Quantitative Saccharification of W o o d and Cellulose JEROME F. SAEMAN, JANET L. BUBL, A N D ELWIN E. HARRIS Wis.
Forest Products Laboratory, U. S. Department of Agriculture, Madison,
A rapid analytical technique has been developed for the hydrolysir of cellulosic materialr to reducing sugar in nearly quantitative yieldr. The method involves treatment of the material with 72% sulfuric acid for 45 minuter at 30" C. followed by a secondary hydrolyrir for 1 hour in a 15-pound autoclave or for 4.5 hours at the boiling point. Data are prerented to show how the yield of reducing sugar varies with the conditions used. A table shorn the "potential reducing sugar" content of 1 5 rpecies of wood and the variation occurring within a speciea.
INis
EXPERIMENTAL work on the hydrolysis of wood, it desirable to determine the percentage of total sugar and of fermentable sugar potentially available in a sample of wood or hydrolyzed wood residue. According to existing techniques, this can be done by means of a cellulose and a pentosan determination, both comparatively long and slow operations. The quantitative saccharification of wood was investigated at the Forest Products Laboratory, in cooperation with the O5ce of Production &search and Development of the War Production Board, in an effort to devise a rapid and convenient technique for determining potential reducing sugars as sugar, rather than indirectly as cellulose, or by difference after a lignin determination. A certain amount of work has been done in this field using methods essentially similar to the Klsson, or Willstatter, lignin determination (8, 9 ) except that the hydrolyzate was analyzed for reducing sugar. Kiesel and Semiganovski ( I ) describe a method for quantitative saccharification using 80% sulfuric acid. Ritter, Mitchell, and Seborg made a study of factors affecting the conversion of an isolated wood cellulose to sugar (6). The work of earlier investigators, too, showed that high yields of sugar are obtainable from cellulosic materials (4,ZO). DESCRIPTION OF EXPERIMENTS
CONDITIONSFOR HYDROLYSISWITH CONCENTRATED ACID. The present ex eriments on the uantitative saccharification of wood were uniertaken using, as atasis, the sulfuric acid lignin analysis. This method, according to a widely accepted cedure (6),involves mixing 72 sulfuric acid with the cellul?zi material at 20' C. for 2 hours, iluting to 3% acid, and hydrolyxing at the boilin point for 4 hours. The chief criterion for a correct 1~ anafysis is that lignin must be obtained in minimum yield. onditions must be severe enough to remove all carbohydratea but not sufficiently severe to cause their recondensation into insoiuble materials. Workers in the field of lignin chemistry appear to be in general agreement that a sulfuric acid concentration of 72% is most satisfactory for lignin analysis from the standpoint of speed and accuracy. For this reason this concentration of sulfuric acid was used for experimenta on saccharification. T o reduce the time required for analysis, it is desirable to have the temperature of the. 72% sulfuric acid as high as is prmissible without causing excessive d e w m y i t i o n of the cell@osicmaterial. Ritter, Seborg, and .Mitchell s owed that the eld of lignjn from wood treated m t h 72% sulfuric acid for 1 rour showed a nearly constant low value when the temperature was varied from 15' to 30' C. (6). These same workers reported that with a %hour treatment with 72% sulfuric acid, Crosa and Bevan cellulose showed a maximum yield of reducm sugars at 35" C. For these reasons it was considered well to &gin expenmenta on saccharification of cellulose using a temperature of 30" C. CONDITIONS FOR SECONDARY HYDBOLYSIS.According to the various methods of lignin analysis with strong acids, a secondary hydrolysis is conducted at the boiling point after dllution of the strong acid with water. It was thou h t that, by carrying out this secondary hydrolysis in an autocfave (sterilizer) at 0.8-kg. (16-pound) s t e m preasure the time necessary for complete hydrolysis could be reduced. Before makin this rno&ication, however, a glucose solution was made, in 4 6 sulfuric acid, of such concentration as to be
(r
similar to the su ar concentration present during secondary hydrolysis. The ckecomposit ion of this solution was determined after heating for various lengths of time at 100' and 121' C. in an autoclave. To avoid changes in volume during the treatment, the samples were sealed in glass bombs. After heating, the samples were shaken with excess chalk and filtered and sugar analyses were made by the Shaffer and Somogyi method, using their reagent 50 with a 30-minute heating period. The sugar concentrations in the original and in the heated samples were as shown in Table I. The data in Table I indicate that sugar decomposition in solutions held a t 100" C. for 3 hours was comparable to that in solutions held a t 121' C. for 1 hour. Although it was difficult to measure accurately such small changes in sugar concentration, Table I shows that there was no apparent disadvantage in conducting the secondary hydrolysis in an autoclave.
