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T H E JOURNAL OF I N D U S T R I A L A N D ENGINEERING CHEMISTRY
THE KREIS REACTION OF COTTONSEED-OIL PRODUCTS By W. B. Smith ARMOUR AND COMPANY, KANSASCITY,KANSAS Received February 24, I920
T h e Kreis test for rancidity, consisting of shaking a fat or oil with equal volumes of hydrochloric acid and phloroglucinol-ether solution, has come i n t o use in this country as a means of determining t h e fitness of t h e fat for food. It is fully described b y Kerr,' who mentions among its limitations t h a t i t is given b y other substances t h a n rancid fats, including t h e .impurities in crude cottonseed oil. T h e purpose of this article is t o show t h a t not only ,crude, but also refined cottonseed oils, may give t h e reaction; and that when the Kreis test is to decide whether a fat is rancid this natural reaction may cause a mistake t o be made. In t h e inspection of animal fats there is not t h e same danger of an erroneous decision as with cottonseed'Oil products' I n the first place> lard> etc., do not contain carbohydrates Or Other impurities such as are found in crude cottonseed oil; and, secOnd, the chemist is aided by free acid, Odor) taste, color, and sediment t o form a decision as t o their soundness. On t h e other hand, t h e refining of cottonseed oil with caustic soda removes t h e free acid and insoluble matter, while t h e naturally disagreeable taste of the oil masks faint rancidity. Consequently t h e Kreis test assumes a much greater importance, a n d is in fact almost t h e only means of detecting incipient rancidity in cottonseed oil, a n d any error in making or interpreting t h e test may have far-reaching results. Kerr's statements in regard t o cottonseed oil are as follows: Fresh, sweet fats do not.give the reaction except in certain special cases. Such a case is that of crude cottonseed oil, which reacts with great intensity. In this case the substance which causes the reaction is removed by refining with caustic soda. The Kreis test is not specific for rancid fats. It is given by aldehydes and ketones, other than those which occur in rancid fats, * * * * * by crude cottonseed oil and probably by other crude oils. In making use of the Kreis test for the detection of rancidity, it is necessary to guard against a reaction due to the presence of any reacting substance, other than those due to rancidity. If such a substance is present any COnClUSiOn drawn from a positive reaction is worthless * * * * all animal fats and all refined vegetable oils are free from reacting substances. I
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As f a r as t h e writer has experienced, these statements are correct except in t h e case of refined cottonseed oil. I t is very reasonable t o expect t h a t t h e chromogenetic substances in crude cottonseed oil, which give a Kreis reaction many times stronger t h a n any color produced by rancidity, might pass t o some extent into t h e refined oil. I n most cases refined cottonseed oils show a negative Kreis reaction, b u t there are often found on t h e market oils which, in color, odor, taste, and appearance are entirely edible, but which give a positive Kreis test strong enough t o mislead t h e chemist into pronouncjng them rancid. The importance of a correct diagnosis of a color test which may lead t o the condemnation of carloads of edible oil can be readily sees, and for this reason the 1
THIS JOURNAL, 10 (1918), 471.
