Fractionation methods countercurrent distribution

Partition Methods. Walter (1952) devised a countercurrent distribution procedure for the fractionation of casein in a two-phase system containing water-ethanol-phenol at pH 8.2. The concentration of /3-casein increased in the phenol phase. Ellfolk (1957) employed a two-phase system consisting of collidine, ethanol, and distilled water at 20 °C. The a-caseins were concentrated in the water-rich phase, while the /3-caseins were concentrated in the collidine-rich phase. 

Perpendicular flow occurs in chromatography, countercurrent distribution, field-flow fractionation, and related methods. Below we explain the basic mechanism by which flow assumes its vital role in these separation techniques. 

In attempts to synthesize the oxazolone-thiazolidine of formula 2, by condensing the dimethylcysteine with the appropriate oxazolone, it was noted that the reaction products always showed a very low antibiotic activity though they had properties similar to penicillin. However, at first all attempts to concentrate the activity by various fractionation procedures, including chromatography, failed and it was doubted in many circles whether or not the activity could really be due to penicillin. This uncertainty was resolved by du Vigneaud and co-workers (S) who employed the method of countercurrent distribution (see Chapter I), and were able to isolate a crystalline sodium salt from the mixture. This salt had full antibiotic activity and was identical in every respect with naturally-occurring benzylpenicillin. The synthetic acid was present in the reaction mixture at a concentration of only about 0.1%. 

The colistins—Colistin has been investigated by several authors and various tentative structures have been proposed. This is because colistin is also a mixture of chemically closely related peptides. In 1953 Japanese chemists separated commercial colistin by paper-chromatography into three different components, and Morito reported in 1961 on the separation of colistin into the components A and Aj by countercurrent distribution. In 1963 commercial colistin was fractionated by the same method into three components , which were designated A, B and C and which corresponded to those found earlier - . Colistin C was found only in small quantities and sometimes it was absent in commercial material. 

One hundred kilogram-moles per hour of an equimolar mixture of benzene B, toluene T, n-hexane C6, and n-heptane C7 is to be extracted at 150°C by 300 kgmole/hr of diethylene glycol (DEG) in a countercurrent liquid-liquid extractor having five equilibrium stages. Estimate the flow rates and compositions of the extract and raffinate streams by the group method. In mole fraction units, the distribution coefficients for the hydrocarbon can be assumed essentially constant at the following values. 

Compared with chromatographic methods, this countercurrent distribution method is probably less efficient, the apparatus required is very much more complicated, and the labor involved is considerably greater nevertheless, it possesses several definite advantages over chromatographic methods. The chief of these is the absence of any solid phase, which may act as an adsorbent for the solutes. In partition chromatographic methods this adsorption may often lead to distortion of the bands and render fractionation very inefficient. The behavior of a solute on a countercurrent distribution depends only on its partition coefficient, which in most cases is constant, so that it is possible to calculate the exact theoretical distribution curve and this may be used as a very sensitive test for purity. 

One of the main requirements at present is for methods that will efficiently fractionate large polypeptides with molecular weights between say 1,000 and 10,000. At present the best method for dealing with them is by countercurrent distribution and possibly by certain ionophoretic methods. While techniques for separating this class of substances would be extremely useful, it seems that the chief difficulty is to find suitable degradation procedures for producing a simple mixture of large polypeptides from a protein. 

Infrared spectra are now widely used in the examination of pharmaceuticals. The sixteenth revision of The Pharmacopoeia of the United States (U.S.P.) and the eleventh edition of the National Formulary (N.F.) have presented identification tests which used infrared spectroscopy, whereas no infrared tests were used in U.S.P. XV or N.F. X. Infrared spectra have attained acceptance in legal considerations and are now given in patent applications as characteristics of antibiotics of unknown structure. In the pharmaceutical industry there are many applications for quantitative infrared analyses in research and development work, pharmacy research, and in various phases of pharmaceutical production. For example, infrared data are used to characterize reaction conditions and yields, to assay the purity of intermediate products, to examine such problems as the stability of a drug in the material in which it is suspended, and to maintain quality control in the chemical production of bulk drugs. A recent review (Papendick et al, 1969) has given many references to fractionation and isolation methods for pharmaceutical analysis, such as the various types of chromatography, electrophoresis, countercurrent distribution, and extraction. The authors presented many references to infrared analyses for a wide variety of compounds (Table 16.1). 