YIELD OF REDUCING SUGU FROM WOODAND W ~ ECELLUR LOSIC MATERIALS. A series of experiments was performed in
which 5 ml. of 72% sulfuric acid were allowed to react at 30" C. for various lengths of time with 0.5 gram of Douglas 6r wood, ound to pass a 30-mesh screen. The samples were then %luted with different amounts of water and autoclaved at 6.8kg. (15-pound) steam pressure for 15, 30, and 60 minutes. Sugar analyses were made after neutralization with chalk. From the data in Table 11, it appears that there is considerable latitude in the choice of conditions for quantitative saccharification of wood. High yields of reducing sugar occurred with the following treatments: (1) 4:minute pnmary hydrolysis, &minute secondary hydrolysis, w!h 4% sulfuric acid; (2) 85minute primary hydrolysis, 60-minute secondary hydrolysis, with 4% sulfuric acid; and (3) 121)-minute rimary hydrolysis, &minute secondary hydrolysis, with 2% supfuric acid. Another experiment w a s conducted in an effort to define more precisel the conditions for quantitative saccharification of wood. KOfurtger work was done on the acid concentration for secondary hydrolysis, since 4y0 acid appeared satisfactory. A different sample of wood was used for this experiment. The results are given in Table 111.
Table 1.
Su ar Concentrrtionr in Samples of Glucore Determined after heating at 100" and 121 C. in an Autoclave
Sample
No.
Heating Period
Temperature
Rugsr Concentrations
Min.
* c.
Mo./ml.
100 100 100 100 100
1.68 1.68 1.b6 1.66 1.66
1 2
3
4 6 0 b
6 7 8 9 10
Table
lb
60 180
II. Yield of Reducin Sugar from Douglas Fir Hydrolyzed under V?arious Conditions
Time of Primary Hydrolysis at 30° C.
Time of Secondary Hydrolysis at 121' C.
Min.
Min.
%
%
%
40
16 30 a60
27.7 38.8 63.0
39.7 51.2 63.4
b0.b 64.0 68.0
85
15 30 60
36.9 46.0 66.4
4b.O b0.4 6b.2
53.7. 64.7 66.8
120
1b 30 60
42.3 61.9 b8.0
48.1 69.2 68.0
b7.0 65.6 64.b
Reducing Sugar Yield 1% acid 2% acid 4% acid for secondary for secondary for secondary hydrolysis hydrolyais hydrolyaia
36
Vol. 17, No. 1
INDUSTRIAL A N D ENGINEERING CHEMISTRY
Table Ill. Reducing Sugar Yield of Samples of Douglas Fir Wood Subjected to Different Periods of Primary and Secondary Hydrolysis Primary Hydrolysia a t 30' C. with 72% Acid Min. 30
Secondary Hydrolysis with 4% Acid Min. 30 00 120
Reducing Sugar
% 08.3 08.0 07.0
45
30 00 120
07.9 08.0 07.0
00
30 00 120
08.7 08.9 00.4
The accuracy of analytical methods for determining reducing sugars is such that optimum conditions cannot be precisely chosen from the data in Table 111. Primary hydrolysis for 45 minutes a t 30" C. and secondary hydrolysis for 60 minutes a t 121' C. appear to give sufficient latitude to ensure reproducibility of the results within the limitations of the sugar analysis. A number of analyses were made identical with those described previously except that primary hydrolysis was carried out a t 20' C. for 2 hours, as in the lignin analysis (6). The yield of reducing sugar was found to be the same as that obtained using a higher temperature and shorter time. This method of saccharification was applied to extracted cotton, to a high-alpha pulp, and to glucose in an effort to determine how nearly quantitative the method was, and where the chief loss of glucose occurred. A glucose sample when given a primary hydrolysis of 45 minutes and a secondary hydrolysis of 60 minutes showed a "potential sugar content" of 96.9%. A similar sample that was autoclaved with 4% sulfuric acid but was not treated with 72% acid showed the presence of 97.373 potential sugar, which indicates that the secondary hydrolysis is more destructive than the treatment with 72y0 acid. Several samples of wood cellulose and extracted cotton showed the presence