Reagents for lodometric Determination of Peroxides in Fats LEOPOLD HARTRIAN AND MARGARET D. L. WHITE Fats Research Laboratory, Department of Scientific and Industrial Research, Wellington, New Zealand As some of the reagents used for the iodometric
other reagents which are supposed to be free from the so-called “oxygen error” (liberation of iodine by atmospheric oxygen) has shown that all reagents are subject to this error to a greater or lesser extent and that a low oxygen error is accompanied by low results. Exclusion of a i r during the estimation of peroxides is essential in any exact work regardless of the reagent used, and the only advantage one can expect from a good reagent seems to be the absence of blanks.
estimation of peroxides in fats give high blanks, and some react with the liberated iodine, an investigation has been carried out to develop a reagent free from these disadvantages. An approximately 107‘ solution of citric acid in a mixture of tert-butyl alcohol and carbon tetrachloride gives results in good agreement with the acetic acid-carbon tetrachloride mixture which is the most frequently used reagent, without giving rise to blanks. Comparison with
T
HE iodometric method of peroxide estimation in both the form recommended by Lea (3) in 1929 and its modifications hm successfully withstood all attempts to replace it by other oxidation-reduction systems. Substitution of stannous chloride and of ferrous salts for potassium iodide has been found unworkable by Stansby ( 8 ) , and a similar result has been experienced in this laboratory with arsenious acid. The ferrie-thiocyanate procedure of Lips, Chapman, and RlcFarlane ( 6 ) gave promise but has been shown by Lea ( 4 ) to suffer from the interference of atmospheric oxygen, and this has been confirmed by one of the originators of the method (1). The iodometric method, however, is not without weaknesses, one of them being the use of acetic acid in most of its numerous modifications As stated by Stansby ( 8 ) , some lots of this acid, even though meeting ACS specifications, give high and irregular blanks, while some may react with iodine. This, and the high “oxygen error” (liberation of iodine from potassium iodide by atmospheric oxygen) which is peculiar to acetic acid, have created interest in alternative solvents. Acetone, acetic anhydride, ethyl alcohol, and isopropyl alcohol have been named as giving no appreciable blanks and as rendering unnecessary precautions against atmospheric oxygen in the determination of organic peroxides ( 2 , 7 ) , but it seems that peroxides in fats constitute a special case. Here even the total absence of a blank titration does not rule out the oxygen error, as there are reasons to assume that the reaction between peroxides in fats and the iodide ion and the reaction between this ion and atmospheric oxygen do not proceed independently. However, the elimination of blanks would in itself constitute a distinct advaptage if the solvents mentioned above were otherwise satisfactory. Actually acetic anhydride, apart from being a poor solvent for fats, has been found in this laboratory to give rise to higher and more erratic blanks than acetic acid. On the other hand, acetone, ethyl alcohol (which is not a solvent for glycerides), and isopropyl alcohol are known to react with iodine in the presence of water and acids, R hich constitutes a source of error. An investigation has theieforr been carried out in this laboratory 15 ith the purpose of developing a reagent free from the disadvantages of the reagentb previously mentioned, and of comparing its performance uith that of others.
time a much better fat solvent than isopropyl alcohol and even its technical grades do not give in the presence of a suitable acid any blank titration after addition of potassium iodide. Citric acid was considered a suitable source of hydrogen ions, as it haa a higher dissociation constant than acetic acid, and being a mild antioxidant, or rather a metal deactivator, it would presumably afford a certain measure of protection to the fat during the estimation. The properties of these two compounds suggested that they could be used jointly to replace acetic acid in the determination of peroxides. A certain amount of chloroform or carbon tetrachloride has been found useful to prevent floating of the fat during titration, and after several tests the following mixture, designated as “citric acid reagent” has been adopted in this work: 10 grams of citric acid B.P. quality dissolved in 60 ml. of hot tert-butyl alcohol followed by the addition of 35 nil of carbon tetrachloride.
Table I.
Reaction between Iodine and Various Solvent6 (10 ml. of solvent i 10 ml. of 0.1 N iodine) 0.1 N Sodium Thiosulfate, MI. -4cetone 9.82 Inopropyl alcohol 10.11 Acetic acid 10.16 Aqetic anhydride 10.16 Citric mid reagent 10.16 terl-Butyl alcohol 10.16 Blank 10.16
Although these amounts are not unduly critical, there is no advantage in using either more concentrated citric acid solutions or anhydrous citric acid. On the contrary, a saturated solution containing approximately 17 grams of citric acid instead of 10 grams tends to give rise to blanks. Conversely, a substantial reduction of citric acid belov the quantity specified results in diminished peroxide values, 5 grams of citric acid giving about 20% lower values. ANALYTICAL PROCEDURE AND R E S U L T S
The interaction between iodine and various reagents mifi measured by adding to 10 ml. of each reagent 10 ml. of an approximately 0.1 N iodine solutio? in acetic acid, diluting with 50 ml. of water, and titrating after 5 minutes on addition of 1 ml. of saturated potassium iodide solution and starch with 0.1 A‘ sodium thiosulfate. Experiments with acetone have not been pursued in the present work because of the appreciwhle reactivity of this solvent with iodine (Table I).
