Bubl, Janet L.

Forest Products Laboratory (U.S.)

Harris, Elwin E.

Saeman, Jerome F.

Undetermined

ScholarsArchive@OSU

TI1E QUANTITATIVE SACCIIAPIFICATIO NOF WOOD AND CELLULOS ESeptember 1944No. 81458egwtUNITED STATES DEPARTMENT OF AGRICULTURELEOREST SERVIC EFOREST PRODUCTS LABORATOR YMadison, Wisconsi nIn Cooperation with the University of WisconsinTHE QUANTITATIVESACCL .RIFICATION OFWOODANDCELLULOS +̂l .1' 2ByJEROME F . SAEMAN, ' ChemistJANET L . BUBL, ChemistandELWIN E . HARRIS, ChemistForest Products Laboratory3 , Forest Servic eU . S . Department of Agricultur eSummar yA rapid analytical technique has boon developed for the hydrolysi sof cellulosic materials to reducing sugar in nearly quantitative yields .The method involves the treatment of the material with 72 percent sulfuri cacid for 45 :_inutes at 300 C . followed by a secondary hydrolysis for 1 hou rin a 15-pound autoclave or for 4-1/2 hours at the boiling point .Data arc presented to show how the yield of reducing sugar varie swith the conditions used .A table is given showing the "potential reducing sugar" content of15 sp ecies of wood and the variation occurxtig within a spopios .Introductio nIn experimental work on the hydrolysis of wood, it is desirable t odetermine thy; percentage of total sugar and of fermentable sugar potentiall yavailable in a sample of wood or hydrolyzed wood residue . According t oexisting t :c .-:_,ioueo, this can bo done by moans of a cellulose and a pontosandetermination, both comparatively long and slow operations .Based on studies of the U. S . Forest Products Laboratory at Madison, Wis . ,in cooperation with the Office of Production Research and Devolopmont o fthe War Production Board .2Preoonted before the American Chemical Society, Now York City, Septembe r11-15, 1944 .-Maintainod at Madison, ?this ., in cooperation with tho University of Wisconsin .R1458 -1-The quantitative saccharification of wood was investigated at th eForest Products Laboratory, in cooperation with the Office of Productio nResearch and Development of the War Production Board, it an effort to devis ea rapid and convenient technique fQr determining potential reducing sugar sas sugar, rather than indirectly as cellulose, or by difference after alignin determination . A certain amount of work has beef done in this field 'using aethods essentially similar to the Klason, or Willstatter, ligni ndetermination (1, 8) except that the hydrolysate was analyzed for reducingsugar . Kiesel•and Semiganovski (2) describe a method for quantitativesaccharification using 80 percent sulfuric acid, Ritter, Mitchell, an dSeborg made a study of factors affecting the conversion of an isolated woo dcellulose to sugar (5) . The work of earlier investigators, too, showe dthat high yields of sugar acre obtainable from cellulosic materials (4, 9) .DfscrIptith of	 oxi,sent sa ea'Conditions for Hydrolysis with Concentrated Acid.The present ex periments on the quantitative saccharification of woo dwere undertaken ueirig, as a basig, the sulfuric acid lignin analysis . Thi smethod, according to a widely accepted procedure S), involves the mixingof 72 percent sulfuric acid with the Gellulos.ie material at 20 0 C . • for 2houxe, diluting to 3 percent acid, and hydrolyzing at the boiling point fo r4 hours . The chief criterion for a correct lignin analysis is that ligni nmust be obtained in minimum yield . Conditions must be severe enough t oremove all carbohydrates, but not sufficiently severe to cause their recan-densation into insoluble materials . Workers in the field of ligni nchemistry appear to be in general agreement that a sulfuric acid concentra-tion of 72 percent is most satisfactory for lignin analysis from the stand -point of speed and accuracy . , For 'this reason this concentration of sulfuri cacid was used for experiments en saccharification .To reduce the time :required for analysis, it is desirable to have th etemperature of the 72 percent sulfuric acid as high as is permissible with-out causing excessive decomposition of the cellulosic•material . Ritter ,Mitchell, and Seborg showed that the yield of lignin from -wood treated wit h72 percent sulfuric acid for e, period of 1 hour showed a nearly constantlow value when the temperature was varied from 15 0 'to 3Oo C . (Q) . 