~Ie OXIDATION of CELLULOSE Witli CHROMIC ACID

~iE OXIDATION OF CELLULOSE WI Tli CHR OMIC ACID

A Thesis

Al ber t Richard Reid M.Sc. (Dalhousie)

Submitted to the Fa cul t y of Graduate Studies and Research in partial ful filment of t he r cquirements for the Degree of Doctor of Philosophy.

Di vi sion of Industrial and Cellulose Chemistry, McGi l l University. Ma r ch , 1953. AC ~iliOVffiEDGE IBNTS

The author wishes to express sincerc appreciation to Dr. C.B. Purves for his constant encouragement and gui dance which was so generously gi ven dur ing the course of t his research.

Sincere appreciation is also cxpr e s s ed to Dr . T.E. Timell and to fe1 low stude~ts of the Department of Indus trial and Cellulose Chemistry for their advice and kind intercst.

Grat ef ul acknow1edgements are made for the financia1 assistance received from the Department of Veterans Affairs, for the funds made availab1e from the Ha r ol d Hi bb er t Memor i al Fe11owship, and for the aid in the form of summer grants received f r om the Pulp and Paper Research Institute of Canada. TABLE OF CONTENTS

GENEPJ~L I NTRODUCTI ŒIJ ...... l HISTüRICAL INTRODUCTION ...... l Analytical ~ethcds for carbcxyl and carbonyl Groups ....•...... •....•... •...... 18 R~SULTS AND DISCUSSION ...... 29 Cxi da t i on s of Cellulose wi t h Chromic Aci d ••••••••• 29 Ob servations on the Estimation of carbonyl Groups.. 50 Application of the Kiliani Reactions to Oxycelluloses •..•.•..•.•.•...... •....••...... •• 61 BXPERItŒ NTAL SECTION ...... 75 Determination of Reduci ng Sugars by Condensation wi th Cyanide •••.•••••••••.•••••.•.• 75 r~1 e thodA. ..•..•.....•...•.•...... • 75

~·,~ e th o d .B •••. III ..••••••••••••••••••• •• •••••••••• • 76 Det er mi n& t i on of CarboÂJl Groups in OX7lce11ulos e...... •. 77 Estimation of Carbonyl Groups in Ox i di zed Cellulos e •...... ••..•...•. ....•.•••.•• 78 Hydr-oxyLam.l.n e Hydr ochl or i de Ii1 e t h od •••••••••••• 78 Cyanide T;:ethod .. •...... •.. •• . 79 E s ~ ima t i on of the Aldehyde Content of Oxidized Cellulose ••••••••••••••••••••••••.•• 80 Copper Reducing Power of O~Jcellulos es •••••••••••• 83 r:l o i s t ur e Content ...... •..•.•...... • 85 A sh Content ...... •...••.. 85 Pretreatments of Cellulose •.•••••••...... ••••.•••. 86 Oxi dations with Sulphuric Acid - Dichromate ••••••• 87 Oxidation of Oxalic Acid by Potassium Dichromate ••• 88 Oxidation of Cellulose by Potassium Dichromate -Oxalic Aci d •..••••••••••••••••••••• 91 Change in carbonyl and Carb o ~Tl Content durf.n g Oxidation •...... •.....••. 94 Large-Scale Oxidation of Cellulose (Oxycellulose V-5) ••• •• •• ••.•.•••••• •• • •• • •• • •• • 96 Ni t r a t i on of Oxycellulose V-5 ••.•••. •.•••••••• 97 Estimation of the Aver age Degree of Polymerization ••••••••••••••••••••••••••• 98 Me t hylat i on of oxycellulose V-5 wi th Dia~omcthane ••••.•••••••••••••••••••••• 99 ii

Table cf Contents (cont'd)

Preparation and Reduction of the Cyanohydrin of Oxycel1u1ose V-5 ...... 101 As cendi ng Me tho d of Paper Chromatography ...... 107 Descen àing Mebh.od of Pa p e r- Chromatography ...... 108 Sm,1IiIARY AND CLAD:IS TO ORIGI NAL RE8EARCH ...... 109 BIBLIOGRAPHY ...... 112 iii

LIST CF Tl-u3LES

T l~13LE Page

l Cupra&~onium Fluidity and Tensile Strength of Various Hydrocelluloses

and Oxyc e l Lu'Lo s e s •• g ••••••••••••••••••• •• ••• 3

II Copper Reducing Power at Va r i ou s Stages of Oxidation 32 III Oxalic Acid - Dichromate System ...... 37 IV Cxidations of Swollen Cellulose with Dichromate and Cxalic Acid at 25°C. 42 v Al d eh yd e and Ket8~e Contents of O~Tcelluloses .... 46 VI Estimation of Gl u co se by the Cyanohydrin Reaction. 51

VII Esti~ation of Fructose by the Cyanohydrin Reaction ...... •. 54

VIII Es t Lmat Lon of t:1.e Carbonyl Content of Oxy - cellulose by the Cyanohydrin Reaction ••••••• 56

IX Recovery of Amraon5a from Aqueous Cyanide Solutions and from the Corre sp onding Unmodified Cellulose Blanks .••....•..•...••• 58

X Comparison of carbonyl Determinations on Oxycellulose by the Cyanide and Hydrozyla.mine Me t h od s ••.•..•...... ••.••••• •• 60

B 3 h~vi o ur of Oxycelluloses during Cyanohydration and Saponificatior...... 64 XII RF Val1.:.es of Acids by the As cending l'iI ethod ...... 69 XIII RF Values of Acid3 by the Dcsccnding I.~c th o d ...... 71 XIV RD Values of Acids by the Des cend Lng I\icthod ... . 72 J. ' .. xv Re-oxidation of Ox y cel l ulo fi e \'V i t h Chlorous FJ. oi d ... 82 XVI Comparis on of Draidy and He y e s Copper Numb e r s •••• 85 iv

TABLE Page

XVII Chan ge in pH of an Oxa1ic Aci è - Potassium Di chroma t e 301'.1ti01J. 'vi th Time of Reaction •• 89 XVIII Fractiona1 Distillation of Aci ds from the Ba.r-Lurn Salt s 106 v

LIST OF PIGURLS

FIGURE

l Oxidations of unswollen linters with chromic acid at 20°C• •.•.•••••••.••..•••• 30

2 Time of oxidations in 0. 2 N oxalic acid - ' 0.04 N po tas s i um di ch r oma te solutions .... 34 3 Rate of oxidation of 0.2 N oxalic acid by 0.1 N p o t a s s i um dichromate ••••••••••••••• 35

1 Changes in the properties of unswollen cellulos e wi t h increased tille of oxidatien ...... •...... ••• 49

5 Paper chromatograms of simple fatty acids by a scending method •••••••••••••••••••••• 70

6 Determination of the intrinsic viscosity of nitra tod ozycellulcse V- 5 ••••••••••••• 100 r

GENERAL I NTRODUCTI ON

Cellulose oxi da t i ons are of interest because t hey

i mpair the ~hys i c a l properties of pap er s and cotton textiles and occur, sometimes in bleaching and dyeing, and always dur ing exposure to lisht, air, and moi s t ure over long periods of time. With the exception of a few cases, the detailed nature of the r eactions involved in mi l d or drastic oxidations is still obscure, although it h~s long been known that oxidation forms acidic and r educing groups in sorne of the glucose units that comprise the long macromolecules of cellulose. The num ber and position of these gr oup s vary with the extent of the oxidation, with the physical sta te of the material and with the oxidant employed. Only Q f ew methods are available for their determination.

This Thesis first examines the oxidation of cellulose wi t h aqueous chromic acid with a view ta pr epa r i ng ~~ oxycellu lose of high reducing p ower in a minimum time. The oxidation of cellulose induced by dichromate in the presence of oxalic rather than sulphuric acid, wa s p articularly investigated and found to be suitable. Mu Ch time was spent in checking the re liability of existing methods for determining total carbonyl, an d also aIdehyde groups, in the oxycelluloses and in de ter mining the optimum conditions for the oxidation. A portion of this work, showing how the ke t one gr oup in fructose could be II quantitatively determined by means of the cyanohydrin reaction, has already been accepted for publication in Analytical Chemis try.

With the experience thus gained, a large batch of the oJ:ycellulose was condensed with the cyanide ion and the result ing cyanohydrin was saponified, the whole procedure involving the conversion of the carbonyl groups to a-hydro~J acid units. Reduction of the adduct with hydriodic acid yielded a mixture of low molecular fatty acids. Contrary to expectation, the i n i t i a l oxidation had beeu intensely localized in the cellulose. HISTORICAL INTRODUCTION

The first attempt to classify cellulose which had been chemically modified by oxidizing agents was made by Witz (1) in 1883-1884. He described such oxidized products, which still retained the fibrous structure, but not the mechanical strength of unmodified cellulose, under the heading of It oxy_ celluloseslt• At almost the same time, this term was applied by Cross and Bevan (2) to the powdery, non-fibrous, and partly water-soluble products obtained from the drastic oxidation of cellulose with nitric acid.

For the next forty years, research on such oxidations was confined largely to empirical tests and observations because of the limited knowledge concerning the chemical structure and the physical nature of cellulose itself. However, the invest igations during this period did indicate that the oxidation of cellulose was a very complex chemical reaction yielding oxy celluloses which varied in their properties according to the reagent employed. The modern phase of research on oxidized celluloses only spans the past twenty to thirty years, and closely parallels the rapid advancement in the general chemis try of cellulose.

In technical literature the term Itoxycelluloselt de scribes a product that has been chemically modified by oxida tive agents in such a limited manner that the fibrous nature of the starting material is still retained, although the mechanical strength is decreased. Thus the definition of Witz (1) is still in use, even though the term "oxycellulose" is actually a misno mer, since it implies an addition of oxygen, whereas an abstrac tion of hydrogen may also be involved. Parsons (3) and more re cently Unruh and Kenyon (4), have suggested the replacement of

the term by the more precise riame, "oxLdd z ed celluloseu • How ever, both terms have been used interchangeably throUghout the great mass of the literature and the custom will be followed in this thesis. Much of the following information parallels that contained in reviews by purves and co-workers (5)(6)(7), the most recent of which was published only three months ago (October 1952).

