The composition and partial structure of silky oak gum

Item Type text; Thesis-Reproduction (electronic)

Authors Harris, Leland, 1924-

Publisher The University of Arizona.

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Link to Item http://hdl.handle.net/10150/319578 THE COMPOSITION AND PARTIAL STRUCTURE OF SILKY OAK GUM

by Leland Harris

A Thesis

submitted to the faculty of the Department of Chemistry in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in the Graduate College, University of Arizona

1952

Approved Seci^-Lucf;/ TABLE OF CONTENTS

Page INTBODWCTION ...... 1 SILKY OAK GUM ...... ' 5 A» Source of the Gum ...... : » . 5 B. Physical Appearance of the Gum 5 C » Qualitative Analysis of the Untreated Gum . . 6 1. Test for the Methoxy Group 0 6 2. Test for Methyl Pentoses . . . .* # . .. . 6 3. Naphthoresorcinol Test for Hexuronic Acid „ J 4. Spectroscopic Analysis of the Ash 7

D, Qualitative Tests for Sugars ...... 7 1. Introduction ...... 7 2. Test for L-arabinose 8

3. Test for D-xylose » ...... 8 4. Test for D-galactose ...... 8 5= Fermentation Test for the Absence of D-glucose, D-msmnose, and D-fructose . . ■ 9 - 6 . Paper Chromatographic Detection of Sugars . 9 E. Methods of Quantitative Analysis ...... 14 1. Moisture Determination...... 14 2. Ash Determination ...... 14

3. Uronic Acid Determination ...... 14 4. Pentosan Determination ...... 14

223111 5. Methylpentosan •Determination « ...... 15

60 Methoxyl Determinations . . . . . » . 16

PURIFICATION, FRACTIONATION, AID ANALYSIS OF SILKY OAK OUD$ ® • ® ® o e . e o - o e . o 6 o e . e e e o * © 1 Y

A® Purification of Silky Oak Gum » ...... 1 7 - B. Fractionation of the Purified Cum . , ® , . 17 0. Preparation of Free Acid from Silky Oak Gum© 18 D. Solubility of the Crude Gum, Purified Frac­ tions# and the Gum Acid . » => . » . » . . . 19 ■ E= Analysis of the Crude Gum, Free Gum Acid, and the Various Purified Fractions » „ . 20 F. Analysis of Unpurified and Purified Silky® . Oak Gum and Gum Acid, Table I® © © ® © . 21 G© Discussion of Results in Table I© © © . © © 22

HYDROLYSIS OF THE GUM, ISOLATION, IDENTIFICATION, AND ANALYSIS OF THE PRODUCTS 23 A. Method Used in Hydrolysis of Gum and Isolation ’ of the Products . v © . . . © © © © . . . 23

B. Hydrolysis for 2.5 Hours at 80° C© in a 4 per . cent Solution of Sulfuric Acid © . © © . . 23

C© Hydrolysis of the Free Gum Acid for 2 Hours at 65° C . in a 2 per cent Solution of Sulfuric Acid © © ...... © . . © . 26 D. Hydrolysis of Fraction A, for 3©5 Hours at 80° C. in a 3=5 per cent Solution of Sul­ furic Acid . . © © © . © © . . . © . . „. © 27

E. Hydrolysis for 13 Hours at 98° C. in a 4 per cent Solution of Sulfuric Acid © © © © 30 Page

F. Hydrolysis of the Free Gum Acid for 15 Hours at 98° C. in a 4 per cent Solution of Sulfuric Acid . „ ...... 31 Q. Hydrolysis of the Gum Acid for 18 Hours at 98° C „ in a 4 per cent Solution of Sulfuric Acid ...... 32 H. Hydrolysis of Barium Salts 3 and 5 for 6 Hours at 98° 0. in a 2 per cent Solu­ tion of Sulfuric Acid ...... 34 I. Analysis of Barium Salts from Hydrolysis of Silky Oak Gum, Table II...... 36 J. Discussion of Results of the Analysis of the Barium Salts...... 37 'K. Sugars-Obtained by Hydrolysis of the Gum 37 L. Identification of the Uronic Acid in Silky Oak Gum ...... „ 38

SXJMMARy o o a o . . o o e e . . . » . a o o » e e 4 l

BIBLIOGRAPHY ...... 43 ACKNOWLEDGMENT

. The author wishes to express his sincere apprecia­ tion and gratitude to Dr. Ernest Anderson for his un­ tiring as s is tance and encouragement - during the course of this investigation. - : THE COMPOSITION AND PARTIAL STRUCTURE OF SILKY OAK GUM

V \ INTRODUCTION , '' v - ' ' ;■

In recent years much work has been done on the prop­

erties and chemical composition of plant gums at this Uni­

versity under the direction of Dr. Ernest Anderson and Dr. Lila Sands. The first of the papers to be published on plant gums from the University of Arizona appeared in 1925. . In this publication the authors described the occur-

rence, appearance/ some physical and chemical properties Ofv c&olla gum and mesquite gum which are found in South- western United States,

Due to the large amounts of mesquite gum available in this part .of the country and the interesting chemistry in­ volved in its investigation, the work continued along this line. In the latter part of 1925 and 1926 articles ap­ peared which described the preparation of L-arabinose from

mesquite gum and the compositloh of this gum. 5 The re-

suits of these articles indicated that mesquite gum consists of 50 per cent L-arabinose, 18.7 per cent D-galactose and

13 per cent of an aldehyde acid belonging to the glucuronic :acid:.group. \ : y ^ ■ - 0.: / c ■ ' r ; . ■ V - ; 4 2 Further investigation by Anderson and Otis showed that mesquite gum is the inorganic salt of an organic acid cons is ting of f bur itqle cule s of L-arabinose, three mole­ cules of D-galactose-, and one molecule of methoxy-D-gluc- uronic acid united with the loss of seven molecules of water. : .. . . - . ■ , : , 5 . \ ' .; - ' , ' In 19^6 E. Vi White at the University of Idaho found that one molecule of methoxy-D-glucuronic acid and one of L-arabinose occupy terminal positions in the repeating unit of mesquite g-um. The remaining units of L-arabinose were found to be joined by glycosidic linkage at the first and second carbon atoms while the galactose residues seemed to be trebly linked at the first, third, and sixth positions.

White stated that the ratio of L-arabinose to D-gdlactose to methoxy-D-glucuronic acid in mesqulte gum is.4;2:1.

In 1932 Anderson published an article summarizing some of the results of the previous investigations of plant gums. He Stated that plant gums are probably pathological products which occur chiefly as salts of. complex organic acids. The hydrolytic products of the gums revealed that ■ they usually consist of either D-glucuronic acid or D-galac turonic acid and several molecules of one or more sugars. The acid and sugars seem to be held together in a large molecule by glycosidic linkages„ In .this publication Ander son described the general method used in separating the ' . .. : ;: 3 products of hydrolysis of the gums. References are also given to the analytical methods used in determining the com­ position of the resulting hydrolytic" products; In 1936 E. Anderson, F . H . Russell and L„ ¥„ Seigle^ suggested that the gum from lemon - tirees is composed of one molecule of a methylated uronic acid plus two molecules of P^galactose plus two molecules of L-arabinose minus five

molecules of water. In this case the simple formula seems to be doubled. The researchers stated that lemon gum is

not formed from pectic substances nor from hemicelluloses in lemon wood but from some of the simple sugars or poly­ saccharides present in the inner portion of the bark of citrus trees. • ' : • ■ / . -; • : : ' : • 8 ' ’ : ■ ' In 1949> J. K. N. Jones and F . Smith reviewed the studies that have been made on gum arable, damson gum, cherry gum, egg plum gum, inesquite gum, and gum tragacanth. The authors state that the uronic acid component found in

gums is usually B-glucuronic acid in the pyranose form.