DEVELOPMEhT OF YEW REAGENT
In view of the reactivity of alcohols such as ethyl alcohol and isopropyl alcohol with iodine, it seems surprising that tert-butyl alcohol, the chemical constitution of n hich precludes such reactivity, has not hitherto been proposed as an alternative solvent. It is miscible with water in all proportions, being at the same 527
528
ANALYTICAL CHEMISTRY
Table 11. Peroxide Values Obtained with Acetic AcidCarbon Tetrachloride Mixture and Citric Acid Reagent in Nitrogen Atmosphere
Butterfat
Muttontallow1
hf utton tallow I1 (from Swift test) Mutton tallow I11 (rancid) Linseed oil (oxidized) e
Hot Cold Cold Hot Cold Cold Hot Cold Cold Hot Cold Cold Hot Cold Cold
2.5 15
60
2.5 15
60 2.5 15
60
3 . 0 (KI) 3 . 0 (NaI) 2.4 2.4
1.1 1.8 0.1 0.3
3 . 2 (KI) 3 . 2 (KaI) 2.7 2.8
2.5 2 . 6 KI) 2 . 5 INaI) 2.6
1.2 0.2 0.2
2.4 2 . 9 (KI) 2 . 9 (NaI) 2.9
14.5 12,7 13.7
0.3 1.5= 0.5“ 0.8a
13.6 13.2 13.6
2 , 5 228.1 15 202.1 215.3
236.9 209.3 215.0
2 . 5 474.9 417.; 464.,
442.5 368.4 428.1
60
15
60
Nil Xi1 Nil Nil Nil Nil Nil Nil Si1
Different lot of reagent.
The following reagents were compared: the citric acid reagent described above; a mixture of acetic acid and carbon tetrachloride ( 2 to 1) using the new Lea procedure slightly modified (6); acetic anhydride according to Nozaki (7); and isopropyl alcohol according to Kokatnur and Jelling ( 2 ) . When using the first two reagents mentioned above, approximately 1 gram of fat was weighed in a 6 X 1 inch test tube, dissolved in 20 ml. of solvent, and deaerated with nitrogen, 1 ml. of a saturated potassium iodide solution was added, the flow of nitrogen was discontinued, and the stoppered tube was left in the dark for 15 and 60 minutes, respectively. The mixture was titrated after dilution with 50 ml. of water with 0.002 N sodium thiosulfate (0.01 ‘V or 0.1 N for highly oxidized fats), using starch as indioator, and the results referred to as peroxide values were expressed as number of milliliters of this reagent used for 1 gram of fat (1 ml. of 0.002 N sodium thiosulfate per gram of fat is equivalent to 1 millimole of peroxide per kilogram of fat). When using Lea’s new “hot” method, the contents of the tube were boiled for 2.5 minutes in a water bath. The Nozaki procedure was carried out by dissolving the fat in a mixture of 10 ml. of acetic anhydride and 5 ml. of carbon tetrachloride, followed by the addition of 1 gram of powdered sodium iodide. FVhen the procedure of Kokatnur and Jelling was used, 1 gram of fat was dissolved in 25 ml. of anhydrous isopropyl alcohol and after adding 1ml. of acetic acid and I ml. of saturated potassium iodide the mixture was heated for 15 minutes to incipient boiling and titrated while hot without starch.
is only limited; the nature of the latter is not fully known. Benzoyl peroxide was purified by dissolving the commercial product in chloroform and precipitating with cold methanol and its analysis was carried out both in nitrogen atmosphere and in air, the mixture being allowed to stand 15 minutes in the cold. When working without nitrogen, the potassium iodide solution was mixed with other reactants by giving a rotary motion to the test tube. Aqueous sodium iodide instead of the potassium salt was used in some analyses of benzoyl peroxide and fat peroxides. The comparison of all four reagents, which was undertaken mainly with the view of establishing their respective susceptibility to oxygen error, offered certain difficulties. The cold method is not applicable to isopropyl alcohol, because boiling is essential for the reduction of peroxides in this solvent, while it is more satisfactory than the hot method for the other three reagents. Furthermore, although best results with these other reagents are obtained on 60 minutes’ standing in cold in an inert atmosphere, this time is too long if the estimation is carried out in the presence of air, because of excessive blanks. It was resolved to apply the cold method with 15 minutes’ standing to the citric acid reagent, acetic acid-carbon tetrachloride mixture, and acetic anhydride, and 15 minutes’ boiling to isopropyl alcohol. Results are shown in Table IV. No blanks were obtained when using the citric acid reagent and isopropyl alcohol, respectively, with and without nitrogen atmosphere. Blanks with acetic anhydride varied from lot to lot of the reagent and ranged from 0.3 to 5.4 ml. of 0.002 N sodium thiosulfate.