Extensive work by Clibbens and Ridge (8) showed the re1ationships existing between tensi1e strength, cuprammonium fluidity, (the reciprocal of the viscosity in poises of 0.5 per cent solutions), copper number, and alkali stability of hydrocelluloses were not valid for several types of oxycellu 10ses. For instance, there was no correlation between the ten sile strength and cuprammonium fluidity of the oxycelluloses shown in Table l. Davidson (9)(10) explained that the observed cuprammonium fluidities of alkali-labile oxycelluloses were of ten high owing to alkaline . scission during the measurement, and not necessarily to chain cleavage during oxidation. TABLE l

CUPRAMMONIUM FLUIDITY AND TENSILE STRENGTH OF VARIOUS HYDROCELLULOSES AND OXYCELLULOSES %Loss of strength Caused by acid attack Caused by Caused by Caused by Fluidity (Hydrocel- attack with attack with attack r~th (Rhes) luloses) NaOCL ~Cr207 + H2S04 K2Cr207 + 00H)2

10 10 7 2 l 20 34 25 Il 6 30 58 47 26 16 from Clibbens and Ridge (8)

Because of differences in their chemical properties, Davidson (11) classified oxidized celluloses into two main groups according to an important generalization by Birtwell, Clibbens and Ridge (12); acidic types, which showed a high absorption of basic dyes and a low copper-reducing power (copper number), and reducing types, which exhibited a low absorption of basic dyes, possessed high copper numbers and were very easily broken down by alkali. The former were prepared by oxidants in alkaline media, and the latter by acidic oxidants. A third type, prepared by oxidants in neutral media, had properties intermediate between these two extremes. The classification was only approximate because there were exceptions, and it was possible to prepare an oxidized cellulose which displayed the properties of aIl three types. 4.

The literature on various oxidations of glucose and other simple sugars (13)(14)(15) indicates that the products range from carbon dioxide and formic acid to relatively mild and specifie changes to substances like gluconic, glucuronic, and saccharinic acids. The oxidation of cellulose ls not en- tirely analogous, because the carbonyl groups and the hydroxyl groups in the fourth position of the anhydroglucose units are b'Lo cked by glycosidic linkages. Nevertheless, a great variety , of products May result from oxidative attack on any or aIl of the three exposed hydroxyl groups remaining in the repeating unit and on the few exposed carbonyl groups at the ends of the Macromolecules. Sorne of the possible oxidized units are as follows; oxidation of reducing end groups (I), of primary hy droxyl units (!l, R = CHO or COOH), of secondary hydroxyl func tions (III and ~), and oxidation by the cleavage of 2,3-glycol units (V, R = CHO or COOH). Mixtures of these forms May also occur, and (VI) represents one suCh possibility.

Perhaps the simplest oxycellulose i8 that represented by (I). This type may be prepared by selectively oxidizing a hydrocellulose with alkaline hypoiodite, under the carefully controlled conditions of temperature and a~falinity reported by Ma r t i n , Smith, Whistler and Harris (16) after a reinvestigation of the work of Bergmann and Ma ch emer (17). A more recent investigation of this reaction has been carried out by Husemann and Weber (18). The terminal aldehyde unit can also be quantitatively oxidized by a dilute aqueous solution of chlorous acid, 5.

H OH C-C ° -0, /OH H\ Il C\ COH HH C 1 CH20H

l II (R = CHa or COOH)

ID IV

V (R = CHO or COOR) VI thus changing the reducing properties of hydrocelluloses to acidic functions (19). This oxidative process--aldose to aldonic acids--is highly selective, and only aldehyde groups are attacked while ketone structures remain unchanged. 6.

Another type of oxidized cellulose, periodate oxycellulose, was prepared in 1937 by Jackson and Hudson (20)(21) with aqueous periodic acid, a reagent which had previously been used in the oxidative cleavage of 1,2-g1ycols (22).

CH20H r + HI04 --~>2H2C=0 + H20 + HI03 ••• (1) CH20H

Jackson and Hudson demonstrated the course of the reaction by hydrolysing the oxycellulose or oxystarch and isola ting glyoxal and D-erythrose (Yll and YI!l) from the hydrolysate.

/ OCH3 0-, RR VII HC '/ '\.H OCH IX C C l"" 3 H"~_o/Lo_ HC- OCH3 1 OCH3 CH20H ""

V (R = CHO)

VIII X (R = CH3 or H)

Hence, periodate oxycellu1ose had the structure 7.

(V, R = CHO) which was formed by the oxidative cleavage of the second and third free hydroxyl groups of the basic glucose unit of cellulose.

Further evidence of a selective glycol cleavage was found by Harris and collaborators (23), who isolated D-erythronic and glyoxylic acids from periodate oxycelluloses after re-oxida tion with alkaline hypoiodite and subsequent hydrolysisj and by Purves and co-workers (24)(25) who submitted the oxidized cel lulose and the corresponding oxystarch to drastic methanolysis which led ta the recovery in high yield of glyoxal tetramethyl acetal (IX) and isomeric cyclic acetals of the general type

(X, R = CH3).

Another type of oxyce11ulose which has become prominent during the past decade is that which results from oxidation by nitrogen dioxide or its dîmer, nitrogen tetroxide, either in gaseous form or in carbon tetrachloride solution. Kenyon and co-workers (26)(27)(28)(29)(30)(31) discovered this type, and conc1uded that the nitrogen oxides preferentially oxidized the primary alcohol group in cotton, cotton linters, and wood pulps to carboxylic acid functions. The oxidation was described as highly uniform, and even at high levels of oxidation, the prod ucts were fibrous. Nitrogen dioxide oxycelluloses consisted of chalns of anhydroglucose and anhydroglucuronic units, (II, R = COOH) whose relative proportions depended on the amount of oxidation. The incompletely, as weIl as the completely, oxi dized cellulose was named "celluronlc acid". These products 8. possessed a high copper number as weIl as a high carboxyl con tent, and samples with 12 per cent or more carboxyl groups dis solved rapidly in dilute aqueous sodium hydroxide solution.

Kenyon and his collaborators found that the carboxyl content, as measured by the evolution of carbon dioxide, tended to a limiting value of 25 per cent. Since complete oxidation should theoretically produce a polyanhydroglucuronic acid with a carboxyl content 25.57 per cent, these investigators conclu ded that the primary hydroxyl group was attacked by the nitro gen oxides to form uronic acid units. The calcium acetate method (see below) for the estimation of carboxyl groups indi cated only slightly lower values for carboxyl groups than those obtained by the uronic acid determination. Supporting evidence for structure (II, R = COOH) was found through the acetylation of tile residual hydroxyl groups in the oxycellulose, which gave a product with an acetyl content near the calculated value.

Kenyon and others showed in a more recent investiga tion (32) that small amounts of carbonyl groups were also present, and account for the observed high copper number and great sensitivity to alkali (26)(27) of nitrogen dioxide oxy celluloses. The fact that oxidation with chlorous acid decreased trle reducing power suggested the presence of aldehyde functions, while the change from yellow to colorless occurring when this type of oxycellulose was alternately dried from the moist state and then left in a moist atmosphere, strongly indicated the

of~-diketones ...uresence of traces (33) as shown in structure (&, R = COOH).

A great deal of literature is available concerning oxidations of cellulose by other reagents to products which

display in varying proportions the properties of both ~le high ly acidic (i.e. periodate-chlorite and nitrogen oxide oxidized celluloses) and the extreme reducing type (i.e. periodate oxy cellulose). These reagents include hypochlorite and hypobromite at various hydrogen ion concentrations, hydrogen peroxide,

ozone, nitric acid, permanganate, and oxygen and alkali, but ~le great amount of research in this field has thrown little light on the detailed structure of the products.

Cllbbens and Ridge (34) were the first to investigate the oxycelluloses produced by the action of hexavalent chromium ion. They showed that a solution of O.04N potassium dichromate at 25°C. did not alter the reducing power of cotton, but when the hydrogen ion concentration was lowered from pH 5 by addition of O.IN or O.2N sulphuric acid, the cotton acquired many reducing groups as indicated by the increased copper number. 10.

Clibbens and Ridge observed that when the sulphuric acid was replaced by an equivalent amount of oxalic acid the oxidation proceeded about one hundred times as rapidly. A linear rela tionship existed between the copper nurnber and the successive small amounts of dichromate added. Later tiley showed that the products formed in the early stages of oxidation, ei~ler with sulphuric or oxalic acid, were of the non-acidic, alkali-sen sitive, highly reducing type (8). Davidson also found such lightly oxidized products to be in the sarne class as periodate oxidized cellulose (35). From the results of ~e former work (11) he reasoned that oxidation with dichromate - oxalic acid was accompanied by little hydrolysis of the glycosidic linkages, because the products possessed muCh greater tensile strength and lower nitrocellulose fluidity than other oxycelluloses of the sarne reducing power. However in later research (36) more drastic oxidation of cellulose with dichromate in the presence of sulphuric acid (up to more than one atom of oxygen per glucose unit) resulted in oxycellulose with acidic properties, as denoted by increasing methylene blue absorption. The more highly oxidized dichromate oxycelluloses were thus found to be ra dically different from those oxidized with periodate. The ash content of this type of oxycellulose, due to retention of chromiurn, increased to a certain value and then became independent of the amount of oxygen consumed by the product. No similar detailed study was carried out on oxalic acid - dichromate products be cause the oxygen consumed by the cellulose could not be deter- Il. mined, owing to the simultaneous oxidation of oxalic acid.

Grangaard, Gladding, and Purves (24) prepared oxycel luloses with chromium trioxide in acetic acid - acetic anhydride media, and in the analysis of the products, found no glyoxal tetramethyl acetal (IX), thus indicating the absence of 2,3 glycol cleavage during the reaction. This work confirmed Davidson's conclusions (36) that periodate oxycelluloses did not resemble products highly oxidized with chromic acid. Hibbert and Parsons (37) had also prepared oxycelluloses with reducing and acidic properties by using chromium trioxide in 90 per cent acetic acid.

Meesook and Purves (38) examined chromic acid oxidized cellulose for the presence of reducing groups. The aldehyde, as opposed to the ketone content, was deter.mined by re-oxidation of the oxycellulose with chlorous acid (pH 2.5), and the increase in carboxyl together with the corresponding decrease in carbonyl groups, were between 46 and 56 per cent of the original total carbonyl content. The remainder of the carbonyl content was assumed to be ketonlc. This assumption was proved partially correct by a similar oxidation of xylan, a pentosan resembling cellulose but possessing no primary hydroxyl groups. The car bonyl value, considered to be totally ketonic, was close to that calculated as ketone groups in the oxycellulose, but the low re coveries and an unexpectedly high acid content rendered the re suIt uncertain. Perlin and Purves who also used chromium trioxide in acetic acid - acetic anhydride (5) found that 15 to 20 per cent 12.

of the carbonyl content was re-oxidized by c~lorous acid to carboxyl groups, and was thus designated as aldehyde. They concluded that the aldehyde Lroups were aIl in the pos i t i on originally occupied by the primary hydroxyl group (II, R = CHO), because the 2,3-dialdehyde structurz (V, R = CHO) was not con sidered to be present in chromium trioxide or/cellulose (24).

Purves (7) suggested tha t_the~ceton~ ~ouns ~otJl.t~ackad_by - -- chlorous acid were either in the form indicated in (III) and (II) or in an equivalent common enolic form as reprcsented in (XII), but a diffcrentiation has not yet been made with certainty.