The hexoses, which are usually B-galactose or B-mannose,

have the pyranose form, while B-arabinose is in the furahose

form and B-xylose is found in the pyranose form 6 The methyl- pentoses found in plant gums are L-rhamnose and L-fucose

and these have the pyranose structure. Jones, and Smith state that the significance of the occurrence of so many sugars united by all the known types of glycosidic linkage :: ^ . : /v ’ v;. ; -v.; X: ■ :;7 ^ 4 is not known; unless: it be that they are so constituted as to be able to resist the action of an organism or a series of invading organisms with their accompanying enzyme systems . ' : - v : v , ' - : ' : : : /Y' Recently E. Anderson and H . D .- Ledbetter invest!- ■. gated the composition of sapote gum,, which IS available in Peru. . The investigation showed that this polyuronide con­ sists of the calcium and magnesium salts of a methoxyuronic acid to which pentosan material is attached. The only sugars present were D-xylose:and L-arabinose. The complex salt contained approximately one methoxy group to two uronic acid groups and seven anhydro pentose units. It was found that the L-arabinose groups were easily hydrolyzed off the polyuronide in this case, while the D-xylose attached to the uronic acid, presumably by a glyeosidic linkage/ was very difficult to remove even under rigorous conditions of hydrolysis. • , -

The present investigation deals with the composition and partial structure of the gum from silky oak, G-revillea . robusta.» The general methods used in the work have been described by Anderson and Bands , and are similar to those used by Anderson and Ledbetter.^ SILKY OAK G1M

SOURCE OF THE GUM Originally the name silky oak was used for the timbers of Orites excelsa and Grevillea robusta which grow on the coastal areas in southern Queensland and northern Hew South 10 Wales in Australia. This source of supply has been prac­ tically exhausted and the name is now the standard trade common name for the timber of Cardwellia sublimus. In the United States it is known as lacewood. The gum used in this investigation was collected from silky oak trees, Grevillea robusta, growing on the grounds of the Biltmore Hotel in Phoenix, Arizona. Many of the vigorous, healthy trees had no gumj however, one fairly young tree about eight inches in diameter had quite a large amount of the gum.' Most of the gum was collected from trees which were injured in some way or appeared to be diseased.

Many of the trees had dead limbs and on these trees gum exudations were found, thus in this case corroborating the theory that gums are pathological products and not normal products of plant metabolism.

PHYSICAL APPEARANCE OF THE GUM . - • . The samples of silky oak gum varied in color from a dark brown to a light reddish brown. The material contained > large ammmts: of bark and dirt?^ which were separated from the gum during- the process of purification. The cleanest and lightest colored grades of crude gum were used In this , investigation. ' ■

AHAhYS IS^ OF THE TMlTSAteB ' ; . Test for the methoxy group

' •: - ' - " IX ' ' ' ■ Von Pellenherg showed that an ester or glycosidic

methoxyl group is easily hydrolyzed off with 10 per cent sodium hydroxide solution, while an ether methoxyl group is only removed hy boiling in . 72 per cent sulfuric acid.

In either case one of the resulting products is free methanol

Which may be detected by a delicate test developed by . 12 ' ' ■ - : ; • Deniges. When this test was applied to silky oak gum,. .the results were negative both for an ether linked methoxyl • group ahd for an ester or glycosidic methoxyl group. Test for me thy1 p entose s

Methyl pentoses or methyl pentosans> on distillation with hydrochloric acid, yield methyl furfural in the dis­

tillate. The presence of methyl furfural may be detected by the method of Toliens and Oshima^ as described by Browne . and Zerban. The hydrochloric acid distillate is treated

with concentrated hydrochloric acid and a small amount of

phloroglucinol added. After a few minutes the solution is

filtered from the greenish blaek precipitate of furfural phloroglucide. If the filtrate is colored yellow or reddish 7 yellow methylfurfural Is present. When this test was ap­ plied to the distillate, obtained by heating an aqueous, slightly acid solution of crude silky oak gum, no color developed, thus no methyl pentose or methylpentosans were present in the crude gum, Naphthoresorcino1 test for hexuronlc acid The presence of a hexuronlc acid in any material may be detected by heating an acid solution of the material with an alcoholic solution of naphthoresdrcinol„ The solution is then cooled and extracted with ether. If either D-gluc- uronic or D-galacturonic acid is present, the ether is col­ ored a violet blue„ Silky oak gum gave a positive naph- thoresorcimal test.

Spectroscopic analysis of the ash

A photograph of the emission spectra of the gum ash indicated that a large amount of calcium and magnesium were present with slight traces of silver, copper, and iron.

Thus, the gum appears to be primarily a calcium and magne­ sium salt of a complex acid.

QUALITATIVE TESTS FOR SUGARS Introduction

It will be shown later that the composition and also the partial structure of the gum may be determined by hydro­ lyzing it for varying lengths of time and at different tem­ peratures in a mildly acid, aqueous solution. The sugars. which result as products of hydrolysis of the gum, are identified by a number of well established tests which Will be discussed in the following paragraphs.

Test for ■L-arabinose L-arabinoseis best identified by converting it to the &s-benzylphenylhydrazone or the diphenylhydrazone. The methods of preparing these derivatives are adequately de- ; 15 ' scribed by van der Haar, Test for D-xylose - One of the best methods for detecting xylose in the

presence of other sugars is Bertrand’s reaction by means of bromine and cadmium carbonate. The bromine oxidizes the xylose to xylonlc acid.. Hydrobromic acid is formed at the same time„ The hydrobromic and xyIonic acids react with : the cadmium .carbonate forming cadmium xylonate and cadmium

bromide. When the solution is evaporated and allowed to

stand, it deposits characteristic boat-shaped crystals of the double bromide and xylonate of cadmium„ The directions for . . ■ ■ ■ ' - • ■ ■ . i4 this, test are given in Browne and Zerban.

Test for D-galactose The test most generally employed for detecting galactose

either lh the free or combined form is the production of •mucic acid upon oxidation with dilute nitric acid; The crys­

tals of mucic acid are identified by their melting point and crystalline fora. Browne and Zerban*^ describe this qualitative test„ .

