Stability of Fats and Oils Applications of the Methylene Blue Test H. D. ROYCE, The Southern Cotton Oil Company, Savannah, Ga.
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HE relationship between
to'measure the a n t i o x i d a n t Recent developments in rancidity and stability effect of carotinoid pigments. oxidative induction tests are reviewed, and additional data on the period and the fading use of methylene blue as an oxidation-reduction EXPERIMENTAL time of methylene blue in oils exindicator are presented. Photoelectric control posed to r a d i a t i o n was first APPARATUS.The a s s e m b l y of the end point has not been found satisfactory pointed out by Greenbank and previously d e s c r i b e d (12)has Holm (4),who made it the basis in testing hydrogenated fats, because of secondary been simplified by omission of of a photoelectric stability test. the p h o t o e l e c t r i c control, so color changes that take place in the reaction mixThe possibilities of adapting the that the unit may be constructed ture. Stability curves for a number of shortening m e t h o d to r o u t i n e keepinga t low cost in any l a b o r a t o r y products and salad oils are given. Light has been quality determinations were outhaving average facilities. Figused to shorten the induction period in the aclined in a previous paper ( l a ) ure 1 presents the arrangement from this laboratory, including a celerated peroxide test, and the relation of methylof the essential parts of the apdescription of a modified apparaene blue fading time to Kreis values and peroxide paratus now in use. tus. Since that time extended values has been determined for cottonseed oil The reaction chamber is an use of the method on a variety air thermostat of asbestos-lined aged to a peroxide value of 250 millimoles. of fats has led to a better underwood c o n s t r u c t i o n 10 X 10 X standine of itslimitations. which. 14 feet lon The sliding sample while n i t detracting from the value of the method for certain rack 2 holds six American Oil Chemists' Bociety oil color tubes 0 applications, necessitated further changes in the apparatus. equidistant from the source of illumination L. This rack pulls out from the side of the box and facilitates the rapid change of Also the use of accelerated oxidation data to supplement in- samples with minimum temperature drop in the thermostat. dividual fading-time determinations will be pointed out, in The magnesia block mounting is cut away under the tubes, so conjunction with comparative figures on peroxide formation. that the fading time may be checked by viewing the sample from as well as by direct transmitted light through The increasing attention being centered on the develop- above throu h W2, Wl. The bfower heater unit B was adapted from a 400-watt ment of improved stability test and evaluation of fat stabi- hair drier by soldering a circulating flue over the intake port, lizers becomes apparent in reviewing recent literature. Of and by insertion of resistances and relay in the heater circuit. especial interest in connection with the methylene blue method Since it was desired at various times to operate over a temperais the procedure recommended by Bruere and Fourmont (S), ture range from 40" t o 90" C., the most convenient resistance was found t o be lam sockets, 81, Sz and Sa, mounted involving the use of a series of decolorized reduction indicators mounting on the side of the box (S, and Z i n parallel, with S1 in series with having graded reduction potentials. Depending on the relay and 8, in series with fan motor, best values for operation a t stage of incipient rancidity in the fat under examination, 70" C..were SI,50 watts, Sz, 100 watts, and &,25 watts). When certain of the indicators revert to the colored form. Probably operating at other temperatures, or to maintain temperature at start of test before the irradiating lamp L is cut on, the necessary a comparison of results by this method with the iodometric changes in resistance may be made easily. peroxide estimations of Lea (8) or Wheeler (IS)would show METHOD. Clean, dry A. 0. C. S. oil