Rosenkrantz (1957) has written one of the earlier articles on the utilization of fractionation procedures with infrared analysis and has listed several types of fractionation techniques chromatography, countercurrent distribution, preferential solvent extraction, sublimation, fractional crystallization, molecular distillation, dialysis, centrifugation, electrophoresis, diffusion, and freeze-drying. He has also given references to work in which these methods have been used to fractionate a large variety of biological compounds. Elvidge and Sammes (1966) have discussed many of the techniques mentioned above. 

Some of the substances that have been separated by this method are given in papers referred to by Morris and Morris (1964) amino acids, peptides (particularly those having molecular weights ranging from 500 to 5000), polypeptide antibiotics, proteins (including enzymes), carbohydrates (although for most compounds in this chemical class other fractionation methods are much more frequently applied), purines, pyrimidines, nucleic acid derivatives, tRNA s that are specific for various amino acids, organic acids, steroids, lipids, antibiotics that are not peptides, porphyrins, pterins, vitamin B,2 and other vitamins, lipoic acid, and alkaloids. The countercurrent-distribution procedure of Holley et al. (1965) is widely used, sometimes with modifications. Korte et al. (1965) have separated three isomers of tetrahydrocannabinol. 

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 ... 

The first detailed studies of amino acid sequences were reported by Hilschmann and Craig (8). These investigators had spent considerable effort in learning how to purify Bence Jones proteins by countercurrent distribution. This, however, proved to be unnecessary. An ammonium sulfate fraction of urine, which contains the Bence Jones protein, finally was used. Although such preparations are not completely pure, methods of peptide analysis can often be successfully applied in the presence of small amounts of contaminants. 

Zamecnik and Stephenson (81) carried the approach previously used by Berg and co-workers (cf. 194) one step further by separating the non-esterified, periodate-treated RNA chains from the amino acid-bearing one, which is not attacked. For this purpose the dialdehyde resulting from periodate action was converted into an RNA carbonyl-hydrazide compound, thereby acquiring a rich, bluish-purple color it could then be separated from dye-free RNA by fractional precipitation with n-propanol, or by countercurrent distribution. VaUne-acceptor RNA of a high specific activity was isolated by this method. 

Results of an experiment to fractionate total sRNA from E, coli by the method of countercurrent distribution (Goldstein et al., 1964) are given in Fig. 4. Clear separation into fractions with different amino acid groups can be seen. 

Once the crude alkaloids have been obtained separation methods are applied which take advantage of previous experience and modem methodology such as column, partition or ion exchange chromatography, countercurrent distribution and the like. The results of the fractionation can be followed by all or as many of the physical methods as need be brought to bear to the problem. 

Fractionation methods related to countercurrent distribution have been reviewed and automatic equipment and techniques described (Dutton and Scholfield, 1963 Dutton et al., 1961 Dutton and Cannon, 1956 Scholfield et al., 1961a,b) Scholfield and Dutton, 1958 Scholfield and Hicks, 1957 Therriault, 1963). 

The basis for the second purpose is the use of a two-phase distribution in conjunction with a countercurrent process of some sort, so thar separation may be made in spite of closely related partition ratios. This method may be called Extraction for Fractionation Purposes... 

Example 12.10. Countercurrent liquid-liquid extraction with methylene chloride is to be used at 25°C to recover dimethylformamide from an aqueous stream as shown in Fig. 12.22. Estimate flow rates and compositions of extract and raffinate streams by the group method using mass units. Distribution coefficients for all components except DMF are essentially constant over the expected composition range and on a mass fraction basis are ... 

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