ANALYTICAL EDITION
202
ensuring that a fairly rapid stream of water passes through the electrode chambers a t all times. The apparatus has proved to be very useful in biochemical procedures. As an example, it was desired to free some aqueous extracts of certain tissues of their inorganic phosphate. It was found that phosphate ion could be reduced to the limits of the method of testing in 2 or 3 hours, whereas it took 48 or more hours t o accomplish this against running water in an ordinary cellophane sack.
LITERATURE CITED (1) Baer, Kolloid-Z., 46, 176 (1928). (2) Bernhard and Beaver, J.Bid. Chem., 69, 113 (1926).
Vol. 4, No. 2
(3) Bradfield, Naturwissenschjten, 16, 404 (1928). (4) D h M , Biochem. Z.,153, 504 (1924); Bull. SOC. chim. biol., 8, 144, 604 (1926); Kolloid-Z., 41,243 (1927). ( 5 ) Ettisch and Co-workers, Biochem. Z., 171, 443, 454 (1926); 195, 175 (1928); 200, 250 (1928); 216, 401, 430 (1929); KolloibZ., 45, 141 (1928). (6) Freundlich and Loeb, Biochem. Z., 150, 522 (1924). (7) Fuohs and Honsig, Ber., 58B, 1323 (1925). (8) Harvey, Am. J. Pharm., 97, 66 (1925). (9) Hoffman and Gortner, J.Bid. Chem., 65,371 (1925). (10) Pauli, Biochem. Z., 152, 355 (1924); 187, 403 (1927). (11) Reiner, Kolloid-Z., 40, 123 (1926). (12) Reitstotter, Ibid., 43, 35 (1927). (13) Rheinboldt, Ibid., 37, 387 (1925).
RECEIVED November 25, 1931.
Factors Affecting Quantitative Determination of Lignin by 7 2 Per Cent Sulfuric Acid Method GEO.J. RITTER,R. M. SEBORG, AND R. L. MITCHELL, Forest Products Laboratory, Madison, Wis.
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d
pretreating such pulps with the M O D I F I C A T I O K of A discussion is given of the discovery of alcohol-benzene solvent. the Ost and Wilkening additional factors that aflect the quantitative (3) method for the dedetermination of lignin in wood. Owing lo a termination of lignin was pubRATIOOF ACID TO SAMPLE knowledge of these factors, the following modiJicalished by M a h o o d (a) of the tions have been incorporafed in the Forest As originally adopted by the Forest Products Laboratory in Forest P r o d u c t s Laboratory, 1922. The modified method was Products Laboratory method for the delermination the method specified 12.5 cc. of designed for the quantitative of lignin in wood: 72 per cent sulfuric acid per 2 determination of lignin in wood. 1. The residue after extraction with alcoholgrams of sawdust. That ratio Since its publication, Mahood's benzene should be extracted with hot water. of acid to sample is sufficient, method has been modified at 2. The concentrated acid-wood mixture should but it requires considerable time v a r i o u s times. The need for for t r i t u r a t i n g the wood-acid changes became necessary soon be kept in a bath at 20" C. mixture. If the ratio of the after 1922, when such materials 3. Two hours should be allowed for solution of acid to the sawdust sample is as Cross and Bevan cellulose, the carbohydrates in the 72 per cent sulfuric acid. increased, as advocated later chemical pulps, and flax were 4. The diluted mixture should be hydrolyzed in this paper, the trituration analyzed for their lignin content. for 4 hours. can be accomplished with More recently the need for addigreater ease and in less time. tional changes became amarent when Sherrkd and Harr&-(4)found that slightly elevated tem- Further, when the method is employed for the analysis of peratures alter the physical and the chemical nature of lignin chemical pulps, which have a much higher cellulose content than wood, it is found necessary to increase the acid-sample prepared by the 72 per cent sulfuric acid method. These modifications may be classified under the following ratio in order to dissolve the carbohydrates (1). headings: (1) treatment of samples with alcohol-benzene; EXTRACTION OF SAMPLEWITH HOT WATER (2) ratio of acid to sample; (3) extraction of the sample with If the