Chapter 9

To separate the hemicelluloses, celluloses, and the constituents of lignin without essential change, the substance, after being freed from fat, is extracted with dilute hydrochloric acid and ammonia, and the residue frequently agitated for a day or two with 5-6 p.ct. caustic soda solution. It is then diluted, the extract poured off, neutralised with hydrochloric acid, treated with sufficient alcohol, and the hemicellulose filtered, dried, and weighed. The residue from the soda extract is washed on a filter with hot water, and extracted with Schweizer's reagent.

When the final residue (lignin) is subjected to prolonged extraction with boiling dilute ammonia (a suitable apparatus is described, with sketch) until the ammonia is no longer coloured, a residue is obtained which mostly dissolves in Schweizer's reagent, and on repeating the process the residue is found to consist largely of mineral matter. The dissolved cellulose-like substances often contain considerable amounts of pentosanes.

According to the nature of the substance, the extraction with ammonia may take weeks, or months, or even longer; the ammonia extracts of hard woods (as lignum vitæ) and of cork are dark brown, and give an odour of vanilla when evaporated down. The residues, which are insoluble in water, but redissolve in ammonia, have the properties of humic acids. Other vegetable substances, when extracted, yielded, besides humic acids, a compound, C_{6}H_{7}O_{2}, soluble in alcohol and chloroform, but insoluble in water, ether, and benzene; preparations from different sources melted between 200° and 210°.

FOOTNOTES:

[4] The original paper is reproduced with slight alterations.

[5] This purple colour would appear to be due to a highly dissociable compound of omega-brommethylfurfural with hydrogen bromide. The aldehyde gives yellow or colourless solutions in various solvents, which are turned purple by a sufficient excess of hydrogen bromide. Dilution, or addition of water, at once discharges the colour.

[6] Other forms of cellulose were also examined--for example, pinewood cellulose--and the substances separated from solution as thiocarbonate (powder and film). All of these gave good yields of omega-brommethylfurfural.

[7] The change is empirically represented as

C_{6}H_{12}O_{6} + HBr - 4H_{2}O = C_{6}H_{5}O_{2}Br.

SECTION IV. CELLULOSE GROUP, INCLUDING HEMICELLULOSES AND TISSUE CONSTITUENTS OF FUNGI

VERSUCHE ZUR BESTIMMUNG DES GEHALTS EINIGER PFLANZEN UND PFLANZENTEILE AN ZELLWANDBESTANDTEILEN AN HEMICELLULOSEN UND AN CELLULOSE.

A. KLEIBER (Landw. Vers.-Stat., 1900, 54, 161).

~ON THE DETERMINATION OF CELL-WALL CONSTITUENTS, HEMICELLULOSES AND CELLULOSE IN PLANTS AND PLANT TISSUES.~

In a preliminary discussion the author critically compares the results of various of the methods in practice for the isolation and estimation of cellulose. The method of F. Schulze [digestion with dil. HNO_{3} with KClO_{3}--14 days, and afterwards treating the product with ammonia, &c.] is stated to be the 'best known' (presumably the most widely practised); W. Hoffmeister's modification of the above, in which the nitric acid is replaced by hydrochloric acid (10 p.ct. HCl) is next noted as reducing the time of digestion from 14 days to 1-2 days, and giving in many cases higher yields of cellulose. The methods of treating with the halogens, viz. bromine water (H. Müller), chlorine gas (Cross and Bevan), and chlorine water, are dismissed with a bare mention, apparently on the basis of the conclusions of Suringar and Tollens (_q.v._). The method of Lange, the basis of which is a 'fusion' with alkaline hydrates at 180°, and the modified method of Gabriel, in which the 'fusion' with alkali takes place in presence of glycerin, are favourably mentioned.

These methods were applied to a range of widely different raw materials to determine, by critical examination of the products, both as regards yield and composition, what title these latter have to be regarded as 'pure cellulose.'

This portion of the investigation is an extension of that of Suringar and Tollens, these latter confining themselves to celluloses of the 'normal' groups, i.e. textile and paper-making celluloses. The present communication is a study of the tissue and cell-wall constituents of the following types:--

1. Green plants of false oat grass (_Arrhenatherium, E._). 2. Green plants of lucerne (_Medicago sativa_). 3. Leaves of the ash (_Fraxinus_). 4. Leaves of the walnut (_Juglans_). 5. Roots of the purple melic grass (_Molinia cærulea_). 6. Roots of dandelion (_Taraxacum officinale_). 7. Roots of comfrey. 8. Coffee berries. 9. Wheat bran.