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following experiments are selected from a large amount of work as tending t o elucidate the subject. I n order t o decide whether a positive Kreis test in a refined cottonseed oil always indicates rancidity, i t is necessary t o find some reaction of rancid cottonseed oils which is different from t h a t of non-rancid oils. Caustic soda, hydrochloric acid, hydrogen peroxide, etc., all act similarly in both cases. Lewkowitsch,l who has a very poor opinion of color reactions, states: Exposure to light destroys those minute traces of chromogenetic substances which give rise to color reactions that were for a long time, erroneously, considered as characteristic. Since pure glycerides are themselves colorless, the light can only affect the foreign substances dissolved in them. This is further proved by the fact that insolated cottonseed oil does not reduce silver nitrate so readily as does cottonseed oil kept in the dark. On t h e other hand, it is well known and easily proved t h a t sunlight does not decrease rancidity as measured by t h e I,reis test, even if air and be excluded, but increases it. Consequently experiments were begun on t h e assumption t h a t an increase in t h e Kreis test after exposure of a sample of oil to direct sunlight would indicate rancidity, while a decrease would indicate natural chromogenetic substances. EXPERIMENTAL
This assumption was first tested out on crude oils which are acknowledged b y Kerr t o contain natural chromogenetic substances giving t h e Kreis reaction. Owing t o t h e intensity of t h e color developed b y pure crude oil, t h e samples were diluted with lard or other neutral fat. Small bottles were filled with t h e mixtures, corked tightly, and exposed t o t h e sun for about three weeks, being opened a t intervals t o determine their Kreis reaction. Table I a n d the accompanying curve show t h e results. These figures prove that mixtures containing from 0.05 t o I 2 . 5 per cent of crude cottonseed oil were affected by t h e sunlight so t h a t t h e ~~~i~test decreased uniformly and markedly, until rancidity began t o set in, when i t increased distinctly b u t more slowly. TABLEI-EFFECT OF SUNLIGHT ON NATURAL KREIS REACTION Kreis Reaction (Expressed in Intensity of Color) Crude Oil 7Per Begin2 4 7 10 14 17 20 TURE cent ning day ddYS days days days days daysdays A , . , , , 12.5 400 300 200 150 100 75 40 25 B . , , , . 3.1 175 ., 125 BO 50 35 25 12 20 C 1.5 100 .. 75 50 30 20 12 2 10 GO 50 40 35 15 5 6 20 20 35 30 20 4 1 2 .. 5 5 1 0 . 10 . . 20 12 5 Trace 2 2 2 7 20 35 ..
..
ED:::: : g:: 2;;;:: g:g5
. . . . ....
I n other words, by simply exposing these samples in a southern window t h e Kreis reaction was decreased from a n intense purplish red, graded I : 400 on Kerr's scale, t o a weak red estimated as I : 2 5 , in sampleiA, i. e., 94 per cent of t h e Kreis-reacting substance disappeared. I n t h e case of Sample C, sunlight changed t h e oil from a Kreis reaction of I : IOO t o I : 2 , i. e., 98 per cent, or all but a mere trace of t h e chromogenetic substances disappeared. All t h e other samples showed a similar decrease of t h e natural Kreis reaction, while t h a t due t o rancidity increased considerably in some cases. 1
"Oils, Fats and Waxes," 5th Ed., I, 46.
Aug.,
1920
T H E J O U R N A L OF I N D U S T R I A L A N D E N G I N E E R I N G C H E M I S T R Y
Although not shown here, certain samples of crude a n d refined oils originally giving a strong Kreis reaction have been found t o give an entirely negative one after exposure t o sunlight. Of course this does not usually happen because t h e exposure produces rancidity before t h e chromogenetic substances are entirely destroyed. So far these results confirm t h e researches of Kerr. They indicate t h a t the Kreis reaction in crude cottonseed oil is not a t all due to rancidity, and also t h a t exposure t o sunlight distinguishes t o a certain ext e n t between rancid and non-rancid oils giving a positive Kreis test. This method was therefore applied t o commercial fats and oils, both animal and vegetable, some of which were edible and some inedible. Representative results are shown in Table 11.
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agrees with t h a t of Kerr, who found t h a t “all refined vegetable oils are free from reacting substances.”
TABLE11-KREIS REACTION OP’ COMMERCIAL FATSAND OILS ,--KREIS REACTIONUntreated Exposed Oil Oil No. COMPOSITION 1:l 1 :5 1 Cottonseed oil refined in laboratory ... 1:3, 1:7 2 Cottonseed oil, commercial product. Negatlve 3 Cottonseed oil, commercial product. Negative Trace 1:4 4 Cottonseed oil commercial product. Negative 1 :2 3 Cottonseed oil: commercial product. 1 : l 1:3 6 Cottonseed oil commercial product. 1 :4 1 :6 7 Cottonseed oil: commercial product. 8 Cottonseed oil, commercial product, 1 : 7 ...... 1 : 10 1 : 10 9 1 :4 10 1 :4 11 1 :3 12 1:5 13 Negative ............. 14 Negative 15 Negative 16 C!rude sesame oil.. Negative 17 1:s 18 Negative 19 Negative 20 1 : 15 21 1 : 12 22 White grease.. . . . . . . . . . . . . . 1 : l 23 Peanut oil. . . . . . . . . . . . . . . . . 1:2 24 Negative 25 1:4 26 Negative 27 28 1:s seed oil and tallow.. . . . . . . . . . . . . 1 :8 29 Compound mixture of cottonseed oil 1:3 and stearin.. . . . . . . . . . . . . . 1:6
.
