Paper Chromatography of Volatile Fatty Acids

Paper Chromatography of Volatile Fatty Acids

Himansu Mukerjee, University College of Science and Technology, Calcutta, India

URING investigations of the fermen- D tation products of certain algae (Chlorella vulgaris and Chlorella pyre- noidosa), it was necessary to separate and identify very small amounts of volatile fatty acids.

It is convenient to use methanol- ammonia and methanol-acetone-am- monia as solvents for the paper chro- matography of formate and acetate, when the methods of Kennedy and Barker (6), Hiscox and Berridge (5) , and Brown and Hall (8) are followed.

Other methods were less suitable because they required large amounts of material (3) or because the volatile free acids cannot be applied as such to the paper (7'). Conversion into hydroxa- mate (4) or ethyl esters (1) before chromatography is inconvenient.

Formate has been satisfactorily separated from acetate by using methanol-ammonia solvent. Bromo- phenol blue, as used by Kennedy and Barker (6), is a very useful indicator.

EXPERIMENTAL

Khatman No. 1 filter paper was used

throughout. To eliminate the forma- tion of ghost spots, it was washed first with 1% oxalic acid, as recommended by Kennedy and Barker (6), then thoroughly with distilled water, and finally dried a t room temperature before use.

For unidimensional descending chro- matography a t 25-27' C., the follow- ing solvents were used: A, 100 ml. of absolute methanol with 1 ml. of 30% ammonium hydroxide; B, 70 ml. of absolute methanol with 30 ml. of ace- tone and 1 ml. of 30% concentrated ammonium hydroxide solution; and D, butanol saturated with 1.5N ammonium hydroxide.

To the paper was applied 0.01 mi. of an aqueous solution containing 2 to 5pmoles of each acid as its ammonium salt.

Solvents C and D used by Kennedy and Reid (8), respectively, take 6 to 8 hours for development. A and B re- quire only 4 hours.

After development of chromatograms, the papers were dried a t room temper- ature to reduce decomposition of the ammonium salts to the minimum. This takes from one to several hours. The spots were then located 11s spraying the

Table I. I?, Values for Ammonium Salts of Fatty Acids at 28" C. Ethanol- Ammonia Methanol-

Ammonium Methanol- Acetone- New Salts Ammonia Ammonia analysk K

Formic 0 56 0 45 0 28 0 31 Acetic 0 65 0 52 0 30 0 33 Propionic 0 70 0 59 0 44 0 44 Butyric 0 73 0 65 0 51 0 54

Butanol- Ammonia

New

0.09 0.10 0.10 0 11 0.18 0.19 0.24 0.29

analysis R

papers with a solution of bromopheno blue (6).

The indicator solution was prepared by dissolving 40 mg. of bromophenol blue in 100 ml. of 10% aqueous alcohol mixed with 200 mg. of citric acid. In- tense blue spots appear against a pale yellow background.

R; values in different solvents are shown in Table I ; some values by Kennedy (K) and by Reid (R) are included to show to what extent they are reproducible under the conditions used in the present work. The values given are the average of four experi- ments

Methanol-ammonia satisfactorily sep- arates formate and acetate, but methanol-acetone-ammonia separates t8he salts of all four volatile acids.

LITERATURE CITED

(1) Boldingh, J., Discussions Faraday SOC.

(2) Brown, F., Hall, L. P., Nature 166,

(3) Elsden, S. R., Biochem. J. 40, 252

7,162 (1949).

66 (1950).

(1946). (4) Fink, K., Fink, R. M., Proc. SOC. Exptl. Biol. filed. 70, 654 (1949).

(5) Hiscox, E. R., Berridge, N. J., Nature 166,522 (1950).

(6) Kennedy, E. P., Barker, H. .4,, ANAL. CHEM. 23, 1033 (1951).

( 7 ) Lugg, J. W. H., Overell, B. T., dus- traliun J. Sci. Research A l , 98 (1948).

(8) Reid: W. W., Nature 166, 569 (1950).

WORE done during a fellowship tenure at the Research Institutes, university of Chicago. FelE Fund, Chicago, Ill.

Simple Apparatus for Backwashing Chromatographic Columns with Inert Solvents

Ralph L. Oannley and Bernard L. Weigand, Morley Chemical Laboratory, Western Reserve University, Cleveland 6, Ohio

ACKWABHING with water has long B been recommended in the prepara- tion of ion exchange columns to remove fines and provide maximum homo- geneity (1-3), but has not been applied with organic solvents to familiar ad- sorbents such as alumina and silica.

To backwash with an organic solvent. a pump must give sufficient liquid veloc- ity to fluidize the adsorbent. Thesol- vents, because of their cost, should be recycled after removal of adsorbent fines, and contamination should be avoided both to permit re-use and to prevent fouling the column. The apparatus dia- grammed incorporates these necessary features and is easy to construct and simple to operate. The diagram is not to scale, for the only critical size is that

of reservoir C, which must hold enough solvent to flood the entire apparatus while retaining a liquid level immersing the tip of M . Glass wool is used for filtering because it offers little hindrance to liquid flow and may he discarded after use.

Apparatus. The solvent is drawn from reservoir C by pump A (Eastern Industries, Model E-1). This pump has performed very satisfactorily, although it requires priming and a needle valve to regulate the flow of liquid. Sufficient heat is generated to vaporize more vola- tile liquids and cause vapor lock; therefore the solvents are passed through condenser B for cooling. Operation of the pump produces finely divided black particles which are removed by a glass

wool plug in an enlargement of the tub- ing a t G. After backwashing column D, the solvent returns to reservoir C. R'hen it enters the base of C it passes through a main glass wool filter held in place by glass beads. To ensure com- plete removal of adsorbent fines, the exit tube, X, from C also contains a glass wool plug.

Tygon tubing was used extensively in earlier models of this apparatus but extraction of plasticizer from the tub- ing contaminated the solvent. Ball joints a t G, N , and J reduced rigidity of the apparatus to permit almost com- plete use of glass tubing. As the pump causes vibration, however, four Tygon joints. F , were retained to give freedom of movement and simplify alignment. Only inch of Tygon is exposed to solvent and plasticizer extracted from

1284 ANALYTICAL CHEMISTRY