of 105.0 to 108.00/, of potential sugar; the theoretical yield of reducing sugar from pure cellulose is 111.1%. The sugar from cotton contained less than 1.5% nonfermentable reducing material, indicating that little decomposition and reversion had occurred. To compare this technique with other methods, the potential sugar present in two samples of Douglas fir was determined by the proposed method and by a method developed by Kiesel and Semiganovski ( I ) and recommended by Luers (S), in which 80% sulfuric acid is used for the primary hydrolysis, and the secondary hydrolysis is conducted at the boiling point for 5 hours after dilution with 15 volumes of water. The values obtained were as followe:
Sample I Sample I1
Kieael snd Semi anovski Mehd, % 08.3 04.4
Propoeed Method, % 71.4 08.0
From these results it is apparent that the proposed method gives a higher yield of sugar than the Kiesel and Semiganovski method. While neither this method nor any other method for the saccharification of cellulose gives truly quantitative yields of reducing sugar, it is adequate for present purposes. If more accuracy is required, an experimentally determined factor can be used to correct for the sugar decomposed in the process. RECOMMENDED METHOD
The method recommended for the quantitative saccharification of cellulosic materials is as follows: The sample is ground to pass a 30-mesh screen. It is then air-dried to a low moisture content, and the moisture is determined. A quantity of the material sufficient to contain appro imately 0.35 gram of cellulose is weighed into a 30-ml. shell vi>,
and to it are added 5 ml. of 72% sulfuric acid that has been cooled to 15" C. It is next mixed thoroughly with a stirring rod and put in a water bath at 30" C. This temperature is maintained for 45 minutes while the mixture is stirred at 5- or IO-minute intervals. Using wood it is best to add all the 72% acid a t once. With cotton or a pul it is desirable to make a homogeneous mixture with 1 ml. of 72% acid and then add the remaining 4 ml. After a total time of 45 minutes the mixture is washed from the vial into an Erlenmeyer flask with 140 ml. of water. The diluted solution is autoclaved at 15 pounds' steam ressure for 1 hour. At the end of this time the sample is coole$ diluted to exactly 250 ml., neutralized with excess chalk, atered, and analyzed for sugar by the Shaffer and Somogyi method (8). While the use of an autoclave is preferred for the secondary hydrolysis, identical yields of reducing sugar have been obtained by boiling for 4.5 hours at atmospheric pressure. If desired, the fermentable sugar present can be determined in the final neutralized solution by the rapid yeast sorption method described in another report (7').
Table
IV.
Sample NO.
Yield of Potential Reducing Sugar from Various Samples of Douglas Fir Potential Reducin Sugar, 71.4 01.7 08.3 68.0 69.3 07.3 08.0 71.4
2
Deacription of Sample
;?y, ,s ow-growth rapid growth, heartwood 70% sapwood Young, ra id growth heartwood Heartwoo$, id-foot diameter tree Sapwood, same tree Mixture of chipped wood Mixture of chipped wood Young, rapid-growth. two-thirda sapwood
POTENTIAL REDUCING SUGAR CONTENT OF WOOD DETERMINED BY PROPOSED METHOD
There are wide variations in the amount of sugar potentially available in samples of wood of different species, and even in difFerent samples of the same species. Table IV illustrates the differences occurring within a species. All percentages are given on an oven-dry unextracted basis. In Table V is shown the potential sugar in samples of representative hardwoods and softwoods. In all tests, samples of wood cut from three different logs were ground together to average partially the differences within the species.