'hisssame workers stowed that with a 2-hour treatment with 72 percent sulfuri cacid, Cross and Bevan cellulose showed a maximum yield of reducing sugar sat 350 O . 'or these reasons it was considered, well to begin ecpQrinents Qnsaccharification of cellulose using a temperature of 300 C .R1458	-2-Conditions for Secondary Hydrolysi sAccording to the various methods of lignin analysis with strong acids ,a secondary hydrolysis is conducted at the boiling point after dilution o fthe strong acid with water . By carrying out this secondary hydrolysis in anautoclave (sterilizer) at 15 pounds steam pressure, the time-necessary fo rcomplete hydrolysis is reduced .Before making this modification, however, a glucose solution was made ,in 4 percent :sulfuric acid, of such concentration as to be similar to thesugar concentration present during secondary hydrolysis . The decompositionof this solution was determined after heating for various lengths of time a t1000 C . and at 1210 C . in an autoclave . To avoid changes in volume duringthe treatment, the samples were sealed in glass bombs . After heating, thesamples were shaken with excess chalk, and filtered and sugar analyses wer emade by the Shaffer and Somogyi method using their reagent 50 with a 30-minute heating period . The sugar concentrations in the original and in th eheated samples were as shown in table 1.The data in table 1 indicate that sugar decomposition in solution sheld at 100 0 C . for 3 hours was comparable to that in solutions held a t121° C . for 1 hour . Although it was difficult to measure accurately suc hsmall changes in sugar concentration, the data given in table 1 show tha tthere was no apparent disadvantage in conducting the secondary hydrolysi sin an autoclave .The Yield of Reducing Sugar from Wood andother Cellulosic Materials Hydrolyze dunder Various Condition sA series of experiments was performed in which 5 milliliters of 7 2percent sulfuric acid was allowed to react at 30° C . for various lengths oftime with 0 .5 gram of Douglas-fir wood, ground to pass a 30-mesh screen .The samples were then diluted with different amounts of water and autoclave dat 15 pounds steam pressure for periods of 15, 30, and 60 minutes . Sugaranalyses were made after neutralization with chalk . The results are shownin table 2 .From the data in table 2, it appears that there is considerabl elatitude in the choice of conditions for quantitative saccharification o fwood, High yields of reducing sugar occurred with the following treatments :(1) 40 minutes primary hydrolysi s60 minutes secondary hydrolysis wit h4 percent H2SON4(2) 85 minutes primary hydrolysi s60 minutes secondary hydrolysis wit h4 percent H 2S0481458	.:3 :.s•(3) 120 minutes primary hydrelysi s60 minutes secondary hydrolysis wit h2 percent ' HSQ4Another experiment was conducted in an effort to define more pre-cisely the conditions for quantitative saccharification of wood . No further -work was done•on the acid concentration for secondary hydrolysis since 4percent acid appeared satisfactory . A different sample of wood was used fo rthis experiment . The results are given in table 3 .The accuracy of analytical methods for determining reducing sugars i ssuch that optimum conditions cannot be precisely chosen from the data i ntable 3 . Primary hydrolysis .for 45 -minutes and secondary hydrolysis for 60minutes appears to give sufficient latitude to insure reproducibility of th eresults within the limitations of the sug analysis .This method of saccharification was applied to extracted cotton, is ahigh-alpha pulp, and to glucose , in an effort to determine how nearl yquantitative the method was, and where the chief loss of glucose occurred .A glucose sample when given a primary hydrolysis of 45 minutes and asecondary hydrolysis of 60 minutes showed a °potential sugar content" o f96 .9 percent . A similar sample that was autoclaved with 4 percent sulfuri cacid but was not treated with 72 percent acid showed the presence of 97 . 