WATER-SOLUBLE ORGANIC ACIDS 41 



Column chromatography of dinitrophenylhydrazones may be carried out on diatomaceous 

 earth ("Celite") with ethanol in ethyl ether as the solvent or on cellulose powder with n- 

 amyl alcohol saturated with ammonia as the solvent. The instability of many keto acids 

 makes it desirable to separate them as derivatives rather than as the free acids. 



The best, generally applicable procedure for separating and isolating the plant acids 

 from a mixture is column chromatography either using ion exchange methods or partition 

 chromatography. Resins have more capacity per gram than the partition absorbents; but 

 the latter have the advantage that an indicator may be incorporated to follow the passage 

 of acid bands through the column, and they apparently permit somewhat cleaner separa- 

 tions than ion exchange resin columns do. Ion exchange separations may be carried out 

 on strongly basic resins in the hydroxide form if sugars have been removed by a prelimi- 

 nary purification step. No general procedure can be recommended since preparation of 

 the column and the method of elution will vary with the acids to be separated. 0. 1 N 

 hydrochloric acid is often used as an eluant, fractions collected arbitrarily, and analyzed 

 for acids by paper chromatography or specific color reactions. Appropriate fractions 

 can then be pooled and concentrated to obtain the pure acids. Several hundred milligrams 

 of mixed acids can be separated using only about 10 g. of resin. Monocarboxylic acids 

 come through first, followed by di - and tricarboxylic compounds. Further discussion 

 of this type of procedure and its application to plant juices may be found in the papers of 

 Owens et al. (5) and Goudie and Rieman (6). Separations by partition chromatography 

 have used as support for the aqueous stationary phase such substances as silica gel, celite 

 or cellulose powder. It is desirable either to suppress the ionization of the acids by mix- 

 ing some dilute mineral acid into the absorbant or to insure complete ionization of the 

 acids by mixing some ammonium hydroxide with the absorbant. In the latter case an in- 

 dicator such as bromcresol green can also be admixed and changes color where acidic 

 bands are present. As the mobile phase mixtures of 1-butanol and chloroform (saturated 

 with the stationary phase) are most commonly used. The proportion of these two solvents 

 is varied according to the polarities of the acids to be separated, and it is often recom- 

 mended to increase gradually the ratio of butanol/chloroform as the development proceeds. 

 In some cases the use of ethyl ether as a mobile phase separates pairs of acids that can 

 not be resolved by butanol-chloroform. Some pairs of acids tenaciously resist separation. 

 About 100 mg of mixed acids can be separated on a 25 g. column of silica gel. General 

 books on chromatographic methods offer much useful information regarding these tech- 

 niques. See also the paper of Wager and Isherwood (7). 



Paper sheet chromatography (see below) can also be used to purify rather large 

 amounts (up to 100 mg. ) of acids if streaks of the acid mixture are applied to thick sheets 

 of prewashed filter paper. 



CHARACTERIZATION 



Before attempting to identify the organic acids in a plant tissue or crude extract it 

 is ordinarily desirable to make a preliminary separation of the acids from other plant 

 constituents using one of the methods described in the previous section. Certain color 

 tests and precipitation reactions may be used to indicate the presence of certain acids. 

 Since each one of these represents a special case, the general references should be con- 

 sulted for their application. The simplest general method for characterization of a mix- 

 ture of organic acids is paper chromatography, and for best results two dimensional 

 chromatography is recommended. The acids must be converted into a single ionic form 

 to prevent streaking. They can be run as the free acids using highly acidic solvents or 

 as ammonium salts by using solvents which contain ammonium hydroxide. The latter pro- 

 cedure must be used for the volatile acids. If a large amount of oxalic acid is present, it 

 tends to smear and obscure other acids. It may be removed by a preliminary precipita- 

 tion with calcium or by chromatography on acidic silica gel. Application of an acid mix- 