Table
V. Yield of Potential Reducing Sugar and Fermentable Sugar from Representative Hardwoods and Softwoods Potential Reducing Sugar
Species Hardwoods American beech Aspen Birch Maple Red oak Sweetgum Yellow poplar Softwoods Douglas fir Eastern white pine Hemlock Ponderosa pine Redwood Sitka spruce Southern yellow pine Sugar pine
Fermentability by Yeast Sorption
%
%
70.1 75.1 09.9 08.2 03.0 08.4 70.9
75.1 70.3 07.8 71.0 03.0 73.8 70.1
00.0 00.5 60.1 08.0 52.4 70.1 04.8 04.3
80.2 80.3 88.2 82.2 77.1 85.3 82.0 82.4
~~
The data in Table V are intended only as a rough indication of the percentage of sugar available from the various species. It is obvious from the data in Table IV that large variations occur within a species; hence, only large ditlerences can be considered of significance. This method for the quantitative saccharification of cellulose has been used successfully a t the Forest Products Laboratow for the past year in the evaluation of material for hydrolysis. It is useful in assaying various wood wastes, such as sswduet, shavings, bark, and slabs, for their potential carbohydrate conten(. The yield of sugar obtained by dilute acid hydrolysis will
ANALYTICAL EDITION
January, 1945
vary, depending on the conditions employed, but *der the same conditions there is good correlation between the relative amounts of sugar obtained from different woods by the two methods. LITERATURE CITED
(1) Kiesel, A., and Semiganovaki, N., Em., 60B,333-8 (1927). (2) Klsson. P..Cellulosechem.. 4, 81 (1923). (3j Luers, H., A n g w . Chem., 45, 369-76 (1932). (4) Monier-Williams, C.W., J. Chem. Soc., 119,803-5 (1921). (5) Ritter, 0.J., Mitchell, R. L., and Seborg, R. M.,J . Am. C h m . SOC.,55, 2989-91 (1933).
(6) Ritter,
37
G. J., Seborg, R. M., and Mitchell, R. L., IND. ENO.
ANAL.ED.,4, 202-4 (1932). (7) Saeman, J. F., Harris, E. E., and Kline, A. A., Forest Products Lab. Report, R1459 (1944). (8) ShafTer,P. A., and Somogyi, M., J . Biol. Chem., 100,695 (1933). (9) WillstLtter, R., and Zechmeister, L., Em., 46,2401 (1913). (10) Wohl, A., and Krull, H., Cellulosechem., 2, 1-7 (1921). CREAf.,
PRESENTEO before the Division of Cellulose Chemistry a t the 108th hfeeting of the A M ~ R I C ACHEMICAL N SOCIETY, New York, N. Y. Based on studiea of the U. S. Forest Products Laboratory in cooperation with the Office of Production Research and Development, War Production Board.
Rapid Volumetric Method for Aluminum L. J. SNYDER,
Ethyl Corporation, Baton Rouge, La.
A rapid and accurate procedure is described for the determination of aluminum in the presence of 1 to 10% of impurities such as calcium, copper, chromium, iron, magnesium, manganese, and zinc. Experimental data are presented showing the accuracy and the effects of impurities commonly associated with aluminum. Five to 10 minutes' time is required per analysis.
HERETOFORE,
it has been common practice to determine aluminum by precipitation as the hydrous oxide followed by ignition to constant weight. This procedure is time-consuming and of such doubtful accuracy that materials high in aluminum content are analyzed by determining the other components and calculating aluminum by difference. However, when the impurities are present to the extent of only a few per cent and an w a y of the impurities is not required, a direct, rapid, and accurate volumetric method is desirable. The use of potassium fluoride in the acidimetric determination of aluminum has been studied by a number of investigators (1-6). The method described by Viebock and Brecher (6) appears to have the widest application. Their procedure consists of neutralizing the aluminum solution with sodium hydroxide in the presence of sodium potassium tartrate to the phenolphthalein end point, adding neutral potassium fluoride, and titrating the liberated potassium hydroxide 3a. a measure of the aluminum present. This reaction does not proceed stoichiometrically, the end point appearing about 10% before the equivalence point. Aluminum hydroxide, formed by the neutralization of a solution of an aluminum salt to the phenolphthalein end point, reacta with potassium fluoride to form the neutral salt, AlFI.3KF, and liberates 3 moles of potwium hydroxide permoleof aluminum present. This reaction is slow even with heating (6),but in the presence of sodium potassium tartrate it is practically instantaneous (e). The acid used to titrate the liberated base is a direct measure of the aluminum present. The following are the reactions for the neutralization of the aluminum ion and the liberation of the base by the potassium fluoride:
+
AI+*+ 30H- +Al(OH), Al(OH)* 6KF +ALFI.3KF 3KOH
+
+
EXPERIMENTAL C.P.