These raw materials were treated for the quantitative estimation of cellulose by the method of Lange (b), Hoffmeister (c), and Schulze (d), and the numbers obtained are referred for comparison to the corresponding yields of 'crude fibre' (Rohfaser) by the standard method (a).

As a first result the author dismisses Lange's method as hopeless: the results in successive determinations on the same materials showing variations up to 60 p.ct. The results by c and d are satisfactorily concordant: the yields of cellulose are higher than of 'crude fibre.' This is obviously due to the conservation of 'hemicellulose' products, which are hydrolysed and dissolved in the treatments for 'crude fibre' estimation. A modified method was next investigated, in which the process of digestion with acid chloroxy- compounds (c and d) was preceded by a treatment with boiling dilute acid. The yields of cellulose by this method (e) are more uniform, and show less divergence from the numbers for 'crude fibre.'

The author's numerical results are given in a series of tables which include determinations of proteids and ash constituents, and the corresponding deductions from the crude weight in calculating to 'pure cellulose.' The subjoined extract will illustrate these main lines of investigation.

___________________________________________________________ | | | | | | Crude Fibre | Pure Cellulose | | |_____________|____________________________| | | | | | | Raw Material | Weende | Hoffmeister | Hoffmeister, | | | Method. | Method. | modified by | | | (a) | (c) | Author. | | | | | (e) | |________________|_____________|_____________|______________| | | | | | | Oat grass | 30.35 | 34.9 | 31.5 | | Lucerne | 25.25 | 28.7 | 20.5 | | Leaves of ash | 13.05 | 15.4 | 13.8 | | Roots of melic | 21.60 | 29.1 | 21.4 | | Coffee beans | 18.30 | 35.1 | 23.3 | | Bran | 8.2 | 19.3 | 9.3 | |________________|_____________|_____________|______________|

The final conclusion drawn from these results is that the method of Hoffmeister yields a product containing variable proportions of hemicelluloses. These are eliminated by boiling with a dilute acid (1.25 p.ct. H_{2}SO_{4}), which treatment may be carried out on the raw material--i.e. before exposure to the acid chlorate, or on the crude cellulose as ordinarily isolated.

~Determination of Tissue-constituents.~--By the regulated action of certain solvents applied in succession, it appears that such constituents of the plant-complex can be removed as have no organic connection with the cellular skeleton: the residue from such treatments, conversely, fairly represents the true tissue-constituents. The author employs the method of digestion with cold dilute alkaline solutions (0.15 to 0.5 p.ct. NaOH), followed by exhaustive washing with cold and hot water, afterwards with cold and hot alcohol, and finally with ether.

The residue is dried and weighed as crude product. When necessary, the proportions of ash and proteid constituents are determined and deducted from the 'crude product' which, thus corrected, may be taken as representing the 'carbohydrate' tissue constituents.

~Determination of Hemicelluloses.~--By the process of boiling with dilute acids (1.25 p.ct. H_{2}SO_{4}) the hemicelluloses are attacked--i.e. hydrolysed and dissolved. The action of the acid though selective is, of course, not exclusively confined to these colloidal carbohydrates. The proteid and mineral constituents are attacked more or less, and the celluloses themselves are not entirely resistant to the action. The loss due to the latter may be neglected, but in calculating the hemicellulose constants from the gross loss the proteids and mineral constituents require to be taken into account in the usual way.

QUANTITATIVE SEPARATION OF HEMICELLULOSE, CELLULOSE, AND LIGNIN. PRESENCE OF PENTOSANES IN THESE SUBSTANCES.

WILHELM HOFFMEISTER (Landw. Versuchs-Stat, 1898, 50, 347-362).

(p. 88) The separation of the cellulose-like carbohydrates of sunflower husks is described.

In order to ascertain the effect of dilute ammonia on the cellulose substances of lignin, a dried 5 p.ct. caustic soda extract was extracted successively with 1, 2, 3, and 4 p.ct. sodium hydroxide solution. Five grams of the 2 p.ct. extract were then subjected to the action of ammonia vapour; the cellulose did not completely dissolve in six weeks. Cellulose insoluble in caustic soda (32 grms.) was next extracted with ammonia, in a similar manner, for 10 days, dried, and weighed. 30.46 grms. remained, which, when treated with 5 p.ct. aqueous caustic soda, yielded 0.96 grm. (3 per cent.) of hemicellulose.