alcohol-benzene extracted residue is extracted with hot water; (4) duration of contact of sample with 72 per cent sulfuric acid; (5) temperature of the mixture containing 72 per hot water and dried before treating with 72 per cent sulfuric acid, very noticeable effects are observed in some cases, as is cent sulfuric acid and wood; and (6) duration of hydrolysis. shown in Table I. The lignin residue obtained is lighter in PRETREATMENT OF SAMPLES color, the yield lower, and filtration and washing facilitated. Apparently these effects are due to the presence, in some To avoid the presence of foreign materials in the lignin residue, pretreatment of the sample is necessary. The oils, woods, of extractives which are insoluble in alcohol-benzene resins, fats, and waxes are removed by extracting the sample but soluble in hot water. Further, these extractives are with a minimum boiling-point solution of alcohol-benzene insoluble in sulfuric acid or converted into insoluble products by the acid treatment. as recommended by Mahood (a). Bray, in determining lignin in chemical pulps ( I ) , found DURATIONOF CONTACT OF SAMPLEWITH 72 PER CENT the alcohol-benzene extraction unnecessary in most pulps, SULFURICACID since any fatty or resinous materials remaining after the The time during which the sample and the concentrated pulping process caused no interference with the subsequent lignin determination. Later the extraction was omitted in acid are in contact has a direct bearing on the quantity of the the determination of lignin in all chemical pulps. Recently lignin residue. Time must be allowed for the carbohydrates it has been found, however, that the extreme difficulty experi- to dissolve, but continued exposure appears to caramelize enced in filtering and washing the lignin residue from sulfite some of the dissolved carbohydrate material partially renderpulps which retain resins and fats may be largely overcome by ing it insoluble. The insoluble material is thus included in
I N D UST R IA L A N D E N G IN E E R IN G C H E M I STR Y
April 15, 1932
the lignin residue. The amount of partially decomposed carbohydrate material and the color of the lignin residue increases with the time of contact between the sample and the concentrated acid. Relationships between times of exposure of the sample to the concentrated acid and lignin yields are shown in Figure 1. A period of 2 hours has been adopted for the duration of contact of the sample with the 72 per cent sulfuric acid. TABLEI. EFFECTOF TEMPERATURE AND EXTRACTION WITH HOT W A T ~ R ON LIQNIN YIELDS OF FIVEWOODS TREATMENT WITH ACID^ FOR 17 H R . AT
SPECIES Sugar maple
.4v. White spruce
Av. Incense cedar
Av.
Catalpa
Av. Mesquite
5
ROOM
TEMP. 23.15 23.50 23.05 23.25 23.24 27.00 27.50 27.70 27.35 27.39 35.85 36.40 36.25 36.10 36.15 20.32 20.35 20.55 20.38 20.40 30.20 30.35 30.60 30.55 30.42
Av. 7 2 per cent sulfurio acid.
TREATMENT WITH ACID"FOR 2 H R . AT 20' c. Without hotWith hotwater water extraction extraction 21.40 21.05 21.70 21.20 21.45 21.10 21.35 21.52 21.18 26.00 25.90 25.95 25.90 26.00 25.95 26.30 25.85 26.06 25.90 34.80 33.85 34.25 33.80 34.05 33.75 34.10 33.25 34.30 33.66 17.67 17.23 17.32 18.12 18.04 17.31 18.40 17.34 18.06 17.30 24.70 27.95 24.70 28.00 25.10 27.75 24.50 27 15 27.71 24.75
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203
finely divided insoluble ligneous material so that filtering and washing of the residue may be facilitated. Working with several woods, it was found that 4 hours are sufficient for the hydrolysis of the carbohydrate portion, giving a clear filtrate. For shorter periods the filtrate from some woods was cloudy and gave a brown residue, whereas some of the woods examined did not require such a long period. Some types of materials to be examined in the future may require even longer periods, in which event the 4-hour period should be modified to suit the conditions.
