Column chromatography constituents

Partition column chromatography for separating several of the primary constituents of the pyrethrum extract has been reported. The elution pattern for some of the constituents of the pyrethrum mixture recovered from the partition column is shown in Table I. 

Adsorption column chromatography has been employed to separate the constituents of pyrethrum. Florisil and aluminum oxide have been used as adsorption columns to retain much of the pigmented materials. The pyrethroids may be caused to elute with several solvents. In our experience mixtures of hexane with ethyl acetate, methanol, ethyl ether, dichloromethane, or acetone have provided different elution patterns. 

Solvent-partitioned mixtures are fractionated further by column chromatography involving Sephadex LH-20, silica gel, or a combination [19]. Quantitative recovery of constituents is often possible with Sephadex chromatography. With a MeOH/CHCl3 (1 1) eluent, pigmented and other polar material is removed in the early fractions, while the nonpolar isonitrile-related compounds elute later. 

Column chromatography yields quantitative results even for constituents present in only small amounts (with the exception of tryptophan, methionine, and glutamine, in which cases the recovery is not complete see below, (b) Dowex 50-X4 columns). It lends itself also to extraction on a preparative scale for further analysis of a particular constituent it does not require previous desalting of urine. The major inconvenience of ion exchange column chromatography for routine analyses is that the technique is elaborate and time consuming, and requires experienced personnel and expensive equipment. Recent developments, however, have allowed for considerable reduction in time consumption, and the latest completely automatic devices open the way to serial analyses. 

Up to the present time, Moore and Stein s 1954 procedure has given the most satisfactory results, both qualitative and quantitative, for the study of amino aciduria. Paper chromatography is useful as a screening process for routine urine analysis prior to the use of column chromatography for more accurate and complete analysis. It is also most useful for the analyses of fractions containing unknown constituents obtained by column chromatography or to check whether such fractions are single substances or mixtures. 

The content of aroma compounds is, in general, low, and compositions of these compounds are often complex. Therefore, at the dawn of analytical chemistry, aroma compounds were extracted from a huge mass of raw material. Fractionation was carried out by means of distillation, and various other classical procedures (e.g., crystallization, pH control in extraction, derivatization) were employed. Quite obviously, compounds revealed using these procedures were inevitably restricted to a set of major constituents, if any. Occasionally, before the 1950s, additional techniques like UV-IR spectroscopy and open-column chromatography were employed and were helpful to some extent. 

This process of separation, also known as column chromatography, was first developed in 1900by Day, an American petroleum chemist. However, a more extensive study was made in 1906 by Tswett, a Polish botanist. He observed that when a solution of plant pigments in petroleum ether is passed slowly through a column packed with alumina, a number of horizontal bands of different colours are produced in the column. This is, evidently, due to the fact that different constituents of the mixture are adsorbed to different extents. The most readily adsorbed constituent is held at the top. The others with decreasing order of adsorbabilities are held up in different zones down the column in the same order. This, of course, gives only a partial separation of the various constituents as some of the less readily adsorbed... 

In order to separate or to estimate the various constituents, the chromatogram, after development, is pushed out of the glass tube and the various zones are cut with a knife at the boundaries. The coloured components are dissolved separately in suitable solvents. This process of recovery of constituents from the chromatogram is known as elution. Since 1930 column chromatography has been used extensively in the separation and purification of plant pigments and other natural products. It is now frequently used in a number of laboratory processes for purification of several other compounds as well. The substance to be purified need not be necessarily coloured. However, a... 

Baru, M. B., Cherskii, V. V., Danilov, A. V., Moshnikov, S. A., and Mustaeva, L. G. (1995) Automatic SynChrom system for solid phase peptide synthesis and liquid column chromatography. I. Principles of design and structural constituents. Russ. J. Bioorch. Chem. 21, 498-505. 

Relatively few truly automated, high-resolution analytical systems are now used in the clinical laboratory. For this presentation, I have arbitrarily chosen only those systems that use column chromatography for separation. This choice is based not only on the ability of these systems to separate literally hundreds of the molecular constituents in a physiological fluid but also because they are directly amenable to a high degree of automation. Obviously, this latter point is extremely important for any future development in the clinical laboratory. Further, only liquid chromatography will be discussed here since there has recently... 

It was only in 1956, a hundred years after the isolation of taxine, that Graf in WUrzburg showed by electrophoresis that taxine is actually a mixture of several compounds [20], By column chromatography on buffered silica gel he obtained three constituents in a pure state taxine A (1%), taxine B (30%) and taxine C (traces), the alphabetical designation presumably referring to their order of isolation [20]. 

Thus the first level of compositional information is group type totals as deduced by adsorption chromatography form the distribution of saturates, olefins, and aromatics in materials that boil below 315°C (600°F) (ASTM D-1319). Adsorption methods (ASTM D-2007) can also be used to determine hydrocarbon types in kerosene, but, with all adsorption methods, allowances must be made for the loss of volatile constituents during the workup procedure. Thus column chromatography would be best done with a stabilized (volatile constituents removed to a predetermined temperature) feedstock. 

Amino acid content is found by complete hydrolysis of the peptide bonds, separation of the constituent amino acids by column chromatography, and quantitation using reagents such as ninhydrin or dansyl chloride. However, this gives us no information about the N- to C-sequence. 

Removal of non-basic impurities. In the work-up of a crude mixture of water-insoluble alkaloids, isolation procedures are often impeded by the presence of persistent non-basic impurities that are difficult to remove by conventional means, including column chromatography phenols and hydroxylated terpenoid constituents are particularly troublesome in this regard. In many instances, removal can be achieved by prolonged ether extraction of a dilute sulphuric acid solution of the crude bases in a continuous extraction apparatus [128]. In one case this also failed, but a counter-current method referred to later removed the impurity. 

From amongst the numerous applications of adsorption column chromatography, an interesting example is provided by the separation of a mixture of two enantiomers into its constituents whose separation cannot be carried out by the other usual physical methods like fractional crystallization or fractional distillation. A well-known illustration of the separation of enantiomers without their having to be converted into diastereoisomers by chemical reaction with optically active acids is the resolution of Troger s base, effected by V. Prelog and P. Wieland in 1944,... 

Prior to structural elucidation and possible eventual synthesis, the isolation of component phenolic lipids in a pure state is essential. The cold methods of thin layer chromatography (TLC), column chromatography (CC), flash chromatography, high performance liquid chromatography (HPLC) and hot methods (GC), often after derivatisation, are well established. Argentation versions of these separatory methods are less common but are desirable for the rapid separation of unsaturated constituents. 

For biological evaluation larger quantities of component phenols and their constituents may be required and where different components such as alkylresorcinols and anacardic acids co-occur, an initial solvent separation may prove desirable. Mild extraction methods, such as supercritical fluid extraction with carbon dioxide, [1,219,220], followed by phase separation [221], preparative TLC [222], or column chromatography [223] may be valuable for complex mixtures. 

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