Fermentation test for the absence of D-glucose ^ P-mannose ?

andiD-fruetOse ., - i: i':.' v / ■ <' An aqueous solution, containing D-glucose, D-mannose, or D-fructose, will ferment within a short time in the presence of' ordinary baker‘s yeast„ .Thus, the absence of

fe rmenfat ion of the unknown sugar solution by this yeast indicates the absence of the above-mentioned sugars. Paper chromatographic detection of sugars ; . ; . . . x? . ; ■; • " - ... ^ Rao and Beri hate recently published a very unique ; method for the detection of sugars by the Horizontal Migra­ tion Method of paper chromatography. In this investigation r this, method was used with a moderate degree of success„ The experimental procedure is described in the following

paragraphs., - 'Whatman No. 1 circular filter-paper, 15 cm. in diameter,

was used for the development of the chromatogram. Perpen­ dicular to the dianietep and at equal distance from the cen­ ter two parallel cuts (about 2 mm. apart and 2 cm. in length) were made in the same direction on the filter paper. The piece lying between the two parallel cuts was cut off at

the end away ffom the center and folded along the diameter and perpendicular to the plane of the paper (Fig.1). The r

tail so formed was cut down to about 1.5 cm. in length. In the formation of the tall it is essential that it be FIG. I

A' and a"= p a r a l l e l c u t s B = "TAIL" C = MICRODROP OF THE SUGAR SOLUTION

A t

B M B —% D “ C * D __

t t E E

FIG. 2 A = GLASS PLATE B = FILTER PAPER C * "TAIL" D = SOLVENT F = PETRI DISH rectangularly perpendicular to the plane of the paper, otherwise the zones may iiot be truly circular „ However, good results may be obtained even if the zones are slightly elliptical, ' : A one per cent solution of the sugar was -placed, by ineans of a capillary dropper as a micro-drop on the joint of the tail and the paper, and air-dried. The solvent to be used for the irrigation was placed in an inverted desic­ cator lid (approximately 13 cm, in inside diameter), which was held in place by inversion in the main portion of the desiccator. The filter paper was placed over the solvent in such a way that the tail hung down into the solvent be­ low. Another desiccator lid was placed over the filter paper in order to retard, evaporation of the solvent. Rao . / . yj - ' ' v : -: ; ' and Beri used'a more convenient apparatus, consisting of a Petri-dish and a glass plate (Fig. 2). The desiccator system was not thermostatically controlled as prescribed in the original method, but was kept at room temperature (20 C. - 25- C .),: This gave satisfactory results if stand­ ards were run at the same temperature as the unknown sugar, solution. \ '

The solvent gradually rose by papillary action up from the tail and irrigated the filter paper, spreading itself uniformly in a halo. When the irrigation had continued sufficiently: long to give a circle 10 to 12 cm. in diameter, the paper was removed and the position of the solvent boundary marked immediately with a pencil. The paper was then placed on a shallow dish serving as a support and dried in an oven at 100° for five minutes. It was then sprayed rapidly and evenly with a solution of aniline hydrogen phthalate in butyl alcohol and dried at 100° for 5 minutes. A brownish circular zone corresponding to the position taken up by the sugar appeared on an almost white back­ ground. From the center four radial lines were drawn to the marked edges of the solvent boundary. The center por­ tion of the brown ring was marked at the point of intersec­ tion of the ring and the line drawn to the solvent boundary. The distance through which the sugar moved divided by the distance the solvent moved was calculated in each case, and the average of the four values gave the circular Bf value.

Many different solvents were used by Rao and Beri.1^ The most satisfactory ones.were found to be phenol, n-butyl alcohol, s-collidine, methyl ethyl ketone, and p-cresol.

In all cases the solvents were saturated with water before their use for the irrigation. The solvents used In the present investigation were n-butanol and methyl ethyl ke­

tone. Moist butanol-1 gave good results in the detection of L-arabinose and B-galactose, since the difference in cir­ cular R|» values of these sugars was 0 .06, the minimum dif­ ference necessary to have two separate rings present, on the 12 chromatogram„ However„ the difference between the Rf values of L-arabinose and D-xylose was only ©.01, using butanol-1 as an irrigant, thus only one ring was observed from a solution containing both of these sugars. Moist methyl ethyl ketone was the best solvent available to re­ solve the two sugars, L-arabinose and D-galactose. The difference in circular values for these sugars in this solvent is 0.08 at 25° C., which is sufficiently large to give a small distance between characteristic rings. It was impossible to separate D-xylose from L-arabinose by using methyl ethyl ketone at 25° C. Another solvent, m-cresol, was tested but gave no separation of the two pentoses. Other solvents were not tested. Possibly one will be found that will separate L-arabinose and D-xylose. In any case it is always advisable to use at least two different solvents for irrigating purposes, since one solvent may not give a ring separation in a mixture, while the other will do so. The Horizontal Migration Method of filter paper chroma­ tography is a very convenient method for the identification of sugars, especially when they are present in mixtures.

Every sugar has a characteristic Rf value in a particular solvent, which may be used for its Identification' It is significant to note that only a very small amount of sugar need be present in solution to give a chromatogram. The irri­ gations take only a short time (from 0.5 to 2 hours), and ' ' . ■ ■ ■ . ■ ■■■■. ■ 13 produce accurate and reproducible results„ This method, along with the other tests previously described was used frequently in identifying the products of hydrolysis of silky oak gum. ' V ' ' '' :' ‘ ■ . i4 METHODS OF QUANTITATIVE ANALYSIS Moisture determination Approximately 0.2 gram samples of gum,' powdered to , pass through a forty mesh sieve, were dried to constant weight in an oven at 100° - 105° Co The per cent moisture was calculated from the loss in weight. The salts were ■ ■ - ■ dried in an Abderhalden vacuum drier. Ash determination . The per cent ash was determined by igniting the samples from the moisture determination to constant weight. The ignition was carried out at the dull red heat of the Bunsen flame after the sample had been charred at a low tempera­ ture . . ; " Uronic acid determination

When a compound containing a uronic acid is heated with 12 per cent hydrochloric acid, one molecule of carbon

dioxide is liberated for each molecule of uronic acid. The

method used for determining the per cent uronic acid present

is a modification of the original method of Lefevre and l8 Tollens. It is described in the Methods of,Analysis, The Institute of Paper Chemistry.^ Complete details of this

method and a diagram of the apparatus used are given in the

MasterSs Thesis of H. D. Ledbetter, University of Arizona.20 Pentosan determination,

The per cent pentosan was determined by the method , giyeti in Methods of Analysis, The Institute of Paper Chemis-

try. .. The method depends upon the conversion of the pen­

tose sngars in the gum into furfural by distilling with 12 per cent hydroGhloric acid. . The furfural in the distillate is precipitated by the addition of phloroglucinol> as first developed by ToHens and Kruger and finally established in its present forq by Tollens and Krober. . The yield of furfural obtained in this determination does not correspond'exactly with the theoretical yield, yet by carrying out the distillation under carefully con­ trolled conditionsit is possible, by means of formulas 7 or tables, which have been established for different weights of pure pentoses, to determine with a very close degree of approximation the amount of pentosan present.

A correction must be made for the furfural given by the uronic acid on. distillation with 12 per cent hydrochloric acid. y¥an der Haar found that.uronic acids yield a weight of furfural phloroglucide equal to one-third the total weight of;uronic acid present.This correction was applied in all determinations of the per cent pentosans. , Browne and Zerban list a number of precautions and ' limitations which; must be observed to obtain satisfactory results using the distillation method. ; Methylpentosan determination

This method is based upon the solubility of methyl furfural phlorogluclde and the Insolubility of furfural phloroglueide in warm 95 per cent ethanol. Haywood’s modi­ fication of the Tollens-Ellet method1^ was used in this determination. When this method was applied to silky oak •gum* no methyl pentoses were found. Methoxyl determinations V

The modified Zeisel method as described in Methods of Analysis, The Institute of Paper Chemistry1^* was used for the determination of per cent methoxyl. Mo methoxyl groups were found in silky oak gum. - . PtfRIFICATIOM, FRACTIONATION, AND ANALYSIS OF SILKY OAK GUM Purification of silky oak gum Three hundred grams of the crude gum were heated In vslx liters of water„ The hot solution was pressed through two layers of cloth. On addition of a small amount of ethanol to the filtrate, some dark gum was precipitated. The solution was decanted from the precipitate, To the

clear liquid was added approximately six volumes of ethanol, This gave a cloudy .solution, ip vfloceulate the suspended gum a small amount'of sodium chloride dissolved in a' few ml. of water was added, A light .colored gum settled out of solution and was isolated hy siphoning away the supernatant

liquid. This was rubbed with 85 per cent ethanol. Approxi­ mately 150 grams of a light colored, granular solid were ob­

tained, ; . - 1

There seems to be two parts to the gum, One part is darker and. less soluble in water than the other. The main

portion of the gum is a white material which can be obtained in granular condition as described above.