PROCEDURE FOR DETERMINATION OF LIGNININ WOOD Since it has been shown that certain factors affect the lignin determination, it is suggested that the sulfuric acid method for the determination of lignin in wood be modified to read as follows: Approximately 2 grams of air-dried sawdust (60 to 80, or 80 to 100 mesh) are weighed in a tared alundum crucible. The crucible and its contents are dried to constant weight at 105" C., cooled, and weighed. The material is then extracted for 4 hours in a Soxhlet apparatus with a minimum boiling solution of alcohol-benzene. The solvent is removed by suction, the residue washed with alcohol by suction to remove the benzene, and then extracted with 400 cc. of hot water in a water bath for 3 hours, filtered, washed with hot water, then with alcohol, and finally dried. (Washing the 42 40
I
I
/I
TEMPERATURE OF CONCENTRATED ACIDAND WOODMIXTURE Room temperature was originafly specified for dissolving the cellulosic material in the 72 per cent sulfuric acid. Since there is usually a wide range in room temperatures during the various seasons of the year, and since Sherrard and Harris (4) have found that the amount and the properties of lignin are altered by slight variations in temperature during its isolation, its seemed advisable to study the temperature factor. From the results recorded in Figure 1, it may be noted that the 17-hour treatment of the sample with 72 per cent sulfuric acid indicates that yields from 22.3 to 28.3 per cent are obtained at temperatures ranging from 17" to 35" C. Shorter treatments of 1 and 2 hours a t the same temperatures indicate that yields from 21.5 to 22.5 per cent are obtained. ''0 5 IO I5 20 25 30 35 40 The 1-hour treatment shows, as a result of undissolved carboT E M P E R l T U R E (DEGREES CPNTIGRADE' hydrates, a rise in yield in going from 15" to 10" C. The 2hour treatment a t 20" C. has been chosen, therefore, in order FIGURE1. EFFECTOF TIMEAND TEMPERATURE ON LIGNINYIELDSIN SUGARMAPLE to ensure complete solution of the carbohydrates with miniWITH 72 PER CENT SULFURIC ACID mum decomposition. If these conditions are not maintained, lignin yields may be obtained from refined pulps or from Cross residue with alcohol aids in the removal of the sawdust from and Bevan cellulose which may be due largely to partially the crucible after drying.) The dried residue is transferred to decomposed carbohydrates, as shown by Sherrard and a glass-stoppered weighing bottle, and is well mixed with 25 cc. Harris (4). Darker color of the lignin as well as increased of 72 per cent sulfuric acid a t 20" C., and maintained a t that yields accompany increasing temperatures of the acid-wood temperature for 2 hours. The resulting mixture is transmixture. ferred to an Erlenmeyer flask, diluted with water to make a 3 per cent acid solution, and then boiled for 4 hours under a DURATION OF HYDROLYSIS reflux condenser. The hydrolyzed residue is filtered on a After the dissolving of the carbohydrates is completed in the tared alundum crucible, washed free of acid by means of hot 72 per cent sulfuric acid, enough water is added to the wood- water, dried, and weighed. The lignin content is calculated acid mixture to obtain a 3 per cent acid solution. During the on the basis of the oven-dry sample. In case a correction for ash is desired, transfer the lignin dilution, a part of the modified carbohydrates in the form of dextrin-like material is re-precipitated. The mixture must residue to a tared platinum dish and ash in the usual way. therefore be boiled in order to hydrolyze and render soluble I n the determination of lignin in chemical pulps, 40 cc. of all the carbohydrates and a t the same time coagulate the 72 per cent sulfuric acid should be used for dissolving the
20
204
ANALYTICAL EDITION
carbohydrates present in a 2-gram sample. The water extraction may be omitted with all chemical pulps and the alcohol benzene may be omitted with alkaline-cooked pulps. If large quantities of lignin are prepared at one time, i t is difficult to control the temperatures developed during the mixing of the acid and the sawdust. It is advisable, therefore, to use a lower concentration of acid and mix i t with the sawdust at a lower temperature than specified above. Sherrard and Harris (4) have found that a 70 per cent sulfuric acid mixed with sawdust a t 10’ C. is satisfactory. Results obtained from lignin determinations in several woods according to the foregoing revised method are recorded in the last column of Table I, The effect of tempera-
Vol. 4, No. 2
ture on the lignin yields may be noted by comparing columns 1 and 2; the effect of hot-water extraction, by Comparing columns 2 and 3.
LITERATURE CITED (1) Bray, M.W., Paper Trade J.,87,5943 (1928). ENQ.CHEM.,14, 933 (2) Mahood, S. A.,and Cable, D. E., J. IND. (1922). (3) Oat, H., and Wilkening, L., Chem.-Ztg., 34,461 (1910). (4) Sherrard, E. C., and Harris, E. E., IND.ENQ. ORRIM.,24, 103 (1932). RECBIVED October 1, 1931. Presented before the Division of Celluloae Chemistry at the 82nd Meeting of the American Chemical Society, Buffalo, N. Y., August 31 t o September 4, 1931.
