The determination and isolation of the organic acids in fruit

Abstract

A new method has been evolved which will separate quantitatively many of the acids commonly found in fruit and if necessary can be readily modified so as to allow the constituent acids to be isolated in a relatively pure state and identified. The essential steps in the procedure are the extraction of the acids from an acidified extract of fruit by 50 % (v/v) n-butanol-chloroform, the preparation of a concentrated solution of the acids in 50 % (v/v) tert-amyl alcohol-chloroforni and their final separation and analysis using a modified partition chromatogram. In each of these steps a column procedure has been used on account of its convenience, mild treatment of the organic acids concerned, and the ease with which the acids can be handled quantitatively. It is necessary to concentrate the dilute solution of the acids to at least 5-10 mg./ml. so that the separation of the acids on the chromatogram will be sharp. The addition of an equivalent amount of the acids in dilute solution gives unsatisfactory separations.In the present study the partition chromatogram used is a very much modified version of that devised by Martin & Synge (1941), the most noteworthy change being the replacement of the aqueous solution of indicator by 0-5N-H2SO4. The bands are then revealed by continuously feeding a small amount of an aqueous solution of an indicator into the solvent emerging from the bottom of the column. 0-5N-H2SO4 is used instead of water in order to depress the ionization of acids such as oxalic and fumaric which would otherwise give very wide bands owing to the rapid increase in the partition coefficient with dilution. EXPERIMENTAL ReagentsPreparation of silica gel. Silica gel prepared from water glass by the method of Gordon, Martin & Synge (1943) has proved to be unsuitable as a supporting medium for the aqueous phase ofthe chromatogram because ofits adsorptive properties, e.g. in the case of oxalic acid the adsorbed acid is held so firmly that it cannot be extracted from the column even if a large volume of solvent is used. Very pure silica gel (Kargin & Rabinovitch, 1935) shows similar properties which suggests that the adsorption is connected with the structure of the gel and not with the presence of impurities.The method described in the present paper for the preparation of a non-adsorbent gel is based upon the fact that treatment with ION-HCl at room temperature for about a day eliminates the ability to adsorb organic acids.The actual procedure consists of three operations.(1) Silica gel substantially free from AIII" and FeIII is prepared from water glass by precipitation with lON-HCl. A large excess of HCI is used and the gel thoroughly washed with 10 N-HCI in order to remove all but traces of All" and Fe"'. Analysis of a dry gel indicated that 0004 and 0.0002 % of Al and Fe respectively remained.(2) The dry gel is allowed to stand for at least 2 weeks. This is necessary because many freshly prepared gels, while showing no adsorptive properties at first, rapidly change in the course of a few days to give gels ...

Organic Acid Metabolism and Ion Absorption in Roots

“…At the end of the CaBr2 treatment, the roots had absorbed a bromide excess of 6.2 meq per kg fresh weight over the Ca absorbed. The total organic acid anion at this point had decreased by 3.7 (6). The ground material was acidified with ed to distilled water for periods up to an additional H2SO4, mixed with silica gel and eluted with 50% urs.…”

Photosynthesis and Metabolism of Organic Acids in Higher Plants

“…This small volume of solvent does not remove the organic acids completely from the silica gel. The succinic and oxalic acid fractions are removed more completely than malic and citric acids; in a second wash the ratio of malic and citric acids to succinic and oxalic acids will be higher than in the initial wash. ISHERWOOD (14) employed a more thorough extraction with the tertiaryamyl alcohol-chloroform mixture and reported almost complete recovery of organic acids. The small volume of solvent was used in our experiments to yield a more concentrated solution for addition to the analytical column and to insure that sufficient organic acids for a C14 analysis would be recovered.…”

“…of silica gel in the column. ISHERWOOD (14) rates the capacity of a 3-gm. column at 10 mg. of mixed acids; we have separated effectively as much as 16 mg. of acids on a 1-gm.…”

“…To the extract in tfie flask was added 1 gmi. of silica gel (prepared according to ISHERWOOD (14)), and extract and gel were mixed by passing them through an 80-mesh sieve. The gel was made into a slurry with about 2 ml.…”

Chromatographic Identification and Determination of Organic Acids in Water

“…Recently numerous studies have pointed to the usefulness of chromatographic separation and identification of many naturally occurring acids (1,2,4,7,9). The technique described here is the method of Bulen, Varner, and Burrell (1) as modified by Mueller,Buswell,and Larson (6) for quantitative identification of volatile acids.…”