OH OH 1 1 -Oi/C=C\lI C C H,\H /L C--O 0-- 1 CH20H

XII

Despite a considerable amount of research on the problem, both in the fields of inorganic and organic chemistry there is still much controversy concerning the mechanism involved in oxidations with chromic acid. Waters and co-workers (39)(40)(41) obs er ved that gaseous oxygen was &bsorbed during the oxidation of alcohols, ethers, ketones, and hydrocarbons by chromium trioxide in glacial acetic acid. Because of the oxygen uptake they concluded that the mechanism involved an initial dehydrogenation by chromium trioxide, resulting in the gcneration of organic 13. free radica1s as unstable intermediate compounds. They de- picted the oxidation of tetra1in (39) by a chain reaction se quence (Equations 2 and 3) the initial step being the abstrac- tion of hydrogen atoms from tetralin by chromium trioxide to produce transient radicals (Equation 4).

R"CH. + 02 ---~ R"CH- 0- O· • •• (2)

R"CH-O-O· + R"CH2,---=> R"CH- O-O-H + R"CH· • • • (3 )

VI ~O v ~O R"CH2 + 0= Cr~ ---) R"CH. + Ho-cfr~ (4) ~O ~O • ••

Mosher and his colleagues (42)(43) advanced the theory that the chromic acid oxidation of some alcohols in aqueous acetic acid took place via the removal of hydride ion from the hydroxyl group of the alcohol, a transitory intermediate being subsequently formed:

H + o o o Il RCR -H: ==> RCR Loss of H+ :> RCR ·.. (5 ) H H

Westheimer (44) ruled out the possibility of the intermediates proposed by Mosher and others, at least in the case of isopropyl alcohol, in favour of an ester mechanism which just as easily explained the oxidation of secondary hydroxy1 groups in dilute acid media. (Equations 6 and 7). More recent work by Westheimer and co-workers (45)(46) showed strong eviden- 14.

HCrO~- H+~\====' + CH3CHOHCH3 + (CH3)2CHOCr03H + H20 • •• (6 )

H20 + (CH3)2CHOCr03H- ~H30+ + (CH3)2CO + HCr03- ••• ( 7) ce of an ester mechanism for this type of oxidation. In parti cular, the kinetics of the decomposition of isopropyl chromate, the intermediate in equations 6 and 7, further substantiated the above mechanism (47). In this investigation the reaction rate was observed to be the sarne either in the presence or absence of oxygene The presence of ketonic functions in aqueous chromic acid oxystarch (6) would suggest that the oxidation of more complex molecules followed lines similar to the mechanism pro posed by Westheimer and collaborators for more simple compounds.

In a review of the research in this field Westheimer (44) concluded that the mechanism of the oxidation of organic acids, such as oxalic, lactic, and malic, was quite different from that for the oxidation of isopropyl alcohol. He considered that the oxidation involved a direct attack of the oxidizing agent upon the organic acid (or ion), and that further detailed studies might weIl reveal the presence of organic free radicals. Induced oxidations (i.e. the oxidation of indigo by dichromate in the presence of oxalic acid) were thought by Westheimer to offer the best evidence for the existence of unstable interme diates in the forro of pentavalent and tetravalent chromium com pounds ,

Theories concerning the oxidation of acids. as weIl as alcohols, are therefore still in an indecisive state. 15.

Eefore c10sing this brief review of the structure of the oÀ7ce11uloses, it seems desirable to discuss two related aspects of general importance, the great influence of the physical accessibility of the cellulose sample to the oxidant, and the instability of reducing oxycelluloses toward alkali.

As a result of X-ray studies and research with the electron microscope, the long macromolecules of which fibrous cellulose is composed were found to run througn submicroscopic crystalline and amorphous regions. The former were considered to be areas of highly geometrical order whereas the latter pos sessed a more open irregular network. While the denser crys talline regions were obviously less accessible to attack by chemical reagents, the amorphous areas were quite vulnerable. Consequently, the extent of the chemical attack (including that by oxidative reagents) upon cellulose would depend a great deal upon the physical condition of the material as weIl as on the nature of the reagent.

studies on the oxidation of cellulose by aqueous periodate (20)(48)(49) and nitrogen dioxide (26)(27)(33) showed that the process took place at two different rates, a fast re action which indicated attack on the amorphous region, and a much slower reaction involving penetration of and attack on the more closely packed crystalline portions. The yield of oxycel lulose remained almost quantitative during oxidative attack by these two reagents, and thus there was very little secondary oxidation whereby water-soluble fragments were formed. During 16. the course of the oxidation of the crystallites, the X-ray dif fraction pattern of the cellulose was destroyed, although at different rates with each oxidant.

Gladding and Purves (50) found that the oxidation of dry, highly swollen cotton linters with chromium trioxide in a non-swelling medium of glacial acetic acid and acetic anhydride produced insoluble oxycellulose almost quantitatively up to an oxygen consumption of approximately 0.3 atoms per glucose unit. When unswollen linters were used, the yield began to decline af ter approximately 0.1 atoms of oxygen per glucose unit had been consumed. Their Interpretation of the results was that the re covery remained high until the accessible portion was oxidized, and decreased when further attack on the oxidized units resulted in the production of fragments which were soluble in the reac tion medium. They also found that there was very little differ ence in the accessible region of swollen and unswollen linters when aqueous sulphuric acid and chromium trioxide were used, the reason being that swelling occurred during the oxidation. They showed also that the carbonyl content of the oxycellulose, as a result of secondary oxidation, passed through a maximum as the rate of formation became less than the rate of loss caused by the further oxidation of carbonyl groups to acid functions and soluble debris. Davidson (36) was the first to report the ex istence of such a maximum for oxycelluloses prepared with aqueous chromic acid. This maximum was near 30 for unswollen cellulose (10 units higher for swollen cellulose) as measured by the

Schwalbe-Braidy copper number method, and was marked by the total 17. disappearance of tensile strength. oxycelluloses made with ni trogen dioxide (51) and periodate (21) were found to attain cop per numbers of 100 and 120 respectively witho ut suffering a com plete loss. Cellulose which was reduced to a powder by chromic a ci d and possessed no fiber Gtructure, still retained the X-ray diagram of the original material (3 6). This observation also supported the i dea that chromic acid penctrated and oxidized only the amorphous portion of the fiber, thereby causing its disintegration, but left the crys t allites almost intact.

Many workers have observed the easy degradation of oxy celluloses by alkali, even under mild conditions. Peat and others (52), for example, found that treatmont of a highly re ducing permanganate oxycellulose with boiling one per cent caus tic soda dissolved the oxidized portions, leaving a residue which w~s largely unmodified cellulose. The soluble portion contained low molecular weight acids including formic, acetic, lactic, sac charinic, and an unidentified acid, C3H5(OH)2COOH. Further sup port has been given by Harris and others (23) who re-oxidized highly alkali-labile periodate oxycellulose with chlorous acid and found that the alkali sensitivity of the product was almost zero. The nitrocellulose fluidity of the product was much less affected by pr evi ous boiling in alkali than was that of the per iodate oxycellulose itself. Studies on glycosides such as turan ose, or 3-«rglucosido-D-fructose (XIII) (53) and glucosidodihydrox yacetonc ( YJV) (54) have shown that in both compounds the glycosi dic linkage is readily cleaved by alkali, and have l ed to the generalization that a ketone gr oup makes an adjacent glycozidic 18.

bond unstable.

XIII --XIV

Head (55) found that diazomethane reacted with the carbonyl groups in periodate and lightly oxidized dichromate oxycelluloses in such a manner as to destroy their reducing power, as measured by copper number and their sensitivity to alkali. In order to account for these results, Head suggested that the groups were converted to ethylene oxides or methyl ketones

(RGHO --:;> RCHOCH3 ) , or were methylated in the enolic form to the H non reducing grouping R-C-OCH3 1:. 0-1

Detailed experimental work on the degradation and solubility of oxycelluloses in alkali, together with possible mechan isms have been summarized and discussed by purves (7).

Analytical Methods for Carboxyl and Carbonyl Groups The deter.mination of acidic and reducing properties in oxycelluloses depend to a great extent on adaptations of the existing methods employed for estimation of carboxyl, aldehyde and ketone functions in simple organic compounds. ~hile there are a great number of methods recorded in the literature for evalua ting these functions in oxycelluloses, very few of them have met with general acceptance. Davidson and Nevell (56)(57) and Nevell (58) published a careful experimental study of the relative mer its of the most promising carboxyl determinations using various types of oxycellulose at different stages of oxidation. Purves and collaborators (5)(6) and Purves (7) summarized various pro cedures for the estimation of both carboxyl and carbonyl func tions in oxycelluloses and oxystarches.

At the present time carboxyl groups are usually estima ted either by ion-exchange reactions with various cations or by modifications of the uranie acid estimation of Lefevre and Tol lens (59). Because the acidic gr oup s in oxycelluloses are ap proximately of the same strengtll as those in glyceric and gluconic acids (60) they show a great affinity for metallic cations and basic dyestuffs.

R- corn + M+ X-'---:> R- COaM + HX ••• (8 )

The estimation of carboxyl groups depends on a quantita tive displacement to the right in equation 8, a condition which is favoured by a high concentration of the salt, MX, a low hydro gen ion concentration (i.e. HX must be di1ute or a weak acid), and the absence of competing or foreign cationi.

The metllod of Neale and Stringfellow (61) involves treating the oxyce11ulose with sodimn chloride solution and ti- 20. trating the liberated hydrochloric acid with alkali. They recom mended the addition of a known excess of alkali to overcome the difficulty of a drifting end-point in the direct titration. Da vidson and Nevell (56) criticized this method as being unsuitable for general use with reducing oxycelluloses because it gave high values of carboxyl group owing to the alkali-Iability of the modified cellulose.

The silver absorption method employing silver o-nitro phenolate (62) or the more effective silver m-nitrophenolate (57) gives satisfactory results only if a correction is made for re duction of silver ion by the oxycellulose. The method is unsuit able for use with dichromate oxycelluloses because of the presen ce of adsorbed trivalent chromium which causes high results.

The absorption of methylene blue (63) is the most gen eral1y applicable method for slightly modified reducing oxycel l1.110ses, but again the results with dichromate oxycelluloses are too high owing to the presence of chromium ash,

Lüdtkels method emp10ying calcium acetate (64) has been widely used , and technical improvements by ~~eesook and Pur-ve e (38), Kenyon and co-workers (30), and Davidson and Nevell (56),have made this procedure of genera1 validity, and the best method available for chromic acid oxycelluloses. However, it is not sensitive enough to give accurate results with very sma11 amounts of carboxyl groups.

