February, 1924 I N D U S T R I A L A N D ENGINEERING CHEMISTRY 139

undecomposed. This would indicate that as long as any cellulose is left in the woods, pentosans will also be present.

COPPER NUMBER-The data obtained show a marked in- crease of reducing power with decrease in the cellulose content.

SOLUBILITY IN HOT WATER AND CAUSTIC ALKALI-The solubility of decayed groundwood in hot water and 1 per cent sodium hydroxide solution, calculated on equal weights of the original sample, increases to a maximum, then decreases rapidly as the cellulose content decreases. This point of

maximum solubility is obtained when the cellulose content (calculated on the original sample) is approximately 25 per cent. This would indicate that the action of the organisms is first to produce alkali-soluble substances, which are de- stroyed by further action.

The solubility in 1 per cent caustic alkali solution, cal- culated on equal weights of the sample analyzed, continues to increase with decrease of cellulose content.

Quantitative Methods for the Analysis of Hemicellulose in Apple Wood'.'

By W. E. Tottingham and F. Gerhardt

UNIVERSITY OR WISCONSIN, MADISON, Wrs.

N A previous article3 consideration was given I to the hydrolyzable

fraction of apple wood, des- ignated as hemicellulose, in relation to physiological aspects of fruiting. The present paper deals with certain results accrued from effortc; to determine quanti- tatively the composition of this fraction. Where not designated otherwise, the wood was derived from branches one year old.

SEPARATION OF EXTRACTIVE

Reference was made in the earlier paper to an e x t r a c t e d fraction, the

The extract obtained in I per cent sodium hydroxide seems to. haoe no physiological significance. In remooing starch from fhe tissues precursory to acid hydrolysis, saliva is preferable over fahadiastase.

Hydrolysis by dilute sulfuric acid appears to be preferable to the use of hydrochloric acid, as the former liberates a smaller proportion of reducing substances remooable by clarification. Discrepancies in results previously reported can now be ascribed to the liberation of furfural from products of hydrolysis other than pentoses. The greater part of the apparent pentose content of unclarijied producis of hydrolysis consists of compounds removable by fermentation with yeast.

Excessioe remooal by basic lead acetate of products of acid hydroly- sis uith reducing power renders this clarifying agent unsuitable for use. The hydrolytic effects of boiling for I or 2 hours with I per cent sulfuric acid are limited largely to cleaoage of pentose com- pounds, but the results gioe little promise of a general method for dissecting the hemicellulose. The destructive action of 4 per cent hydrochloric acid upon the products of hydrolysis causes its use in the determination of hemicellulose to be of highly questionable value.

could hardly be due to aging of the enzyme preparation in this case, for it had been received from the producers only a few weeks before use. The results are suggestive of the difficulty encountered by H ~ r t o n , ~ who ascribed inefficient activity of taka- diastase to the persistence of dextrins produced in the preliminary phases of starch digestion. They led to pref- erence for saliva in clearing woody tissues of starch, RELATION BETWEEN CON- CENTRATION OF ACID AND RECOVERY OF HEMICELLU-

Samples of a p d e spur LOSE

constituents of which resembled those derived by mild acid hydrolysis. The best method of extraction as regards yield of extractive consisted of digestion in cold 1 per cent sodium hydroxide subsequent to removal of the calcium soluble in 1 per cent hydrochloric acid. Recovery of the dry extractive by precipitation in acidified alcohol varied from 5 per cent in apple wood to over 20 per cent in young plum branches. However, inasmuch as the yield of this fraction showed no correlation with physiological treatment of the trees, this phase of the investigation has been discontinued.

REMOVAL OF STARCH From the standpoint of convenience it would be more de-

sirable to use takadiastase than saliva in preparing woody tissues for hydrolysis of the hemicellulose; yet it seemed de- sirable to compare results by enzymes from these two sources in the present case. After cessation of the usual starch re- action with iodine and filtration of the soluble products, the residue was redigested for 20 minutes with additional enzyme preparation. The results appear in Table I.

