Determination of Fatty Acids by Potentiometric Titration Microgram Analysis BENJAMIN W. GRUNBAU-M, FREDERICK L. SCH-iFFER, AND PAUL L. KIRK Division of Biochemistry, University of California, Berkeley, Calif.
Microgram quantities of fatty acids were titrated with dilute base produced by a n i o n exchange column. End points were determined potentiometrically with a glass electrode i n a carbon dioxide-free atmosphere. .ipplication of the procedure to the analysis of liver fatty acid was tested.
F
AIicrocheiiiical Specialties Co., Berkelev, Calif.) in which were mounted the electrodes and the buret tip, and a capillarv buret ( 4 ) of 0.05-nil. capacity which was supplied with standard alkali from an ion exchange column ( 2 ) . The upper portion of the chamber, holding the calonirl electrode (Beckman No. 270) and the buret tip, nas mounted rigidly on a two-post buret stand ( 2 ) . The buret was mounted on the upright of the stand and was connectrd to the tip assembly bv means of a short length of gum rubber tubing. The lower, movable portion of the chamber held the glass electrode (Beckman No. 290-82) and was provided with a wrll containing saturated sodium hydroxide solution to absorb carbon dioxide in the apparatus a t the time of sealing. The closure of the t n o portions of the chamber was by means of a lightly greased ground joint, which was held together with springs or rubber bands. A wooden block was provided to hold the lower portion in an upright position when it was removed from the upper part. After proper alignment was achieved, the electrodes and buret tip were sealed into the chamber n i t h a cement made of 3 parts of rosin and 1 part of beeswax. The electrodes and the exterior of the buret tip were treated with Desicote ( 1 ) every 2 or 3 weeks. Stirrer. A rotating magnetic stirrer (.A. H. Thomas Co.) was employed. Stirring bars of 5- to 6-mm. length were constructed by sealing fine iron wire into glass capillaries. pH Meter. A Beckman Model G pH meter was employed. The length of the lead of the calomel electrode wa5 increased with ordinary insulated wire to allow easy placement of the meter. The pH meter, the buret stand, and the magnetic stirrer were all connected to a ground wire t o avoid erratic readings. Special Pipet. A special pipet (X), shown in Figure 2, was designed to deliver 25 pl, of fatty acid solution, followed by rinsing with 50 pl. of alcohol. The bore of the pipet beyond the calibration mark terminated a t the bottom of a small glass bulb fitted with a ground joint. A duplicate bulb was also provided. The pipet was filled to the mark and emptied as usual ( 4 ) . Then the second bulb, which had been previously charged with a given quantity of solvent, was attached and while the pipet was held a t a 60' angle, the solvent was expelled through the bore, thus rinsing out any remaining contents of thc pipet.
OR the analysis of lipide fractions from needle biopsy samples
in studies of infectious hepatitis and other conditions i t became necessary to determine minute quantities of fatty acids The total fat content of liver is considerable, but after fractionation of the sample, which is of the order of l-mg. dry weight, the quantities of fatty acids available were in the microgram range. Schmidt-Xielsen ( 5 ) titrated IO-s-gram quantities of fatty acids in alcoholic solution with alcoholic tetramethylammonium hydroxide, using thymol blue as an indicator. It was felt that greater sensitivity and accuracy could be obtained with potentiometric titrations using a glass electrode. A titration assembly has been described by Sisco, Cunningham, and Kirk (6) in which the glass electrode served as the titration vessel. More recently this principle has been applied in the titration of organic acids by Ingold ( 3 ) . The titration assembly described in the present paper provides isolation from atmospheric carbon dioxide and makes use of commercially available electrodes and p H meter.
REAGENTS
Potassium hydroxide solution, carbonatefree, was prepared from 0.001 N potassium chloride on the strong base anion exchange resin (2). Ethvl alcohol, 95%, was twice redistilled and stored over copper pellets. Primary standard solutions were prepared by weighing benzoic acid and potassium acid phthalate and dissolving in 95% ethyl alcohol and water, respectively.
eFigure l. Glass Electrode Titration Vessel Attached to reagent supply and buret
PROCEDURE
The procedure described is for application t o known fatty acids or as the final step in the analysis of tissue samples. The details of preparation of fatty acids of tissue sample3 will be reserved for a future publication on the fractionation of minute tissue samples. For the estimation of the total fatty acid content of liver samples, application was made of the well-known procedure of
Using a rotating magnetic stirrer, alcoholic solutions of fatty acids were titrated with carbonate-free aqueous potassium hydroxide of constant normality provided by an ion exchange column ( 2 ) . The method was applied to pure fatty acids in quantities of 0.005 to 0.025 microequivalent and to the fatty acids from liver digests. APPARATUS
Titration Assembly. The titration assembly, shown in Figure 1, consisted of a special two-piece glass chamber (obtained from
480
Figure 2. S p e c i a l Pipet for Alcoholic Solutions
V O L U M E 25, NO. 3, M A R C H 1 9 5 3
481 solvent. In blank titrations, appreciable amounts of alkali were required for the adjustment to end-point reading. For this reason it was important to use a reproducible volume of alcohol. A preliminary titration was always performed in each series of titrations to condition the electrodes to the alcohol-aqueous potassium hydroxide system. After each titration the electrodes were rinsed with alcohol and water. Flexible plastic tubing of small diameter, connected to an evacuated waste bottle, was used to remove the sample and washings from the glass electrode cup. The results of the titrations were recorded as a plot of pH meter reading versus volume of base. The exact end point was determined after subtraction of the blank curve from the curve for the sample. The mean blank curve was used if different blanks mere found in the same titration series. I n practice, only the endpoint region need be plotted.