Nevell (58) has investigated the uronic acid method 21. using nitrogen dioxide oxyce11uloses. He concluded that the metilod can only give a rough estimate of the uronic acid groups in reducing oxycelluloses and points out that even simple keto sugars such as fructose, sucrose, and ascorbic acid, yield as much carbon dioxide as uronic acids themse1ves. A modification of the standard procedure, using 25 per cent sulphuric acid for trle decarboxylation instead of the usual 12 per cent hydrochloric acid, did not improve the method.

Among the earlier methods summarized by Purves (7) for the estimation of carbonyl groups in oxycelluloses the most wide

lt ly employed is the Itcopper number , which is defined as grams of copper reduced from the cupric to the cuprous state by 100 grams of dry, modified cellulose in strictly standardized conditions. There are many modifications, each giving reproducible results except possibly in very highly reducing oxycelluloses or hydro celluloses.

Gladding and Purves (50) determined the carbonyl groups in chromic acid oxycelluloses and oxystarches in a more stoichio metric manner by treatment with hydroxylamine hydrochloride and titration of the acid liberated as a result of oxime formation.

• •• (9 )

This reagent reacts also with acidic groups in oxycel luloses, and tberefore estimates total carbonyl and carboxyl func tions. Because aqueous solutions of the reagent are unstable af ter standing at room temperature for several hours, the estimation 22.

must be done in ninety minutes. The question as to whether this was sufficient time for aIl the carbonyl groups to react was clarified in the affirmative by Meesook and Purves (38) who checked the estimation with methyl hydroxylamine hydrochloride (CH30NH2.HCL) a reagent which is stable for days in aqueous solu

tion. The me~lod works weIl with highly reducing oxycelluloses but not with slightly oxidized materials. It is unsuitable for chromic acid oxycelluloses and oxystarches of high ash content since the ash interferes to give fictitiously high results (5)(6).

Two methods are in general use for the estimation of aldehyde groups in oxycelluloses; the alkaline hypoiodite proce dure employed by Bergmann and Machemer (17) in their study of hydrocelluloses, and the selective oxidation with chlorous acid used by Harris and co-workers (23) for periodate oxycelluloses. The latter method has been used in the study of chromic acid oxy celluloses and oxystarches by purves and co-workers (5)(6)(38). Estimations of the aldehyde content of oxycelluloses with hypo iodite have been criticized because of the alkalinity of the re agent, its tendencies to over-oxidize the cellulose, and its swift reversion to iodide and iodate. The re-oxidation of oxycellulose with chlorous acid at pH 2.5 and 25°C and estimation of the addi tional carboxyl groups, is considered by Purves (7) to be the most promising general method for determination of aldehyde func

tions.

During the past few years some attention has been focused on the cyanohydrin reaction (Equation 10) as a means of measuring 23. the total carbonyl groups in hydrocelluloses and oxycelluloses•

...... -C=O + • • • (10)

The determination of the reducing groups in simple sugars by the Kiliani reaction was foreshadowed by Hudson, Hartley, and Purves (65) and Coombs (66), and they established suitable conditions for the reaction. In 1951 Yundt (67) estimated the carbonyl groups of various sugars by noting the consumption of cyanide, and concluded that the method was satisfactory for glucose and other aldoses. He found that his conditions gave erroneously high results with fructose. Militzer's similar estimation (68) (69) gave good results for aIl aldoses and apparently also for the keto-sugars investigated. Frampton and co-workers (70) estimated the reducing groups in aldoses and hydrocelluloses by titration of the ammonia expelled when the corresponding cyanohydrins were saponified in boiling alkali. They freed the system of unreacted cyanide prior to the saponification, and gained in accuracy by eliminating a large blank.

Preliminary comparisons of the relative merits of the cyanide and hydroxylamine hydrochloride methods for the determina tion of total carbony1 groups in chromic acid oxycel1uloses (5) and oxystarches (6) indicated that the former offered promise, but the investigations were hampered by the interference of the chromium ash with the hydroxy1amine method. However, recent work by McKil1ican and Purves (71) indicated that both methods gave similar results when applied to a hypochlorite oxystarch in which 24.

chromium ash was absent.

In an effort ta locate the ke t on e groups in o)~celluloses and oxystarches the above investigators (5)(6)(71) applied the complete Kiliani series of reactions to the oxidized prod ucts. Iüliani's researches which have been reviewed (5)(72) depended on the conversion of sugars to their cyanohydrins, which wcre then saponified and the resulting higher carbon sugar acids were reduced with hydrogen iodide and red phosphorus. Identifi- cation of the final fatty acid proved the location of the carbonyl group in the original sugar molecule. These crude fatty acids were contaminated with small amounts of combined iodine, which were removed with zinc and hydrochloric acid. Perlin and pur- '7 ves (5) repeated the work wi th glucose (structures XV to XVIII) and fructase (structures XIX to XXI) and, by finding optimum conditions for each reaction, improved and overall yields of the aliphatic lactones. The improvement in the de-iodination step resulted from the use of the Schwenk reduction with Raney nickel - aluminium alloy and caustic soda (73) or of catalytic hydrogenation over Raney nickel at high pressure. Glucose gave the lactone of 4-hydroxy heptanoic acid (XVII) and n-heptanoic acid (XVIII) in a combined yield of 73 per cent of theory, the lactone com prising 90 per cent of the product. In the case of fructose, 2-methyl-4-ethyl butyrolactone (LXI) was recovered in almost 74 per cent of the theoretical yield, but the reduction with hydrio die acid had to be slightly more drastic than that for glucose. 25.

li ,0 ,f] 1,0 110 C'I C-OH C-OH 1 1 1 ECOH HCOR ~::I CH2 1 1 1 0 1 ROCH HCN Hcon HI + Red P CH2 :> ::> rF~ 1 then 1 then 1 HCOH hydrolysis HOCH de-iodination HC CH2 1 1 1 1 HCOH HCOH CH2 CI{2 1 1 1 1 H2 COH HCOH CH2 CH2 1 1 1 H2 COH CH3 CH3

]0/ ]0/1 ]0/11 ]0/111

R2COH H2COH 1 1 OH c = a C""""" 1 l 'COOH HOCH HCN HOCH HI + Red P > 1 then 1 then > ECOR hydrolysis HCOH de-iodination 1 1 RCOH HCOH 1 CH20H

XXI

Perlin (5) was unsuccessful in isolating any of the lactone (XXI) from cellulose oxidized by chromium trioxide in acetic acid - acetic anhydride, although he found sorne evidence of the presence of this lactone arnong the products. As mentioned previously, in the similar research on chromic acid oxystarch, Ellington and Purves (6) succeeded in isolating and identifying 2-methyl-4-ethyl butyrolactone (XYJ) und methyl-n-butyl acetic acid. This result showed that oxidation had occurred at the second position of the glucose units in oxystarch. Since these investigators failed to isolate ethylpropylacetic acid or deriva tfves thereof, there was no evidence as to whether or not the chromic acid oxidized the third positions of the glucose units to ketone groups. Since any aldehyde groups would be in the sixth positions of the basic glucose units of the oxycellulose, the cyanohydrins would yield carboxylic acids of the uronic acid type which would probably be decarboxylated during reduction with boiling hydriodic acid. McKillican's (71) isolation of 2 methyl-4-ethyl butyrolactone from starch oxidized with hypo chlorous acid at pH 4 showed that 2-keto units were formed in this case a Ls o,

During and since the time the above work was carried out, the development of paper chromatography, a powerful new analytical tool, has made great advances, which have been reviewed elsewhere (74). The advances of interest to the present research concern the detection and identification of organic acids.

Lugg and averell (75) separated dibasic hydroxy acids with the help of a two-phase system, n-butanol and aqueous acetic acid. By the use of acetic acid (which is quite volatile) in the mobile phase, they eliminated "tailing" of the carboxylic acid spots. This "tai1ing" was caused by the adsorption of.acids by the paper and by their partitioning in favour of the aqueous phase as dilution increased. After running the chromatograms for approximately 30 hours at 17°0., and drying at 60°C., the 27.

acids were located as yellow spots by spraying the paper with a dilute solution of bromophenol blue or bromocresol green and then re-drying. However, this method was unsuitable for organic acids which were highly volatile. Fink and Fink (76) were able to separate volatile acids by converting them into their potassium OK hydroxamate derivatives (R--C==N--OH) and using suitable solvents. Employing butanol, ethanol and various concentrations of ammonium hydroxide, Brown and Hall (77) separated the Cl to Cs simple ali phatic acids, but dicarboxylic acids did not move along the paper.

Brown (7S) later overcame the i~mobility and tendency of the lat ter acids ta "streak" by developing the chromatogram with ethanol and ammonia. A more recent method for separating the aliphatic acids (Cl to CS) was that of Kennedy and Barker (79), who made use of the ammonium salts and developed the chromatograms with ammoniacal ethyl alcohol, a one-phase system. The ascending method was used by these workers, and location of the acid spots was considerably improved in comparison with other procedures by spraying with a sp ecial bromophenol blue - citric acid indicator. Another advantage was the absence of confusion caused by spots originating with the alkali.

In recent investigations concerning the formation of carboxylic acids during the photosynthesis of radio-active car bon dioxide (C1402) by plants, Benson and his colleagues (SO) employed two-dimensional paper chromatograms and were successful in separating the acid products and recognizing them by their

Rf values. 28.

Up to the present time paper chromatography has appa rently not been applied to the separation of the acidic products resulting from the saponification and reduction of oxycellulose cyanohydr-Lns, 29.

RESULTS AND DISCUSSION

Oxidations of Cellulose with Chromic Acid

The initial ob ject of the research was to find op timum conditions for the preparation of a chromic acid oxy cellulose with a high carbonyl and a low carboxyl content. In addition, the yield of the oxycellulose had to be nearly quan titative, in order ta provide some assurance that neither second ary oxidation nor hydrolysis had produced excessive degradation. These requirements suggested that optimum conditions would be those that produced the highest reducing power or the most rapid reduction of chromic acid in the shortest period of time.

Aqueous sulphuric acid, 0.2 N and 0.4 N, containing various amounts of potassium dichromate was employed in preli minary work. Experiments 3, 4 and 5, Figure l, gave the highest oxygen consumption based on the decrease in the concentration of dichromate. The product of Experiment 4 attained a copper number of 2.76 in 140 minutes for an oxygen consumption of 0.056 atoms per glucose unit. After treatment of cotton for five and one half hours with 0.2 N sulphuric acid solution, 0.1 N with respect to dichromate, Davidson (48) obtained a product with

a copper number of 2.41. He found, however, that the high ini tial rate of decrease in the concentration of dichromate was

for the most part caused by adsorption by cellulose and not by reduction. It was probable, then, that the fast initial reac- 0 CD 0 o _ --0 ------0-4. 1------::>0Z 0/-- --- o 0 0-3 . 10 ,/ wC? ,/ ~t""~ --i Cf) 0 • .-5. 0 / o 1 ::J ...J ,..-0 (90 V 0 '1 0::: 6 w '; n, wrt)c 0 ::1 0 , ::J 6 CI) z o o o e- 2. Cf) (\1 1---. ~ C? o 0 ..... ct z 0 Wc(!) . >- 0 <5

FIGUlŒ 1. Oxidations of unswollen lint ers with chromic aci d at 20°C.