.Fractionation of the purified gum - - . /Fifty grams of the purified granular gum were mixed with thfee liters of water and heated in a boiling water bath.

Not all of the gum dissolved in the hot water. The insoluble

portion was removed by•filtering the solution through a double layer of cloth. The insoluble portion was■mixed with ethanol 18

and allowed to stand for* a short time. It was then rubbed with ethanol in a mortar, filtered off at the pump, washed with ethanol and -ether1 and dried in vacuo . This portion of the gum is called fraction B. It weighed approximately 7 .5 grams ,or 15 per cent of the purif ied gum used. The filtrate from fraction B;’was treated for a short . time with 'bromine. Then f our liters of ethanol were added. The resultant precipitate was filtered., . washed with ethanol,

and dried in the oven at 100° C . , This portion of the gum

is called fraction Ay, It weighed approximately 33,7 grams or 67,4 per cent of the purified gum .used.. . When; more ethanol was added to the filtrate from frac­ tion At, a second fraction Ao slowly formed. It weighed :

approximately flye grams or 10 per cent of the original gum used. This is the most soluble portion ■of the gum. The

specific rotation, of fraction Ar, was -8 .1°, ' . ■ D ■: . . ' ^ ■ : -;. ■ : Preparation of free acid from silky Oak gum Fifty-three grams of crude gum were stirred with hot water until most of it dissolved. The solution was filtered through qualitative filter paper. The total volume of the

filtrate with washings was three liters. One hundred fifty ml. of concentrated hydrochloric acid were added to this

solution with stirring, The resultant acidic solution was immediately diluted with seven volumes of 85 per cent ethanol, Whereupon a gummy acid was precipitated from the solution', , ’ The ; solwnt: was decanted from the residue, and the gum acid was washed with 95 cent ethanol arid dried at .100° C „ ■ i ; The yield was 42- grams or 80 per cent of the crude gum used. Solubility of the crude gum, purified fractions, and the. gum acid ' ' . • / /i r i f v

\ - . Approximately G.-l grams of the crude gum/ fraction A]_5 .fractipn:Agf and the gum acid were placed in 100 ml „ of ■

waiter at 25 C . Fraction Ag> was shaken constantly and dis­ solved in a few minutes. The crude gum,, fraction Ai, and the gum acid did not appear to be soluble imder these con­ ditions * The mixtures were allowed to stand for three days with occasional shaking. After this interval the crude gum was a viscous milky solution which contained a suspension

of small Swollen particles,: Fraction A^, on shakirig in its - -aqueous solution, gave very large, swollen semi-solid par­

ticles in an almost clear solution. The mixture containing

'fraction B was slightly cloudy and when shaken, small swollen

particles were visible in the solution.. Fraction B was so slightly soluble that some of the particles did not even swell in the water solution. The free gum acid was very simi lar to fraction in that it gave large swollen particles ; in aqueous; solution. A . '

, . C. L. Mantell states that the gums belong to. that ; class of colloids called emulspids which form hydrophilic

.'■water loving" dispersions. The gums swell or disperse in -^water>:. and they generally. tolerate the presence of large amounts of electrolyte. The gums form highly viscous solu­ tions' or absorb large quantities of water In order to swell Analysis of the crude gum, free gum acid, and the yarlous purified fractiohs ■ ::v:^ 'Vv:'v : \ ' v \

: The crude gum, free gum acid and the various purified

fractions were analyzed by the. methods previously described The results obtained are given in Table 1„ ; :

Only the lightest and cleanest grade of crude gum was used for analysis. The samples in all cases were ground fine and passed through a forty-mesh sieve. fa) TABLE I. - ABALYSIS OP UHPURIFLED AWD PURIFIED SILKY OAK GUM AID GUM AGID 1

Col. 1 Cols. 2, 3 and 4 Col. 5 Purified Gum Fractions

Crude Gum Fraction A% Fraction Ag Fraction B Gum Acid

• Moisture j, fo 11.87 5.27 5,29 5.37 6.68

Ash, % 3o03 1.13 0 2.03 0.22

Garbon Dioxide, ^ ■'■•"4.79 4.42 4.18 4.58 3.74

Uronic Acid, fo 21.01 19.39 18.33 20.09 16.40

Pentosan, fo 31.96 28.48 30.03 25.08 31.66

Equivalent Weight 918 995 1052 961 1176

Anhydro Pentose Units per Uronic Acid 2.22 2.15 2.39 1.83 2.82 Anhydro Hexose Units per Uronic Acid 2.66 3.20 3.35 3.25 3.77 (a) The results have been corrected for moisture and ash. . , The equivalent weights are calculated from the uronic acid. ro H ; ;: ; : : ■ ; ; ■ c : ; ; \ - : : 22 Pis etas sion of Results in Table I . ’

The results in Table I indicate that the crude gum contains 2.22.pentose units and 2.66 hexose units per

uronlc acid groups| fraction Ai contains 2.15 pentose units >nd 3.20 hexose units per uronic acid groupj fraction Ac '

contains 2.39 pentose units and 3.35 hexose units per uronic

acid group; fraction B contains 1.83 pentose uhits and 3.2$ hexOse.units per uronic acid group; and the gum acid con-

; tains 2.8,2.. pentose unit a, and .3«77 hexose units per uronic acid group. There are an.average of approximately 2.28

anhydro pentose.units and 3.25 anhydro hexose units per uronic acid group in the gum; ' The ratio .vof uronic acids to pentoses to hexoses is in small whole numbers 4:10:13. It will be shown later, by methods of hydrolysis, that the gum is composed 0# P-glucuronic acid, P-galaOtpse ^ and L-arabinOse. It was also found that L-arabinose was easily hydrolyzed off by treatment with a warm, dilute solution of ;

'suifuric acid. & .combination of this information with that obtained front Table I indicates; that the gum is composed of

, 2.28 L-arabihose units and 3.25 D-galactose units per D-glu- curonic acid group. Further the gum probably contains L- arabinose in the furanose format the ends of the large gum

. molecule, since it is readily removed by-gentle hydrolysis In acid solution. ' ' ; i- ;' . . ' ; - v ■ ; -- ' . HiraOLIBIS OF THE GUM,, ’ISOIATIOM# IDENTIFICATION, AND

ANALYSIS OF THE PBODFCTS 4' - " V ^

Method Used in Hydrolysis of Gura and Isolation of the Hr

Products^ ' \ ' / h

For hydrolysis the gum was dissolved in water and the •solution made 4 per cent acid with sulfuric acid.J The solu­

tion was heated for varying lengths of time and degrees of temperature. It was then neutralized by addition of barium "carbonate, decolorized with activated carbon, filtered at the pump,; and the f iltrate concentrated in vacuo to a thin syrup. The barium salts were precipitated by addition of

the syrup■to ethanol. The ethanol solution of the sugars,

free Of barium salts, was concentrated in vacuo and the sugars identified.14^15'17'29 : V t -f. :

Hydrolysis for 2.5 Hours at 80° C. in a 4 per cent Solution of Sulfuric Acid v . 't'' /' ; Fifty-six grams of thelight; colored silky oak gum,

which had been purified once, were mixed with 1 liter of a . 4 per cent solution of sulfuric acid and .heated at 80° C .

for 2.5 hours. After removal of the sulfuric acid and de- colorizafion of. the solution, 23 grams of barium salts were .

obtained as described above. These were, purified by dis- ■ solving in •water and: reprecipitating by addition to ethanol.

