144 METHODS OF ANALYSIS [Chap. 



stand for at least 2 hours, shaking frequently, centrifugalize if necessary, and 

 filter through a large folded filter. 



(C) Cider containing alcohol, and similar products. — Make 250 cc. of the sample 

 alkaline to litmus paper with sodium hydroxid solution and evaporate on the steam 

 bath to about 100 cc. Transfer the sample to a 250 cc. graduated flask, add 30 grams 

 of pulverized sodium chlorid and shake until dissolved. Dilute to the original 

 volume, 250 cc, with saturated salt solution, allow to stand for at least 2 hours, 

 shaking frequently, and filter through a folded filter. 



(d) Salted or dried fish. — Wash 50 grams of the ground sample into a 500 cc. gradu- 

 ated flask with water. Make slightly alkaline to litmus paper with strong sodium 

 hydroxid solution and dilute to the mark with water. Allow to stand for at least 

 2 hours, shaking frequently, and then filter through a folded filter. Pipette accu- 

 rately as large a portion of the filtrate as possible (at least 300 cc.) into a second 

 500 cc. flask. Add 30 grams of the pulverized sodium chlorid for each 100 cc. of 

 solution. Shake until the salt has dissolved and dilute to the mark with saturated 

 salt solution. Mix thoroughly and filter off the precipitated protein matter on 

 a folded filter. 



8 DETECTION AND ESTIMATION. 



Extract benzoic acid as directed under 2 or 4. If benzoic acid is present in 

 considerable quantity, it will crystallize from the ether in shining leaflets having 

 a characteristic odor on heating. Dissolve the residue in hot water, divide into 

 2 portions, and test according to 9 or 10. 



9 Ferric Chlorid Test. — Qualitative. 



Make the solution from 8 alkaline with ammonium hydroxid, expel the excess of 

 ammonia by evaporation, dissolve the residue in water, and add a few drops of a 

 neutral 0.5% ferric chlorid solution. A brownish precipitate of ferric benzoate 

 indicates the presence of benzoic acid. 



10 Modified Mohler Test.^ — Qualitative. 



Add to the water solution, prepared as described under 8, 1-3 cc. of N/3 sodium 

 hydroxid and evaporate to dryness. To the residue, add 5-10 drops of concentrated 

 sulphuric acid and a small cr3^stal of potassium nitrate. Heat for 10 minutes in 

 a glycerol bath at 120°-130°C., or for 20 minutes in a boiling water bath. The 

 temperature must not exceed 130°C. After cooling add 1 cc. of water and make 

 distinctly ammoniacal; boil the solution to decompose any ammonium nitrite which 

 may have been formed. Cool and add a drop of fresh, colorless ammonium sulphid, 

 without allowing the layers to mix. A red-brown ring indicates benzoic acid. 

 On mixing, the color diffuses through the whole liquid and, on heating, finally changes 

 to greenish yellow. This differentiates benzoic acid from salicylic acid or cinnamic 

 acid. The last two form colored compounds, which are not destroyed by heating. 

 The presence of phenolphthalein interferes with this test. 



11 Quantitative Method. 



Pipette a convenient portion (100-200 cc.) of the filtrate, obtained in 6 or 7, into a 

 separatory funnel. Neutralize the solution to litmus paper with hydrochloric 

 acid (1 to 3) and add an excess of 5 cc. of the same acid. In the case of salted fish 

 a precipitation of protein matter usually occurs on acidifying, but the precipitate 

 does not interfere with the extraction. Extract carefully with chloroform, using 

 successive portions of 70, 50, 40, and 30 cc. To avoid an emulsion, shake cautiously 