3percent potential sugar, which . indicates that the secondary hydrolysis i smore destructive than the treatment with 72 . percent acid. Several sample sof wood cellulose and extracted cotton showed the presence of 105 .0 to 108, 0percent potential .sugar ; the theoretical yield of reducing sugar from pur ecellulose is 111 .1 percent .To compare this technique with other methods, the potential suga rpresent in two samples of Douglas-fir was determined by the propos-ed metho d'and by a method developed by Kiesel'and Semiganovski (2) and recommended b yDuero (.q), in wMMch 80 percent sulfuric acid is used for the primaryhydrolysis , , -and the .secondary hydrolysis is conducted at the boiling poin tfor 5 hours after dilution with 15 volumes ' of water .  The values obtainedwere as follows :	'Zieeeland Sem1ganovski method	Proposed method 'Sample I	68 :3 percents •	71 .4 percentSample II	64 .4 percent	68 .0 percentProm these results it is apparent that the proposed method €:ives• . ahigher yield of sugar than the Kie^el and Semiganovski method : Whileneither this method nor any other method for the saccharification o fcellulose gives truly quantitative yields of reducing sugar, it is adequat efor present purposes . If mere accuracy is required, an experimentall ydetermined factor can be used to correct for the sugar decomposed in th eprocess .R1458	-4--Recommended Method for the Quantitative Saccharificatio nof Cellulosic 'Material sThe method recommended for the quantitative saccharification o fcellulosic materials is as follows :The sample is ground to pass a 30-mesh screen . It is then air driedto a low moisture content, and the moisture is determined . A quantity ofthe material sufficient to contain approximately 0 .35 gram of cellulose i sweighed into a 30-milliliter shell vial . To this is added 5 milliliters o f72 percent sulfuric acid that has been cooled to 15° C . It is- next mixedthoroughly with a stirring rod and put in a water bath at 30°	This tem-perature is maintained for 45 minutes while the mixture is stirred at 5- o r10-minute intervals . Using wood it is best to add all of the 72 percen tacid at once . With cotton or a pulp it is desirable to make a homogeneousmixture with 1 milliliter of 72 percent acid and then add the remaining 4milliliters . After a total time of 45 minutes the mixture is washed fromthe vial into an Erlenmeyer flask with 140 milliliters of water . Thediluted solution is autoclaved at 15 pounds steam pressure for 1 hour . Atthe end of this time the sample is cooled, diluted to exactly 250 milli -liters, neutralized with excess chalk, filtered, and analyzed for sugar b ythe Shaffer and Somogyi method_ (7) . While the use of an autoclave i spreferred for the secondary hydrolysis, identical yields of reducing suga rhave been obtained by boiling for 4-12 hours at atmospheric pressure. I fit is desired, the fermentable sugar present can be determined in the fina lneutralized solution by the rapid yeast sorption method described i nanother report	(10) .The Potential Reducing Sugar Content of Various Sample sof Wood Determined by the Proposed Metho dThere are wide variations in the amount of sugar potentially avail -able in samples of wood of different species, and even in different sample sof the same species . Table 4 illustrates the differences occurring withi na species. All percentages are given on an oven-dry unextracted basis .In table 5 is shown the potential sugar in samples of representativ ehardwoods and softwoods. In all tests, samples of wood cut from thre edifferent logs were ground together to average partially the difference swithin the species .The data in table 5 are intended only as a rough indication of th epercentage of sugar available from the various species . It is obvious fromthe data in table 4 that large variations occur within a species ; hence ,only large differences can be considered of significance ,R1458	~3=Literature Cite d(1) !lason, P ., Cellulosechemie 4, 81 (1923) .