This gave 18 grams of white.barium salt 17 The results of the analysis of this salt are given in Column 1 of Table IT. 24

These results show that during hydrolysis under these con­ ditions, all of the pentose groups and slightly more than one hexose group were removed„ The resulting barium salt had approximately 1.88 anhydro hexose groups per uronic acid group. The ease with which the L-arabinose was hydro­ lyzed off suggests that it is present in the gum in the furanose form. The ethanol solution of the sugars in the filtrate from barium salt 1 was concentrated in vacuo and the gum

sugar found to weigh 30 grams. This was dissolved in hot glacial acetic acid and seeded with crystals of L-arabinose. On standing, 10 grams of white crystalline sugar were ob­ tained. These were recrystallized once from glacial acetic acid and 5.8 grams of crystals were obtained. These showed

'|^]250= 8 5 .0°. A small amount of these crystals was converted to the diphenyIhydrazone and found to melt at

196° c., which is the melting point of L-arabinose diphenyl- hydrazone. When some of this diphenylhydrazone was mixed with a known sample of L-arabinoSe diphenylhydrazone, there was no change in the melting point. Another small sample

of the crystalline sugar was converted to the benzylphenyl- hydrazone. After recrystallization from 90 per cent ethanol,

it melted at 174° C ., which is the melting point of L-arabin­ ose benzylphenylhydrazone. A mixed melting point of these crystals with a known sample of L-arabinose benzylphenyl­ hydrazone showed no change in the melting point. A paper ' 2 5 chromatogram of some of the crystals was prepared accord- Ing to the method of Rao and Berx . and the sugar was found to be a mixture of L-arablnose and D-galactose. These tests establish the presence of L-arablnose In the gum, A mucic acid test for D-galacto.se was ma.de on a small amount of the 5.8 grams of crystals and mucic acid melting at 214 :C. was obtalned. This establishes the presence of r D-galactose in the gum. ; The 5 .8 grams of crystalline sugar which showed ^5° = 4, 8 5 .0° was evidently a mixture, of L-arabinose,

which has 104.5° &nd D-galactose, which has

■ M l 09-

. 1 ' : ■ ' - . : : . 14 " When Bertrand’s test' for D-xylose was made on some of the.gum sugar, no characteristic boat shaped crystals were obtained. D-xylose was evidently not present. A fermentation test, with ordinary baker’s yeast> on some of the gum sugar was negative but the control test was positive. This proves the absence ofDB-glucose, D-mannose- ’

and D-fructose . > -g.h ' / : V1' ':: ; Evidently both L-arabinose and D-galactose are hydro­ lyzed off the gum "under these conditions. Since L-arabinose

in the furanose form is probably removed first' by hydrolysis^,

it was decided that milder conditions of hydrolysis might ' yield a sugar which is pure L-arabinose. - Hence,» the free

gum acid was hydrolyzed under the conditions described in . < • ;-- . j 26 the following paragraph, . ; Hydrolysis of the Free Gum Acid for 2 Hours at 65 C in a ,2 per cent Solution of .Sulfuric Acid. Fifty grams of the dry5 free acid, of the gum Were mixed with i o5 liters of water- and;allowed to stand over- . •

- ■ ' ^ o • night, The mixture was then heated to fc>5 C* and to it was added with vigorous shaking a solution of 16 ml. of .concentrated sulfuric acid which had been diluted with water . This .gave a 2 per cent solution of sulfuric acid. The heating at 65° was continued for 2 hours. Then 7 volumes .of 85 per cent ethanol were added to precipitate the unhy- -drolyzed gum. The colloidal solution was coagulated by ad­

dition of one gram of sodium chloride dissolved in 5 ml. of water. After it had settled the insoluble gum residue was filtered off and found to weigh approximately 40 grams.

The filtrate frbm the unhydrolyzed gum was neutralized by addition of barium and calcium carbonates to remove the sulfuric:acid. After filtering off the sulfates, the clear

filtrate containing the sugars was concentrated in vacuo ‘ and found to weigh approximately 20 grams. This was dis- • .solved In hot methanol and filtered. A small amount of ■ - ’

ethanol was added to decrease- the solubility^ and the solu­

tion was seeded with' L-arabinose . After standingy 7 grams .of crystalline sugar were /obtained.. The filtrate from the crystalline sugar was concentrated In vacuo and found to ' V ■ ■ 27 ' , weigh 12.5 grams. Some of the gum sugar gave a positive ' mucic acid, test for D-galactose/ but a negative fermenta­ tion test. A paper chromatogram was made on the 7 grams of crystalline sugar. It showed the presence of large amounts of L-arabinose and small amounts of D~galactose. ■;

Hydrolysis of Praction for 3-5 Hours at 80° C. in a 3.5 ;

per cent Solution of Sulfuric Acid

Twehty-five grams of fraction A^ of the purified gum were mixed with 1 liter of water and allowed to stand for 48 hours at room temperature. The resulting colloidal solu­ tion was warmed to 80° 0. and 21 ml. of concentrated sulfuric acid, which had previously been dissolved in 100 ml. of ' water, were added. This makes a 3.5' per cent solution of sulfuric acid. The solution was heated at 80 C . for 3.5 hours. It was then neutralized with barium carbonate, de­

colorized with activated carbon, and filtered from the pre­ cipitate. The filtrate was concentrated in vacuo to a small volume, and the barium salts were precipitated by the addi­ tion of ethanol. After drying, these weighed 11.5 grams. The barium salts were purified by dissolving them in 220 ml. of water,: filtering the solution, and adding the clear fil- ,

trate to 1 liter of 95 per cent ethanol. This gave a cloudy,

colloidal solution which was coagulated by addition of a

few ml. of sodium chloride solution. After standing, the

white precipitate was centrifuged out, washed with ethanol. dried and found to weigh 10 grams. This is barimm salt 2.

