George H. CurtisEdward S. West, Charles L. Hoagland and  HYDROXYLATED FATTY ACIDSVALUES OF LIPIDS APPLICABLE TODETERMINATION OF ACETYL AN IMPROVED METHOD FOR THEARTICLE:

1934, 104:627-634.J. Biol. Chem. 

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AN IMPROVED METHOD FOR THE DETERMINATION OF ACETYL VALUES OF LIPIDS APPLICABLE

TO HYDROXYLATED FATTY ACIDS*

BY EDWARD S. WEST, CHARLES L. HOAGLAND, AND GEORGE H. CURTIS

(From the Laboratory of Biological Chemistry, Washington University School of Medicine, St. Louis)

(Received for publication, January 10, 1934)

None of the methods that have been proposed for the determina- tion of the acetyl values of lipids is generally satisfact.ory and none is applicable to free hydroxylated fatty acids. Benedict and Ulzer (1) first proposed a method in which the insoluble fatty acids of a fat are acetylated and the acetyl value determined by estimating the KOH bound before and after saponification of the acetylated acids. Lewkowitsch (2), however, showed this method to be in- accurate and to give high results by demonstrating that non-hy- droxylated acids, as palmitic, stearic, and oleic, give considerable acetyl values when treated according to Benedict and Ulzer’s method. This he found to be due to relatively stable mixed an- hydrides formed by boiling the acids with acetic anhydride. Lew- kowitsch (3) then proposed the method which in essentials is the one most commonly used at the present time, and which consists in acetylating the fat (or other lipid, not fatty acids) by boiling with acetic anhydride, removing excess anhydride by boiling with water, and determining the acetic acid liberated (as mg. of KOH equivalent) per gm. of acetylated material. A determination of volatile acids must be run on the original unacetylated material and subtracted from the above results to give the so called true acetyl value. The method is laborious and time-consuming as well as subject to a number of errors. If the material acetylated contains free fatty acids, the results may be too high, as in the

* Presented in part before the meeting of the American Society of Bio- logical Chemists at, Cincinnati, April, 1933.

627

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Acetyl Values of Lipids

method of Benedict and Ulzer. The prolonged boiling of the acetylated product with water may lead to hydrolysis and loss of acetic acid with low results (4). The titrations of small amounts of acid in the large volumes required are not accurate. This is an important source of error when the acetyl value is low and the volatile acid correction of rancid oils may be equal to or greater than the true acetyl value.

Roberts and Schuette (4) have recently published an acetyl method which consists in heating the sample with a weighed amount of standardized acetic anhydride in a sealed tube at 120’ for an hour, followed by refluxing at reduced acidity, and final ti- tration with alkali. The writers have found the method tedious and have been unable to obtain consistent results with it.

The evident need, stressed by Andrews and Reed (5), for a simple accurate method applicable not only to fats, waxes, and sterols but also to free hydroxylated fatty acids has prompted the writers to devise the procedure reported below. It is being used in this laboratory in a study of the distribution of hydroxylated fatty acids in the animal body and their metabolism.

Verley and Biilsing (6) determined the hydroxyl groups of alco- hols and phenols by acetylating with acetic anhydride and pyri- dine, decomposing excess anhydride with water, and titrating with aqueous alkali. Peterson and West (7) showed the method to be applicable to hydroxyl groups of sugars and sugar derivatives. A thorough study of the method in attempts to apply it to accurate determination of the acetyl values of lipids in this laboratory has failed, except with castor oil and some of the waxes. The failure was found to be due to the presence of free fatty acids in the acety- lated mixtures which could not be efficiently removed by filtration and which do not titrate accurately in aqueous solution, especi- ally in the large volumes necessary. These difficulties have now been overcome in the procedure outlined below.

The method consists in acetylating the sample with a measured quantity of a mixture of acetic anhydride and pyridine (either hot or cold), decomposing excess anhydride with a little hot water, and titrating with alcoholic alkali after the addition of sufficient butyl alcohol to give a homogeneous solution. The acidity of the material is determined in the same way on a sample treated with pyridine only and this value is subtracted from the above titration.