color tubes, graduated a t that the color change upon the presence of peroxides the 25-ml. level, are filled t o the mark with the oil or melted fat - depends t o be tested. One milliliter in the sample. of a freshly prepared 0.025 The l i m i t a t i o n s of the per cent solution of methylKreis test applied to fats ene blue in a n h y d r o u s stored in hermetically alcohol is added, and quickly mixed with the oil by shaks e a l e d c o n t a i n e r s have ing. The tubes containing been pointed out by Pool the mixture are then brought (IO), who uses oxygen abto 70" C. by holding for a sorption t o s u p p l e m e n t few m o m e n t s in a steam b a t h , and the time is rethis t e s t . R i c h a r d s o n , corded when the s a m p l e s Eckey, and Andrews (11) are placed in the i r r a d i a also employ oxygen absorption c h a m b e r . The end tion measurements to estabpoint is observed t h r o u g h the windows W1 and WZ and lish the stability of salad with most vegetable oils and oils supplied to the mayonhydrogenated fats the color naise i n d u s t r y , Grettie change is quite abrupt at the and Newton (6) have comend of the reduction period. The fading time is recorded bined certain f e a t u r e s of in minutes, and unless otherthe B a i l e y (2) a n d t h e wise indicated in the followIssoglio (7) tests t,o arrive FIGURE1. APPARATUS FOR THE ESTIMATION OF FATSTABILITY ing data, the d e t e r m i n a a t a method for following tions have been m a d e a t BY THE METHYLENE BLUE-LIGHTREACTION 70" C., 0.001 per cent dye oxidative d e c o m p o s i t i o n RT. Reotifier transformer A . Bimetallic thermoregulator concentration, w i t h 100under a c c e l e r a t e d aging SI. Resistance in relay circuit B . Heater-hlower watt in si de-f r o s t ed daySa. Resistance in heater circuit M . Magnesia block conditions. More recently, 8s. ResiRtance in blower circuit 0. Oil color tube oontainingsample l i g h t M a z d a l a m p at 15 L. 100-watt daylight lamp V. Adjustable ventilator Newton (9) has used the cm. from the center of the W I ,We. Windows R. Mercury relay sample. rate of peroxide formation 2. Drawer elide T Thermometer 244
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July 15, 1933
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INDUSTRIAL AND ENGINEERING CHEMISTRY
A few precautions are necessary to obtain consistent and reproducible results, Moisture must be excluded in all operations, and if the original sample contains more than traces of water it should be dried, care being taken to minimize oxidation in this operation. The methylene blue reagent should be prepared from aldehyde-free anhydrous alcohol, and can be kept satisfactorily for a few days in a stoppered bottle placed in a desiccator. A number of determinations have been made using aldehyde-free anhydrous propanol, but it has no apparent advantage over the lower alcohol. Newton (9) has emphasized the importance of thoroughly cleaning glassware which has been in contact with rancid fat, in connection with the incubation accelerated rancidity test, and it is likewise imperative to clean the color tubes carefully in the present method. It has been found that rinsing with a fat solvent, followed by a 30-minute boil in 20 per cent sodium hydroxide and overnight soaking in cleaning solution, serves the purpose very well. With most fat samples, the degree of accuracy of the method is determined by the personal factor in reading the end point. Colored oils rich in yellows and reds naturally give a green color with the reagent, and as the blue fades the green shades rather gradually to the original tint. However, visual observation of the end point has been found to be more satisfactory and reliable than the photoelectric method, for in spite of the use of color filters in con-
FADING TIME- MINUTES AND METHYLFIGURE 2. EFFECTOF TEMPERATURE ENE BLUECONCENTRATION ON FADING TIME
Teats made on freshly deodorized ohoice cottonseed oil. 1. Temperature-fading time curve 2. Concentration-fading time curve 70" C. and 0.001 per cent methylene blue conoentration adopted as stanaard for oontrol work.