Table 111. Analysis of Benzoyl Peroxide (Cold method, time of standing atmosphere) 15 minutes, with and without nitrogen Citric Acid Reagent doetic Acid-Carbon Tetrachloride Nitrogen No nitrogen Witrogen No nitrogen % ’ Peroxide Found 100.9 X I ) 9 9 . 7 (KI) 99.7 (KI) ”” 101.3 [NaI) 9 9 . 8 (NaI) 9 9 . 9 (NaI) 9 9 . 5 (K1) (NaI)
Some difficulty was experienced with all reagents in assessing the end points of titrations when 0.002 N sodium thiosulfate waa used, but with some experience results. reproducible to hO.1 ml. were obtained. The results shown in tables represent the mean of duplicate and triplicate analyses.
DISCUSSION The fats investigated were butterfat, beef tallow, mutton tallow, and linseed oil. Peroxide values obtained when the Of the solvents examined, acetic anhydride has been found citric acid reagent was compared with the mixture of acetic acid less satisfactory than acetic acid. It gives high blanks and a and carbon tetrachloride as the standard reagent are shown in high oxygen error and is unpleasant to use. Isopropyl alcohol ia Table I1 and were on the whole in good agreement for fats which the only solvent that does not show a considerable oxygen error. went through the process of autoxidation at room temperature This and the absence of blanks would make it satisfactory, but or were subjected to the “Swift test.” For fats that were oxifor the low peroxide values obtained with it as compared with dized at temperatures above 100’ C. or polymerized, higher the Lea method (Table IV). The low results may be due to the peroxide values were obtained with the acetic acid-carbon tetrasuppression of ionization in isopropyl alcohol or to its reactivity chloride mixture, but as has been pointed out by Lea (6) such with iodine. fats may contain groupings other than true peroxides and conseA low oxygen error being associated with low results, it would quently in this case the highest peroxide values are not necesseem that the only advantage one can expect from a reagent is sarily the most correct ones. In no instance was a blank titration obtained with the citric Table IV. Peroxide Values Obtained Using Various Reagents, w-ith and without acid reagent. Nitrogen Atmosphere 15 Minutes’ -4s a check, determinations of active oxygen in benzoyl peroxide were carried out using both above mentioned 3.5 1.3 3.8 0.5 0.5 3.1 1.5 1.4 Beef tallow reagents (Table 111), although 2.8 2.5 4.8 2.8 6.5 Mutton tallow IV 3.6 6.2 3.9 2 0 . 1 2 1.1 the analogy between benzoyl Linseed oil 23.6 28.7 23.8 25.8 24.4 27.0 peroxide and peroxides in fats
V O L U M E 2 4 , NO. 3, M A R C H 1 9 5 2 the absence of a blank titration. Thus a suitable reagent is one whose hydrogen ion concentration, while not facilitating a blank, ie sufficient t’o effect the reduction of peroxides within a reasonahle time. A solution of citric acid in a mixture of tert-butyl alcohol and carbon tetrachloride, which gives results in good agreement with the Lea hot and cold methods but produces no blanks, seems t o fulfill these requirements. The absence of blanks is of particular value in the analysis of fats lvith a low peroxide content. In view of the uncertainty regarding the end point in the titration with 0.002 N sodium thiosulfate, only in the total absence of iodine liberation can a fut sample be safely declared as having zero peroxide value. Precautions against atmospheric oxygen are essential for accur:tte results, regardless of the solvent used. The use of sodium iodide instead of potassium iodide, which lintl been advocated in conjunction with the acetic anhydride nit+hod (Y), and with a modified isopropyl alcohol procedure (IO) deserves a brief comment. .-ilthough both iodides give similar rewlts, sodium iodide appears preferable because of its greater solubility whether in the form of powder or aqueous solution. This applies particularly to Lea’s original hot method (S), where the presence of undissolved potassium iodide powder has given rise to doubts regarding the quantity to be used (9). Furthermore, aqueous solutions of sodium iodide have been found t o kerp longer than those of potassium iodide.
529 ACKNOWLEDGMENT
The authors are indebted to F. B. Shorland, director of the Fats Research Laboratory, Department of Scientific and Industrial Research, for permission to publish and for advice and to R. A. Chapman, Food and Drug Laboratories, Department of National Health and Welfare, Canada, for constructive criticism and suggestions. LITERATURE CITED
(1) Chapman, R . A,, and SIackay, I