When cellulose is dissolved in Schweizer's solution, the residue is, by repeated extraction with aqueous sodium hydroxide, completely converted into the soluble form. On evaporating the ammonia from the Schweizer's extract, at the ordinary temperature and on a water-bath respectively, different amounts of cellulose are obtained; more hemicellulose is obtained, by caustic soda, from the heated solution than from that which was not heated. In this operation the pentosanes are more influenced than the hexosanes; pentosanes are not always readily dissolved by caustic soda, and hexosanes are frequently more or less readily dissolved. Both occur in lignin, and are then undoubtedly indigestible. These points have to be considered in judging the digestibility of these carbohydrates.

A comparison of analyses of clover, at different periods, in the first and second years of growth, shows that both cellulose (Schweizer's extract) and lignin increase in both constituents. In the second year the lignin alone increased to the end; the cellulose decreased at the end of June. In the first year it seemed an absolutely as well as relatively greater amount of cellulose, and lignin was produced in the second year; this, however, requires confirmation. The amount of pentosanes in the Schweizer extract was relatively greater in the second than in the first year, but decreased in the lignin more in the second year than in the first: this result is also given with reserve.

DIE CONSTITUTION DER CELLULOSEN DER CEREALIEN.

C. F. CROSS, E. J. BEVAN, and C. SMITH (Berl. Ber., 1896, 1457).

~THE CONSTITUTION OF THE CEREAL CELLULOSES.~

(p. 84) Straw cellulose is resolved by two methods of acid hydrolysis into a soluble furfural-yielding fraction, and an insoluble fraction closely resembling the normal cellulose. (a) The cellulose is dissolved in sulphuric acids of concentration, H_{2}SO_{4}.2H_{2}O, H_{2}SO_{4}.3H_{2}O. As soon as solution is complete, the acid is diluted. A precipitate of cellulose hydrate (60-70 p.ct.) is obtained, and the filtered solution contains 90-95 p.ct. of the furfuroids of the original cellulose. The process is difficult to control, however, in mass, and to obtain the latter in larger quantity the cellulose (b) is digested with six times its weight of 1 p.ct. H_{2}SO_{4} at 3 atm. pressure, the products of the action being (1) a disintegrated cellulose retaining only a small fraction (1/12) of the furfural-yielding groups, and (2) a slightly coloured solution of the hydrolised furfuroids. An investigation of the latter gave the following results: By oxidation with nitric acid no saccharic acid was obtained; showing the absence of dextrose. The numbers for cupric reduction were in excess of those obtained with the hexoses. The yield of ozazone was high, viz. 30 to 40 p.ct. of the weight of the carbohydrate in solution. On fractionating, the melting-points of the fractions were found to lie between 146° and 153°. Ultimate analysis gave numbers for C, H, and N identical with those of a pentosazone. The product of hydrolysis appears, therefore, to be xylose or a closely related derivative.

All attempts to obtain a crystallisation of xylose from the solution neutralised (BaCO_{3}), filtered, and evaporated, failed. The reaction with phloroglucol and HCl, moreover, was not the characteristic red of the pentoses, but a deep violet. The product was then isolated as a dry residue by evaporating further and drying at 105°. Elementary analysis gave the numbers C 44.2, 44.5, and H 6.7, 6.3. Determinations of furfural gave 39.5 to 42.5 p.ct. On treating the original solution with hydrogen peroxide, and warming, oxidation set in, with evolution of CO_{2}. This was estimated (by absorption), giving numbers for CO_{2}, 19.5, 20.5, 20.1 p.ct. of the substance.

The sum of these quantitative data is inconsistent with a pentose or pentosane formula; it is more satisfactorily expressed by the empirical formula

O / \ C_{5}H_{8}O_{3} CH_{2}, \ / O

which represents a pentose monoformal. Attempts to synthesise a compound of this formula have been so far without success.

UEBER EINIGE CHEMISCHE VORGÄNGE IN DER GERSTENPFLANZE.

C. F. CROSS, E. J. BEVAN, and C. SMITH (Berl. Ber., 1895, 2604).

~THE CHEMICAL LIFE-HISTORY OF THE BARLEY PLANT.~

(p. 84) Owing to the presence of 'furfuroids' in large proportion as constituents of the tissues of the stems of cereals, these plants afford convenient material for studying the problem of the constitution of the tissue-furfuroids, as well as their relationship to the normal celluloses. The growing barley plant was investigated at successive periods of growth. Yield of furfural was estimated on the whole plant and on the residue from a treatment with alkaline and acid solvents in the cold such as to remove all cell contents. This residue is described as 'permanent tissue.' The observations were carried out through two growing seasons--1894-5--which were very different in character, the former being rainy with low temperature, the latter being abnormal in the opposite direction, i.e. minimum rainfall and maximum sunshine. The barley selected for observation was that of two experimental plots of the Royal Agricultural Society's farm, one (No. 1) remaining permanently unmanured, and showing minimum yield, the other (No. 6) receiving such fertilising treatment as to give maximum yields.