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nection with the latter, many natural oils and all-hydrogenated fats which have been examined give secondary color changes that interfere with photoelectric control. Since the methylene blue reduction period for most refined edible fats and oils does not exceed one hour, and the average is less than 30 minutes, it is not particularly tedious or inconvenient to record the end points visually. The duration of the methylene blue fading time may be varied within certain limits by changing the reaction temperature, dye concentration, and light intensity. Figure 2 gives the values for freshly refined and deodorized cottonseed oil under the maximum range of conditions which have been found practical with the present apparatus. For control work on the estimation of stability of salad oils and shortenings, 70" C. and 0.001 per cent methylene blue concentration were adopted as standard conditions. Figure 3 shows the comparative stabilities of four types of hardened cottonseed oil shortenings, aged in friction top cans a t 90" to 95" F. Organoleptic manifestation of rancidity is indicated at the points designated by R on the methylene blue fading-time curves. All-hydrogenated shortenings are
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FIGURE 3. VARIATION OF FADING TIME (REDUCTION PERIOD) WITX AGEFOR DIFFERENT TYPESOF HYDROGENATED SHORTENING 1 2:
All-hvdrogenated Pack'age vegetable compound
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Bulk vegetable corn ound Bulk oleo compouna
generally conceded to have superior keeping quality in comparison with compound types, and the fading-time curve 1 illustrates this point very well; 2 and 3 are two different grades of hydrogenated vegetable compound shortening, and having similar composition, the stability curves follow the same general trend. Curve 4 may be interpreted to show that the original sample was very close to a state of incipient rancidity, since the slope of the curve corresponding to the initial stage of the aging period resembles that of the other fats at a later stage in the test. While this inferior keeping quality of the oleo compound cannot be attributed entirely to the oleostearin content, i t has generally been found that strictly vegetable compounds show a higher initial fading time. If, however, the curves are to be interpreted for practical purposes, it should be pointed out that the smaller slope and higher fading time during the later stages of the test on the oleo compound indicate that, although the original conditions of such fats may not be very good, the rate of change under ordinary storage conditions is slightly slower than the rate for vegetable compound. Certain metals in oil-soluble form are known to have a marked effect on the stability of fats, and Figure 4 gives the fading time of methylene blue in cottonseed oil to which very low percentages of some of the common metals have been added. Copper is a poverful pro-oxidant for autoxidation reactions (1) and the accompanying curve shows that only 20 p. p. m. reduce the fading time from 24 to 8 minutes. Manganese in somewhat higher concentrations likewise has a strong pro-oxidant effect, but ferrous iron,x divalent tin, and nickel a t the low concentrations plotted on the graph have no effect on the fading time. Zinc apparently has a stabilizing action, but this point has nqt been checked by aging tests, and it is probable that the prolonged fading time in this case is due to some interaction of the zinc with methylene blue. Further work is being conducted on the applicability of the methylene blue reduction method in the presence of pro-oxidants and antioxidants. Figure 5 presents keeping quality curves for a number of freshly refined and deodorized vegetable oils. Coconut oil 1 Under oxidizing conditions, low concentrations of iron may shorten the induotion period of fats and oils considerably. I n one experiment, 0.00026 per cent of ferric stearate was dissolved in cottonseed oil, and the sample was aged by aeration and irradiation a t looo C. The rate of peroxide formation was accelerated greatly. I n the present experiment, it is thought that oxidation of F e + + to F e + + +does not occur in the limited test period (24 min.). Generally speaking, the methylene blue test is noc always reliable when applied to fats containing substances having a high reduction or oxidation potential.
ANALYTICAL EDITION
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has a high original fading time, but this falls rapidly to a value only slightly higher than that for corn oil after aging 5 weeks in glass a t room temperature. Soybean oil gives an aging curve similar to corn oil, while the remaining oils are grouped quite closely. Peanut oil, a t least this particular sample, proved to be the most unstable of the group. Since the histories of these oils prior to refining and deodorizing are
FIGURE4. EFFECTOF METALSON FADING TIMEIN COTTONSEED OIL Metals added as freshly prepared soaps of' fully hydrogenated cottonseed fatty acids
not known, it is not possible to generalize from the data given on the relative stabilities of these oils as a class. For example, some of the oils may have been prepared from old or damaged seed, and some may have been extracted with solvents, as against pressing for others. This graph is merely presented to show that methylene blue reduction affords a convenient method for following the progress of oxidative rancidity in various kinds of oils. The relation of fading time to Kreis number and peroxide value is shown by Figure 6. A sample of cottonseed oil was aged rapidly by aerating at 100' C. under intense illumination, so that a rancid odor developed in 2 to 2.5 hours. Six hours under these conditions sufficed to complete a test, sending the peroxide value (millimoles of peroxide) to 250. (A comparison of accelerated aging tests, with and without photocatalysis, has been included in another paper.) It may be said
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FICURE 5. STABILITYCURVESFOR FRESHLY REFINED VEGETABLEOILS FROM DIFFERENT SOURCES Samples aged at 90° to 95O F. in loosely stoppered glass bottles, exposed t o diffused daylight. 1. Coconut 5. Cottonseed 2. Corn 6. Peanut 3. Soybeen 7. Sunflower 4. Rape
here, however, that for pure cottonseed oil the shape of the aging curve is approximately the same in both cases, so that the light mny be considered as a catalyst which does not markedly alter the type of reaction products which are operative in bringing about the color change in methylene blue.
