Microdetermination of Unsaturated Fatty Acids by Alkali Isomerization LEAH COHADAS BERK', NORMAN KRETCIIhlERf RALPH T. HOLMANa, A N D GEORGE 0. BURR' Uninersity of M i n n e s o t a , Minneapolis, M i n n . Alkali isomerization of the polyene fatty acids produces characteristic ultraviolet absorption bands which may be used for quantitative estimations. When ethylene glycol-potassium hydroxide is used as reagent for this purpose, the background absorption produced during the heat treatment becomes a large portion of the total absorption and is variable, prohibiting use of 100-microgram quantities of fatty acids. T h i s difficulty is avoided by the use of aqueous alkali at high temperature and pressure. Analytical data are presented for linoleic, linolenic, and arachidonic acids.
S
ISCE its introduction as the medium for conjugation of
fatty acids (6),ethylene glycol-potassium hydroxide has been widely used in the spectrophotometric analysis of lipides ( 1 , 4,8, 9). Brice and Swain ( 3 )have strongly criticized the use of glycol as the solvent for fats containing very small amounts of polyenoic acids, because of the strong and variable ultraviolet absorption developed on heating it in the presence of air. However, this high and variable background can be reduced by protection of the reagent by an atmosphere of nitrogen (IO). In preliminary experiments with glycol-potassium hydroxide under air using the desired small sample size (100 micrograms), the blank absorption was variable and so high in comparison to that of the isomerized fatty acids that accurate measurement of the unsaturated fatty acids was prohibited. Substitution of glycerol for glycol did not materially improve the results. Another serious disadvantage common to these two reagents is that the low dilutions required for spectral examination in the mcasuremeut of 100-microgram quantities of fatty acids require the measurement of light absorption in very concentrated viscous solutions. In view of the fact that conjugation of fatty acids takes place readily in aqueous potassium hydroxide a t high temperatures (t),this was finally adopted as the solvent for the spectrophotometric determination of 100-microgram quantities of fatty acids. The absorption of potassium hydroxide in water rises rapidly a t wave lengths shorter than 2300 A. A t 2350 A . , the position r of the maximum for conjugated diene, the E! 9,.is approximately 0.05, depending upon the concentration a t which reading is made. This value is highly reproducible. Therefore, if the final reading is made in an alkali concentration of less than 170, the background density is virtually eliminated as a source of error. Absorption due to potassium hydroxide approaches a zero value a t 3000.4. APPARATUS
The equipment consists of a source of high pressure steam, an upright, electrically heated boiler which feeds steam through a needle valve into the horizontal autoclave or bomb shown in Figure 1. The bomb, 10 cm. in diameter and 63 cm. deep, has a metal thermometer, a stainless steel lining, and a stainless steel sliding tray with eight holes of the right diameter to hold snugly the 30-ml. nickel crucibles that act as reaction vessels. The bomb is provided with an inlet copper pipe running the length of the bomb, which feeds steam through small lateral holes spaced throughout the length of the chamber. An outlet drain with a large valve is so placed to permit rapid release of pressure and
drainage of condensate. The front lid is provided with a soft metal gasket and is easily opened and closed to admit samples. Nickel crucibles stand up well under prolonged use. The temperature of the bomb can be easily held within 1' C. by controlling the flow of steam with the needle valve. The bomb is mounted in a sheet iron tray with a water drain, and a spraying device is mounted above the bomb, permitting rapid cooling of the apparatus a t the end of the heating period. PROCEDURES
Preliminary work with linseed oil indicated that maximum conjugation of diene and trieve was reached in 10 to 15 minutes a t 180" C . , using 6.0% potassium hydroxide as reagent. At least 20 minutes were required a t 170" to attain the same degree of conjugation. At 160" conjugation did not reach a maximum in 40 minutes. The effect of potassium hydroxide concentration in the reagent upon the degree of conjugation a t the end of 15 minutes a t 180" is shown in Figure 2. Linolenic acid was used, and it was found that 4 to 6% of potassium hydroxide was required to produce mauimum conjugation, of both diene and triene. The 6% level of alkali was chosen for use in all subsequent studies. Using 6% potassium hydroxide, a time study of the development of chromophores from arachidonic acid was made. Figure 3 shows these results together with the preliminary results on linseed oil. Triene conjugation from linseed oil remains relatively constant and high after 10 minutes' treatment. Diene conjugation, however, reaches a maximum a t 10 minutes and decreases progressively thereafter. With arachidonic acid the characteristic tetraene band increases progressively, but has nearly attained its maximum a t 15 minutes. Triene absorption from arachidonate also reaches its maximum by 15 minutes. A 15-minute reaction time was selected as the best compromise. During the preliminary studies there was considerable variability, which was thought to be due to oxidation of the samples as the carrying solvent was evaporated from the nickel crucibles. A trace of hydroquinone in alcohol, amounting t o 0.1% of the fatty acids, was added to the solution. This reduced the variability among replicates, presumably by its antioxidant effect. Tests showed that this material has no effect on the absorption spectra after the alkali "cook." On the basis of the above preliminary work, the following find procedure was adopted for the study of purified lipides.
