Countercurrent chromatography general

Ito, Y., W.D. Conway, and Y. Ato, Editors High-Speed Countercurrent Chromatography (Chemical Analysis. Vol. 132) John Wiley Sons, New York, NY, 1995. Kenneth, W.W., R,E Davis, and PM. Larry General Chemistry with Qualitative Analysis, 6th Edition, Saunders College Publishing, Philadelphia, PA, 1999. Kohlmann, F. Electrical Conductivity Measurements, in Process/lndustrial Instruments Controls Handbook, D.M. Considine, Editor, 4th Edition, McGraw-Hill, New York, NY, 1993. 

One of the most effective methods for the separation and purification of preparative amounts of dyes and of their subsidiary colours is high-speed countercurrent chromatography (HSCCC) (Conway, 1990 Ito, 1996), both in its conventional and in its modified modes. A general approach to the separation of dyes by HSCCC is presented in Figure 4.2.4. The experimental conditions for this approach were given earlier (Weisz and Ito, 2000). 

Figure 4.2.4 General approach to the preparative separation of dyes by high-speed countercurrent chromatography, (adapted from Weisz and Ito, 2000).

Amino acids have high melting or decomposition points and are best examined for purity by paper or thin layer chromatography. The spots are developed with ninhydrin (see Lederer and Lederer, p.44). Customary methods for the purification of small quantities of amino acids obtained from natural sources (i.e. l-5g) are ion-exchange chromatography (see p. 20) or countercurrent distribution (see p. 28). For general treatment of amino acids see Greenstein and Winitz [The Amino Acids, Vols 1-3, J.Wiley Sons, New York 1961]. 

The main limitation of the technique is the problem of achieving radiochemical, as distinct from chemical, purification of the final labeled product. Purification of the irradiated sample is necessary to remove radiation-induced degradation products and labile tritium. Many of the degradation products are not only chemically similar to the parent compound, but also possess much higher specific activities. For complete purification it is necessary to use multistage processes, such as gas and column chromatography, countercurrent distribution, and fractional distillation. Distribution of isotope within a molecule is generally random and nonuniform however, in some circumstances useful specificity can be achieved.17... 

The Isolation and Identification of free ecdysteroids from Manduca and insects in general has not been too difficult, because we had partition systems that effectively separated free ecdysteroids from their impurities. For example, from processing 130 g of pupae, the ecdysteroids which partition into the butanol phase are now present in only 413 mg of residue (Fig. 3). This material could be further purified by column and thin-layer chromatography and countercurrent distribution. On the other hand, the ecdysteroid conjugates are present in 3.74 g of residue that is water-soluble which presents additional obstacles to further purification. The nature of the conjugation or the impurities present quite often prevented successful column or thin-layer chromatography of the conjugates. More recently, however, a method was described for the separation of... 

A most revealing information that can readily be obtained about a peptide is its amino acid composition. Yet, the results of amino acid analysis are really meaningful only if the sample consists of a single peptide. Analysis of mixtures is usually an unrewarding effort. Thus, purification should precede analysis and this generalization is valid for most other methods of structure determination as well. Homogeneity as a prerequisite of analysis can not be overemphasized. Purification is sometimes possible simply by crystallization but in most cases chromatography, electrophoresis or countercurrent distribution or a combination of these techniques is needed. 

The general theory of countercurrent distribution is still of general interest since manual liquid-liquid extraction remains a common laboratory technique. In addition, countercurrent distribution models often provide a starting point for the explanation of separations by chromatography and continuous liquid-liquid extraction processes. A favorable feature of countercurrent distribution systems is that separations are completely predictable, once the solute distribution ratios and the phase ratio are known. 

The separation of nucleotides and deoxynucleotides, previously a formidable task involving the fractional crystallization of heavy metal and alkaloid salts 102) has been made much easier by developments in analytical techniques. Ion-exchange methods may be used for the purification, isolation, and identification of both classes of nucleotides from hydrolysis mixtures 103), Countercurrent distribution 104) and starch 106) and cellulose-column 106) as well as paper-strip chromatography 107) have also proved to be useful in separating nucleotides from natural sources. Spectro-photometric procedures based on the characteristic ultraviolet absorption spectra of the purines and pyrimidines have been the most convenient method to locate, estimate, and identify the fractions obtained in the previous separations. Since the nucleotides are acid in nature, they are often named as acids, e.g., adenylic acid, cytidylic acid. The general constitution of the purine nucleotides (and by analogy the pyrimidine nucleotides) is demonstrated by their hydrolysis by acids to a purine and ribose (or 2-deoxyribose) monophosphate and by alkalies to the nucleosides and phosphoric acid. The order of the constituents in a purine nucleotide must, therefore, be ... 

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