40
z 30 0 c 5
-I
0 v,
g 20 Y
v, K
30
w
g t=
i
10
s
z (9
0
/
20
0
5
6 7 8 9 1 0 pH METER READING
I1
8 15 v)
K
p
Figure 3. Titration of Benzoic Acid A . Titration curve B . Blank curve C. Corrected curve
0 0:
2
5
saponification with alkali, acidification, and extraction \\.it11 a water-immiscible fat solvent,
-4stirring bar was first placed in the cup of the glass electrode and then a 25-pl. aliquot containing an appropriate amount of the fatty acid dissolved in 95% ethyl alcohol was transferred to the cup. This transfer v a s accomplished with the special pipet. The empty bulb of the pipet was replaced by the other bulb containing 50 pl. of ethyl alcohol and the pipet was rinsed by expelling the alcohol carefully into the electrode cup. Alternatively, an ordinary 25-pI. transfer pipet ( 4 ) was used and was rinsed carefully with two 2 5 4 . portions of alcohol. The calomel electrode and buret tips were rinsed 11-ith mater and dried with absorbent t,issue, and the previously filled buret was adjusted to the zero mark. The lower ortion of the titration assembly was raised into position and t l e magnetic stirrer, resting on a wooden block, moved as close as possible to the titration assembly. A second stirring bar, placed in the alkali n-ell, kept the surface of the alkali renewed and aided the absorption of carbon dioxide in the chamber. The titration was carried out 11-hile the buret and pH meter readings were recorded, large increments of alkali being added until the end point was ap]roached. S e a r the end point, scale increments of 5.0 mm. (0.62 PI.) \yere generally used. Blank titrations were carried out ir, the same manner, using the s:mr pipcat and alcohol from the same hatch as that used for the
I * Titrations Of 25 Of Fatty Acids 0.00106 ,% Potassium Hydroxide Acid Stearic Palmitic
Theoryo, c
x o . of
Titrationa 5 3
10
3
Found, €
0,0230 0.0250 0.0249 0.0248 0 0125 5 0.0125 0.0050 3 0.0053 Nixed acidab 0.0256 , 3 0.0254 t , microequivalents. b Mixture of stearic, palmitic, myristic, and lauric.
Std. Deviation, e
0.00074 0.00060 0.00024 0.00021 0.00018
5
,
I
6
7
8 9 IO pH METER READING
I
I1
12
Figure 4 . Palmitic Acid Titrations 25-111. samples A . 0.000998 N B . 0.000499 N c. 0.000200 N
The potassium hydroxide produced by the column was stand-. ardized by titration of standard solutions of benzoic acid and potassium acid phthalate. A 25-p1. aliquot of the benzoic acid was titrated as above, except that water was substituted for the alcohol in rinsing the pipet. The phthalate was titrated in aqueous solution RESULTS
Standardization of the base by titration of seven 25-pl. samples of 0.00105 .V benzoic acid gave a value of 0.00107 X, with a standard deviation of +0.000015 .V. Titration of six 5.02-pl. samples of 0.00492 N potassium acid phthalate gave a value for the alkali of 0.00105 =k 0.000014 N . The mcan value of 0.00106 L I T was used. The difference in the values given by the two primary standards cannot be readily explained, but may be due to volumetric errors. Alcoholic solutions, which have a laree coefficient of thermal expansion, were handled a t varying-room temperatures. (Tested p = 0.03 for the probability of such values occurring in random sampling.) On diluting the benzoic acid with water for the titration, and using a similarly treated blank, a well defined titration curve resulted. An esample of such a curve is shown in Figure 3. Replicate titrations were carried out with pure stearic and palmitic acids and with a niisture of stearic, palmitic, myristic, and lauric acids. The results are shown in Table I. Figure 4 s h o w typical titration curves for three different quantities of palmitic acid after correction for the blank. Similar curves were
482
ANALYTICAL CHEMISTRY
Table 11. Titrations of 25 @I. Aliquots of Liver Fatty Acid Preparations with 0.00106 N Potassiuni Hydroxide Sample Sample 1 (liver -4)
mrniJle 2 (liver B)
Sample 3 (liver B) Snmple 4 (liver B)
KOH, pl. 14.6 14.4 14.4 14.3 15.5 15.9 17.2 17.7 15.5 15.8
Liver (Dry), €
0 , 0 13.5 0.01533
.
0.0133 0.0152 0.0164 0.0169 0.0187 0.0188 0.0164
0.0168
./Mg.