The results of its analysis are given in Column 2 of Table IlV When some of this salt was oxidized with nitric acid, it gave muelc acid melting at 216° C . .The ethanol filtrate from barium salt 2 contained the sugars liberated during the hydrolysis of the gum. This was concentrated in vacuo and the gum sugars found to weigh 13 grams. This was dissolved in hot glacial acetic acid and seeded with L-arabinose <, The solution became solid

with crystals within a short time, After standing for a time the crystals were filtered off and found to weigh 6

grams„ There were 7 grams of non-crystalline gum sugar re­ maining in solution. The 6 grams of crystals were again dis solved in hot glacial acetic acid and the solution seeded

with L-arabinose, After standing, 3.5 grams of white crys­ tals were obtained „ These showed 4= 96.3° „ When 0 .3

gram of these crystals was mixed with 0.3 gram of sodium ace tate and 0.6 gram of benzylphenylhydrazine hydrochloride and warmed in 8 ml. of 70 per cent ethanol <, the solution soon became solid with crystals. After standings the crystals were filtered off, dried and recrystallized from 90 per cent ethanol. They melted at 174° 0. When a mixed melting point of these crystals with a known sample of the benzylphenyl­

hydraz one of L-arabinose was made s there was no change

in the melting point. This establishes the presence of ■ ' 29 L-ara'binose in the gum. This was confirmed by conversion

of some of the 3=5 g2?^ms> of crystalline sugar to the di- phenylhydrazone which melted at 196° CU, the known melting- point of this derivative of L-arabinose. Also, a T per cent, aqueous solution of the 3.5 grams of crystalline sugar gave a paper chromatogram the same as that of L-arabinoge1^ in-- . dieating the presence of this sugar.

The filtrate from the 3-5 grams of crystalline L-ara- binose was concentrated down and the mucic acid test for D-galactose was made on it. This gave mucic acid, melting at 215° C., which proves the presence of D-galactose In the gum. When the mucic acid test was made on some of the 7 grams of gum sugar previously described, mucic acid, melt­ ing at 215° 0,., wad obtained - ; ; V

Fermentation tests were made with ordinary baker's yeast oh the sugars from hydrolysis at 80° G. and at no time was there an7 evidence of fermentation within a few hours,

though control experiments showed fermentation within a very short time. This establishes:the absence of D-glucose, D- ,:

mannose, .and D-fructose . ' ' ^ :. v / ;v; ' V " ‘ ; 't' ’ . . ' ' Ik - : \ . ' -"V - When Bertrand's test was made on the sugars, it was:.:: .negative.- The analysis of the crude gum as well as of ali- fractions of it showed the absence of methyl pentoses.

In another hydrolysis at 80° C. of 56 grams pf the pur­

ified silky oak gum for 2.5 hdurs in 4 liters of a 4 per cent solution of sulfuric acid, essentially the same results were ■ . - :y;:-' V ' v 30 pbtained, v: The ana lysis of barium salt 5, Table II shows that all of the L-arablnose is hydrolyzed off under the conditions of this experimentwhile approximately 1.5 anhydro p-galaetose uzLits are removed for each uronic acid group present. . ' : More vigorous hydrolysis of the gum, i.e. hydrolysis - - at'a higher temperatmre in"a mildly acid solution, may liberate more sugar units which are attached to the gum. . Identification of these sugars will lead to a better under­ standing of the composition and structure of the gum. With this in mind, the gum was hydrolyzed for varying lengths of time in a boiling water'bath. , A description of the pro­ cedures used and the results obtained follows.

Hydrolysis for 13 Hours at 98° C , In a 4 per cent Solution of ..Sulfuric Acid ' ,v -

v Forty ‘grams of the free acid,: which had previously been hydrolyzed for 2 hours at 65 C. in a 2 per cent solution of sulfuric acid:, were mixed with 1 liter of a 4 per cent solution of sulfuric acid and heated for 13 hours in a bath of boiling water. It was then neutralized with barium car­ bonate, decolorized' with activated; carbon, and filtered at : the pump. The f 11 trate was concentrated and poured into ethanol to precipitate-13 grams of white barium salt 3. The results of the inalysis of this salt are given in Golumn 3 of Table II. During this hydrolysis all of the pentose Units, - and:-2 i>31 -^tiydiro' teexidse units were removed„■1 The salt still retained 1.46 anhydro hexose units per- uronic acid. 1 - :■ : " - The ethanol -filtrate' from the 13 grams of barluiri salt 3 contained the sugars liberated during the hydrolysis„; It 1 was cdhcentrated in vacuo and found to; weigh :20. grams; By ' -dissolving-it in warm- glacial acetic acid and some ethanol, 10 grams of crystaliine sugar were ^ obtained. : The filtrate , from this was 'concentrated in vacuo., and 12 grams of a gum

sugar remained. : i ‘ v - ' - ’ ' - - ' A mucic: acid test ‘fdr D-galactose was made on some of • the gum sugar and on some of the crystal1ine sugar. • In both cases tha test was positive« A fermentation test with baker1s yeast was made‘on the gum sugar and was negative. Bertrand1s test for D-xylose, using some of the crystalline sugar/ gave negative results. A circular chromatogram^^ . was made on the gum sugar and also on the crystalline sugar.

In both cases the results showed the presence of B-arabinose

and D-galactose. In the gum sugar the L-arabinose and B- galactose^ seemed to be present in equal amounts but in the

crystalline sugar there appeared to be more galactose than. ardbinose . The crystalline sugar showed T«<1 ^ = -f 83.8Q . Hydrolysis of the Free Gum Acid for 15 Hours at 98° C. in a 4 per cent Solution of 'Sulfuric Acid. : ' , One hundred grams of the free acid fromVsilky oak gum- •

was mixed with 3 liters . of'a 4 per cent solution of sulfuric'-

acid and heated-for 15 hours in a bath of boiling water i f he ; ' ■ ■ ■ ■■ ; ; V ; . . ■ ■ . 3 2 solution was neutralized by addition of barium carbonate /and-the barium sulfate was filtered.off. The filtrate was concentrated in vacuo, and the solution was poured into ethanol. This precipitated 22.5 grams of barium salt 4. The. results of the analysis of this barium salt are given in Table II. These results show that all of the pentose

units and/ approximately 2 hexose units, had been removed dur­ ing the hydrolysis . The salt still contained approximately 1.22 anhydro hexose mi t s per uronic acid. The filtrate from barium salt .4.was concentrated in vacuo and approximately JO grams of gum sugar were obtained„ This showed ^5° - 4. 9 3 ,6°. This suggests that it is a mix-

ture of D-galaotoSe which has ^ = J - 80.5° and L-arabin-

ose which has f0*! ^ t 104.5°. When a circular ehromato- - : . ;L. ■ d :/'. h ■-. - : - v - - ■ y v . -. gram was made on the crystalline sugar, both L-arabinose -and D-galaotose were found present . This ^ sugar did not give Berfrand!s test for D-xylose. Hydrolysis of the Gum Acid for 18 Hours at 98° C. in a 4 ; per cent Solution of Sulfuric Acid

Barium salt 5 was prepared under these conditions and

isolated as in previous experiments. It was purified twice by dissolving in water and repreclpitating by addition to

ethanol. The results of the analysis of this Salt are given in Column 5 of Table II. These results show that approxi­

mately one anhydro hexose unit remains attached to. the uronic v :: . ■ ; ; . ; ■■■■ ■ 33 acid under these conditions and that all of the pentose units have been liberated. They also show the great diffi-

culty met in liberating the last sugar that is attached to. the uronlc acid. This confirms the observations of other investigators of some aldobionlc acids.

■"O ' ; ; ' / ' ' ' ’ ' ' p g ' From the results reported by Link and Niemann in their study of the destruction of uronic acids by hot mineral acids > it is certain that much of the uronic acid present was destroyed during this and previous experiments by the

long heating with sulfuric acid. Thus, much of the last

sugar attached directly to it would be present in the sugars liberated during hydrolysis under these conditions. The filtrate from which barium salt 5 had.been removed contained some of all of the sugars that had been present i n 'the gum. This filtrate was concentrated to a gum sugar.