Vol. 5, No. 4
By the use of a 200-watt lamp a t 15 cm. from the oil, employing a modification of Wheeler's (15) accelerated aging apparatus, the aging period has been reduced 100 per cent and more. The peroxide value curve in Figure 6 shows the extent of the oxidative induction period, while the fading time and Kreis numbers follow a fairly constant inverse proportion. This may be interpreted to indicate that the oxidative decomposition products causing the Kreis reaction are responsible for the methylene blue reduction, whereas the concentration of peroxide as measured by Wheeler's method is better adapted to register the termination of the induction period, when fat samples are aged by aerating a t high temperature. Like the Kreis test, the methylene blue method is most satisfactory when used on fresh nonrancid fats, or fats in a condition of incipient rancidity, in which a rancid flavor has not become pronounced. Interpretation of methylene blue fading time for practical application to problems of processing and storage of fats and oils may best be indicated by referring again to Figure 3. All the vegetable shortenings used in this series were freshly bleached and deodorized a t the beginning of the test. Thus, knowing the history of the fats, the initial fading times would
FIGURE6 . COMPARATIVE STABILITY CURVESON COTTONSEEDOIL SUBJECTED TO ACCELERATED OXIDATIONAT 100 C. Irradiated by 200-watt lamp at 16 om. distance
be sufficient to classify the vegetable shortenings according to stability, in the order 1-2-3, with 1 standing considerably superior to 2 and 3. The oleostearin used in making the oleo compound had not been freshly deodorized, so that, although the initial fading time is the lowest of all, it would not be safe to conclude that this type of compound has the lowest stability. Subsequent values on the aginp, curve tend to show that if the oleo compound had had the same treatment as the other samples, the initial fading time would have been equal to or higher than 2 and 3. For the estimation of stability when testing samples of known history, the methylene blue method ranks with direct oxygen absorption ( I S ) , with the added advantage of a much shorter test period and simplicity of apparatus. Additional practical information on fat stability may also be obtained by using the methylene blue test in conjunction with accelerated aging, or low-temperature aging, when time is not the dominant factor. Wheeler's accelerated oxidation apparatus in a modified form is being used at present in this laboratory, and the reaction is followed by measuring the change in methylene blue fading time as well as increase in peroxide values.
ACKNOWLEDQMHNT The author wishes to express his appreciation to M. C. Kibler for assistance in the experimental work, and to W. S. Love11 for making Figure 1.
July 15, 1933
INDUSTRIAL AND ENGINEERING CHEMISTRY LITERATURE CITED
(1) Alyea, H. N., and Backstrom, H. L. J., J . Am. Chem. Soc., 51, 90 (1929). (2) Bailey, H. S., and Ebert, H., Cotton Oil Press, 7, 35 (1923). (3) Bruere. P.. and Fourmont. A,. Ann. fals.. 25. 91 (19323. (4) Greenbank, G. R., and Holm, G. E.; IND.ENO.CHEM.,Anal. Ed., 2, 9 (1930). (5) Ibid., 17, 625 (1925). (6) Grettie, D. P., and Newton, R. C., Ibzd., 8, 291 (1931). .
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(7) Issoglio, G., Ann. chim. applicata, 6 , 1 (1916). (8) Lea, C. H., Soap, 7, 83 (1931). (9) Newton, R. C., Oil Soap, 9, 247 (1932). (10) Pool, W. O., Oil Fat. Ind., 8, 331 (1931). (11) Richardson, A. S., Eckey, E. W., and Andrews, J. T. R., Ibid., 8, 409 (1931). (12) Royce, H. D., Soap, 7, 25 (1931). (13) Wheeler, D. H., M. M. A., Bull. 121 (Jan., 1932).
RECEIVED February 6, 1933.
Determination of Formic, Acetic, and Propionic Acids in a Mixture 0. L. OSBURN,H. G. WOOD,AND C. H. WERKMAN Ames Field Station, Bureau of Chemistry and Soils, in Coijperation with Bacteriology Section, Iowa Agricultural Experiment Station, Ames, Iowa
P
REVIOUS publications (6, 10-13) have reviewed the literature and discussed the need for rapid and accurate methods for the quantitative determination of the volatile fatty acids in fermenting liquors. I n these papers the application of the partition method to two acid mixtures was shown, together with theoretical discussions of the possibilities of the partition method in general. The method presented in this paper is intended to deal specifically with the problems presented under the special conditions involved in the fermentative production of propionic acid. In the procedure described, the partition method is combined with a modified mercuric oxide oxidation for the separate determination of formic acid.