,4 sample of fatty acid or oil is weighed, dissolved in Bloor's mixture of purified alcohol and ether (3 to l), and made to a volume that contains approximately 100 micrograms of lipide in each milliliter and hydroquinone in an amount equal to 0.1% of the lipide. One-milliliter aliquot portions of lipide solutions are pipetted into the nickel crucibles and, without evaporation of the solvent, 1 ml. of 6y0potassium hydroxide is added to each. Blanks are prepared containing 1 ml. of Bloor's mixture and 1 ml. of 6% potassium hydroxide, Two blanks and six samples
Present address, i Z 4 South Van Buren St., Green Bay, Wis. Present address, Long Island College of Medicine, Brooklyn, N. Y . Present addrese, Texss A . and M . College System, College Station. Tex. 4 Present address. Experiment Station, Hawaiian Sugar Planters Association, Honolulu, T . H. I
1
718
V O L U M E 2 2 , NO. 5, M A Y 1 9 5 0
719
used, thus increasing the sensitility by a factor of 5. Similarly, with plant tissues containing fatty acids no more unsaturated than linolenic acid, it is possible to work accurately with s a m p l e containing as little as 50 micrograms of either linoleic or linolenic acids. Mixtures of purified linoleic and arachidonic acids were analyzed using this microI 5 method, the 2700 A. band 3 2 1 being used as a measure of arachidonic acid. Results on Figure 1. Bomb or Autoclave Used for Alkaline Isomerization three such mixtures (Table 1. Steam inlet tube with lateral holes 4. Lid with lead gasket 2. Stainless steel tray for crucibles 5. Metal thermometer 11), show method is applica3. Nickel crucibles with covers 6. Stainless steel shield ble to the determination of these acids in mixtures. The constants given in this paper are only,indicative of the type of results possible with the method described. They should not be used as constants with other appazatus or by other investigators who may be working under different conditions. The degree of conjugation of these substances is dependent upon many factors, one of which certainly is the type of construction of the apparatus. During the development of this technique, this was very apparent; the constants fluctuated whenever a change of de-
END
CROSS SECTION
n
\ 4
VIEW
7
\
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I
Table I.
at Maxima of Absorption Bands Used for Analysis
Acid Linoleic Linolenic Arachidonie ~
Figure 2. Effect of Potassium Hydroxide Concentration upon Degree of Isomerization of Linolenic Acid during 15 Minutes' Treatment at 180" C.
(Calculated for free acid) 2350 A. 2700 A.
3025 A.
...
627 487 425
... ...
328 258
63.6
Table 11. Determination of Linoleic and Arachidonic Acids in Known Mixtures Present,
Y
Linoleic Acid Calcd., %
31.6 62.4 93.6
23.6 47.6 73.2
Obsd., % 29 :5 26.7 75.7
Present.
