METHODS OF SOLVENT DETECTION AND TESTING

Chemical separations may first be accomplished by partitioning on the basis of polarity into a series of solvents from non-polar hexane to very polar compounds like methanol. Compounds may also be separated by molecular size, charge, or adsorptive characteristics, etc. Various chromatography methods are utilized, including columns, thin layer (TLC) gas-liquid (GLC), and more recently, high pressure liquid (HPLC) systems. HPLC has proven particularly useful for separations of water soluble compounds from relatively crude plant extracts. Previously, the major effort toward compound identification involved chemical tests to detect specific functional groups, whereas characterization is now usually accomplished by using a... 

The reaction with permanganate constitutes a valuable and much-used test for unsaturation in an organic compound. The substance is dissolved in cold alcohol, a few drops of sodium carbonate solution are added, and then a drop.of dilute permanganate solution. Rapid disappearance of the red colour indicates the presence of a double bond. The Baeyer test can also be carried out in pure glacial acetic acid, which is stable towards permanganate. Another method of detecting double bonds is by the decolorisation of bromine. As a rule, chloroform is used as solvent. 

Based on the 96-well format, OCT-PAMPA was proposed and has proved its ability to determine (indirectly) log Poet [87]. PAM PA is a method, first developed for permeability measurements, where a filter supports an artificial membrane (an organic solvent or phospholipids) [88, 89]. With this method, the apparent permeability coefficient (log P ) of the neutral form of tested compounds is derived from the measurement of the diffusion between two aqueous phases separated by 1-octanol layer (immobilized on a filter). A bilinear correlation was found between log Pa and log Poct> therefore log Poet of unknown compounds can be determined from log Pa using a calibration curve. Depending on the detection method used a range oflog P within —2 to +5 (with UV detection) and within —2 to +8 (with LC-MS detection) was successfully explored. This method requires low compound amounts (300 pi of 0.04 mM test compound) and, as for the previous method, samples can be prepared in DM SO stock solutions. For these experiments, an incubation time of 4h was determined as the best compromise in term of discrimination. The limitation of the technique lies in the lower accuracy values... 

If we measure a residual current-potential curve by adding an appropriate supporting electrolyte to the purified solvent, we can detect and determine the electroactive impurities contained in the solution. In Fig. 10.2, the peroxide fonned after the purification of HMPA was detected by polarography. Polarography and voltammetry are also used to determine the applicable potential ranges and how they are influenced by impurities (see Fig. 10.1). These methods are the most straightforward for testing solvents to be used in electrochemical measurements. 

Method. 0.2 ml of an ethanolic solution of hydrochloric acid (0.65 ml of concentrated hydrochloric acid per litre of absolute ethanol) is added to the dry keto steroid in a small test-tube [103,104]. 0.2 ml of a solution of DNS-hydrazine (2 mg/ml in absolute ethanol) is then added. The contents of the test-tube are heated in a bath in boiling water for 10 min for hydrazone formation. The tube is cooled and 0.2 ml of sodium pyruvate (5 mg/ ml in absolute ethanol) is added to destroy the excess of DNS-hydrazine. The tube is permitted to stand at room temperature for 15 min. 6 ml of diethyl ether and 3 ml of Q.5-N aqueous sodium hydroxide are then added and the tube is shaken. The diethyl ether layer is removed and evaporated to dryness. The residue is dissolved in a small volume of chloroform (0.2-0.5 ml) for TLC analysis. The keto steroid derivatives are separated on layers of Alumina G (Woelm) (thickness, 250 fim) which have been activated at 120 °C for 30 min. The solvent consists of dioxane-chloroform (1 9). The separated derivatives are observed under UV light at 366 nm. The limits of detection are in the 1-2 nmole range for each steroid. 

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