642
ANALYTICAL CHEMISTRY
9. The temperature of the sample does not affect the result of the titration. A sample of chlorine solution buffered a t pH 7 and containing potassium iodide was divided into two parts and titrated a t 0' and 23 O C., respectively. Both titrations showed 0.240 p.p.m. Both the actual microamperes a t corresponding points and the gradient microamperes per milliliter of arsenite were much lower a t the lower temperature, indicating lowered sensitivity, but the amount of arsenite required was the same in both cases. 10. The rate of hydrolysis of chlorine dioxide decreases with temperature. T o reach completion of hydrolysis a t 0" C. requires a t least 10 minutes at pH 12. A solution which contained approximately 1.3 p.p.m. of chlorine dioxide and 0.34 p.p.m. of chloramine was prepared. One part was completely hydrolyzed a t 25" C. pH 12.1, and on titration showed 0.337 p.p.m. of chloramine and no chlorine dioxide. A second and third sample were hydrolyzed at 0 O C. and p H 12, 1for 5 and 10 minutes, respectively, then neutralized and titrated for chloramine and residual chlorine dioxide. After hydrolyzing for 5 minutes the sample still contained 0.029 p.p.m. of chlorine 'dioxide. After hydrolyzing for 10 minutes 0.009 p.p.m. of chlorine dioxide remained, which is just within the accuracy of the method. For Titrations I and I1 a t 0" C. a t least 10 minutes and preferably longer should be allowed for hydrolysis.
ACKNOWLEDGMENT
The authors wish to express their sincere appreciation to G . D. Byrkit and R. S. Aston for the valuable assistance, constructive criticism, and encouragement generously given the conduct of this work. LITERATURE CITED
(1) Griffin, A. E.. J . Am. Water Works Sssoc., 27, 688 (1935). (2) Hopkins, E. S.. Ind. Eng. Chem., 19,774 (1927). ., Pan, Yu-Djai, J . Am. Chem. Soc., 61, 3402 (3) Kolthoff, I. JIand (1939). (4)Maiks, H. C., and Glass, J. R., J . Am. Water Works Assoc., 34,1227 (1942). ( 5 ) Ridenour, G. M., and Ingols, R. S.,Ibid., 39,561 (1947). (6) Scott, R. D., Ibid., 26, 634 (1934). (7) Treadwell, W. D., Helv. Chim. Acta, 4,396 (1921). (6) Washburn, E. W., J . Am. Chem. Soc., 30, 31 (1908). '
RECEIVED Ootobcr 11, 1947. Presented before the Division of Water, Sewage, and Sanitation Chemistry a t the 112th Meeting of the AUERICAN N e u liork, ?i. T. CHEMICAL SOCIETY,
Estimation of Hemicelluloses in Holocellulose from Nonwoody Plant Material EMMETT BENNETT Massachusetts Agricultural Experiment Station, A m h e r s t , Mass. A method is described for the determination of hemicelluloses from holocellulose of nonwoody plant material, based on quick extraction of hemicellulosegwith an alkaline solution that is completely oxidized by an acid chromate solution. The reduced chromate is a measure of the organic matter. which is composed principally of hemicelluloses.
T
HE determination of hemicelluloses is made infrequently, despite the fact that they are probably present in all plants and in relatively large quantities in cereal straws. The heniicelluloses usually recognized are of two general types. The incrusting type consists of pentosans, hexosans, mixtures of these two, and polyuronides. The other type, referred to as cellulosans, are believed to be constituents of the cellulose pattern and do not contain uronic acid groups ( 5 ) . The former group is the more readily soluble and probably corresponds most nearly to the 8and y-cellulose of industry. The lat'ter may be relatively insoluble. .?I portion of this fraction together &-iththe cellulose corresponds approximately to t,he a-cellulose of industry. The hemicelluloses determined by the present met,hod are believed to be chiefly of the polyuronide type. Because some cellulosans as well as pentosans and hexosans may be extracted along wit,h polyuronide hemicelluloses, the use of recognized terminology is abandoned and the whole fraction extracted is referred to as hemicelluloses, although the fract,ion extracted under the conditions prescribed is essentially similar to the 8- and ycellulose fraction. I t is felt that such a differentiation, while empirical, has certain advantages. The methods ordinarily used for t'he estimation of hemicelluloses are based on the determination of the reducing values of t,he sugars formed during hydrolysis, the determination of furfural, and direct isolations (3,7,9). The older methods of isolations are reasonably specific but are rather laborious for routine procedures. -1 critical discussion of various procedures has been offered by Xorman (6). Essentially t'he present method is a modification of Launer's procedure for p- and y-cellulose in pulps and papers (4). The variations are designed t'o shorten the time of operat,ion and to