VJ Plot 1. H2SO4' 0. 2 N; K2Cr207, 0.0 52 N. Plot 4. H2S04, 0. 2 N; K2Cr207, 0.718 N. o 2. 0. 2 N; 0.102 N. 5. 0. 2 N; 0.386 N. . 3. 0.4 N; 0.20 2 N. 6. 0.4 N; 0.050 N. 31.

tions observed in Figure l were due to adsorption and that the data were only of qualitative significance.

Although Experiment 4 gave a product with a slightly higher reducing power in ashorter time than the others, it was evident that long periods of oxidation would be required to yield an oxycellulose containing a really high carbonyl content. Perlin and Purves (5) prepared oxycellulose with a very high re ducing power (0.1 to 0.4 moles of carbonyl per glucose unit) in periods of time ranging from 45 to 180 minutes. However, their method involved oxidation of the cellulose with a solution of acetic acid and acetic anhydride containing chromium trioxide, resulted in products whose high ash content (1 to 4 per cent) interfered with the estimation of carbonyl groups. The oxida tion of cellulose by chromic acid in the presence of aqueous oxalic acid was reported by Clibbens and Ridge (34) also to be rapid, and this method was therefore investigated next.

Results of three preliminary oxidations with oxalic acid and potassium dichramate are shawn in Table II. Oxycel lulose III gave the highest reducing power, but the relatively large amount of reactants (see b el ow) made this method of oxi dation unsuitable for large-scale work. A quantitative yield was desirable since there was no other means of estimating the amount of oxygen consumed by the cellulose, as opposed to the oxalic acid other than by analysis of the product itself. The preparation of oxycellulases l or II seemed ta offer the best 32.

TABLE II

COPP ER REDUCI NG PO\VER ATVARIOUS STAGES OF OXIDATION

Oxidation of Unswollen Linters(a) Time Equivalents of of Di chromate Oxidation per Gram % Copper Oxycellulose (hours) Cellulose Yield Number

r 6 0.005 99 16.5 II 3 0.050 100

III 8 0.500 97

(a) l - Initial concentrations: 0.2 N oxalic acid and 0.1 N potassium dichromate. II - Initial concentrations: 1.2 N oxal i c acid and 0.5 N potassium dichromate. Excess oxalic acid added a. s required to keep the pH less than 2. III - Potassium dichromate added in instalments over six hours, oxalic acid being added as required to keep the pH less than 2.

possibility for combining a practically quantitative yield with a high carbonyl content. Experiment 2 also suggested that the best yield could be obtained by decreasing the time of exposure of the cellulose to ~le reaction mixture.

Davidson (36), employing 0.005 equivalents of dichromate per gram of material in 0.2 N sulphuric acid, found that a copper number of 16.2 was attained by cellulose only 33.

after a reaction time of 94 hours. Since o~Jcellulose l was

prepared b y a similar amount of dichromate in 0.2 N oxalic acid

in a period of six hours, it seemed likely that the me chanism

of oxidation depended on the acid. It was decided t o investi

gate the oxidation of oxalic acid by dichromate in the pres

ence and absence of cellulose, p artly in the hope of finding

sorne measurable difference which could be used to determine

the consumption of oxygen by the cellulose, and partly to gain

information on the type of oxidation.

The p r ep a r a t i on of oxyc e l l ul o s e IV followed that of

Clibbens and Ridge (34) wher-eby each gram of cellulose was

treated with 80 ml. of a 0.2 N solution of oxalic acid made

0 . 0 4 N with respect to dichromate. Employing copper numbers as a measure of oxidation, the rate plots for unswollen and

swollen linters (Figure 2) revealed a rapid initial reaction lasting for approximately 30 minutes, and followed by a slow rise in copper reducing p owe r f o r the r emainder of the time.

The rate of oxidation, more rapid for swollen cellulose, showed that this method resembled other chromic acid oxidations in being dependent on the accessibilit y of the cellulose sam pIes. The r atio of the accessibility of sw ollen to unswollen cellulose, as measured by copper numbers, was approximately

1.4:1 over the range from 20 to 120 minutes of oxidation.

Figure 3 shows how 0.2 N oxalic acid was oxidized by

0.1 N potassium dichromate, t he rate curves for the consumu

tion of both reagents bein s shown. The results indicated that 0 cD A-- 0 ~ ySWOLLEN

0 -c.\l UNSWOLLEN 0 7 a:: ./ w 6 III ~ / . / ô --- - ._ e------· --- ~ 0 .>:e e Zoo

œw 0. Q. CD Q. /... j- °00 ~ At.'1

9 /1· (\j ï·

o 1 ! 1 1 1 1 1 J 1 o 20 40 60 80 100 120 140 MINUTES TIME OF OXIDATION

FIGURE2. Time of oxi dations in 0. 2 N oxalic aci d - 0.04 N potassium dichromat e solutio ns. Plotted against incre ase in copper number. \....) Unswollen celbllose • Swollen cellulose. •-1='- o ~~ 1 1 1 1 1 1 1 1 6

o o CDo 6

1 1lJ a: 0 ... 0 - CD .\ ....J 0 6 a: \ 1lJ Q.

U> ... g~\ Z g ' \ iL!.J c( 6 (1 \ > :5a w o o~. o 0- '"o '\-..00_ o 6 '-"e_ ------e e- o 81 1 1 go-- 20 40 60 80 100 120 140 TIME ( HOURS)

FIGURE 3. Re.te of oxidation of 0. 2 N oxali c a cid by 0.1 ,N potassium di.chr-omat.e

'vJ Concent rat ion of dichromate 0 Concentration of oxalic acid • .V1 36. most of the reactants were consumed during a period of three to six hours, and thereafter the reaction became exceedingly slow or ceased. A comparison of Figures 2 and 3 indicated that the greater part or aIl of the cellulose was oxidized dur ing the period when the rate of disappearance of the oxalic acid and dichromate was most rapide Since direct oxidation of cellulose by dichromate, as in aqueous sulphuric acid, was a very slow reaction, the above results supported the view that some other mechanism had operated.

It was noted in Figure 3 that the reaction apparently ceased after approximately 96 hours, even though quantities of both reactants were still present in the solution at that time. This observation suggested that the oxidation of oxalic acid by dichromate was not complete in water. In a further investiga tion of the dichromate - oxalic acid oxidation-reduction system, using 50 ml. of 0.20 N oxalic acid made 0.10 N with respect to dichromate, the evolution of carbon dioxide was measured after six and 24 hours in separate experiments. The data for this re action and also those for the filtrate from the preparation of oxycellulose lare summarized in Table III. From the quanti ties of reactants present after 24 hours or 96 hours, as deter mined by titration of aliquots, it was found that the amount of oxalic acid to that of dichromate consumed, corresponded very closely to a molar ratio of 7:1 in aIl cases. It was noted in Experirnent 1 that the quantity of oxalic acid consumed in 24 hours was 92 millimoles per litre of solution, while the amount of carbon dioxide evolved during the sarne period in Experiment 2 TABLE III

OXALIC ACID - DICHROMATE SYSTEM(a)

Reactantl} After 96 Hours Consumedt b) Molar Ratio C02 of Expt. Evol ved( b) Reactants No. (6 hours) ~ Acidity(C) Dichromate(d) (COOH)2 K2Cr207 Consumed

1 • • • 2.5· 15· 21}J 9~ 13- 7.1:1- 2.5 7 18 96 14 6.9:1 2 52 2.5 la 17 97 14 6.9:1 64" 3(e) 66 2.5 14 17 95 14 6.8:1

(a) 50 ml. of solution containing 200 mi11iequiv. of oxa1ic acid and 100 mil1i- equiv. of dichromate. (b) mi11imo1es per litre. (c) Mi11iequiv. per litre; estimated by titration with alkali, and corrected for the amount of dichromate present. (d) Mi 11 i eq ui v. per litre; estimated by titration with thiosu1phate. (e) Unswo11en cellulose (1 g.) in system. ·Values recorded after 24 hours.

Vl •...:I 38. was 64 millimoles per litre. Since the consumption of acid after 96 hours was observed to be 96 millimoles per litre, the amount of carbon dioxide evolved in that time was calculated to be 64 x 96 or 67 millimoles per litre of solution. The results 92 therefore showed that only about 67 per cent of the available oxalic acid was oxidized to carbon dioxide, indicating that the reaction did not go to completion in an aqueous medium. This observation was in agreement with that of Snethlage (81) who concluded from kinetic studies that the oxidation of oxalic acid by chromic acid was incomplete in water, and in dilute sulphuric acid up to 13.4 per cent. He believed that the incomplete reaction was due to the formation of a complex or compound between oxalic acid and the reduction product of chromic acid. It was also observed in the present work that when aliquots of the final solution of Experiment l (Table III) were acidified with 20 ml. of 0.2 N sulphuric acid and left at room temperature for 44 hours, the dichromate concentration remained unchanged at 18 milliequivalents per litre. It was therefore apparent that the end point was independent of the hydrogen ion concentration between pH 2.5 and about pH 1. The final violet colour of the present mixtures was evidence that a soluble complex of some sort had formed, but the nature of this complex was not investigated.

Sidgwick (82) reviewed the available research on chromioxalates and showed that compounds such as M3 [cr(C204)3] and M[ Cr(C204)2(H20)21 existed as very soluble complexes which were blue and red respectively. Solutions of the former salt gave no reactions for chromium or oxalate. The "simple" oxalate Cr2(C204)3x H20 was also considered to have a complex structure because of its great solubility in water (83). The failure of these chromioxalates to ionize was conclusively established by Long (84) who made use of oxalate ions containing radioactive carbon. The fact that these compounds were very soluble in wa ter perhaps accounted for the peculiar ease by which chromic acid oxycelluloses were de-ashed with dilute oxalic acid (48).

