Test for Bile Acids: Pettenkofer s test for sucrose Materials: concentrated H2SO4 and 0.

5ml sucrose Procedure: A concentrated sulphuric acid was slowly added to the side of a test tube containing 0.5ml of sucrose (5% solution to 2ml of diluted bile acid). The color reaction at the point of contact was observed. Result: (+) Formation of red color at the point of contact Discussion Max Josef von Pettenkofer,was the first toisolate creatinine in urine and determinedits elemental composition. He worked outa method for determining bile acids whichstill bears his name. He first started an attempt to demonstrate that sucrose can be converted to fatin vitro; he combined sucrose, sulfuric acidand bile, which he hoped might containan active principle of the liver, but addedno reducing substance. The bile and sulfuric acid produced a brilliant red or violet colorwhich he used as the basis of the widelyapplied Pettenkofer test for bile acids. There are two main groups of bile acid: the glycoholic and the taurocholic acids. However, there are several varieties of bile acids. The resulting primary bile acid in humans is 3-alpha,7-alpha,12-alpha-trihydroxy-5-beta-cholan-24-oic acid, or as it is commonly called, cholic acid. Inouye and Ito (1908) reported that when vanillin and concentrated sulfuric acid are added to solutions of bile acids a red line is formed at the line of contact. If the fluids are then mixed, the solution changes to a red-brown and then violet. When this solution is diluted with glacial acetic acid, an adsorption band is shown at B. This reaction is sensitive with taurocholicacidin a dilution of 1: 11,000 and with cholic acid 1:22,000. Thus, this red reaction which was formed at the point of contact shows the presence of bile acids. Since, it was proven by Pettenkofer that sucrose can be converted to fat in vitro; bile acid took its action. Thus, the addition of the chemical sulphuric acid shows the positive result on the bile acid s presence by the formation of a red color at its point of contact by the reaction of cholic group of bile to the sulphuric acid.
Sources: JN: the Journal of Nutrition: Max Josef von Pettenkofer. (1989). http://jn.nutrition.org/content/107/9/1567.full.pdf Inouye, K., and Ito, H., 2. physiol. Chem., 1908, lvii, 313.