include certain desirable feat,ures of other procedures (1). Holocellulose, freed of pectic compounds, is extracted with aqueous alkali and t,he resulting solution is oxidized completely with an acid chromate solution. The reduced chromate serves as an index of t,he organic matter present and is determined spect'rqphotometrically. Holocellulose from nonwoody plant tissue ordinarily cont.ains some lignin, from 30 to 50% of the original percentage content of nitrogen, and substantially all the furfural-vielding subst,ances (e). PREPARATIOS OF HOLOCELLULOSE
The materials used in this work were beet pulp, citrus pulp, cranberry pulp, cornstalks, oat straw, and mixed hay. All samples were ground to pass a 25-mesh screen but were retained bj- a 50-mesh screen. Five holocellulose determinations Tere made on a 3-granl sample ( 2 )of each type of tissue. Samples were first extracted with a solution of alcohol-benzene (1 to 2) for 6 to 8 hours, then twice with 0 . 5 5 ammonium citrate a t 80" C., over a period of 24 hours; the first extraction period vias for 4 to 6 hours. Samples were then subjected to the sodium chlorite-acetic acid treatment. The holocellulose produced was filtered through poplin in the usual manner and allowed to dry a t room temperature before removal from the cloth. Hemicelluloses viere then extracted from this material. ESTIMATlON OF HEMICELLULOSES
Preliminary extractions of holocellulose from cornstalks, using solutions of sodium hydroxide varying in concentration from 0 to 24%, indicated that a 12% solution, specific gravity 1.1309 at 20" C. or approximately 3.39 S,was most effective. The solution which contained the gieatest amount of organic matter was considered the most effective. In order to determine the extent of the removal of pentose
V O L U M E 20,
Np.
643
7, J U L Y 1 9 4 8
Table I. , Hemicellulose as .4nhydroglucose in Plant Tissues on an Ash- and Moisture-Free Basis Trials
Beet Pulp 70
5.4
5.8 5.6 5.2 5.2 6.0 6.1 5.4 6.2 6.5
Citrus Pulp
Cranberry Pulp
Cornstalks
7i
170
"0
9.1 9.1
12.9 12.9
26.6
9.6 8.9
11:8 13.4
26:3 26.6
13:4 13.4 13.1
27: 9 27.9 25.7 15.2
...
...
9.3 9.3 8.8 9.; ... 8.t 6.8 9.3 6.6 9.3 Av.0 5.9*0.8 9,2*0,5 a Weight basis.
..
Oat Hay
Mixed Hay
7i
%
27.3 26.3 26.5 28.9 28.0
2 3 . .5 23.2 23.2 24.3 23.4 22.9 24.4
27 9
2i:4 26 4 26 0 22.4 26 4 26:7 22.5 13:2 28 7 25.9 22.5 13.5 28 4 1 3 . 1 1 0 . 4 26.5*1 4 2 7 . 5 i 1 . 4 2 3 . 1 j = 1 . 0
..
units from the holocellulose by the above treatment, a furfural determination was made on an aliquot of the alkaline extract. In the case of cornstalks, over 857' of the furfural-yielding substances in the holocellulose were removed. More drastic means are usually necessary to remove so large a fraction (5).
A 0.2-gram sample of air-dried holocellulose was treated with 20 ml. of l2y0 sodium hydroxide in a 100-ml. volumetric flask and placed in a constant-temperature bat,h a t 20" C. for 10 minutes. An equal volume of water a t the same temperature was ,then added and the contents were agitated. The flask was left in the bath for a total of 90 minutes, the contents being agitated once more a t about the half-way period. The contents were then diluted to volume, thoroughly mixed, and filtered by suction through a plug of glass wool jammed into the stem of a funnel just below the apex. Twenty-five milliliters of the filtrate were transferred to a 300-ml. Erlenmeyer flask together with 5 ml. of distilled water, 30 ml. of concentrated sulfuric acid, and 5 ml. of 1 S potassium dichromate. The solution was boiled gently for 2 minutes a t 140 to 150O C. The passage of a slow current of air through the solution facilitated boiling considerably. After quick cooling under a tap, the transmittance of the reduced chromate was measured in a Coleman Universal spectrophotometer. This instrument operates on a spectral band of 35 millimicrons; the cuvettes were approximately 13 mm. thick. A wave length of 600 millimicrons and a PC-4 filter xere used. Distilled water was used as the reference solution. The yield of holocellulose must be recorded if the percentage content of hemicellulose is to be reported on the original basis. Results are reported on an ash-, moisture-, and nitrogen-free basis. The nitrogen is converted to protein by use of the factor 6.25. -4s t,he alkaline extract,ion procedure removed about 80 to 10070 of the nitrogen of the holocellulose, it is believed to be in the form of protein. Samples bearing small percentages of nitrogen in the original material, such as n-ood, probably would not, retain a sufficient, amount, of nitrogrn to be significant. The extracts used in t,his work contained up to about 12 mg. as protein. Trial tests using casein to det,ermine the osidation values of proteinaceous materials indicatrd that 1 mg. is equivalent, to about 0.94 mg. of glucose.