Arachidonic Acid Calcd., Obsd.. Y % %
102.6 68.6 34.3
76.4 52.4 26.8
70:O 47.1 28.0
are run each time. The crucibles are covered with nickel covers. The bomb is closed and steam is allowed to enter slowly until the temperature reaches 100" C. with the outlet valve open for Bushing out condensate, alcohol, ether, and air. The valve is closed and steam let in rapidly until the temperature reaches 180" C., when the timing is started. The temperature is held constant for 15 minutes, when the steam is cut off, the outlet valve opened wide, and the cold water spray turned on. As soon as the chamber is cool, i t is opened and the contents of the crucibles are washed with water into 10-ml. volumetric Basks and diluted to volume. Spectral readings are taken immediately with a Beckman spectrophotometer, for the absorption was observed to decrease over the space of several hours. RESULTS
Typical ultraviolet absorption curves were obtained by this method of alkali conjugation of linoleic, linolenic, and arachidonic acids. Working constants for the absorption a t the maxima produced from these acids are given in Table I. Inasmuch as the practical limits of the method are determined by the densities of the band produced, it is clear that 0.8 mg. of arachidonic acid is required to give a density of 0.5 a t 3025 A., using a 1-cm. cell. However, it is not necessary to dilute to 10 ml. for the readings a t the longer wave lengths. By diluting only to 3.0 ml. and by making readings at densities of 0.1 to 0.2, it is possible to work with samples containing only 60 micrograms of arachidonic acid. In mixtures of linoleic and arachidonic acid (linolenic being absent) the 2700 A . absorption band of arachidonate may be
100
30 MINUTES
Figure 3. Effect of Time of Heating upon Degree of Isomerization with 6.0qo Potassium Hydroxide at 180' C. 1. Conjugated diene from linseed oil
2. 3. 4.
5.
Conjugated triene from linseed oil Conjugated diene from arachidonic acid Conjugated triene from arachidonic acid Conjugated tetraene from arachidonic acid
720
ANALYTICAL CHEMISTRY
sign or repair was made on the bomb. It is recommended that investigators determine their own constants and check these periodically. The technique described was developed for application to the microanalysis of biological matter where only minute samples are available. Emphasis was laid on the determination of linoleic and arachidonic acids, which are important components of animal material. The method does not appear to be useful for the analysis of acids more unsaturated than arachidonic, where another technique is of far greater advantage ( 5 ) . However, the method has been used successfully in the characterization of the lipides of liver particulate fractions ( 7 ) . LITERATURE CITED
(1) Beadle, B. E-., and Kraybill, H. R., J . Am. Chem. S o c . , 66, 1232 (1944). (2) Bradley, T. F.. and Richardson, D., Ind. Eng. Chem.. 34, 237 (1942).
(3) Brice. B. A . , and Swain. AI. R., J . Optical SOC.Am., 35, 532 (1945). (4) Hilditch, T. P.,>lorton, R. A , . and Riley, J. P., Analus(, 70, 62 (1945). (5) Holman, R. T., and Burr, G . 0.. A r c h . B i o c h e m . , 19, 474 (1948). (6) Kass, J. P., Miller. E. S.. Hendrickson. J., and Burr, G. 0 . . Abstracts 99th Meeting CHEM.SOC., p. 11, Cincinnati, 1940. (7) Kretchmer, N.,A r c h . Biochem. (in press). (8) Mitchell, J. H., Jr.. Kraybill, H. R., and Zscheile, F. P., IND. EKG.CHEM.,ANAL.ED., 15, 1 (1943). (9) hlowry, D.,Bmde, W.R., and Brown, J. B., J . Biol. Chem., 142, 671 (1942). (10) O'Connor, R. T.,Heinaelman, D. C., and Dollear, F. G., 01'1 & Soup, 22, 257 (1945). R E C E I V E DAugust 8. 1949. P a r t of thesis presented by Leah Cohadas t o t h e Graduate Faculty of t h e University of Minnesota in partial ftilfillrnent of t h e requirements for t h e M.S. degree. J u n e 1945. The work u a 8 aided by grants froni t h e S a t i o n a l Dairy Council on behalf of t h e Ameriran D a i r y Association and from the National Livestock and Meat Board.