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West, Hoagland, and Curtis 629

This result subtracted from the titration of a blank on t.he acetic anhydride-pyridine mixture gives the necessary data for calcu- lating the acetyl value. The titration of all of the acids, both acetic and higher fatty acids, is very accurate w’hen carried out in the butyl alcohol solution with alcoholic alkali. A large number of determinations may be run simultaneously and the execution of the method leaves little to be desired from the standpoint of sim- plicity, accuracy, and time required. Preliminary experiments indicate that the method may be applied satisfactorily to semi- microquantities.

The definition of acetyl value as “the milligrams of KOH re- quired to neutralize the acetic acid from 1 gm. of acetylated fat”

TABLE I

Acetyl Values of Lipids

Substance

Castor oil.. Cottonseed oil Linseed oil.. . Croton oil. Neat’s-foot oil. Coconut oil

-

, I

c

-

- Acetyl

value by the Lew- kowitsch letinition

146.1 5.5 6.3 8.3

20.0 4.4

d -

-

Acetyl value by xoposed efinition

126 4.3 5.0 6.4

15.4 3.4

I *

-

Substance

Olive oil.. Carnauba wax. Cholesterol. Ricinoleic acid., Lithium lactate..

-

(

-_

-

3.5 41.2

130.9 160.1 395.3

I

,d --

-

Acetyl ralue by mpoacd efinition.

2.7 32.6

111.5 134.7 431.7

* According to the proposed definition, acetyl value = mg. of acetyl (CH&O) taken up per gm. of lipid = (titration difference (cc. of 0.1 N

alkali) X 4.3)/(weight of sample in sm.).

is not satisfactory as a general definition. It has specific refer- ence to the Lewkowitsch procedure of determining the acetyl value, and the necessity of calculating the weight of acetylated material when other methods are used is an unnecessary incon- venience. Calculation of the number of hydroxyl groups in a compound of known molecular weight from such acetyl values is

also somewhat complicated. The writers propose as a more suit- able definition of acetyl value, the mg. of acetyl taken up per gm. of substance. Such values have a definite chemical meaning which should be applicable to all methods of determination and easily used in related calculations. A comparison of values according to the present and proposed definitions is given in Table I.

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630 Acetyl Values of Lipids

Methods

Reagents Pyridine. Mallinckrodt’s medicinal grade is dried by refluxing

for several hours with good barium oxide and then distilled. The fraction passing over above 114” is used.

Acetic anhydride, Mallinckrodt’s reagent quality, redistilled. Alcoholic NaOH. This is prepared by dissolving sufficient 60

per cent aqueous NaOH (60 gm. per 100 cc.) in 95 per cent alco- hol to make a 0.3 to 0.35 N solution. The precipitated carbonate is easily removed by adding a little norit, shaking, and filtering. It should be standardized against standard acid (phenolphthalein) daily. The solution remains colorless for a long time if kept be- low 25”.

Butyl alcohol, ordinary commerical butyl alcohol (Commerical Solvents Corporation). It generally contains a trace of acid but this is taken care of in the blank titration.

Procedure

Two samples (0.5 t,o 1.0 gm. of materials with high acetyl values, as castor oil, and 2 to 3 gm. of those having low acetyl values, see Table 11) of material are weighed into each of two 250 cc. ground glass-stoppered Pyrex Erlenmeyer flasks.’

5 cc. of acetic anhydride-pyridine mixture (1 volume of anhy- dride and 7 volumes of pyridine) are added to one of the above samples from a Folin-Ostwald blood pipette, with careful measure- ment. To the other, 5 cc. of pyridine only are added. A blank flask is set up with 5 cc. of the acetic anhydride-pyridine mixture. The stoppers are moistened with pyridine and placed loosely in the flasks. The flasks are then placed over well fitting holes (to insure a minimum escape of steam around the flasks we have used rubber rings over the holes) of a steam bath, and allowed to heat about 5 minutes to permit expansion, the stoppers being slightly unseated if necessary. The stoppers are then turned in firmly and the heating continued 40 to 45 minutes.