per 100 ml. There is a slight tendency for the data for the laqt two concentrations to deviate from Beer's law, according to which one should obtain a straight-line relationship. The tendency. however, does not seem to be consistent or very significant; hence a straight line was drawn which satisfied all points. As the sugars in the hemicellulose are in the anhydro form, the concentrations of glucose were recorded as anhydroglucose. Table I shou-s that, in general, agreement within any one set of determinations is better than that between sets of duplicates or triplicates, This is due to the fact that in each trial a nem sample of holocellulose was prepared. The control of the latter procedure is not so good as the subsequent procedures that involve extractions with alkali and oxidation with chromic acid. The results indicate about as wide deviations as are likely t o occur. It is essential that the holocellulose determinations be made under very definite conditions as regards time of digestion and temperature. More precise results can also be obtained if filtrations are made through crucibles, rather than cloth, although the latter facilitates filtering considerably. DISCUSS105
In relatively small quantities starch n-as found to be removed from plant tissue-by sodium chlorite. Starch treated with sodium chlorite as in the regular tests was not destroyed, as evidenced by the reaction with iodine. A recent patent covers the solubilizing action of sodium chlorite on starch (8). Pectic materials are not completely removed by treatment with sodium chlorite. Treatment of commercial pectin with chlorite did not result in a destruction sufficiently great to prevent the formation of gels n-ith calcium salts. I n some instances incomplete removal of pectic substances may result in a slight enhancement of the percentage content of the hemicelluloses. This statement becomes more significant when one considers that citrus pulp yields about 9V0 of furfural, about 49yo of which may be credited to pectic substances. The yield of furfural from cornstalks is practically unaffected by the use of ammonium citrate. The evtensive removal of pectic substances from beet pulp and citrus pulp followed by a subsequent treatment with chlorite produces a residue which to a great extent has lost the characteristics of the particle and has acquired a somewhat sticky nature. The fibers from the cereal straws, being Ion in pectic materials, do not appear to have been alterrd. I t was found expedient to filter the residues from the hemicellulose extracts after dilution to volume. There was no significant difference in the reducing value of the filtrates whether filtered before or after dilution to volume. Solutions diluted to volume and allowed to stand before filtration did not change significantly in reducing value until after about 4hours. S o correction in oxidizing values n as made for the presence of uronic acids or for differences in uronic acid content of the hemicelluloses oi different plants. The manipulations drscribed above are believed t o make it possible to cut the time of operation down to about one fourth of the time used for the older gravimetric methods. If uniform conditions are maintained, the reproducibility of the method is good.
STANDARDIZATION OF REFERENCE CURVE
A curve was established Tvhich related the reduced chromate to glucose. .ilthough pentose sugars are usually predominant in hemicelluloses, a reference curve can be made using the cheaper and more easily availablr glucose because the amount of reduced chromate produced by equal amounts of glucose and sylose under these conditions is not significantly different. Data were obt,ained by t,aking the average of five or six det,erminations on each of eight solutions of glucose (expressed as anhydroglucose) t,hat varied in concentration from 0 to 140 mg. per 100 ml., and oxidizing 25-ml. aliquots of these solutions in the manner described above. Percentage transmission values obtained were then plotted against milligrams of anhydroglucose
LITERATURE CITED (11 Barton, C. J., and Prutton, .1.J.. ISD.ENG.CHES., . ~ S I L . ED., 16.429 (1944'1. .~~ (2) Bennett, Emmett. I b i d . , 19, 215 (1947). (3) Buston, H. W., Biochem. J . , 28, 1028 (1934). (4) Launer, H . F., Bur. Standards J . Research, 18, 333 (1937). (5) Korinan, A . G . , Biochem. J . , 30, 3055 (1936). ~
(6) Xorman. A. G., "Biochemistry of Cellulose Polyuronides, Lignin, etc.," London, Oxford University Press, 1937. ( 7 ) Preece, I. A., Biochem. J . , 25, 1304 (1931). ( 8 ) T-incent, G . P., U. S. Patent 2,409,085 (1947). (9) Weike, H . D . , and Phillips, M a x , J . Agr. Research, 7 4 , 77 (1947). RECEIVED .\ugust 25, 1947. Contribution 636 Agricultural Experiment Station.
of
the
Massachusetts