(2.) iiesel, A. and Semiganovski, 1 ., Ber . 60B, 333-8 (1927) .(3) Luers, H ., Angew. Chorale 45, 369-76 (1932) .(4) Monier--Williams, C . W ., J . Chem. Soc . 119, 803-5 (1921) .(5) Ritter, G. J ., Mitchell, R . L ., and Seborg, R . M .,	J . Am . Chem .Soc . 55, 2989-91 (1933) .(6) Ritter, G . J ., Seborg, R . M ., and Mitchell, R . L ., Ind . Eng . Chem.Anal. Ed . 4, 202-4 (1932) .(7) Shaffer, P . A . and Somogyi, N .,	J . Biol . Chem . 100, 695 (1933) .(8) Willtatter, R . and Zechmeister, L ., 3er . 46, 2401,	(1913) .(9) Wohl, A. and gull, H ., Cellulosechemie 2, 1-7 (1921) .(10) Saeman,	J. F ., Harris, E . E ., and Kline, A . A ., FPL Mimeo . 81459Table l .--Sugar concentrations in samples of glucose determine dafter heating at 1000 C . and 121° C . in an autoclaveSample	: Heating period	: Temperature	: SugarMinutes °	o .concentration sMilligrams pe rmilliliter1 0 0 1 .58► 2 2 121 1 .583 8 121	: 1 .554 30 121 1,565 .60	. 121	: 1 .546 0 100 1 .587 _ 5 100 1 .568p	: 15 100 1 .569 60 100 1 .5610 180 100 1 .55Table 2 .---Yield	 of reducing sugar from Douglas-fir hydrolyzed unde rvarious condition sTime of	Time of	Reducing sugar : Reducing sugar : Reducing sugarprimary : secondary : yield using 1 : yield using 2 : yield using 4hydroly^is. : hydrolysis	percent acid : percent acid : percent aci dat .30° C .	at 121 C . : for secondary : for sebondary : for secondaryMinutes T1linuteshydrolysisPercenthydrolysi sPercenthydrolysis--Percent40 15 27 .7 39,7 50 . 530 38 .8 51 .2 64 . 060 53 .0 63 .4 68 . 085 15 36 .9 45 .0 : 53 . 730 46 .0 56 .4 64 . 760 56 .4 65 .2 66 . 8: t120 15 42 .3 48 .1 57 .030 51,9 59 .2 65 . 560 58 .0 68 .0 64 .5R1458Tablo 3 .--RedViAgsugaryield of samples of Douglas-fi rwood qOjectedto differentperiodsof primaryandsegondaKyhydrolysisTime of primary	: Time of secondary :hydrolysis at 300 C . 4 hydrolyg ip wi:th 4 : Reducing sugarwith 72 percent acid : . percent acid	: .-------------------- -Minutes-------------------Einutes----------- ----- -Percent30 30 68 . 330 60	• 68 . 630 120	- 67,045 . 30 67 . 945 ' 60 68 . 645 120 67 .060 30 68 . 760 60	: 68 . 960 12Q . 66,4Table 4 .-A.Ytp1d, sf potential redu'eing sugar from .Arrieus samples of Douglas-fi rSample 4 Potential Description of sampl e1 reducing sugar	 ,:	 ------------ -------------------- -1 Percent1 71 .4 Young, rapid growth, 70 percent61 .74 .	sapwood .Old., slow growth heartwood .3 : 68 .3 1 Young, rapid-growth heartwood .4 t 68 .0 Heartwood, 1 .5-foot diameter tree ..5 69 .3 Sapwood., same tree .6 67 .3 Mixture of chipped woo&7 68 .0 Mixture of chipped wood.8 : 71 .4 Young, rapid growth, two-third st	sapwood .41458Table 5 .--Yield of potential reducing sugar and fermentablesugar from samples of representative hardwood sandsoftwoodsSpecies	: Potential re- : Fermentability by: ducing sugar	:	yeast sorptionPercent PercentHardwoodsAmerican beech	 : 70 .1 75 . 1Aspen	 : 75 .1 76 . 3Birch . . .	 :	. 69 .9 67 . 8Maple	 68 .2 71 . 0Red oak	 : 63 .6 63 . 0Sweetgum	 66 .4 73. 8Yellow-poplar	 :Softwoods70 .9 76 . 1Douglas-fir	 : 66 .6 86 . 2Eastern white pine	 : 66 .5 86 . 3Hemlock	 66 .1 88 . 2Ponderosa pine	 : 68 .0 82 . 2Redwood	 : 52 .4 77 . 1Sitka spruce	 : 70 .1 85 . 3Southern yellow pine	 : 64 .8 82 . 0Sugar pine	 : 64 .3 82 .4R1458

The quantitative saccharification of wood and cellulose

https://ir.library.oregonstate.edu/downloads/8049g924s

The quantitative saccharification of wood and cellulose

Abstract

Similar works

Full text

Available Versions

ScholarsArchive@OSU