmonary metabolites of prostaglandins in several simple thin-layer chromatography and bioassay systems

The behaviour of the pulmonary metabolites of prostaglandins E₁, E₂ and F_2α were examined in several thin-layer chromatography (TLC) systems commonly used to differentiate parent prostaglandins. Although the systems chosen readily distinguished between a prostaglandin and its own metabolites, they often did not differentiate between a parent prostaglandin and the metabolites of another. In particular, 13, 14-dihydro-PGF_2α was virtually indistinguishable from PGE₂, and 13, 14-dihydro-PGE₂ was similarly indistinguishable from PGE₁, in all systems investigated. These pairs of prostaglandins could not be distinguished by bioassay on the rat stomach strip alone. Although distinction could be made by parallel assay on the rat stomach strip, chick rectum and rat colon, the differential assay obtained would not be enough to allow identification of these prostaglandins and metabolites in samples containing their mixtures. The 13, 14-dihydro-prostaglandin metabolites were also active in contracting the isolated rat uterus. The findings indicate that TLC and bioassay together may not permit identification of prostaglandins in biological fluids.     

Preparative separation of nebivolol isomers by improved throughput reverse phase tandem two column chromatography

Development of preparative methods for the isolation of chiral molecules has been considered challenging by conventional unit operations due to their identical physical and chemical properties. This has evolved chiral stationary phases for the separation of chiral components using chromatography technique. However, separation method using chiral adsorbents requires high pressure, are expensive, and have low productivity. Generation of bulk quantities purified nebivolols using the available high pressure chiral separation methods is impractical and operating cost-intensive. Thus, there is a need to develop economical methods using nonchiral adsorbents for the purification of nebivolols or similar active ingredients. The present work demonstrates a unique and scalable tandem two-column method for the separation of isomers of nebivolol using inexpensive reverse phase adsorbents. The first column of the scheme causes removal of charged and nonisomeric impurities whereas tandem operation of second column increases resolution of d-nebivolol and l-nebivolol. The maximization of separation due to tandem operation of second column causes enhancement of the throughput of the process. The developed preparative process produces >98% purity of both d-nebivolol and l-nebivolol with overall loading capacity of 56 g (L of adsorbent)⁻¹ and productivity of 20 g L⁻¹ day⁻¹.     

Separation of prostaglandins A, B and C by thin-layer chromatography on ferric chloride impregnated silica gel

A convenient thin-layer chromatographic method for separating and isolating the isomeric prostaglandins A1, B1, and C1 under non-alkaline conditions was developed as a prerequisite for the in vivo investigation of the metabolism of PGA1. The method was based on thin-layer chromatography on ferric chloride impregnanted silica gel. The resolution of prostaglandin A from prostaglandin B proved to be superior to that on silica-gel thin-layer chromatography plates impregnated with silver nitrate. Thin-layer chromatography has the advantages of simplicity, speed, and amenability to radioactivity scanning. The method should find application in the separation of other prostaglandins, as well as a number of other classes of compounds.     

Preparative fractionation of DNA restriction fragments by high pressure column chromatography on RPC-5.

High pressure liquid chromatography on the RPC-5 reversed-phase ion exchange system has been shown to have several potential applications as an initial high capacity step in the isolation of specific DNA restriction fragments. The fractionation of the Hinc II digest of lambda DNA, which contains 35 fragments with "flush ends" ranging in size from 3 x 10(6) to 7 x 10(4) daltons, has been used as a model system. Under certain conditions there are some restriction fragments whose elution relative to other fragments is different on RPC-5 chromatography than it is on gel electrophoresis. In some special circumstances it is possible to obtain satisfactory yields (60-70%) of a pure restriction fragment after a single passage through an RPC-5 column.     

Preparative chromatography and natural products discovery

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Preparative and analytical size-exclusion chromatography of chitosans

Two chitosan samples (fraction of acetylated units (F_A) 0.15 and 0.52) were fractionated by preparative size exclusion chromatography (SEC). The molecular weights and molecular weight distributions of the fractions were analyzed by analytical size exclusion chromatography coupled to an on-line low angle laser light scattering detector and a differential refractive index detector (SEC-LALLS-DRI), and their intrinsic viscosities were determined. The exponent (a) of the Mark-Houwink-Kuhn-Sakurada (MHKS) equation was found to be 0.92 ± 0.07 and 1.1 ± 0.1, respectively, at I = 0.1 and pH 4.5. No variation in F_A related to molecular weight was found. Reversible interaction between chitosans and different column packings strongly influenced the log M-V relationships. This interaction was generally most pronounced for the low-F_A chitosan, suggesting that the protonated amino groups are involved. Ammonium acetate buffer reduced this effect and the use of a new type of SEC-packing seemed to eliminate it. The more highly acetylated chitosan also had a more pronounced tendency towards concentration dependent self-association, which most probably involve intermolecular hydrophobic interactions between the acetyl groups.     

Preparative fractionation of lignin by gel-permeation chromatography

The combination of an agarose gel (Bio-Gel A) and a dioxane–water (1:1) solvent system allowed the fractionation, on a preparative scale, of a very polydisperse, non-derivatized lignin preparation (enzymatically liberated lignin prepared from sweetgum sapwood with Lenzites trabea). Three fractions differing markedly in molecular weight were obtained. A gel of crosslinked alkylated dextran (Sephadex LH-20) with the same solvent system allowed division of the lowest molecular weight fraction into two fractions. These materials were characterized by measurements of intrinsic viscosity and number-average molecular weights in dimethylformamide and dioxane–water. It was established that the two highest molecular weight fractions were associated in an average trimeric form in dioxane-water (1:1) as compared to the form (considered to be molecular) that occurred in dimethylformamide. Molecular size distributions and eluant volumes of the fractions were determined with a Sephadex G-100–formamide system, the latter being one of the most powerful nonaqueous solvents for lignin. Adsorption effects were known to be absent in this case, and the lignin molecules were considered to be unassociated in formamide. The four fractions were distinguishable with the formamide–G-100 system, thus indicating that the original fractionation was based on molecular size. The enzymatically liberated lignin contained molecules that comprised a continuum of molecular weights from approximately monomeric to molecules that were at the limit of the solvating power of dioxane–water (1:1) and dimethylformamide. Limited physicochemical data were consistent with a compact, approximately spherically symmetric shape of the lignin in solution.