When some of this sugar was Oxidized with nitric acid, mueic acid was obtained, thus proving the presence of D-galactose .. as in previous experiments. The diphenylhydrazone of L-ara-

binose, melting at 198 C .3 was also obtained from the sugar.. A paper chromatogram was prepared> which showed circular . values corresponding to both D-galactose and L-arabinose..

Some Of the sugar was subjected to a fermentation test with

ordinary baker's yeast with negative results, though the control experiment was positive. This establishes the ab­

sence of D-glucose, D-mannose, and D-fructose„ Bertrand's ' ■ ' ' ' . . 34 l6 test for D-xylose was made on the sugar.gum with negative results. These, results agree with all the previous experi­ ments . The only sugars present in the gum are D-galactose and L-arabinpse. The latter sugar is readily liberated by treatment with sulfuric acid and is almost certainly present in the furanose form.

Hydrolysis of Barium Salts 3 and 5 for 6 Hours at 93° C. in a 2 per cent.Solution of Sulfuric Acid

Twenty-seven grams of the combined barium salts 3 and 5 were dissolved in 500 ml. of water. To this solution were added 7 ml. of concentrated sulfuric acid. This was enough acid to make the solution 2 per cent with sulfuric acid after precipitating all the barium as barium sulfate.

The solution was heated in a bath of boiling water for 6

hours, neutralized with barium carbonate, decolorized with

activated carbon, and the barium sulfate and carbon filtered from solution. . The filtrate was concentrated in vacuo,

filtered again, and the barium salts precipitated by addi­

tion to 95 per cent ethanol. The resulting barium salt 6,

after purification by dissolving in water and reprecipitat- ing by addition to ethanol, weighed 13 grams. The results

of its analysis are given in Column 6 of Table II. Calcu­

lations show that 1.11 anhydro hexose units still remain for each uronlc acid group.

Before hydrolysis the salts contained an average of 1.2 anhydro hexose units per uronic acid group. -•/ . V. ' ' 35 . The alcoholic solution from barium salt 6 was concen­ trated in_vacuo to a gum sugar. This was dissolved in hot glacial acetic acid and seeded with D-galactose. Imme­ diately some crystals settled out of solution. After stand­ ing, the solution was decanted from the crystals. They were moistened with a small amount of water and alcohol added. On cooling, the white crystals that formed were filtered off, washed with ethanol, and dried in the oven. These crystals showed = 4- 81,4°, More crystals obtained from the filtrate, showed ah £04] ^ ° =.+•8.0.6°.

' , ■■■ : ■ • ' ■■ ; ■ D ‘ ■ • ' '' ■ Paper chromatograms indicated the presence of D-galactose alone in each of the sugars. It is evident that D-galactose is present in large amounts in the salts before hydrolysis, along with a slight tract of L-arabinose„ TABLE II. - ANALYSIS OF BARIUM SALTS FROM HYDROLYSIS OF SILKY OAK GUI

Col. 1 Col. 2 Col. 3 Col. 4 Col. 5 Col. 6 Salts from Salts from Salts from Salts from Salts, from Salts from Gum Heated Gum Heated Gum Heated Gum Heated Gum Heated Barium Salts for 2.5 Hr. for 3.5 Hr. for 13 Hr. for 15 Hr. for 18 Hr. 3 & 5 Heated for 6 Hr. .at. 80° at 80° at 98° at 98° at 98° at 98° Barium Barium Barium Barium Barium Barium Salt 1 Salt 2 Salt 3 Salt 4 Salt 5 . Salt 6

Barium, 0 7.29 9.79 13.18 18.12 15.55 15.40

Carbon Dioxide, 0 7.78 7.89 8.84 9.59 10.68 10.01

Uronic,Acid, 0 34.13 34.61 38.78 42.06 46.85 43.80

Equivalent Weight 566 ' 558 498 459 412 440

Anhydro Hexose Units per Uronic Acid 1.88 1.83 1.46 1.22 0.93 1.11

Specific Rotation $= f ■ .• 57.80 * 50.3° - 12.5° - 19.4° - 1 9 .2° ... H i s (a) Barium,Salts 15 3, 4 and 5 were hydrolyzed in a 4# Solution of Sulfuric Acid. Barium Salts 2 and 6 were hydrolyzed in a 3.50 and 2^ Solution of Sulfuric Acid, respectively. No pentosans or anhydro pentose units were present. Discussion of Results of the Analysis of the Barium Salts The results of analysis of barium salt 1 show that when the gum is heated to 80° C „ for 2.5 hours in a 4 per cent solution of sulfuric .acid, all the L-arabinose and some of the D-galactose are removed, k little less than two anhydro D-galactose units remain for each uronic acid - group present„ More vigorous hydrolysis removed additional amounts of D-galactose. Finally hydrolysis of the gum acid in a boiling water bath for 18 hours removed all but one anhydro.hexose unit per uronic acid group. The remain­ ing hexose attached to the uronic acid is difficult to re­ move , since a mixture of barium salts 3 and 5 on hydrolysis with 2 per cent sulfuric acid in a boiling water bath for 6 hours yielded barium salt 6, which still contained approxi mately one anhydro hexose unit per uronic acid group. The difficulty in liberating the last sugar has occasionally been reported. The great change in specific rotation of some of the barium salts is probably best explained on the assumption that some of these salts are of poly-aldobionic acids and have not been hydrolyzed to simple aldobionic acids.

Sugars Obtained by Hydrolysis of the Gum

The sugars, separated as hydrolytic products of the gum contained B-arabinose and D-galactose with no traces of

D-xylose, D-glucose, D-mannose, or D-fructose. The specific rotations of the sugars ranged from -o- 80.0° to ^ 96.3° 38 indicating that a mixture of L-araMnose and D-galactose, whose rotations are respectively f 104.5° and ^ 8 0 .$°^ is present. The sugars from barium salt. 6 contained D-galac- tose in large amounts with a possible trace of L-arabinose, This was to be expected since analysis of barium salts 3 and 5 indicated that only hexose units remained linked to the uronic acid. Identification of the Uronie Acid in Silky Oak Gum Eighty-four grams of the free acid from silky oak gum were mixed with 2500 ml. of a 4 per cent solution of sul­ furic acid and heated for 16 hours in a bath of boiling water. The solution was then neutralized with barium car­ bonate, decolorized with activated carbon, and concentrated in vacuo to a syrup. This was again filtered and 24 grams of barium salts obtained by addition of the filtrate to . ethanol.

