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Glycosides Analytical methods 1.

Separation Procedure a)Extraction separation of glycosides from such substance as tablet excipients( starch, lactose, sorbitol) may take advantage of the solubility of the desired material in water immiscible organic solvent. b)paper chromatography applied to separation of small quantity of glycosides. This technique used to advantage in establishing criteria of purity for the more highly purified glycosides such as digitoxin. Solvent systems are used as separating media such (29,79). There are also paper partition methods using chloroform and saturated formamide. The example used here is Digitoxin Identification of Digitoxin For the test the sample (2mg.in 5ml.0f chloroform-methanol (1:1))and standard (same concentration) are compared by placing 10mcl. Aliquots of each as separate spots on a filter paper strip, approximately 10 cm from one end. The chromatography is developed in a closed chamber according to the descending technique. For instance the end strip nearest the sample is placed in a trough containing equilibrated solvent,and the through is located in the upper part of the chamber so that the solvent may flow downwards through the paper. To maintain a saturated atmosphere, small amountof both phaseof the equilibrated solvent is placed in the bottom of thechromatographic chamber When the solvent reaches the point within 5cm. of the bottom edge of strip,the chromatogram is removed and allowed to dry in air. Location of glycosides zones in both sample and standard accomplished by spraying the strip with a 25% solution of trichloroacetic acid in methanol, followed by heating at 100 C for 1 minute. The presense of digitoxin in the sample is seen by the colour of grayish green spot having an Rf value as the spot appears in standard.

Paper chromatography It showed that a ghost spot remained on the origin. That this was nt due to an impurity, and later identifitied was due to a partially irrrevisible absorption of solute on cellulose phase when mobile solvent was not present. (Hassall and Magnus) Fluorometric reaction to Paper chromatography

Separated glycosides and aglycones of Digitalis purpurea. The fluorescence was elicited by adding hydrochloric acid and hydrogen peroxide and its intensity showed for digitoxin concentration. Paper partition chromatography Separation with formamide-impregnated paper. Position of digitoxin and digitoxose used 2 sprays. Keller- killiani reagent (ferric chloride with sulfuric and glacial acetic acids) identify digitoxin . c)adsorption chromatography d)column partition chromatography separation and analysis of digitoxin The method of Banes and Carol No correalation existed between biological and chemical analysis. Sample contained 62,8% digitoxin and 37,2% gitoxin total 100% U.S.P. XIV chemical analysis value 99% and it is said to be pure or a mixture of cardioactive glycosides consisting chiefly of digitoxin.(nt less than 95% by assay)

Hazard et al No correalation btw certain physical properties of digitoxin and the biological assay

Colour reactions of cardiac glycosides Keller- Killiani reaction is available. Reaction with digitoxose by measuring colour intensity at 600mu %purity of digitoxin = (A(sample)/A(standard))X 100

Determination of gitoxin in a mixture of cardiac glycosides Offers a simple fluorometric method to measure gitoxin in caidiac glycosides. Advantage is increased conjugated ynsaturation which results in dehydration of gitoxigenin to form dianhydrogitoxigenin. Other analytical methods Spectrophotometry value in identification and determination of glycosides Electrochemical methods useful in determination