Table A1 Added Qrom 0.1282 0.1028
0.0789 0.0513 0,0266 0.1236 0.1236 0.1236 0.1238 0.1236 0.1236
1.
Analysis of Known Aluminum Solutions Difference AI Found Qram Gram 0.1282 0.1025 0.0768 0.0613 0.0266 0.1237 0.1237 0.1235 0.1238 0.1235 0.1236
hlean of differenae
The purpose of this paper is to show that the reaction between aluminum hydroxide and potassium fluoride in the presence of sodium potassium tartrate, with certain modifications of previous procedures, can be used for the quantitative determination of aluminum in the presence of small quantities of some of the usually interfering ions.
REAGENTS.All chemicals used were of Baker's with no further purification.
Barium hydroxide, saturated solution. Hydrochloric acid, 0.3 N solution standardized with reference to a.known aluminum chloride solution, Standard aluminum chloride solution, 0.3 N . Weigh accurately 2.5 to 2.7 grams of pure aluminum wire (Baker's c.P., 99.9970) and dissolve in 200 ml. of approximately 1 N carbonatefree sodium hydroxide, make the solution just acidic with hydrochloric acid (1 to l), and boil slowly to expel carbon dioxide before diluting to 1 liter. Use aliquots of theatandard solution to standardize the hydrochloric acid by the procedure described below. Sodium potassium tartrate, 30% solution. Potassium fluoride (KF.2H20),30% solution, neutral to phenolphthalein and stored in a wax-lined bottle. PROCEDURE. T o the aluminum chloride solution free of ammonium ion and other interfering substances and containing 25 to 130 mg. of aluminum, add standard barium hydroxide solution until the free acid is neutralized as indicated by a slight precipitate of aluminum hydroxide. Introduce 30 ml. of the sodium potassium tartrate solution and continue the titration to the phenolphthalein end point. Add 30 ml. of neutral (to phenolphthalein) potassium fluoride solution and titrate the liberated base with 0.3 N hydrochloric acid to the disappearance of the red color. The end point is considered permanent when the color does not return after 30 seconds. The standard hydrochloric acid used to titrate the liberated base is equivalent to the aluminum present. TESTS ON KNOWNSOLUTIONS. The reliability of the method was determined by analyzing known aluminum chloride samples, prepared by dissolving pure aluminum wire in 1 N sodium hydroxide (carbonate-free). The sodium aluminate was then made slightly acidic with dilute hydrochloric acid and the solution diluted to R known volume. Aliquots were withdrawn and the aluminum was determined by the procedure described. The accuracy was found to be t0.1 mg. Data showing the analysis of known solutions are presented in Table I.
quality
0.0000
-0.0001 0.0001
- 0.0001 0.000
$0.0001 +0.0001 +0.0002 -0.0001
-0.0001 -0.0001
0.0001
ANALYSISOF ALUMINUM I N PRESENCE OF INTERFERINQ SUBThe effect of impurities was determined by adding the ions in three concentrations to solutions containing known amounts of aluminum and then analyzing the solutions for nluminum (mole ratios of aluminum to impurity were 2, 10, and 100). At an aluminum-impurity ratio of 100, only phosphate, sulfate, and silicate interfered, while calcium, chloride, carbonate, chromium, copper, iron, magnesium, manganese, and zinc did not STANCES.