It appears that with the woody tissues diastase gives a smaller recovery of starch than does saliva. This difference

1 R.eceived September 22, 1923. 8 Published with the permission of the Director, Wisconsin Agricultural

8 Tottingham, Roberts, and Lepkovsky, J . B i d . Chem., 45, 407 (1921). Experiment Station.

tissue weighing 10 grams each were freed from &rch -by diastatic digestion. Hydrolysis was then effected by digest- ing different samples 1 hour with 100 cc. of boiling sulfuric acid a t various concentrations of acid. The extracts were filtered, neutralized, and subjected to determinations of re- ducing power and furfural yield before and after fermenting the hexoses.6 Analyses were conducted in this case without clarification of the solutions. The results are given in Table 11. TABLE I-COMPARISON OB THE YIELDS OR STARCH BY DIASTATIC TREATMENT

-DIGESTION- Control, DIGESTION BY SALIVA B Y TAKADIASTASE

Boiled Only Period 1 Period 2 Period 1 Period 2 KIND OF TISSUE Per cent Per cent Per cent Per cent Per cent Apple wood 1.4 3 . 3 0.0 2.2 0.0 Plum wood 1.2 3.9 0.2 3.2 0.2 Maize stalks 1.1 3.3 0 . 1 3.4 0.1

TABLE 11-RELATION BETWEEN CONCENTRATION OB ACID AND PRODUCTS OB HYDROLYSIS OF APPLE SPUR TISSUE

-Concentration of Sulfuric Acid, Per cent- 1.0 2.0 3 . 0 4.0 5.0 6.0

Reducing Powev in Percentage Equivalents of Glucose Before fermentation 19.0 18.5 22.8 27.6 32.5 38.0 After fermentation .. 5.2 .. 5.8 ,. 6.9

Furfuval Yield in Percentage Equivalents of Pentose Before fermentation .. 11.4 .. 11.9 .. 16.9 After fermentation .. 2.6 .. 2.9 .. 4.3

Inspection of the data shows that above the first interval of acid concentration the yield of reducing substances in-

' J . Agr. Sci., 11, 240 (1921). 6 E. B. Fred, of the Department of Bacteriology, supervised the addition

of nutrient media and inoculation with a pure strain of Carlsberg yeast.

140 INDUSTRIAL AND ENGINEERING CHEMISTRY Vol. 16, No. 2

creases about 5 per cent with each successive increase of acid concentration. On the other hand, the liberation of non- fermentable reducing substances, and of furfural-yielding substances both before and after fermentation, increased relatively little with increasing concentration of the acid.

The results of the pentose determinations show that a large part of the furfural yield preceding fermentation is due to fermentable substances, these presumably being hexose in character. SpoehrG has previously stressed the necessity for removal of hexose compounds for accurate determination of pentosans. Special emphasis is placed upon the abnormal yields of furfural in proportion to pentose compounds here reported, as they offer a t least partial explanation of the dis- crepancy between reducing power and other sugar values observed with products of hydrolysis in earlier work.' It appears probable that the values there attributed to xylose are entirely too high.

In this connection it should be recalled that with simple mixtures of arabinose and glucose Davis, Daish, and Sawyers found no interference with the usual method for determining pentose when the proportion of glucose was not excessive. The writers prepared a solution containing 1 per cent glucose with 0.25 per cent xylose, and found no interference from the former in determining the latter sugar by the usual method based upon yield of furfural. These results support the view that their products of acid hydrolysis contain hexose components, possibly in conjugated forms, which yield furfural in the procedure for determining pentose compounds.

CLARIFICATION OF PRODUCTS OF HYDROLYSIS Brownes regards the use of basic lead acetate for clari-

fication as undesirable with sugar extracts. It seemed de- sirable, however, to compare it with the neutral salt for use upon the products of hydrolysis from apple wood. Spur tissue freed from starch was hydrolyzed in one instance by the action of boiling 2.5 per cent hydrochloric acid for 1 hour and in other cases by the use of 1 per cent sulfuric acid for different time periods. The filtered extracts were neutralized by sodium hydroxide, clarified by neutral lead acetate, and de- leaded by sodium sulfate. Table I11 shows the analytical results. TABLE 111-RELATION OF CLARIFICATION TO YIELD OF REDUCING SUB-

STANCES FROM HYDROLYSIS OF HEMICELLULOSE REDUCING POWER IN PERCENTAGE EQUIVALENTS OF GLUCOSE IN DRY

Time of TISSUE HYDROLYZING Hydrolysis Clarified by Neu-

AGENT Hours Sample Unclarified tral Lead Acetate 14.9 15.0 B

C 15.3 n 14 9

HCI, 2.5 per cent 1 A- 5 . 0 8 .0 9 . 4 5 .3 - -~

8 . 9 8 . 9 12 .9 12.0 2

3 16.0 14.4 4 21.2 15.3

H ~ S O ~ , 1 per cent ' 1 E

It is clear that hydrochloric acid liberates large proportions of precipitable reducing substances. The use of weak sul- furic acid, however, has little effect in this respect, except with prolonged action. Further data assembled in Table IV show that the action of basic lead acetate is more ex- tensive than that of the neutral salt. With increase of hy- drolytic effect the proportion of products precipitable by the former salt becomes excessive. The earlier failure to identify fructose among the products of hydrolysis of apple wood, together with a comparison of the results here with those of Bryan10 derived from solutions of glucose and fruc- tose, led to the belief that compounds other than simple sugars are involved in this effect of clarification. It seems

6 Carnegze Inst . Pub., 287, 36 (1919). 2 J . B i d . Chcm , 46, 413 (1921). 8 J . Agr. Sci., 6, 410 (1914). 8 "Handbook of Sugar Analysis,'' 1912, p. 207.