PREPARATION OF FERMENTATION LIQUORS To prepare a fermentation liquor for the quantitative determination of the volatile acids, a quantity containing about 60 cc. of 1 N volatile acid is placed in a distilling flask, made neutral to phenolphthalein, and distilled until the residue in the flask reaches a volume of about 150 cc. The residue is then made just acid to Congo red paper with 1to 1 sulfuric acid, boiled under a reflux condenser to remove carbon dioxide, and subjected to steam distillation. A constant volume of liquid in the distilling flask should be maintained. Two liters of distillate should be collected. From Dyer’s work (3)it is evident that under these conditions about 95 per cent of the formic and substantially all of the acetic and propionic acids will be removed from the fermentation liquors. Prolonged steam distillation should be avoided, because under these circumstances volatile acid may be produced from unfermented carbohydrate. Lactic acid also distills over to an appreciable extent, and for this reason the fermentation liquor should not be saturated with salt before steam distilling. Under the conditions imposed by Olmsted, Whitaker, and Duden (6) lactic acid in 600 cc. of distillate has been found equivalent to 22 per cent of that present in the original liquor. Under the conditions of ordinary steam distillation some substances, such as lactic acid, distill over and may interfere with the analysis. A much more accurate analysis can be obtained if the first steam distillate is neutralized, evaporated to 150 cc., made just acid to Congo red, and redistilled according to Olmsted ( 5 ) . Under these conditions the volatile acids will all come over in 1liter of distillate. This distillate is then adjusted by dilution t o an acid concentration of approximately 0.03 N , and this 0.03 N acid solution is used for the determination of the volatile acids.
DETERMINATION OF FORMIC ACID Results obtained in this laboratory show that strong oxidizing agents, such as chromic acid or permanganate, cannot be used for the oxidation of formic acid in the presence of propionic acid, since some of the propionic acid is oxidized to acetic acid. For example, a solution of 25 cc. of 0.1 N formic acid, 25 cc. of 0.1 N acetic acid, and 50 cc. of 0.1 N propionic acid was oxidized with a dilute permanganate solution for 30 minutes at boiling temperature. The formic acid was completely oxi-
dized by this treatment. (The solution should remain acid, since ropionic acid is oxidized t o oxalic acid in alkaline solution, 4,) l f ter acidifying and distilling the residual solution subsequent t o oxidation, the distillate should have contained 25 cc. of 0.1 N acetic acid and 50 cc. of 0.1 N propionic acid. Analysis showed, however, 52.5 cc. of 0.1 N acetic and 22.5 cc. of 0.1 N ropionic acids-that is, about half of the propionic acid had gee, converted to acetic acid. The use of dichromate (9) for the oxidation of formic acid in the presence of propionic is to be avoided for the same reason. The most convenient method of determining formic acid is the mercuric chloride method of Auerbach and Zeglin ( I ) , but serious errors are likely to be encountered by its indiscriminate use with such complex mixtures as fermentation liquors. It has been found by Weihe and by Wood in this laboratory, working independently with widely different substances, that the mercuric chloride method gives considerably higher results than are obtained by oxidation of the formic acid with mercuric oxide (unpublished data). These high results are apparently due to substances other than formic acid which reduce mercuric chloride, such as acetylmethylcarbinol. I n consequence, it seemed advisable to use mercuric oxide for the oxidation of formic acid and to determine the formic acid by the carbon dioxide evolved, inasmuch as it is highly improbable that carbon dioxide would be evolved by action of mercuric oxide from substances other than formic acid. If circumstances permit, any convenient method of determining formic acid, other than those involving oxidation with strong oxidizing agents, may be substituted.
PROCEDURE FOR DETERMINATION OF FORMIC ACID The formic acid is oxidized with mercuric oxide. The carbon dioxide is absorbed in Bowen potash bulbs and weighed (7), or, if a mixture is being analyzed from which volatile substances may be carried over into the potash I