The numerical results are given in the annexed tables:

Table Headings:

A: Date B: Age of Crop C: Plot D: Dry Weight E: Furfural p.ct. of dry weight (a) F: Permanent tissue p.ct. dry weight G: Furfural from permanent tissue H: P.ct. of tissue I: P.ct. of entire plant J: Ratio a : c

BARLEY CROP, WOBURN, 1894.

________________________________________________________________________ | | | | | | | | | | | | | | | | [G] | | | | | | | | |_____________| | | | | | | | | | | | | [A] | [B] | [C] | [D] | [E] | [F] | [H] | [I] | [J] | |_________|__________|_____|______|______|______|______|______|__________| | | | | | | | | | | | May 7 | 6 weeks | 1 | 19.4 | 7.0 | 53.4 | 12.7 | 6.8 | 1.03 : 1 | | | | 6 | 14.7 | 7.0 | 55.9 | 10.3 | 5.7 | 1.23 : 1 | | June 4 | 10 weeks | 1 | 17.6 | 7.7 | 52.9 | 11.6 | 6.1 | 1.26 : 1 | | | | 6 | 13.5 | 8.1 | 58.5 | 13.4 | 7.8 | 1.04 : 1 | | July 10 | 15 weeks | 1 | 42.0 | 9.0 | 65.7 | 9.8 | 6.4 | 1.40 : 1 | | | | 6 | 32.9 | 10.6 | 65.7 | 12.5 | 8.2 | 1.30 : 1 | | Cut | 21 weeks | 1 | 64.0 | 11.9 | 70.0 | 14.5 | 10.1 | 1.18 : 1 | | Aug. 21 | | 6 | 64.6 | 13.4 | 70.5 | 15.0 | 10.6 | 1.26 : 1 | | Carried | 22 weeks | 1 | 84.0 | 12.7 | 75.0 | 16.5 | 12.4 | 1.02 : 1 | | Aug. 31 | | 6 | 86.4 | 12.4 | 78.4 | 15.1 | 11.8 | 1.05 : 1 | | | | BARLEY CROP, WOBURN, 1895. | | | | May 15 | 7 weeks | 1 | 20.6 | 6.6 | 53.9 | 10.2 | 5.5 | 1.20 : 1 | | | | 6 | 17.8 | 5.8 | 56.7 | 9.6 | 5.4 | 1.07 : 1 | | June 18 | 12 weeks | 1 | 34.6 | 8.0 | 38.2 | 14.7 | 5.6 | 1.42 : 1 | | | | 6 | 33.4 | 7.6 | 44.5 | 15.0 | 6.7 | 1.14 : 1 | | July 16 | 16 weeks | 1 | 52.8 | 12.1 | 55.6 | 16.3 | 9.1 | 1.33 : 1 | | | | 6 | 54.4 | 10.6 | 46.2 | 19.1 | 8.8 | 1.20 : 1 | | Aug. 16 | 20 weeks | 1 | 66.8 | 9.2 | 49.1 | 17.0 | 8.3 | 1.10 : 1 | | | | 6 | 65.0 | 9.8 | 49.8 | 19.1 | 9.4 | 1.04 : 1 | | Sept. 3 | 22 weeks | 1 | 84.3 | 10.4 | 45.7 | 17.6 | 8.0 | 1.31 : 1 | | | | 6 | 86.3 | 10.2 | 45.3 | 17.3 | 7.8 | 1.30 : 1 | |_________|__________|_____|______|______|______|______|______|__________|

The variations exhibited by these numbers are significant. It is clear, on the other hand, that the assimilation of the furfuroids does not vary in any important way with variations in conditions of atmosphere and soil nutrition. They are essentially _tissue_-constituents, and only at the flowering period is there any accumulation of these compounds in the alkali-soluble form. It has been previously shown (ibid. 27, 1061) that the proportion of furfuroids in the straw-celluloses of the paper-maker differs but little from that of the original straws. For the isolation of the celluloses the straws are treated by a severe process of alkaline hydrolysis, to which, therefore, the furfuroid groups offer equal resistance with the normal hexose groups with which they are associated in the complex.