10 Browne, loc. cat. p 215.

quite possible that products of partial hydrolysis of the hemi- cellulose, weakly acid in character, are here precipitated by the basic lead acetate. Inasmuch as it appears desirable to include these with the products defined by reducing power, the use of basic acetate for clarification is here deemed ob- jectionable. Sulfuric acid also appears preferable to hydro- chloric acid because of the smaller proportion of reducing substances produced by it which are precipitable by lead acetate. TABLE IV-CLARIBYING EFFECTS OF NEUTRAL AND BASIC LEAD ACETATES

REDUCING POWER IN PERCENTAGE EQUIVALENTS OF GLUCOSE IN DRY TISSUE

Clarified by Time of HYDROLYZING Hydrolysis Neutral Basic

AGENT Hours Samule Unclarified Acetate Acetate HzSOI, 1 per cent 3 A- 14 .0 13.4 10.9

B 10.9 10.3 7 . 9 HzSO~, 4 per cent 1 C 20.4 13.9 HCI, 2.5 per cent D 3i:5 26.9 9.2

SELECTIVE ACTION OF 1 PER CENT SULFURIC ACID That pentosans hydrolyze readily is well known.ll By

selective fermentation of the products of hydrolysis from treating spur tissues for 3 hours with boiling 1 per cent sul- furic acid (with clarification), it was found with a series of samples that an average of 85 per cent of the hydrolyzed material was of pentose character. I n seeking a method to shorten the time requirement, the writers obtained the same yield of reducing substance by 4 per cent sulfuric acid acting 20 minutes as by 1 per cent acid for 1 hour, but did not de- termine whether or not the ratio of hexose to pentose products was the same in the two cases. The results of the same hydrolytic treatment varied so widely with different samples of tissue as to render doubtful the possibility of developing for general use any conditions of acid hydrolysis which would quantitatively recover only the pentose complexes. DESTRUCTIVE ACTION OF 4 PER CENT HYDROCHLORIC ACID

The hydrolysate from the use of 4 per cent sulfuric acid, described previously3 with reducing power equivalent to 70 per cent glucose, upon further hydrolysis with 4 per cent hydrochloric acid for 3 hours gave products equivalent to only 39.4 per cent glucose. Tests showed the presence in the final products of a small amount of levulinic acid and traces of furfuraldehyde. Further evidence of the destructive action of 4 per cent hydrochloric acid appears in the data of Table V. In these cases, with other properties remaining quite constant, the reducing power of the alcohol-soluble products of hydrolysis decreases as the yield increases. Table VI presents data in which 1 per cent sulfuric acid and 2.5 per cent hydrochloric acid give closely similar quantitative results, but the destructive action of 4 per cent hydrochloric acid is again apparent. These results render the use of the latter reagent, as employed by duSablon,12 of extremely doubtful significance.

8

TABLE V-COMPARISON OF CONSTANTS WITH PRODUCTS OF DIRFERENT

(Time of Hedrolvsis. 2 Hours) HYDROLYTIC TREATMENT OF THE SAME TISSUE

- - . CONSTANTS OF ALCOHOL-SOLUBLE FRACTION

Alcohol- Reducing Power, Furfural Soluble Glucose Yield Pentose

Per cent Per cent Degrees Per cent HYDROLYZINO Products Sp. Rot. Equivalent Equivalent

AGENT HzS04, 4 per cent 12.2 20.0 67.8 51.8 HCI, 4 per cent 19.4 20.1 40.7 50.8

TABLE VI-RECOVERY OF REDUCING SUBSTANCES FROM VARIED HYDROLYTIC TREATXENT

Reducing Power in Percentage Equivalents Time of

HYDROLYZING Hydrolysis of Glucose AGENT Hours Sample Per cent

A 6.4 8 . 9

1 B

1 A 5 . 0 9 . 4 B

B 4 .9

HZSO4, 1 per cent

HCl, 2.5 per cent HCI, 4 per cent 3 A 2 . 2

11 Browne, Zoc. c d . , pp. 547, 553. 12 Rev. gln. botan., 16, 341 (1904); 18, 1, 82 (1906).