The German official test for glycerine for pharmaceutical purposes is much more stringent, 1 c.c. of glycerine heated to boiling with 1 c.c. of ammonia solution and three drops of silver nitrate solution must give neither colour or precipitate within five minutes.
~Nitration.~ Fifty grms. of the glycerine are poured from a beaker into a mixture of concentrated nitric acid (specific gravity 1.53) and sulphuric acid (1.84), mixed in the proportions of 3 HNO_{3} to 5 H_{2}SO_{4} (about 400 c.c. of mixed acids). The mixed acids should be put into a rather large beaker, and held in the right hand in a basin of water, and the glycerine slowly poured into them from a smaller one held in the left. A constant rotatory motion should be given to the beaker in which the nitration is performed. When all the glycerine has been added, and the mixture has been shaken for a few minutes longer, it is poured into a separator, and allowed to stand for some time. It should, if the glycerine is a good one, have separated from the mixed acids in ten minutes, and the line of demarcation between the nitro-glycerine and the acid should be clear and sharp, neither should there be any white flocculent matter suspended in the liquid. The excess of acids is now drawn off, and the nitro-glycerine shaken once or twice with a warm solution of carbonate of soda, and afterwards with water alone. The nitro-glycerine is then drawn off into a weighed beaker, the surface dried with a piece of filter paper, and weighed; 100 parts of a good glycerine should yield about 230 of nitro-glycerine. A quicker method is to take only 10 c.c. of the glycerine, of which the specific gravity is already known, nitrate as before, and pour into a burette, read off the volume of nitro-glycerine in c.c. and multiply them by 1.6 (the specific gravity of nitro-glycerine), thus: 10 grms. gave 14.5 c.c. nitro-glycerine, and 14.5 x 1.6 = 23.2 grms., therefore 100 would give 232 grms. nitro-glycerine. The points to be noted in the nitration of a sample of glycerine are: the separation should be sharp, and within half an hour or less, and there should be no white flocculent matter formed, especially when the carbonate of soda solution is added.
~Total Acid Equivalent.~ Mr G.E. Barton (Jour. Amer. Chem. Soc., 1895) proposes to determine thus: 100 c.c. of glycerine are diluted to 300 c.c. in a beaker, a few drops of a 1 per cent. solution of phenolphthalein and 10 c.c. of normal caustic soda solution are added; after boiling, the liquid is titrated with normal hydrochloric acid (fatty acids are thus indicated and roughly determined).
~Neutrality.~ The same chemist determines the neutrality of glycerine thus: 50 c.c. of glycerine mixed with 100 c.c. of water and a few drops of alcoholic phenolphthalein[A] are titrated with hydrochloric acid or sodium hydroxide; not more than 0.3 c.c. normal hydrochloric acid or normal soda solution should be required to render the sample neutral; raw glycerines contain from .5 to 1.0 per cent. of sodium carbonate.
[Footnote A: Sulman and Berry prefer litmus as indicator.]
~Determination of Free Fatty Acids.~ A weighed quantity of the glycerine is shaken up with some neutral ether in a separating funnel, the glycerine allowed to settle, drawn off, and the ether washed with three separate lots of water. The water must have been recently boiled, and be quite free from CO_{2}. All the free fatty acid is now in the ether, and no other soluble acid. A drop of phenolphthalein is now added, a little water, and the acidity determined by titration with deci-normal baryta solution, and the baryta solution taken calculated as oleic acid.
~Combined Fatty Acid.~ About 30 grms. of the glycerine are placed in a flask, and to it is added about half a grm. of caustic soda in solution. The mixture is heated for ten minutes at 150° C. After cooling some pure ether is added to it, and enough dilute H_{2}SO_{4} to render it distinctly acid. It is well shaken. All the fatty acids go into the ether. The aqueous solution is then removed, and the ether well washed to remove all H_{2}SO_{4}. After the addition of phenolphthalein the acid is titrated, and the amount used calculated into oleic acid. From this total amount of fatty acids the free fatty acid is deducted, and the quantity of combined fatty acids thus obtained.
~Impurities.~ The following impurities may be found in bad samples of glycerine:—Lead, arsenic, lime, chlorine, sulphuric acid, thio-sulphates, sulphides, cyanogen compounds, organic acids (especially oleic acid and fatty acids[A]), rosin products, and other organic bodies. It is also said to be adulterated with sugar and glucose dextrine. Traces of sulphuric acid and arsenic may be allowed, also very small traces indeed of lime and chlorine.
[Footnote A: These substances often cause trouble in nitrating, white flocculent matter being formed during the process of washing.]
The organic acids, formic and butyric acids may be detected by heating a sample of the glycerine in a test tube with alcohol and sulphuric acid, when, if present, compound ethers, such as ethylic formate and butyrate, the former smelling like peaches and the latter of pine-apple, will be formed.