The furfuroids of the cereal straws are therefore not pentosanes. They are original products of assimilation, and not subject to secondary changes after elaboration such as to alter either their constitution or their relationship to the normal hexose groups of the tissue-complex.

(1) CONSTITUTION OF THE CEREAL CELLULOSES

(Chem. Soc. J. 1896, 804).

(2) THE CARBOHYDRATES OF BARLEY STRAW

(Chem. Soc. J. 1896, 1604).

(3) THE CARBOHYDRATES OF THE CEREAL

STRAWS (Chem. Soc. J. 1897, 1001).

(4) THE CARBOHYDRATES OF BARLEY STRAW

(Chem. Soc. J. 1898, 459).

C. F. CROSS, E. J. BEVAN, and CLAUD SMITH.

These are a series of investigations mainly devoted to establishing the identity of the furfural-yielding group which is a characteristic constituent.

This 'furfuroid' while equally resistant to alkalis as the normal cellulose group with which it is associated, is selectively hydrolysed by acids. Thus straw cellulose dissolves in sulphuric acids of concentration H_{2}SO_{4}.2H_{2}O - H_{2}SO_{4}.3H_{2}O, and on diluting the normal cellulose is precipitated as a hydrate, and the furfuroid remains in solution. But this sharp separation is difficult to control in mass. By heating with a very dilute acid (1 p.ct. H_{2}SO_{4}) the conditions are more easily controlled, the most satisfactory results being obtained with 15 mins. heating at 3 atm. pressure.

(1) Operating in this way upon brewers' grains the furfuroid was obtainable as the chief constituent of a solution for which the following experimental numbers were determined:--Total dissolved solids, 28.0 p.ct. of original 'grains'; furfural, 39.5 p.ct. of total dissolved solids, as compared with 12.5 p.ct. of total original grains; cupric reduction (calc. to total solids), 110 (dextrose = 100) osazone; yield in 3 p.ct. solution, 35 p.ct. of weight of total solids.

Pentosazone Analysis N 17.1 17.3 17.07 C 62.5 62.3 62.2 H 6.4 6.5 6.1 Melting-point 146°-153°

From these numbers it is seen that of the total furfuroids of the original 'grains' 84 p.ct. are thus obtained in solution in the fully hydrolysed form, which is that of a pentose or pentose derivative. It was, however, found impossible to obtain any crystallisation from the neutralised (BaCO_{3}) and concentrated solution, the syrup being kept for some weeks in a desiccator. It was noted at the same time that the colour reaction of the original solution with phloroglucol and hydrochloric acid was a deep violet, in contradistinction to the characteristic red of the pentoses. On oxidation with hydrogen peroxide, in the proportion of 1 mol. H_{2}O_{2} to 1 mol. of the carbohydrate in solution, carbonic anhydride was formed in quantity = 20.0 p.ct. of the latter.

Fermentation (yeast) experiments also showed a divergence from the resistant behaviour of the pentoses, a considerable proportion of the furfuroid disappearing in a normal fermentation.

(2) The quantitative methods above described were employed in investigating the barley plant at different stages of its growth. The green plant was extracted with alcohol, the residue freed from alcohol and subjected to acid hydrolysis.

The hydrolysed extract was neutralised and fermented. In the early stages of growth the furfuroids were completely fermented, i.e. disappeared in the fermentation. In the later stages this proportion fell to 50 p.ct. In the earlier stages, moreover, the normal hexose constituents of the permanent tissue were hydrolysed in large proportion by the acid, whereas in the matured straw the hydrolysis is chiefly confined to the furfuroids. In the early stages also the permanent tissue yields an extract with relatively low cupric reduction, showing that the carbohydrates are dissolved by the acid in a more complex molecular condition.

These observations confirm the view that the furfuroids take origin in a hexose-pentose series of transformations. The proportion of furfuroid groups to total carbohydrates varies but little, viz. from 1/3 in the early stages to a maximum of 1/4 at the flowering period. At this period the differentiation of the groups begins to be marked.

Taking all the facts of (1) and (2), they are not inconsistent with the hypothesis of an internal transformation of a hexose to a pentose-monoformal. Such a change of position and function of oxygen from OH to CO within the group --CH.OH-- is a species of internal oxidation which reverses the reduction of formaldehyde groups in synthesising to sugars, and appears therefore of probable occurrence.