Microdetermination of Formaldehyde with Chromotropic Acid CLARK E. BRICKER AND W. AUBREY V A l L Princeton University, Princeton, N . J . The use of chromotropic acid as a specific reagent for determining formaldehyde has been extended 80 that it can be applied in the presence of large concentrations of various organic compounds. In the presence of chromotropic acid, these interfering organic compounds are removed b y evaporation, whereas the formaklehyde is retained by the reagent. Subsequent addition of sulfuric acid develops the true purple color due to the formaldehyde present.
I
S SEVERAL previous publications ( 2 , 3, i , 8) chromotropic acid, 1,8-dihydroxynaphthalene-3,6-disulfonic acid, has been suggested as a suitable reagent for a spectrophotometric method to determine small amounts of formaldehyde. More recently, Speck (10) found that diacetyl yields formaldehyde when heated with sulfuric acid and applied chromotropic acid to determine this compound. Boos ( 1 ) developed a method for meth:. io1 in which the methanol is oxidized to formaldehyde, which is then determined by this same colorimetric reaction. In endeavoring to determine terminal unsaturation by an indirect chemical method based on a formaldehyde determination, Bricker and Roberts ( 4 ) have pointed out that certain organic substances, such as benzene and acetone, inhibit the color formation of formaldehyde-chromotropic acid. I n order t o eliminate this interference, two color reactions for formaldehyde, Schiff's reagent and the potassium ferricyanidephenylhydrazine method of Schryver (9) were studied extensively. These methods were proved to be no better and in most respects inferior to the chromotropic acid reaction. Furthermore, the volumetric method using the optimum conditions with hydroxylamine hydrochloride (6) was investigated and was found to lack the specificity and sensitivity of colorimetric methods. Consequently, a thorough study of the chromotropic acid reaction was undertaken to improve the specificity and reliability of this reagent for formaldehyde in the presence of organic compounds. All previous methods using chromotropic acid as a formaldehyde reagent have developed the purple color by adding a definite volume of the solution to be analyzed to a solution of chromotropic acid. Sulfuric acid is then added and the resulting solution is heated for about 30 minutes in a boiling water bath. No positive interferences, except diacetyl ( 10) and possibly glycerylaldehyde, furfural, and some sugars ( 5 ) , have ever been reported.
However, if certain organic compounds are present with the formaldehyde, a less intense color is produced, This has been at.tributed to the fact that formaldehyde couples with many compounds in strong sulfuric acid and thereby prevents its color development with the reagent. Because there is no apparent color change when chromotropic acid and a formaldehyde solution are heated, it has been assumed previously that there is no reaction between these two compounds unless sulfuric acid is present. I n this investigation it has been found that chromotropic acid does react with or retain formaldehyde when a solution is evaporated t o dryness. During this evaporation the volatile organic compounds which inhibit the purple dye formation are removed. When the sulfuric acid is added and the resulting solution is heated, the correct amount of the purple dye is produced. RECOMMENDED PROCEDURE
10 mg. of chromotropic acid (obtained from Weigh 100 Paragon Division of The hlatheson Company, Inc.) into a 30-nd. beaker. Add a definite volume of the solution to be analyzed, no more than 1 ml. in volume or 100 micrograms in formaldehyde content. If less than 1 ml. of solution is taken, add sufficient water to make 1 ml. Evaporate the solution to dryness on a low temperature hot plate or in an oil bath whose temperature does not exceed 200" C. and heat the residue for at lesst 5 minutes after the last traces of liquid have disappeared from the sides of the beaker. Allow to cool and then add 5 ml. of concentrated sulfuric acid. Heat the resulting solution in boiling water for 30 minutes. Cool and dilute to 50 ml. with water in a volumetric flask. Allow the diluted solution to reach room temperature and then measure the optical density against a reagent blank a t 570 mp. Several known quantities of formaldehyde are carried through this procedure in order to obtain a calibration curve. Because this curve is linear, the number of micrograms of formaldehyde
