Partition chromatographic separation of aromatic acids


Published in: Journal of the American Chemical Society, 1955, 77, 166-168


During the course of work on the metabolism of dibenzanthracene-9, 10-C14 in mice, purification and resolution of mixtures of radioactive organic aromatic acids was sought through a column partition chromatographic procedure. A survey of the literature2 indicated that little work3, 4 had been done on the separation of aromatic acids (excepting the naturally occurring aromatic amino acids) using this technique. We have found a mixture of 90% aqueous methanol and 0.5 N sulfuric acid (9 : 1) as the stationary phase on a silicic acid column, and ligroin (Skellysolve B) as the mobile phase (both solvents equilibrated with each other), to be satisfactory for the separation of several aromatic acids. The following solvent systems (mobile phase/ stationary phase, each phase equilibrated with the other unless otherwise stated), some of which have been described elsewhere, were tried also but failed to give the desired separation: (a) 35:65 butanol + chloroform/ 0.5 N sulfuric acid 6 ; (b) benzene/ 0.5 N sulfuric acid; (c) 1:1 ethyl acetate + chloroform/ 0.5 N sulfuric acid; (d) butanol/ammonium carbonate + ammonium hydroxide; (e) 1:1 ligroin + benzene/90% aqueous methanol; (f) ligroin/0.5 N sulfuric acid; (g) ligroin/ 90% aqueous methanol, both unequilibrated ; and (h) ligroin (equilibrated) /9: 1 smethanol + a formic-acetic acid mixture (unequilibrated). 3 With these systems, the acids that were tried (benzoic, 2-naphthoic, p-hydroxybenzoic and 2-phenylphenanthrene-3, 2′-dicarboxylic acids) were either eluted with the solvent front (a to e), were not eluted at all (f), or were dispersed too widely in the eluant without any separation and with irreproducible results (g). In case of the last system (h), the acids were eluted along with a very large excess of extraneous acid (presumably acetic or/ and formic) making estimation of the organic acids by titration and adaptation to radioactive metabolites impractical.

We have used two different silicic acid column lengths, 23 ± 0.5 cm. and 51 ± 3 cm. The results were found to be reproducible within ± one 2-ml. fraction, as long as the column length was constant within ±0.5 cm. The quantities used were ordinarily of the order of a few milligrams, the recovery being almost quantitative; when a sample of C14– labelled 2-naphthoic acid was run in microgram quantity, the radioactivity followed the titration curve of the acid eluted when milligram quantities were run. It was of interest to note that when certain insoluble acids (or soluble acids in large quantities) were put on the column as a suspension, they were eluted quantitatively in the same chromatographic fashion as smaller quantities put on the column in solution. It is possible that in such cases, the acid eluted in the peak fractions, passes through the column as a supersaturated solution. An increase of the quantity of acid beyond a certain limit (apparently depending on the solubility of the acid in the external phase solvent), and also an increase in the length of the column, caused an increased dispersion of the acid in the eluant, particularly beyond the peak fraction. When a very large excess of the acid was used, it no longer followed a chromatographic pattern (see Table I).

The results obtained are shown in Table I for acids tried singly and in Table II for mixtures. Even though only a few mixtures of acids were tried, it is apparent from Table I that mixtures of acids could be separated on a small (23 cm.) column; a longer column increases the resolution of mixtures of acids which are eluted in about the same fractions on a shorter column (see Table II, footnote c). Re-use of the same column gave unpredictable results.

Phenylalanine, terephthalic acid and 2-phenylphenanthrene- 3,2′-dicarboxylic acid were not eluted till the 400th fraction on a 23-cm. column, presumably due to their comparative insolubility in the external phase.

DOI: 10.1021/ja01606a050


Partition chromatographic separation of aromatic acids. P M BHARGAVA & C HEIDELBERGER. Journal of the American Chemical Society, 1955, 77, 166-168


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