The existence of a violet complex in the present re action mixtures seriously hampered the determination of di chromate and of acidity, but dilution of aliquots clarified the end point sufficiently so that reproducible results could be obtained. The great difference in the consumption of oxalic acid, as measured by the evolution of carbon dioxide (previously calculated to be 67 millimoles per litre), and as estimated by final acidity (97 millimoles per litre for Experiment 2, Table III), was attributed to the existence of a complex chromioxalate in the solution. Since the latter method only determined free acid, and the former was considered to measure only the quanti ty of oxalic acid which was oxidized, the difference between the two estimations was presumed to represent the amount of oxalic acid taken up in the formation of a complex with trivalent chr-omf.um,

According to ~le data in Table III, the final condi tions were not greatly changed when cellulose was present in 40.

the reaction mixture for six hours. A slightly greater amount of carbon dioxide was evolved in Experiment 3 and was perhaps due to degradation of part of the oxidized cellulose whose presence might account for the slight increase in the final acidity. The results again indicated that little, if any, of the dichromate was reduced by the cellulose. A later analysis of a sample of oxycellulose l (see below), prepared by immersing swollen linters in a solution which was 0.2 N with respect to oxalic acid and 0.1 N in dichromate, showed that an oxygen con sumption of 58 milliatoms of oxygen per glucose unit was reached in a period of 75 minutes. This rapid oxidation again coincided with the fast decomposition of oxalic acid illustrated in Figure 3, and provided further evidence that direct attack on the cel lulose by chromic acid was negligible.

Although the exact nature of the oxidation was unknown, Westheimer (44), as previously mentioned, suggested the possibi litY that organic free radicals or transitory intermediates such as pentavalent and tetravalent chromium compounds were respon sible. It has long been known that during the reaction between oxalic acid and potassium permanganate a peculiar oxidation in duced by air occurred, resulting in the formation of percarbonic acid, which then deconwosed to hydrogen peroxide and carbon di oxide (85). By analogy, the rapid oxidation of cellulose by chromic acid in the presence of oxalic acid might be explained by the formation of percarbonic acid and hydrogen peroxide as intermediates: 41.

o II ( COOH)2 + ( 0) ---~) H-O-C-O- OH + CO ------+) H202 + C02 + CO.

This speculation was n o t Lncon s I s t errt wl th the I'ac t t h a t hydro-

gen p er oxi de i n an acidic medium was a much more p owe r f u l oxi- dizins a g ent th an dichromate; t h e standard E values (86) hei n g o = 2H20 + 1.77 volts

HOW 8ver, since Westheimer (44) concluded that the oxidation p o- tential of per ch r omi c a ci.d (Gr5+ + 2e------;.> Cr3+ ) was b e t we en

1.75 a n d 2.0 volts, it was quite p os s i ble that a combination of s e v e r-aL t yp e s of oxida tion, including that by free r adicals, was i nvolved in the oxidation of cellulos e by dichromate in the presence of oxalic a c i d .

As a l r eady mentioned, c ons i der a t i on of the conditions u s e d to prepare o xyceLLu.l.o s e a l, II, a n d III sug gested that there was no need ta add t h e oxalic acid in instalments or to prolong the time of oxJ dation beyon d two hours at 25°C. Sub s eq u en t oxida t i on s (Table IV) were accordin g to these inferences, but car- b onyl gr oup s in the products were estimated by the more stoi- chiometric cyanid e method i nstead of by the less qua ntitative copper r educing me t.hod , VIith the exception of oxycel Lul.os e L, aIl products in Table IV were solvent-exchanged through aleohol and benzene and dr:l.ed over phosphorus pentoxide before analyses were carried out.

The very close correspondence between the properties TABLE I V

OXI DATI ONS OF SWOLLEN CELLULOSE WITH DI CHROWlliTE AND OXALIC ACI D AT 25°C.

Rea gerit Recov err Norma l i t i e s ( a ) of Oxygen e ) Milliequiv.{b) Oxi d Oxy- at ion Mil limo l es Mil l i mol e s Total a s Cr 0 Cr % Cellulose (C OOH)2 K2 2 7 :K:2 207 (mins.) Carbo nyl(c) carboxyl(d) Mil l i a toms car b o xy l

I(f) 0.2 0.1 810 75 24 17 58 59 VII 0.2 0.1 1620 120 24 16 56 57 VI 0.4 0.1 1620 120 3 2 10 52 39 V-l( g) 2 0.5 81 00 60 51 20 91 44 V-2( g ) 2 0.5 8100 120 47 19 85 45 V-3 2 0 . 5 8100 60 80 26 132 3 9 V-4 2 0.5 81 00 120 64 29 122 48

(a) 10 0 ml . of s ol ution p e r gm. of cellulose. ( b) Per gl u co s e unit. (c) Per gl uco s e uni t ; cyan ide method ( d) Per gl u co s e unit; ca lc ium ac e tate (e) Pe r gl u co s e unit; a ssum i n g COOH r equ ir es met hod. t wo a toms , CO, on e ato m, of oxy gen (f) Only 50 ml. of s ol ut i on p er- gm, of ( g) Unsw ollen l i nt ers used. c ell ulose

~ ro • 43.

of' oxycelluloses l and VII 8howed that doubltng the volume oi' oxidant, while increasing the time, had a n egligible ei'fect on the proportion of carbonyl to carboxyl gr oup s . On the other hand, an increase in the ratio of oxa.lic acid to dichromate (oxy

cellulose VI) increased the carbonvlv content at the exnense~ of the carboxyl content, which then accounted for 39 instead of 57 per cent of the oxi dant recovered (column 9). This observation supported the conclusion of Clibbens and Ridge (34) that oxalic acid tendeà. to prevent the further oxidatian of aldehyde to car boxyl groups, which presumably was the normal fate of aldehyde g r ou p s in the p r e s en ce of di chromate and sulphuric act d , Th e re maining runs retained the molar proportions of oxalic acid to dichromate at the more favourable ratio of 4:1, but the concen trations were increased five-fold, a change which élid not g rea t l y diminish the f a vou r a b l e ratio of carbonyl to carboxyl ~r o up s

(column 9). Comparisons of oxidations V-l and V-2, and of V-3 and V-4, showed that in these experiments a one-hour was defini t ely better t han a two-hour perioel of oxidation; in the same way, comparisons of V-l and V-3, and of V-2 an d V-4 revealed the su periority of highly swollen to unswollen linters as a starting material. These observa.tions showed that the accessible portion ai' the cellulose underwent the initial stag e of oxidation within one hour, and in the second hour further oxidation produced molecular debris. The yield of 99 per cent obtained from the unswollen linters, when contrasted wi th the 101 p e r- cent yield from the swollen samples, showed that the f urther oxidation was more pronounced in the former case. Gladding and Purves (50) ob- 44. tained similar results in their oxidations with chromium tri oxide dissolved in acetic acid - acetic anhydride, a non swelling system.

Table IV also showed that the oxygen recovered (col umn 8) in the swollen samples after both one-hour and two-hour oxidations was about 1.4 times the amount recovered from the corresponding unswollen samples (V-l and V-3; V-2 and V-4). This ratio was in close agreement with that round previously by the copper number method (oxycellulose IV, Figure 2), and was in the range of 1.5:1 often found for mercerized, as op posed to native celluloses, observed in media like water (87) which swelled the native sample considerably during the estima tion.

A comparison of the data in columns 4 and 8 (Table IV) suggested that the oxidation of cellulose by dichromate and oxa lie acid was not very efficient. The amount of oxygen found in product l corresponded to a recovery of only 14 per cent, based on the quantity of dichromate present in the solution. For prod ucts VI and VII the recoveries of oxygen were six and seven per cent respectively~ In the preparation of oxycelluloses V, in volving muCh greater quantities of reactants, the oxidations were even less efficient, as the recoveries of oxygen in the products from unswollen and swollen linters were only two and three per cent respectively. These results indicated that the the oxidation of cellulose in the oxalic acid - dichromate sys tem was relatively insignificant compared to the main reaction 45. involving the chromic acid oxidation of oxalic acid.

In order to differentiate aldehyde from ketone groups, samples of the oxycelluloses in Table IV were re-oxidized with chlorous acid at pH 2.5 and 25°C. for one hour, and the increase in carboxyl groups was equated to the aldehyde content. The re-oxidized products were recovered in practically quantitative yields. The ketone contents were accepted as the difference be tween total carbonyl, as d~termined by the cyanide method, and aldehyde. Table V, which summarizes the results, shows that in the lightly oxidized samples l and VII aIl the carbonyl groups were aldehyde within the experimental error. A small amount of ketone, however, appeared in oxycellulose VI which was prepared with a 4:1 molar ratio of oxalic acid to dichromate instead of the 2:·1 ratio empl.oyed in the other two cases.

~Vhen the 4:1 ratio was used, and the concentration of oxidant was increased five-fold (oxycelluloses V), the ketone became roughly equal to the aldehyde content, the amount being slight ly greater in the samples V-3 and V-4, prepared from swollen cellulose. These results explained Davidson's early conclusion (35) that cotton lightly oxidized witll chromic acid resembled periodate oxycelluloses of similar levels of oxidation in physical and chemical behaviour, because the carbonyl groups present were aIl aldehyde in both cases. The differences he later noted (36) between the two series of samples at higher levels of oxidation probably originated, in part at least, in the acquisition of the ketone groups by the dichromate oxycel luloses. TABLE V

ALDEHYDE AND KETONE CONTENTS OF OXYCELL ULOSES

Total Oxy- carbonfl Aldehyde Ketone % Cellulose Content a) content(b) Content(c) Aldehyde

r 24 25 -1 100 VII 24 30 -6 100 VI 32 25 7 78 V-l 51 26 25 51 V-2 47 27 20 58 V-3 80 38 42 48 V-4 64 35 29 55

(a) Millimoles per glucose unit; cyanide method. (b) Millimoles per glucose unit; chlorous acid and calcium acetate methods. (c) Millimoles per glucose unit; total carbonyl minus aldehyde content.

The presence of ketone groups in the oxycelluloses V clearly showed that they contained 2- or 3-keto units of the types noted in the Historical Introduction. There was also qualitative evidence that 2,3-diketo structures like XI (R = CH20H or CHO) were also present. When a sample of the moist oxycellulose V-3 was dried over phosphorus pentoxide at 100°C. for 48 hours, the colaur changed from white ta pale lemon-yellaw. On re-expasure ta maist air at room temperature, the colour slowly changed ta white again, and more quickly so, when part of the caloured sample was immersed in distilled water. 47.

Repetition of the process showed that the colour change was re- versible. As previously mentioned, Nevell (33) observed similar results with nitrogen dioxide oxycellulose alternately dried over phosphorus pentoxide in vacuo and exposed to a moist atmosphere. Nevell inferred that 2,3-diketone structures were p r e sen t , since 1 1 the conjugated system O=C-C=O normally imparted a yellow colour to compounds containing it (i.e. gl yoxal , diacetyl, and benzil.). The results with oxycellulose V-3 similarly indicated the existence of the 2,3-diketone structure, although they diverged from those of Nevell in that very little change of colour was observed when oxycellulose V-3 was dried at room temperature. Even at elevated temperatures, no change in colour was perceptible when the lightly oxidized cellulose samples, l and VII, were dried under similar conditions.