0.302 0.298 0.298 0.296 0.288 0.294 0.370 0.382 0.317 0.325
obtained with stearic acid, the mixed fatty acids, and the fatty acids from liver. The results for the titrations of the liver fatty acids are shown in Table 11. DISCUSSION
Standardization Lvith a capillary buret was preferred to the more accurate method using a larger buret (4)because of technical difficult'iei of obtaining and t,ransferring a large volume of base from the column. Because of the ease of production of dilute standard potassium hydroside free of carbonat,e, and the satisfactory results obtained with this alkali with the electrode used, it was chosen in preference to tetramethylminionium hydroxide as used by others ( 3 , 5 ) . The results of the fatty acid titrations showed good agreement \Tit11 each other and with the theoretical quantities. The niinimum quantity of palmitic acid tested by the present method was 1.3 micrograms, which n-as slightly less than the minimum quantity of 1.7 micrograms reported by Schmidt-Xielsen ( 5 ) . Although directly comparable data are not available, the present method appears to yield definitely greater precision than the method of the above author with replicate titrations of small quantities of pure acids. Good reproducibilitj- \\-as shown among the aliquots of the alcoholic solutions of fatty acids from single liver digests, but large variations were shown among the samples of the same liver, as seen in Table 11. These liver digests (in 30% aqueous potassium hydroxide) were prepared for ot,her purposes in the laboratory and the fatty acid titrations were made only as a demon* stration of the technique. Undoubtedljp, the variations were due t o the lack of a tested procedure for handling the preparations. The aliquots taken represent, about 50 micrograms of dry liver.
The p H readings of the meter were used as a matter of convenience without attempting to interpret them in terms of hydrogen ion activity in alcoholic solution. The end point with fatty acids was always a t or near a p H reading of 9.0. It is believed that 0,0050 =k 0.0002 niicroequivalent represents the lower limit of sample size analyzable with the present apparatus. Further reduction might be achieved by the use of smaller, specially constructed electrodes, thereby reducing the volume of the acid solution from the present 75 PI. needed to cover the electrodes. Larger quantities of fatty acids could be titrated by increasing the concentration of the base. A fivefold increase in the concentration should produce little change in the ultimate sensitivity, as the limiting accuracy of the buret was not approached. The present method is by no means limited t o fatty acids, b u t could readily be applied to alcoholic or aqueous solutions of other weak acids or amphoteric compounds requiring titration to albaline pH's where carbon dioxide absorption must be avoided. The disadvantages of indicator titrations, which are generally magnified with very small quantities, such as lack of suitable indicators in certain ranges, large blanks due to titration of the indicator, and personal variations, are avoided by potentiometric titrations with the glass electrode. ACKNOWLEDGMENT
This investigation was supported by the Veterans hdministration under contract VlOOlM-1979, the Office of Saval Research, and the Research Committee of the University of California. The authors are indebted to Wolfgang Schoniger for helpful suggestions and assistance. LITERATURE CITED
(1) Gilbert, P. T.. Jr., Science, 114, 637 (1961). (2) Grunbaum, B. IT., Schoniger, IT., and Kirk, P. L., ASAL. CHEM., 24, 1857 (1952). (3) Ingold. If-,, Afikrochemie, 17, 276 (1951). (4) Kirk, P. L., "Quantitative Ultramicroanalysis," K e x York, John Wley & Sons, 1960. ( 5 ) Schmidt-Xielsen, K.. C o n p t . rend. trar. lob. C'orlsberg, 24, 233. (1942). ( 6 ) Sisco, R. C., Cunningham, B., and Kirk, P. L.. J . Bid. Chem., 139, 1 11941). RECEIVED for review August 1, 1952. Accepted S o r e m h e r 10, 1932. T h e opinions contained herein are t h e private ones of the writer a n d are not to be construed as official or reflecting t h e views of the Savy Department or t h e naval service at large.
Titration of Uranium(1V) by Electrolytically Generated Ceric Ion ?
N. HOWELL F U R X i S , CLARK E. BRICKEK,
ASD ROBERT V. DILTS Frick Chemical Laboratory, Princeton C'nicersity, Princeton, JV.J.
S
EVERAL methods have been proposed for the determination of uranium, both on the macro scale and in the microgram region (18). However, the majority of these methods involve the use of a standard solution and i t would be advantageous to employ a procedure in which this is not necessary. Because coulometric analysis providesa rapid and reliable method that does not require standard solutions, and the range of coulometric titrations has been successfully extended dov-n to the submicrogram range (6),it appeared desirable to use this technique for the microdetermination of uranium. Preliminary studies indicated that uranium( I V ) could be
determined by coulometric titration using electrolytically generated ceric ion in a sulfate medium. The reaction involved is the oxidation of quadrivalent uranium by ferric ion, according to the equation: LT+T+~ + 2Fe+++ = U + + t + + ++ 2Fe-+ (1) This reaction proceeds rapidly and stoichiometrically a t 90" C., but at Ion-er temperatures the end point is reported to be sluggish ( 2 0 ) . It was found that through the addition of a considerable excess of ferric ions the reaction is catalyzed t o such an extent that it can be quantitatively run a t 'room temperature.