The 24 grams of barium salt were dissolved in water and the barium precipitated by careful addition of sulfuric acid so as to leave no barium and no sulfate in solution. The filtrate was concentrated in vacuo and 18 grams of the gum acid were obtained. This was dissolved in 150 ml. of nitric acid, specific gravity 1 .15, and evaporated almost to dry­ ness in an evaporating dish on the boiling water bath. The residue was dissolved in a small amount of water and allowed to stand in the refrigerator so that the oxalic and mucic acid that had been, formed could crystallize out of solu­ tion . The crystals were filtered off, recrystallized from ; water, and found to melt at 215° C= The filtrate from the mucic •acid was neutralized with potassium carbonate, made acid with acetic acid, and con­ centrated to a small volume„ It was cooled and seeded with a known sample of potassium hydrogen saccharate. On stir­ ring, the solution -became solid with crystals, After standing in the refrigerator, these were fi1tered off and : found to weigh 1 gram; These crystals were dissolved in a

small volume of hot. water, the solution filtered, and a small amount of ethanol added. On seeding the solution with a known sample of potassium a.cid saccharate, crystal- lizationlbegan at once. After standing in the refrigerator 0.5 grams of crystals were obtained. These showed jo<]^0= . ^ 3.lv Fischer25 reported the tW]|0°= >. 7 for pure potas­ sium acid saccharate. To explain the- low rotation it should be noted that these crystals had been recrystallized" only ' • once and were Certainly not perfectly pure. The potassium ' acid salt was now converted to the silver salt as described v _ . ,v; r V ;.■■■ z. ■■ ■ ■ V . ' : ; „ ' by Brown and Zerban. .The silver in this salt was found to be 48.99 per cent. The theoretical amount of silver in silver saceharate 18 50.91 per cent„ The positive rota­ tion of the potassium: salt, the per cent silver ih the 'silver, salt, and the way the solution of the potassium salt. ■ ' ' •: . ' " ' ’ ' ' ' ■ . - y 40 precipitated when seeded with potassium acid saccharate leave little douht hut that, it was potassium acid sacchar­ ate and that D-glucuronic acid is the uronic acid that is present' in the gum. v :;v . , r'. ■ ■ -/ SUMMARY

The silky oak, Grevillea rebusta, is an Australian tree that has been introduced into this country for orna­ mental purposes. The silky oak gum used in this investi­ gation was collected from trees growing on the grounds of the Biltmore Hotel in Phoenix, Arizona. The crude gum contained a small amount of a water insoluble resin. . It was purified by dissolving the gum in warm water and re­ moving the resin, bark, and dirt by pressing the solution through cloth. The gum was then precipitated by addition of ethanol. The purified gum was separated into three fractions which differ in their solubility in water. The free acid, of which the gum is a salt, was also prepared by addition of hydrochloric acid to a water solution of the gum and immediately precipitating the gum acid by addition of ethanol.

Analyses are given in Table 1 of the lightest colored . and cleanest samples of the crude gum as well as of the three different fractions into which it was separated, and of the free gum acid. These analyses show that the gum consists of the. calcium and magnesium salts of a complex - ' acid. There are 2.28 anhydro pentose groups and 3.25 anhydro hexose groups on the average to 1 uronic acid. In small ; ' 42 whole numbers there are 4 uronic acid units to 13 anhydro hexose units .and 10 anhydro pentose units. the gum was hydrolyzed severa.1 times by heating with ,

dilute sulfuric acid under varying .conditions of tempera- ture^ strength of acid, and duration, of heating. The • products of hydrolysis were separated info the barium salts of a uronic acid combined with one or more sugar units and a mixture of sugars. ' , , . - The uronic acid was identified as D-glucuronic acid and the sugars as D-galactose and L-arabinose.„ It was found that the L-arablnose was hydrolyzed off first and easily. It probably was .present in the furonose form. The D-galactose was less readily hydrolyzed off. The last D-galactose unit which was attached directly to the D-glu- euronic acid.was Very difficult to hydrolyze off, Some evi­ dence was ■ found which indicated that-the.gum is not a simple chain of carbohydrate units with D-glucuronic acid at one end and L-arabinose units at the other, with D-galactose units making up the middle portion but that it consists of branching chains in which more than one D-glucuronic acid unit is present. ' . ' . ’ - V i BIBLIOGRAPHY

1. Anderson, B., Sands, L,, and Sturgis, N., Am. J. Pharm.,

; ; x : ^ :' ''' " ■ .2;:: Anderson, Eand Sands, L ., Ind» Eng, Chem., 17,125?

: : : <1925>: V \ V ; ■ :■ ■ v; : 3. Anderson, E.., and Sands, L., J. Am. Chem. Soc c, 48, . 3172 (1926) 4. Anderson, E.^ and Otis, L., J. Am. Chem. 80c ., 52? 4461

; :: , : (#30)::: ' : : - - . -V \ " 'v '', : ; ^ A ' A; ', A" 5. White, E. V„, J. Am. Chem. Soc., 6 8 , 2?2, (1946) 6 . Anderson, E ., J. Chem, Education, 9, 857 (.1932)

7„ Anderson, E ., Russell, F . H., and Seigle, L,. W-., J. Biol,

Chem.,: 115^ 683 (1936) 8 . Jones, J. K. N., and Smith, F., Advances in Carbohydrate Chemistry, Vol. 4, Academic Press, 1949, p. 243 9. Anderson, E ., and Ledbetter, H „ P., J. Am. Pharm. Assoc.,

• ; 4o, 623 (1951) • '' ' . ■ ' ■ ■■■_ 10. Boas, I. H ., The Commercial Timbers of Australla, TheIr Properties and Uses.' Melbourne: J. J. Gourley, Gdvern-

Printer, 226 (1947) ' x ' -p; \ / r

11. von Fellentierg, T ., Bio chem. ' 2 . 8 5 , 44 (1918 ) ; 12. PenigeS,; M.; G. j. Comp . Rend ., 150, 529 (1910) 13. To liens, B., and. Oshima, K ., Ber. , 34, 1925 ,-(1901) : : \ -'":: :;:' .T"i ■■ 'V' :■: ::-;V ' V ; ' w

14. : Browne, G.A :.'# and Zerban, F. ¥.> Physical and Chemical- Methods of Sugar Analysis; John Wiley and Sons,' I n c .. New. York, 3rd.: ed. (194.1) : ,' ' ;'.r : 15. ’ van den . Haar., ;-A. W. > Anleltung . zura Nachweis zu.r -Tnennung und Bestiramung den Monosaccharide Und Aldehydesan'nen,- Gebnuden Bonntnaegen, Beniin, 1920.

16. Bertrand, M. G., Bull soc. ohim., 3, 5, 546 (1B9I) : 1 7 . Bao P „ S-. and- Beni, R. M., Pnoc . Indian Acad. Sci., 33,

, 368 (1951) n ; 4 ' ' 18.. Le Fevne, K. B., and Tollens B., Ben., 25, 2569 (1892)1 .

40, 4153 (1907) 1 9 . Methods of Analyses, The Institute of Eapen Chemistry, Appleton, Wisconsin (1944) 20 . Ledbetteh, H. D., Master of Science Thesis,- University

■_ 9 of Arizona (1950) . 9 9 ; ' 7 9. .. 21. Tollens, B., and Krober, J. Bandw., 48, 355 (1900); 49, 9

■ 7 (1901); • 9 < -;" ^ : n;-- 22. Haywood, J. K., Ball; 105, U.S. Bun. Chem,, ll6' 23.• Mantell, C . E ;, The Water-Soluble Gums, Reinhold Pub­ lishing Corporation,.Mew vYonk, 1947^ p .6 24. Anderson, E., and Sands, E.', Advances in Carbohydrate

: Chemistry, Vol. I, Aoedemlc Press, 1945, P .329 ■•.9 :

25. Fisohery':E. Ber t, 23, 2623 (1890) : 2.6:>;; Eink>: K. P., .and Niemann, C. J. A m ... Chem; Soc., 52,

.9/9': 2 4 7 4 - '' t / :t'; '/ ■ . 9,- ■ ■