The decline in the proportion of carbonyl to carboxyl groups with increased time of oxidation, (Table IV), was due to a decrease in the carbonyl content. The results shown in Table V indicated that this decrease was mostly in the apparent ketone content, since the aldehyde values remained approximately constant when the time of oxidation was extended from one to two hours. It therefore seemed that tile formation of ketone groups in the glucose units of cellulose caused such units to be more vulnerable to hydrolysis or to secondary oxidation with conse- quent dissolution in the reaction medium. Overend and co I workers (88) observed that replacement of the H-C-Œ group 1 1 with Œ2, the methylene group, in pentoses and hexoses greatly 1 decreased the stability of the glycosidic linkages which these sugars formed. If a keto group had the same effect, cleavage of the oxycellulose at this point would cause a 108s of ketone in the product, although the loss was not large enough to af fect the observed yield.

\Vhen studied in greater detail, ~le variation in the propertie8 of unswollen cellulose with increased time of oxida tion in a solution which was 2 N in oxalic acid and 0.5 N in

dichromate, gave the results shown in Figure 4. The carboxyl and aldehyde contents (plots land 2) attained maximum values

in approximately 30 minutes, and thereafter remained relatively constant over a two-hour periode The total carbonyl content, determined by the cyanide method (plot 4), showed a distinct maximum for unswollen linters after about one hour of oxidation. The alternative estimation, in which total carbonyl plus carboxyl was determined by hydroxylamine hydrochloride (plot 5) and car boxyl groups subtracted (plot 3) showed a similar maximum, al though the two plots did not coincide. The significance of this difference is discussed later. The ketone content (plot 7), found by subtraction of aldehyde (plot 2) from the total car bonyl (plot 4), also showed a maximum near 60 minutes, so that the rise in total carbonyl groups was attributed to the oxida tion of secondary hydroxyl groups. A comparison of plots 6 and 4, Figure 4, showed that the maximum carbonyl content occurred somewhat earlier in the oxidation of swollen linters, but a de cline in reducing power, similar to that observed in the case of unswollen cellulose, took place after 60 minutes of oxidation. It was decided, therefore, to retain one hour as the time lOOI i 1 Iii 1- Cl ~O -Z ~ 8T oJ' w Cf) 0 • , u ,~ ::> , ...J 6er <..9 / / 0 O~ a:: W 40~ Cl. / • • .~ , //, Cf) w -- ...J ~------~----- A..:5- 0 ... - - -_ A ~ --0-- 1 ...J 20 • . ---- oc _ .- ...J ------0 -7 ~

30 60 90 120 TIME (MINUTES) FIGURE 4. Changes in the properties of uns~~llencellulose with increased time of oxidation. Flot 1. Carboxyl content Plot 4. Carbonyl by cyarride method 2. Aldehyde content 5. Total carbonyl plus carboxyl 3. Carbonyl by hydroxylamine method 6. Carbonyl by cyanide method ~ (plot 5 minus plot 1) (swollen cellulose) • 7. Ketone content (cyanide carbonyl minus aldehyde content) 50.

of oxidation of swollen linters. The results of the present investigation were in agreement with the observations of David son (36) and Gladding and Purves (50) who also noted a carbonyl maximum in other chromic acid oxidations of cellulose.

Observations on the Estimation of Carbonyl Groups'

As previously pointed out, a great deal of research has been devoted ta the condensation of cyanide with reducing sugars (65)(66)(67)(68)(69)(70). Coombs (66) found optimum con ditions for the reaction and was able ta determine glucose and fructose quantitatively on a semi-micro scale. However, attempts ta estimate the number-average degree of polymerization of hy drocellulose by this method gave very high results. Frampton and co-workers (70) developed a method which was suitable for the estimation of aldoses but ketoses gave high results. They also applied the method to hydrocelluloses, but observed cyano hydrin number-averages which were greater than, instead of being less than, the viscosity weight-averages. Since a reliable means of determining the total carbonyl content of oxycelluloses was required in the present research, it seemed necessary to confirm and extend the work of Coombs and Frampton on aldoses and ketoses.

The results with glucose are shown in Table VI. Met h od A was similar to that employed by Frampton and colleagues (70), whereby the sample was treated with cyanide, acidified at the appropriate time to expel unreacted hydrogen cyanide and the arr@onia was determined by steam distillation after the addi- TABLE VI

ESTIMATION OF GLUCOSE BY THE CYANOHYDRIN REACTION

NH3 Recovered Millimoles Temp. Mil1imoles % Method(a) Glucose Hours E! (OC.) Corr.(b) Theory

With 0.039 M Cyanide Solution

B 0.555 72 8 26 0.438 79 0.555 72 8 26 0.431 78

B 0.764 42 8 49 0.746 98

A 0.747 68 8 49 0.732 98

B 0.533 68 8 49 0.518 97

With 0.30 !VI cyanide Solution

A 0.670 24 8 30 0.683 102 0.839 24 8 30 0.855 99

A 0.883 96 8 30 0.899 99

With 0.39 M Cyanide Solution

B 0.587 66 10 26 0.587 100 0.642 66 10 26 0.628 98

(a) Method A. Removal of excess hydrogen cyanide prior to distillation of ammonia. Method B. No p r i or re moval of excess hydrogen cyanide.

(b) Method A. Corrected by simple subtraction of blank. Method B. Blank proportioned to unused cyanide by the formula given in the Experimental Portion (P. 76 ). tion of an excess of 20 per cent sodium hydroxide solution. Since, however, compressed air was found to cause the results to be inconsistant, purified nitrogen was bubbled through the acidified reaction mixture in order to expel unreacted hydrogen cyanide. A rigidly controlled direct distillation was used in place of steam distillation. Method B was similar to that of Coombs (66), but various concentrations of cyanide were used. Both methods gave close to quantitative recoveries of ammonia when the samples were immersed in 25 ml. of 0.039 M cyanide solution at pH 8 for 68 hours at a temperature of 49°C.

The condensation of glucose took place very slowly under tile above conditions when the temperature was 26°C., as indicated by the low yields of ammonia even after a reaction time of 72 hours. However, at the higher concentrations, quantitative re coveries were realized at temperatures of 26°C. and 30°C. The times of reaction at the higher concentrations were excessive for glucose, but were employed in order to obtain a more accu rate comparison between the cyanohydrin reaction with an aldose sugar and that with oxycellulose (see below). The evidence in Table VI showed that neither the concentration nor a1kalinity of the aqueous cyanide solution was critical provided that suf ficient time was al1owed.

The results with fructose (Table VII) confirmed the fact that this, and presumably other keto sugars, gave erroneous ly high recoveries of ammonia at pH 8 and 10 with 0.30 M potassium cyanide solution. At a temperature of 29°C. and an alkalinity of pH 8, the yield remained approximately constant at 133 per 53. per cent between 70 and 96 hours. Yundt (67), who employed 0.11 N potassium cyanide solution at pH 8.3 for the estimation of fructose by noting the amount of cyanide consumed, rather than the ammonia formed, also obtained results high by 25 per cent. In their studies of fructose and sorbose, Frampton and co-workers (70) observed recoveries of ammonia which were 110 to 196 per cent of theory, depending on the time allowed for the condensation. They used a 0.30 M solution of potassium cyanide and a reaction temperature of 39°C. The anoma1ous behaviour of fructose, however, could be avoided by increasing the a1kalinity of the 0.30 M cyanide solution and decreasing the time of reaction. Both methods for the estimation of am monia gave similar results at pH 10, the yields being approxi mately 107 per cent of theory. When unbuffered 0.30 M soll~tions of potassium cyanide at pH 11.2 were employed, close to quanti tative yields were obtained even at times which were excessive for the condensation. The reaction proceeded normally within the range pH 8 to 10 with 0.03 M cyanide, the results being in agreement with those of Coombs (66), who emp10yed the sarne con centration. Militzer (68)(69) also found that fructose re acted normally and completely with 0.1 M cyanide at pH 9, no differences being observed between aldoses and ketoses. It was thus apparent that fructose reacted simi1arly to a1dose sugars in solutions of low cyanide concentration over the range pH 8 to Il, but behaved in an anomalous manner at high concen trations of cyanide, the deviation decreasing to zero with in creasing alkalinity. No explanation of the curious behaviour 54.

TABLE VII

ESTIMATION OF FRUCTOSE BY THE CYANOHYDRIN REACTION

Condensation at 29°C.

:r-m 3 Recovered Mi11imo1es Mi11imo1es % Method( a) Fructose Hours ~ Corr. (b) Theory

With 0.30 M Cyanide Solution

A 1.019 70 8 1.449 142 0.601 70 8 0.823 137

B 0.979 70 8 1.301 133 0.688 70 8 0.903 131

B 1.065 96 8 1.431 134 0.679 96 8 0.905 133

A 0.926 42 10 1.011 107 0.853 42 10 0.915 109

B 0.934 44 10 0.998 107 0.712 44 10 0.751 106

A 1.023 75 11.2 1.018 99 0.677 75 11.2 0.694 103

With 0.03 M cyanide Solution

B 0.561 96 8 0.573 102 0.474 96 8 0.488 103

B 0.592 96 10 0.571 97 . 0.431 96 10 0.429 100

(a) Method A. Remova1 of excess hydrogen cyanide prior to distillation of ammonia. Method B. No prior re mova1 of excess hydrogen.

(b) Me t h od A. Corrected by simple subtraction of B1ank. Method B. B1ank proportioned to unused cyanide by the formula given in the Experimental Portion (p. 76). 55.

of tbe keto sugars appears to be available at the present time.

The results of the cyanohydrin determination on an

oxycellulose, which was ~ r ep a r e d by a modified version of pro

cedure VII, are sho~m in Table VIII. The samples were weigh8d

on 3. hydrostatic balance and left in the moist state for tests.

Assuming that the carbonyl content of 35 millimoles

per glucose unit, found by the hydroxylamine method from the

calcium salts was correct, 0.03 NI cyanide solution at pH 8 con

densed incompletely even after long reaction periods at an

elevated temperature. The reaction was also incomplete at pH 8

and 30°0. when the concentration of cyanLde wa s increased ten

fold. However, the employment of the latter conditions at pH

10 gave an average carbonyl content, between 22 and 118 hours,

of 35 millimoles per glucose unit, in good agreement with the hydroxylamine method. One higher result at 118 hours was ex

cluded from this average. The high results observed at 140 hours might have been due to a further addition of cyanide

similar to that which, for unknown reasons, caused high values

in the condensation between fructose and cyanide at pH 8 and 10.

The behaviour of oxycellu10se at pH Il.2 was not investigated

bec~use the substance was known to be sensitive to, and readily

degraded by, alkali (7)(23)(52). In order to limit degradation

caused by long exposure to alkaline media, tlle best conditions for the condensation were considered to be 30°C., 0.30 NI cyanide 56.

