40 WATER-SOLUBLE ORGANIC ACIDS 



Lactones may be recognized by their titration behavior. Unlike the free acids they 

 do not neutralize sodium hydroxide rapidly in the cold but do so on heating or with long 

 standing. On reacidifying, the lactone ring usually reforms. Unsaturated lactones of 

 higher plants are reviewed by Schmid (4). 



ISOLATION METHODS 



In the standard procedure for isolating low molecular weight acids, the plant ma- 

 terial is made strongly acidic (pH 1. 0) with sulfuric acid and then extracted thoroughly 

 (sometimes for several days) with peroxide-free ether. The ether extract contains the 

 free acids which may be purified further. This procedure is most conveniently applied 

 to dry material, but the difficulties of drying plant material safely may overcome the 

 simplicity of the extraction procedure. Drying with heat removes volatile acids, destroys 

 keto acids, causes ester formation, etc. Freeze drying is doubtless the best method 

 since it precludes any chemical change, but volatile acids or esters may be lost. If the 

 plant material is neutralized before drying, all acids will be present as non-volatile salts. 

 Interfering lipids and esters may also be removed by a preliminary extraction of the neu- 

 tralized material with ether. 



Rather than ether extraction, the plant acids may be concentrated by the use of 

 anion exchange resins. When an aqueous plant extract is passed through a column of 

 weakly basic anion exchange resin in the hydroxide form, anions are absorbed. After 

 washing the column, free acids may be eluted with 0. 1 N HCl. If several fractions of 

 eluate are taken, some separation of the acids can be achieved. If strongly basic anion 

 exchange resin is used in the hydroxide form, there is danger that sugars in the plant ex- 

 tracts will be decomposed to form such acids as lactic and glycolic. Weakly basic resins 

 avoid this difficulty but have a lower exchange capacity. 



After preparation of a concentrated extract containing the total organic acids by one 

 of the methods described above, separation of the individual components may be undertaken. 

 If the acid mixture has been obtained by ether extraction, it must be transferred from 

 ether to water by shaking with sodium hydroxide solution. Sodium sulfate may be removed 

 by adjusting the aqueous solution to pH 1 H2SO4, adding two volumes of ethanol, and allow- 

 ing to stand in the cold overnight. The solution of organic acids is then separated by filtra- 

 tion from precipitated sodium sulfate. The lower fatty acids up to caproic (Cg) are vola- 

 tile with steam and may be prepared by a steam distillation. Caprylic (Cg) and capric 

 (Cio) acids are slightly volatile with steam but require long periods of distillation for com- 

 plete removal. Separation of the different volatile fatty acids can be achieved by gas 

 chromatography of the mixture or partition chromatography on silica gel using as solvents 

 butanol in chloroform for the Ci - C4 acids and methanol in isooctane for the C5 - C^o acids. 

 Fractional distillation is inefficient for the separation of acids differing by only two carbon 

 atoms and useful only when dealing with rather large quantities of material. 



Separation of the remaining non-volatile acids from each other has often been carried 

 out by converting the free acids to methyl esters and fractionally distilling. It is, however, 

 difficult to achieve complete separations this way unless large amounts are involved. 

 Another method of separation relies on the different solubilities of lead, barium, and cal- 

 cium salts in water and in alcohol; but, again, this method is suitable only if it is known 

 that a limited number of acids is present so that the strategy can be based on knowledge 

 of the solubilities concerned. Special methods for separating individual acids by such 

 procedures will be found in the general references. In particular, oxalic acid may be 

 separated from all others by the great insolubility of its calcium salt in water. The keto 

 acids may be removed from an aqueous solution by adding 2, 4-dinitrophenylhydrazine to 

 precipitate them as hydrazones. The 2, 4-dinitrophenylhydrazones may be extracted with 

 ether and further purified by crystallization or subjected to a chromatographic separation. 



