42 WATER-SOLUBLE ORGANIC ACIDS 



ture to the paper should carry about 10 - 100 iig of each acid unless very thick filter 

 papers are used. One of the best general-purpose acidic solvents is the upper phase of 

 an equilibrated mixture of l-butanol/90% formic acid/water, 10:3: 10, with the lower 

 phase placed in the chamber. A common basic solvent mixture is 1-propanol/conc. am- 

 monium hydroxide, 7:3 or 3:2. Two dimensional chromatograms are generally run using 

 a basic solvent in the first direction and an acidic solvent in the second, but innumerable 

 combinations are possible. One of the most extensive studies is that of Carles el al.. (8) 

 who chromatographed about sixty acids two dimensionally using as the first solvent 95% 

 ethanol/conc. ammonium hydroxide, 95:5, and as the second solvent 1-butanol/formic 

 acid/water, 4:1:5, equilibrated. Howe (9) has reported the chromatographic behavior of 

 about 100 acids and has attempted to correlate their migration with structures. Even 

 using two-dimensional chromatography some acids are not completely separated from 

 each other, and specific spray reagents may be used to distinguish among the possibilities. 

 The most generally used detection reagents are acid-base indicators such as bromcresol 

 green or bromthymol blue. Background color may be adjusted by exposing the paper sheet 

 to ammonia vapor to make the best distinction of acidic areas. Spots indicated by this 

 method usually fade rapidly. Another general detection procedure has been developed by 

 Burness and King (10). In this method the chromatograms are developed in a solvent con- 

 taining ethylamine. When the paper is dried, ethylamine remains as a salt where acids 

 are present, and may be indicated with ninhydrin. Other general reagents will be found 

 in the paper of Carles et al., (8). Special detection reagents are also available for differ- 

 ent acids or classes of acids. Keto acids can be first reacted with 2, 4-dinitrophenylhy- 

 drazine and the dinitrophenylhydrazones. chromatographed (11). This procedure is better 

 than chromatographing the free keto acids which are subject to decomposition, although 

 reagents have been developed to detect free keto acids on chromatograms. The dinitro- 

 phenylhydrazones can be eluted from the paper and identified by their absorption spectra 

 in sodium hydroxide solution as well as by their Rp values. Other specific reagents will 

 be found described in the general references and in a paper by Buch el al. , (12). 



The gas chromatography of organic acids also promises to be of much value in their 

 analysis. Details of this technique can be found in the chromatographic references cited 

 in Chapter 1. 



METABOLIC PATHWAYS 



A review of organic acid metabolism in plants has been presented by Davies (13). 



As stated in the beginning of this chapter most of the plant acids are either in or 

 closely related to the citric acid cycle. Several other major pathways may be discussed 

 in term of their relationship to the citric acid cycle: 



1. The so-called glyoxylate cycle was first found by Kornberg and Krebs (14) in 

 several microorganisms. Evidence for its occurrence in higher plants has 

 since been presented (15, 16). It provides a by-pass between isocitrate and 

 malate, a route for the synthesis of glyoxylate (and possibly oxalate), and a 

 point of entry for acetate. The fact that acetate derived from breakdown of 

 lipids can enter the glyoxylate by-pass and lead to a net synthesis of pyruvate 

 provides a pathway for the synthesis of carbohydrate from fat since pyruvate 

 can enter the glycolytic pathways leading back to starch. Pyruvic decarboxy- 

 lation which forms acetate plus carbon dioxide is an irreversible reaction. 



2. The dark fixation of carbon dioxide into organic acids accounts for acid synthe- 

 sis in some plants. In particular, the succulents (Crassulaceae) are noted for 

 their accumulation of malic acid at night via this pathway (17, 18). Such CO2 

 fixation is not restricted to the succulents, however, and for example, rhizomes 

 of Equisetum also actively fix CO2 into organic acids (15). 



