Chromatography of Organic Acids with Nonesterifying Solvents RALPH
W. SCOll
Department o f Plant Pathology, University o f Wisconsin, Madison, W i s .
the capillary tube sealed into the chromatographic tube. The column was filled with solvent, and air pressure was applied to sweep air bubbles out of the filter paper disk. The slurry of silicic acid was poured into the glass tube hefore the last of t,ht. solvent used to displace air had passed through the paper disk. The column was packed under 3 pounds per square inch air preisure. The samples were introduced into the column in aqueous solut,ion from a paper disk by a slight modification of an earlier method ( 9 ) . The sample of free nonvolatile organic acids wan generally taken to dryness or near dryness on a spot plat'e. Small volumes of acet'one and a glass tube with a capillary tip were used to transfer the sample onto a 0.03-inch-thick filter paper disk (Eaton-Dikeman). The disk was dried in an air stream and put on the silicic acid column after the organic solvent had juFt drained down to the silica. Any residual sample in the spot plate was then picked up in two successive 0.025-ml. portions of 0 . 5 S sulfuric acid with the same capillary tube used before. Each 0.025-ml. portion was picked up in three or four passes of the capillary and alloTved to drain into the column by resting t h r capillary tip on top of the paper disk. Uneven hydration of the column was avoided by moving the capillary tip about the filter paper disk. The addition of the aqueous phase to the undrrsaturated column facilitat.ed complet,e transfer of the sample. It also decreased the tailing compared to that observed when t!ie acids were displaced from the paper dijk by the eluent. Elution by Changing Solvents. Twenty fractions, about 1 ml. each, were collected from the initial solvent. 25 volume 70, of Pmethyl-2-pentanone in methylene chloride. The remainino; solvent was poured off by inverting the column. The second solvent, 60% of 4-methyl-2-pentanone in methylene chloridr. was used for fractions 21 to 50. The second solvent was siniilady replaced by 100yo 4-met'hyl-2-pentanone, and about 50 additional fract'ions n-ere collect,ed. A time-flow fraction collector was used, and air pressure was maintained (0.75 to 2 pounds per square inch) to collect each 1-ml. fraction in about 2 minutes. The eluted acids were titrated in the test tubes with 0.01.V sodium hydroxide; a stream of carbon dioside-free air was used for mising during titration.
During the chromatography of plant acids by elution analysis, it became desirable to isolate some of the free acids. The simplest procedure was to use an eluent which could be easily evaporated from effluent fractions. For this purpose several ketones were tried as eluents. lIost procedures for organic acid chromatography described eluents containing alcohols, which would esterify the untitrated organic acids upon distillation of the effluent. The ketones in combination with halogenated hydrocarbons gal e good separations of many organic acids. Except for the separation of citric from isocitric acid the ketones were useful substitutes for alcohols. Circular paper chromatographj was used to check the purity and identity of acid fractions. The latter technique was found to be rapid and capable of detecting 0.5 to 1 y of most nonvolatile organic acids. The drying time of the developed paper chromatograms \+asreduced from several hours to 20 minutes by heating the papers at 170" to 180" C.
T
HE use of ketones has been developed for the chromatog-
raphy of organic acids, particularly the nonvolatile acids found in plant tissues. Such acids have often been chromatographpd on silicic acid columns with alcohols in the eluents. It has Iieen ieported that alcohols may esterify small amounts of organic acids during their slow passage through a long column (3)and during the preparation of acid samples (4,8). Ketones have good solvent propel ties without this disadvantage during the separation and subsequent recovely of organic acids. Furthermore, when eluents must be changed during the operation of a column, the concentration of ketone may be increased abruptly without eluting inorganic acid from the internal phase as often happrns when alcohols aIe used (3, 8). Lower toxicity and frequently lol?-ercost also may be cited as advantages of ketones over alcohols. Honever, the ketones did not make some separations as well as alcohols did, particularly the srparation of citric acid from isocitric acid.
Table I.
Representative R,' Yalues of Organic Acids on Circular Paper Chromatograms
Solventsb -~ Acid A B C Oxalic