The flasks are removed from the bath and the stoppers quickly loosened (to prevent sticking due to cooling) and placed at an angle in the mouths of the flasks. 5 cc. of water are then added, care being taken to rinse well the stoppers and necks of the flasks. The stoppers are loosely replaced and the flasks heated 1.5 to

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West, Hoagland, and Curtis 631

TABLE II

Acetyl Values of Lipids, Etc., by Pyridine-Acetic Anhydride Titration Method

SUbSt3IlCQ

Castor oil.. . . . . . “ I‘

. . . . .

Cottonseed oil, fresh. . . . .

“ “

Cottonseed oil, old............

Linseed oil, fresh

“ “ “

Croton “

“ “ . .

Nest’s-foot oil. .

Cocoanut oil, fresh. . . . . . .

Cocoanut oil, old

Olive oil.. . . . . .

Carnauba wax. . Cholesterol. . . . . Ricinoleic acid. .

“ “ . . Lithium lactate Palmitic acid. . .

Tem-

Kit- W~i~htOf A&y1 values

(in order of sample)

am.

Hot 0.596, 1.155 146.1, 144.6 Cold 0.630, 1.226 147.1, 147.3

Hot 2.069, 1.542, 5.3, 5.5, 5.4 2.940

Cold 1.383, 1.835 5.9, 5.2

“ 3.670, 2.083, 31.4, 30.6, 1.465 29.8

“ 3.424, 2.515, 5.8, 6.2, 6.1 1.694

Hot 2.692, 1.770, 6.3, 6.5, 6.3 3.288

Cold 3.156, 2.264, 8.2, 8.8, 8.7 1.563

Hot 2.350, 3.044, 8.2, 8.3, 8.3 1.609

“ 3.055, 2.303, 20.4, 20.0, 1.545 19.8

Cold 2.052, 1.974, 4.4, 4.1, 4.8 1.932

“ 3.612, 2.482, 19.4, 20.4, 1.150 20.1

“ 3.498, 2.556, 3.5, 3.9, 3.3 1.774

“ 2.822, 1.228 41.2, 40.9 Hot 0.3352, 0.3826 130.9, 133.5

“ 0.3401, 0.3339 158.5,158.8 Cold 0.3463, 0.3463 159.6, 160.1 Hot* 0.3734, 0.3000 394.4, 395.3

I‘ 0.4755,0.3135, No acetyl 0.3547 value

AWI- age

45.3 47.2

5.4

5.5

30.6

6.0

6.31

8.51

8.21

20.1

4.4

20.0

3.6

41 32.2 58.6 59.8 94.8

-

_ -

6

6

6

-

Given in literature

146 -150.5

5.5- 25

4 - 8.5

19.8- 32

22

o.!+- 12

4.9- 10.6

44.7- 55.2 131.1 (theory) 164.6 “

406.1 “ None

* Heated 65 minutes on the steam bath.

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632 Acetyl Values of Lipids

2 minutes on the steam bath, after which they are allowed to cool 10 minutes with the stoppers at an angle. The stoppers and necks of the flasks are rinsed down with 25 cc. of butyl alcohol (more if necessary to give a homogeneous solution) and the solutions ti- trated with 0.3 to 0.35 N alcoholic NaOH from a good 50 cc. burette, with phenolphthalein as indicator (3 to 4 drops of 0.1 per cent in alcohol). The titrations are all reduced to cc. of 0.1 N alkali.

Instead of heating on the steam bath, the stoppers of the flasks may be sealed in with pyridine and the flasks permitted to stand 24 hours or longer at room temperature and the procedure con- tinued as outlined above. Table II shows that results obtained with heating and in the cold agree well. The values here are ex- pressed according to the Lewkowitsch definition.

Calculations

According to Lewkowitsch’s definition, the acetyl value equals the mg. of KOH required to neutralize the acetic acid from 1 gm. of acetylated substance. If we let A represent the weight of the sample acetylated, B the acidity (cc. of 0.1 N) of the blank, C the acidity (calculated from titration of the sample treated with pyridine alone) of the sample used, and D the acidity of the ace- tylated sample, the calculations are as follows: acidity equivalent of anhydride bound, E = B - (D - C) ; weight of acetylated sample, F = A + (E X 0.0042) ; mg. of KOH equivalent to acetyl bound, G = 5.61 X E; acetyl value = G/F.