The control samples consist of both rancid and nonrancid oils. I n all cases (excepting cottonseed) where the original oil gave a positive Kreis reaction, i t did not decrease on exposure t o sunlight but usually increased, This indicates t h a t with olive, peanut, sesame, coconut, palm, and neat’s-foot oils, and white grease, tallow, and lard, a positive Kreis test is due t o rancidity; a t least, no indication of naturally reacting substances was obtained. T h e samples of cottonseed oil shown in the table include most classes of refined oils, but nearly all of them fall into one of two groups, those in which t h e Kreis test is wholly or partly due t o chromogenetic substances, and those in which i t is chiefly due t o rancidity. All of the samples showed some lessening of t h e Kreis test after exposure t o sunlight, but while in some i t is nearly or quite complete, in others i t is slight or negligible. Of course, in a really rancid oil t h e reaction would increase just as in t h e case of any other vegetable or animal oil. The evidence summarized in this table, therefore, can be explained only on t h e hypothesis t h a t refined cottonseed oil may give a positive Kreis reaction although i t is not rancid. I n this respect alone this work dis-
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The natural Kreis reaction of cottonseed oils is more of a purplish red t h a n the rancidity reaction. It is also more slow t o develop, sometimes taking many hours t o reach a maximum. As was pointed out by Kerr, the test is so delicate t h a t much care must be taken t o avoid false conclusions. For instance, a positive test has been known t o be due t o the age or quality of t h e phloroglucinol-ether solution. Again, the oil used as a dilution medium may cause an unsuspected error. I n one extreme case a petroleum distillate .showing negative t o Kreis gave, when mixed in equal proportions with a rancid fat, a more intense color t h a n the f a t gave by itself. It was positive in a I : 40 dilution, while the substitution of coconut oil for t h e petroleum caused t h e reaction t o show only I=: IO. The error appeared t o lie in the presence of cracked compounds in the petroleum, which turned brown when allowed t o stand with hydrochloric acid, or even in strong sunlight. Temperature appears t o affect t h e intensity of t h e
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color t o some extent, so t h a t irregular results may be obtained with such substances as hydrogenated cottonseed oil, which melts a t a high temperature. The natural Kreis-reacting substances in cottonseed oil are not glycerides, and bear no relation t o t h e decomposition of t h e glycerides, from rancidity or any other cause. The work from which t h e above observations have been selected covers a period of eleven years’ experience with the Kreis test, during which thousands of samples of edible fats have been examined. The portion which was done recently was carried out with t h e assistance of Mr. Isaac N. Jordan, CONCLUSIONS
Refined cottonseed oils and their products sometimes contain chromogenetic substances carried over from t h e crude oil. These substances sometimes cause a positive Kreis reaction in a non-rancid oil, and hence interfere greatly with t h e detection of rancidity by this means. For this reason t h e Kreis test is not entirely dependable for t h e detection of rancidity in cottonseed oil. MODIFICATION OF THE HOWARD METHOD FOR COUNTING YEASTS, SPORES, AND BACTERIA IN TOMATO PRODUCTS By Harry M. Miller NATIONAL CANNERSASSOCIATION, Los ANGELBS,CALIFORNIA Received April 8, 1920
