Separation of digoxin and dihydrodigoxin by thin-layer or paper chromatography.
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A simple one-dimensional paper chromatograph technique is described for the separation of radioactive histamine and some of its metabolites in blood and tissues. After removal of protein, the supernatant fluid (200 microliter) is subjected to two-stage one-dimensional paper chromatography. This enables large numbers of samples to be chromatographed simultaneously. Using this technique in conjunction with liquid scintillation counting, it was possible to monitor the small amounts (0.02 nCi) of radioactive histamine and its metabolites in serial blood samples.
Bovine pineal glands were extracted according to the methods reported by Bensinger et al. (1973) and Cheesman and Fariss (1970). Isobutanol soluble COH-inhibiting activity was further separated by chromatography on Sephadex G-15 and paper chromatography. With the Bensinger method, different active pineal fractions were obtained from Sephadex G-15 columns. Certain of those fractions were further separated by paper chromatography in butanol : acetic acid : water (4 : 1 : 1) and the COH-inhibitor was localized. The pineal COH-inhibitor could also be localized by high pressure, reverse phase liquid chromatography. More COH-inhibiting activity was extracted with the Bensinger method than with aqueous and acetic acid extraction methods used earlier by us. The Cheesman extraction method for arginine vasotocin gave less regular results in our hands than the Bensinger extraction method.
Acetic acid extracts of bovine pineals and cerebral cortex were separated on Sephadex G-25 columns. Subsequently two low molecular weight fractions, F2 and F3, were ultrafiltered through the membranes UM2 and UM05. The UM05 residues were gel filtered on Sephadex G-15 columns or chromatographed on Dowex W50-X4 columns. Fractions from these columns were tested and those which showed COH-inhibiting activity were separated by preparative paper chromatography in different solvents. The absorption spectra of those fractions were recorded and tested for COH-inhibiting activity were separated by preparative paper chromatography in different solvents. The absorption spectra of those fractions were recorded and tested for COH-inhibition. By these methods, a COH-inhibitor was localized in three different solvents. Some active paper chromatography fractions were studied in high pressure, reverse phase, liquid chromatography. This latter method showed that the active fractions obtained by paper chromatography contain several orthophthlaldehyde (OPT) positive compounds. Key words: acetic acid, bovine pineal extracts, compensatory ovarian hypertrophy (COH), paper chromatography, high pressure, reverse phase, liquid chromatography.
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A group of moderately polar C21 steroids (3-5 oxygen functions) has been chromatographed in 9 solvent systems. Using the concept that standard deviation of the mean RF is an index of chromatographic resolution, and that coefficients of correlation between sets of RF data can be used to quantify the similarities of chromatographic systems, we have evaluated the resolving properties of the systems when used individually, and also when used in combinations of two, three and four. The discriminating powers of some of the most effective individual systems, and some of the sequences of systems which are most efficient, are shown graphically as chromatography trees. The relationship between the total effective standard deviation of a group of systems which are used in sequence and the probability that a pair of compounds will be separated by more than 0.10 RF is discussed.
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