According to the proposed definition, the acetyl value equals the mg. of acetyl taken up per gm. of substance. With the neces- sary symbols above the calculation is acetyl value = (E X 4.3)/A.

DISCUSSION

Our statement that the method is applicable to free hydroxy- lated fatty acids and not subject to the error of the Benedict- Ulzer procedure is based upon several points of evidence. We prepared ricinoleic acid of neutral equivalent 298.5 (theory 298.36) according to the directions of Rider (8) and determined its acetyl value by our method both with heating and at room temperature with good agreement between the two determinations as shown in Table II. Our values, however, are a little lower than the theo- retical, amounting to about 97 per cent of the theory. This is

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West, Hoagland, and Curtis

probably due to the presence of a small amount of impurities and possibly to a slight polymerization of the ricinoleic acid similar to that pointed out by Meyer (9). The fact that the results obtained with heating and in the cold checked within the limits of error of the method indicates that no undecomposable mixed anhydrides were formed, because if this were true the heated samples would have undoubtedly been most affected and given higher acetyl values. This source of error also would have caused the acetyl values to be higher than the theory instead of lower, as found. In order to check this point several samples of palmitic acid were run with heating and in every case no acetyl value was found, the titration values checking closely with the acidity of the anhydride plus that of the acid. We have found very low acetyl values on a sample of ricinoleic acid kept under conditions favoring poly- merization.

Carnauba wax and lithium lactate did not dissolve well in the acetylating mixture, the latter rather gelatinizing. Despite this fact both were completely acetylated as shown by the concordant results on different sized samples. The value found for lithium lactate is 97.33 per cent of the theory. Lactic acid determination on the salt by oxidation to nldehyde (10) showed it to be 96.9 per cent pure. This result also shows the method to be applicable to hydroxylated acids.

In most of our determinations we have purposely used samples of widely varying weights. This was done in order that we might be sure that acetylation proceeded to completion. We consider this, along with the general agreement with published values re- corded in the literature as sufficient check on the method. The acetyl value for castor oil is essentially a constant owing to the presence of a rather definite large proportion of hydroxylated acids. Lewkowitsch (11) has pointed out that this is not true for most fats or oils since they may contain sterols and mono- and diglycerides owing to rancidity in varying proportions in addition to hydroxylated fatty acids. Table II shows the large effect of rancidity upon the acetyl values of cottonseed and coconut oils.

We believe that the above method of analysis now makes it possible, by determination of the acetyl values of the fatty acids in fats, to arrive at values which may be considered as true char- acteristics. This is being investigated.

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Acetyl Values of Lipids

SUMMARY

A new method for the determination of the acetyl values of lipids has been developed. It is simple, accurate, and rapid. The method differs from those previously proposed in that it is applicable to free hydroxylated fatty acids.

A new definition of acetyl value more suitable for calculation than the present one is proposed.

BIBLIOGRAPHY

1. Benedict, R., and Ulser, F., Monatsh. Chem., 8, 41 (1887). 2. Lewkowitsch, J., Proc. Chem. Sot., 8, 72, 91 (1890). 3. Lewkowitsch, J., .I. Sot. Chem. Ind., 18, 503 (1897). 4. Roberts, W. L., and Schuette, H. A,, Ind. and Eng. Chem., Anal. Ed.,

4, 257 (1932). 5. Andrews, J. T. R., and Reed, R. M., Oil and Soap, 9, 215 (1932). 6. Verley, A., and Bijlsing, F., Ber. them. Ges., 34, 3354 (1901). 7. Peterson, V. L., and West, E. S., J. Biol. Chem., 74,379 (1927). 8. Rider, T. H., .I. Am. Chem. Sot., 63,413O (1931). 9. Meyer, H., Arch. pharm., 236, 184 (1897), quoted in Lewkowitsch ((11)

p. 164). 10. Wendel, W. B., .I. Biol. Chem., 192, 47 (1933). 11. Lewkowitsch, J., Chemical technology and analysis of oils, fats and

waxes, London, 4th edition, 1, 345 (1909).

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