The modification here described is based on t h e fact t h a t by boiling tomato pulp with Loffler’s methylene blue and Ziehl-Neilsen’s carbofuchsine the microorganisms are stained a slightly deeper color t h a n t h e tomato tissues. Aside from producing this slight differential effect, the combination of t h e two stains facilitates t h e observation of t h e yeasts, spores, and bacteria. Methylene blue, used alone, fails t o properly stain some of t h e organisms, while carbofuchsine, without t h e addition of methylene blue, produces a reddish color which makes it hard t o see clearly t h e outline of t h e bacteria when artificial light is used. Take 2 0 cc. of tomato juice, pulp, or ketchup, or 5 cc. of paste or other highly concentrated products. Transfer the sample t o a I O O cc. beaker using sufficient water t o make t h e total volume about 30 cc. Heat t o boiling, and add 2 t o 4 cc. of Loffler’s alkaline methylene blue. Stir well and continue t h e boiling for 3 min. The amount of methylene blue used depends on the concentration of t h e product under examination, enough being added t o color t h e sample blue. A green color shows t h a t insufficient stain has been used. Next add 2 cc. of Ziehl-Neilsen’s carbofuchsine, stir well, and again boil for 3 min. Let cool a minute, and add 5 t o I O drops of formaldehyde t o reduce any excess of carbofuchsine; stir well and dilute with water t o 60 or 1 2 0 cc., depending upon t h e concentration of t h e product. Shake well, t r a n s f 6 some of t h e sample t o a beaker, prepare t h e microscopic slide in t h e usual manner and examine b y t h e Howard meth0d.l 1 Association of Official Agricultural Chemists, Report of Committee on Editing Tentative and Official Methods of Analysis, 1916, 324.
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It is necessary t o use artificial light since t h e blue color of t h e slide makes examination by daylight very difficult. .
THE BEHAVIOR OF PHENOLPHTHALEIN WITH IODINE AND A METHOD FOR THE DETERMINATION OF PHENOLPHTHALEIN By Samuel Palkin DIVISION OF DRUGS,BUREAUOF CHEMISTRY, Received March 31, 1920
WASHINGTON,
D.
e.
The question has frequently arisen as t o t h e actual therapeutic strength of medicinal preparations containing phenolphthalein. Though phenolphthalein has had widespread use in medicine for many years, no method for its quantitative determination has been found satisfactory. Among those investigated is a titration method proposed by Kollo,’ based on t h e well-known reactions of phenolphthalein with iodine in which tetraiodophenolphthalein2 is formed Numerous experiments with Kollo’s method failed t o give concordant results and made it apparent t h a t t h e reaction was subject t o a number of errors, among which oxidation and incompleteness of conversion t o the tetraiodo compound were later found t o be most important. I n t h e present investigation a study was made of t h e effect upon this reaction of varying successively t h e concentration of iodine, temperature, and concentration of alkali, while t h a t of t h e phenolphthalein was kept constant. EXPERIMENTAL
The general method of carrying out t h e experiments enumerated in Tables I, 11, and I11 consisted quite uniformly in t h e addition of a solution of iodine in potassium iodide t o an aqueous alkaline solution of phenolphthalein. I n nearly every case a n approximately quantitative estimation of t h e principal reaction product, tetraiodophenolphthalein, was made by extraction of t h e reaction mixture with chloroform in a separatory funnel, after t h e excess of iodine had been removed with sodium sulfite and t h e whole acidified with hydrochloric acid. The use of chloroform as an extraction medium was determined upon from a few preliminary experiments with a number of organic solvents immiscible with water. When freshly precipitated with acid from t h e cold alkaline solution, tetraiodophenolphthalein was found t o be readily soluble in chloroform. The color of t h e weakly alkaline solution of t h e pure compound is wine-red by transmitted light and blue by reflected light, Phenolphthalein is only sparingly soluble in chloroform and t h e color of its alkaline solution needs no description. I n each experiment observations were made as t o : ( I ) The character of precipitate formed. ( 2 ) The character, amount, and color of t h e alkaline solution of t h e portion of t h e precipitate remaining insoluble in chloroform. (3) Appearance, weight, and color of t h e alkaline 1
Pharm. Praxis, Bucharest, 7 (1908), 341. Ber., 28 ( 1 8 9 5 ) , 1605.
* ClaSsen and Lob,
