Microextraction and Microtitration of Fatty Acids DEWITT STETTEN, JR., AND GODFREY F. GRAIL Department of Biochemistry, College of Physicians and Surgeons, Columbia University, New York, N. Y.
T
HE difficulties inherent in the titration of minute
amounts of the higher fatty acids with alkali arise from two main sources: T h e end point, on the alkaline side of neutrality, is affected b y absorbed carbon dioxide; this is particularly manifest when dilute reagents are used. T h e t i n t of the indicator at t h e end point is dependent upon t h e composition of the solvent, which is usually a n alcohol-water mixture.
TABLE:^.
DUPLICATE STANDARDIZATIONS O F ALCOHOLIC ALK.4LI STEARIC ACIDON THREE SUCCESSIVE DAYS
AGAINST
Stearic Acid
Displacement Mm.
Normality
MQ. A
16.543 10.066
10.80 6.56
B
13.679 13.999 13.131 13.092
9.34 9.54
0.1699 0.1700 0.1625 0.1628
C
9.06 8.99
0.1608
0.1615
The authors’ buret, calibrated according to Scholander, delivered 3.171 X lo-* ml. per mm. displacement. For purposes of standardization, samples of pure stearic acid were weighed on the microbalance into ordinary 15-ml. centrifuge tubes. Duplicate standardizations of alkali on successive days showed a decrease in normality, indicating the necessity of daily standardization (Table I). In view of the fact that this technique was investigated for the purpose of determining the total (free plus esterified) fatty acids in plasma, a liquid microextractor was employed (Figure 1). Into the lower chamber, A , whirh had a capacity of about 5 ml., 13.314 mg. of stearic acid were transferred in 1 ml. of ethanol and 1 ml. of 3 N alcoholic hydrochloric acid was added. In tube B were placed 10 ml. of petroleum ether, previously distilled from alkali, a reflux condenser was attached, and the whole assembly was set on the steam bath to extract for one hour. Tube B was then disconnected, and after evaporation of the petroleum ether, was kept overnight in vacuo over solid alkali, to remove traces of volatile acids. The residue was dissolved in neutralized alcohol and titrated precisely as described above. Standardization of alkali: 15,490 mg. of stearic acid required 10.66-mm. displacement, N = 0.1613. Titration of extract: acid required 9.16-mm. displacement 9.16 X 3.171 X X 0.1613 X 284.3 = 13.32 mg. stearic acid recovered, corresponding to 100.1 per cent
In an attempt to minimize these difficulties, samples of fatty acids weighing 8 to 20 mg. have been satisfactorily titrated, using relatively small volumes of fairly concentrated alkali (0.16 N ) . Both the acid and alkali were dissolved in 90 per cent methanol, to obviate any variation in the composition of the solvent. a-Kaphtholphthalein (S), 0.5 per cent solution in methanol, has proved a satisfactory indicator, and a micrometerdriven microburet of the type described by Scholander ( 2 ) has been used to deliver the alkali. The titration mixture was stirred b y means of a long stainless steel wire fastened to the blade of a n electric buzzer. Alkali, approximately 0.16 X, was prepared by dissolving appropriate amounts of solid sodium hydroxide in freshly boiled 90 per cent methanol. This solution was well mixed and then allowed to stand, to permit settling of the sediment. Solvent for the acid to be titrated was made up by adding 10 drops of indicator solution to 10 ml. of 90 per cent methanol, boiling for a minute or so, and then adding sufficient alkali from t h e microburet to produce an olive greencolor. Two to 4 ml. of this solvent mixture were used to dissolve each sample of fatty acid, and the titration was carried out 9 s rapidly as practicable. The end point taken was the match of t h e original solvent mixture. Fading of the indicator on standing, due to absorption of carbon dioxide, was noted. After a half hour the change in the color of the solvent was so marked as to warrant its being discarded. However, it was felt that only small errors would be introduced i f each titration was carried out t o the same tint as the solvent mixture a t that time. There was abviously no necessity for a blank titration.
FIGURE 1. LIQUID
MICROEXTRACTOR 1
In the analysis of plasma samples, lipides were extracted from 5-ml. samples of plasma by the alcohol-ether method of Bloor (1). The clear alcohol-ether filtrate was taken to dryness under a bell jar in a stream of nitrogen and the residue redissolved in ether. The solution was filtered into a small centrifuge tube and evaporated to dryness. The residue, in 1 to 2 ml. of ethanol, was transferred to chamber A , of the extractor with the aid of a long slender pipet; 0.2 ml. of 2 N sodium hydroxide in methanol was added and the mixture was gently heated on the steam bath until all the solvent had boiled away. Then 0.4 ml. of 2 N aqueous hydrochloric acid and 1 ml. of ethanol were added, tube B containing 10 ml. of petroleum ether was fitted on, and extraction under reflux was carried out on the steam bath. The extract, in tube B, was dried and titrated as described. Duplicate analyses on three different samples of plasma are given in Table 11.
ACIDSI N TABLE 11. DUPLIC.4TE ANALYSESO F PLASXA FATTY THREE DIFFEREKT SAMPLES Sample A B C
Fatty Acids Microequiaalents per m l . 8.31 6.46 7.77
8.30 6.38 7.66
Summary
A method for the titration of 8- to 20-mg. samples of fatty acids has been described. The microestimation of plasma fatty acids is feasible b y this method, in conjunction with a suitable extraction procedure.
Acknowledgment This work WRS carried out with the aid of a grant from the Josiah Macy, Jr., Foundation.
Literature Cited (1) Bloor, W. R.,J. B i d . Chem., 77, 53 (1928). (2) Scholander, P.F., Scieme, 95, 177 (1942). (3) S@rensen,S. P.L.,and Palitzsoh, S., Biochern.
Z.,24, 381 (1910).
April 15, 1943
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