New Test for Fat Aldehydes Resulting from Oxidation of Fats and Oils HELGESCHIBSTED, The Borden Company Research Laboratory, Syracuse, N. Y. Oxidation of edible fats and oils leads to talXIDATION of fats and amounts of oxygen are absorbed oils leads to the developby butter fat at a temperature lowy flavors and odors. These are considered to ment of tallowy odors of 95” C. It would not be quite be due to the formation of various aldehydes. and flavors whose measurement safe to a s s u m e that the same A new test for f a t aldehydes has been developed, is exceedingly difficult, being quantitative reIationships exist and experimental data are presented to show the subject to the senses of smell a t lower t e m p e r a t u r e s and best composition of the new reagent and the and taste which vary greatly during the early stages of oxidain different people. Numerous tion. method of procedure which will give the optimum attempts h a v e been m a d e to Powick (6) and later Muncolor eflect. The new rosaniline reagent is over measure the degree of oxidation dinger (5) in G e r m a n y have twenty times as sensitive as the Kreis test or the by determining the products of done a great deal of work on the Schiff reagent. It is speciJic for fatty glyceride oxidation. Kreis test. Powick applied the aldehydes. The results of the new test are exThe most widely used test for test to a large number of compounds found in oxidized fats tallowiness in fats, including pressed in arbitrary speciJic color units which f a t t y oils, is t h e Kreis test. and oils. The only one which are called ‘Ifat aldehyde values.” Special apg a v e t h e characteristic color This test depends on a color plications are given. Oxidation tests with butter with phloroglucinol was epihyreaction between phloroglucinol fat at 50” C. gave correlation between the amount a n d t h e oxidized f a t . A drinaldehyde. T h e formation of oxygen absorbed and the f a t aldehyde value. known amount of fat is shaken of this aldehyde from oleic acid bv a comDlicated s e r i e s of vigorously for 30 seconds in a Perhaps the fact that test tube with concentrated hydrochloric acid. A 0.1 per reactions is suggested by P;wick. cent solution of phloroglucinol in ether is added and the the color reaction of the Kreis test depends on the presence of mixture is again shaken. It is then allowed to stand. If the one special aldehyde explains why this test is often unreliable. Richardson (7), as chairman of a Kreis test committee, fat is tallowy, a red or pink color appears in the acid layer. Kerr (4) stated in 1918 that the Kreis test is only roughly reports on the use of the test on wintered cottonseed oil. He proportional to the rancidity. He agrees with Winkel (9) that prepared six samples of this oil with varying exposures to the it is too delicate and not specific. The expression “rancidity” air. These samples were sent to three different laboratories is used by these investigators in a broad sense which covers to be tested according to Kerr’s directions. The conclusion other flavors than those due to oxidation. This has led to was that the average grading of the samples by this method confusion among other workers who have tried to apply the failed to disclose any correlation between intensity of the Kreis test and rancidity as judged by flavors. Kreis test to all types of deterioration in fats and oils. Von Fellenberg (1) in Switzerland and Inichof and SchoschI n recording the results of the Kreis test, it has been customary to designate each as positive or negative. This is ine (3) in Russia used the Schiff reagent as a test for aldehydes not entirely satisfactory, for it fails to take into account the in fats and oils. The Schiff reagent was the customary yellow fact that the same degree of oxidation in different fats and oils solution which is made by adding a sodium sulfite or bisulfite may result in the formation of oxidation products widely solution and hydrochloric acid to a fuchsine solution. Von differing in character, with corresponding variations in flavor Fellenberg’s modification is the least acid and also the most and odor. It is therefore important to judge each type of fat sensitive. Mundinger states that the von Fellenberg test is or oil on its own merits. I n comparing flavor scores with the better than the Kreis test, but neither of these is as sensitive readings of any test for oxidation products, it is important as an organoleptic test when applied to butter fat. to have for each fat or oil an individual scale, varying according EXPERIMENTAL PROCEDURE to the use for which the fat or oil is intended. The purpose of the present investigation is to develop a Holm and Greenbank (2) have shown that the Kreis test can be applied in a quantitative way when relatively large sensitive and reliable test for measuring fat aldehydes. The
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