TABLE VIII

ESTI1~TION OF THE CARBONYL CONTENT OF OXYCELLULOSE(a) BY THE CYANOHYDRIN REACTION

Temp. Millimoles carbonyl Hours I?1i ~~ per Glucose Unit

Potassium Cyanide 0.03 M(b) Method B

84 8 49 13 15 108(C) 8 49 23 l32(c) 8 49 21

Potassium Cyanide 0.30 M Method A

22 8 30 8 48 8 30 13

22 10 30 35 37 48 10 30 30 35 95 10 30 39 118 10 30 37 45 140 10 30 44 50

(a) Samp16 oxidized in 0.2 N oxalic acid, which was 0.1 N in dichromate, for 75 minutes.

(b) A vol ume of 100 ml. per gram of cellulose was used, (c) 48 hours at 26°C., and remainder of the time of reaction at 49°C. 57.

near pH 10, and a time between 22 and 48 hours.

During the investigation it was noted that the pres ence of umnodified cellulose tended to diminish the hydrolysis of the cyanioe. Since the oxycellulose consisted for the Most part of unmodified cellulose, a more suitable blank would con tain sV'follen or unswollen linters, depending on the nature of the product under investigation. For this reason, one or more blanks conta:lning cellulose, of app r-oxuna t eLy the same weight as the samples, were employed with every set of oxycellulose samnlef'1 analyzed. A comparison between reagent and cellulose blanks, each pair being from the same freshly p r-epa r-e d solution of potassium cyanide (Table IX), indicated in aIl csses that the latter blanks yielded le38 arnmonia on distillation than did the former. While the differences were small, the use of re agent blanks would nevertheless decrease appreciably the ob served number of carbonyl groups in very lightly oxidized cel luloses or in hydrocelluloses. In consequence, the number average degree of polymerization of hydrocelluloses found by the cyanohydrin method would be too hi~h, as was probably the case in the determinations made by Coombs (66) and by Frampton and co-workers (70).

Previous attempts to compare the cyanohydrin and hy droxylamine methods of determining carbonyl groups in chromic acid oxycelluloses and oxystarches (5)(6), were hampered be cause the high content of chromium ash interfered with the lat ter Methode McKilllcan and Purves (71), as previously mentioned, 58.

TABLE IX

RECOVERY OF AMMONIA FROM AGUEOUS.- CYANIDE SOLUTIONS AND FROM THE CORRESPONDTNG UNMODIFIED CELLULOSE BLANKS I\Tfl3 Recovered (Millimoles) Cyandde 'l'emp. Reagent Cellulose concn, Method (-c. ) Hours Iili. Blank Blank

0.039 B 49 84(a) 8 0.036 0.030 0.30 A 29 73 8 0.082 0.069 0.30 A 29 43 10 0.090 0.056 0.39 B 26 64 10 0.388 0.363 0.39 B 42 80(b) 10 0.608 0.571

(a) 48 hours at 26 -c. and 84 hours at 49°C. (b) 64 hours at 26°C. and 16 hours at 42°C.

found that both methods gave similar results with a hypochlorite oxystarch in which chromium ash was absent. Since the most highly oxidized product used in this work, oxycellulose V-4, had an ash content of only 0.17 per cent, after de-ashing in 0.4 N oxalic acid, this interferenee with the hydroxylamine estimation would be negligible. The agreement between the two methods (Table X) was very good when the determinations were done on wet samples of oxycellulose. With samples whieh had been previously dried through alcohol and benzene, and then over phosphorus pentoxide, the hydroxylamine method gave much lower results, although mueh better reprodueibility was attained with the dried samples. The latter consisteney would be expected, sinee 59. the weight of drled samples could be more accurately determined.

A comparison of the apparent carbonyl content of wet and dried samples of a 10-g. batch of oxycellulose V-3 (5 g. being dried) showed that the difference was about 10 millimoles per glucose unit for the cyanide estimation, while the hydroxylamine determination gave about 40 millimoles per glucose unit less for the dried sample. The decrè A.s e in the carbonyl content for the latter method corresponded closely ta the ketone content of this particular produ ct (42 millimoles p er glucose unit), but this result was p er-hap s f'or-bul t ous , However, failure of the hy droxylamine method with alcohol-benzene dried samples was of ir regular occurrence, because the discrepancy with the cyanide method wa s only slight for product V-4, which was prepared by the oxidation of a smaller sample (2 g.) of swollen linters. Since the great difference between the two methods of estimation was repeated Witll oxy cel l ulos e V-5, prepared by the oxidation of a 30-g. batch of swollen linters, of which 4 g. were dried through alcohol and benzene, the variable results were most probably due to variations in the accessibility of the oxycellu loses produced by variations i n the amounts dried at one time. Any such changes interfered to a very slight extent, if at aIl, with ~le cyanohydrin reaction, which was therefore a more re liable method of estimating carbonyl gr oup s. The results shown in Figure 4 (plots 3 and 4) for alcohol-benzene dried products V-l and V-2 and also the oxycelluloses prepared by the similar oxidation of unswollen linters for 30 and 90 minutes, also sup ported this conclusion, as the carbonyl contents measured by 60.

TABLE X

COMPARISON OF CARBONYL DETERMINATIONS ON OXYCELLULOSE BY THE CYANIDE AND HYDROXYLAMINE METHODS CarbonylContent (mi11imoles per glucose unit)

Oxycellulose CN Method NH20H·HCl Method

With wet samples

l 24 24 22 V-3 93 91 86 86

With alcohol-benzene dried sanroles V-3 80 49 79 49 V-4 65 58 63 57 V-5(a) 89 65 90 69

(a) Samples from large scale oxidation of cellulose. Time of oxidation 60 minutes. (See Experlmental Portion p. 96 ).

the cyanide method were always greater than those given by the hydroxylamine estimation. 61.

Application of the Kiliani Reactions to Oxycelluloses

The foregoing work indicated that oxycellulose V-3, from swollen linters (Table IV) was the most suitable for cyano- dration, because approximately 50 per cent of the high carbonyl content was apparent ketone. The high ketone content was required in order to isolate derivatives of the corresponding keto-glucose units in sufficient yield for identification.

Larger-scale cyanohydrations were carried out on an oxycellulose V-5, prepared from four 30 g. batches of moist, highly swollen linters as described for sample V-3. A repre- sentative portion of approximately one gram were taken from each batch, thoroughly mixed in water, and then dried through alcohol and benzene for analyses. The carbonyl and carboxyl contents as determined on these dried samples, were 89 and 44 millimoles per glucose unit by the cyanohydrin and calcium acetate methods respectively, the ketone content being 43 millimoles. The carbonyl content based on the undried oxycellulose would then be 99 millimoles per glucose unit (see Table X). A portion of the dried product was nitrated in a fuming nitric acid - phosphorus pentoxide mixture which was not considered to cause further degradation. The average degree of polymerization found from the intrinsic viscosity of the nitrate in acetone solutions, appeared to be 271 although this estimate assumed that the relationship between D.P. and intrinsic viscosity was not affected by the carbonyl and carboxyl groups present. If the original cellulose chain had been of infinite 62.

length, instead of having the D.P. of 1675 observed, Kuhnts formula (89) showed that the fraction of the original glycosidic bonds c1eaved during the oxidation was 2 or less 271 + l' than one per cent. Rough as this ca1culation was, it showed that the contribution of any reducing end groups, formed by degradation during oxidation, to the total carbony1 content was small. The nitrating mixture used gave a nitrate substitution of 2.91 with the original cellulose, and of 2.72 with the oxycellulose. The difference of 0.19 corresponded reason- ably weIl with the total carbonyl plus carboxyl substitution of 0.14 moles per glucose unit (0.099 + 0.043) f ound for the oxycellulose. As noted in similar cases (7) the nitrated oxycellulose was difficult to dissolve in acetone, and the speci- fic viscosities of 0.25 and 0.125 per cent solutions (0.893 and 0.387 respectively) seemed abnormally high. These anoma- lies might indicate some cross-linking caused by acetal-formation or trans-esterification in the product.

A portion of the dried oxycellulose was methylated for 24 hours in dioxane saturated with diazomethane and was recovered with the total carboxyl plus carbony1 content de creased from III to 62 millimoles per glucose unit as measured by the hydroxylamine hydrochloride method. The methylated product still showed strong reducing power comparable to that of the original oxycellulose when tested with Schiff's fuchsin sulphurous acid reagent. This observation, together with the fact that the decrease of 49 millimoles per glucose unit nearly corresponded to the 44 millimoles of carboxyl groups ori- 63.

ginally present, suggested that only the carboxyl groups had been methyla.be d,

The conditions established for the estimation of carbonyl groups by the cyanohydrin mebho d, employing an ex cess of 0.3 M pot a s s i um cyanide a.t 25°C. and pH 8 or la, were used in larger-sca.le preparations. In general, the cyanohy drins were saponified and the ammonia was expelled by concen tration of the alkaline solution, and further concentration, after acidification with oxalic acid, removed the excess hy drogen cyanide. The insoluble product was recovered by fil tration and was analysed for carboxyl gr oup s . If the yield by weight of insoluble product was quantitative, the number of carboxyl groups it contained would equal the sum of the carbonyl and carboxyl groups in the original oxycellulose (Table XI, column 2). Columns 4 and 5, however, show that the actual yields ranged from 61 to 85 per cent, and the car boxyl contents were nearly zero, in the preliminary experi ments with oxycelluloses VII, and V-2. Since the carbonyl groups in these lightly oxidized samples were aldehydes (Table V) it appeared that 6-aldehyde glucose units were very readily cleaved from the cellulose macromolecules to give soluble pr-o duct s ,

The fact that a greater 10S8 of product occurred when the cyanohydration took place at pH 8, instead of pH la, strongly indicated that alkali scission of the oxidized glu cose units tended to precede cyanohydration at the lower pH. TABLE XI

BEHAVIOUR OF OXYCELLULOSES(a) DURING CYANOHYDRATION AND SAPONIFICATION

Original pH % Carboxyl % Carboxyl Oxy CO + COOH of Cyanide Yield Content of Water-Soluble Cellulose Content (b) Solution of Product Product(C) Portion

VII(d) 40 10 75.0 Nil 100 73.4 Nil VII(d) 40 8 65.5 1 96 61.5 2 V-2(d) 66 10 84.4 2 97 85.2 2

V-3{d) 106 10 79.4 •• • •• • V_5(e) 143 10 85.7 47 68 46 (a) With 100 ml. of 0.3 M potassium cyanide per gm. of oxycellulose. (b) Mi11imoles per glucose unit; sum of carbonyl by cyanide method and carboxyl by calcium acetate method. (c) Mi l l i.mol e s per glucose uni tj calcium acetate method. (d) Saponification at 100°C. and atmospheric pressure. ( e ) saponification at 70°C. and reduced pressure.

(J) .;:. • 65.

It was only with the heavily oxidlzed sampleV-5, which contained ketone groups and in which many aldehyde groups had presumably been further oxidized to uronic acid units, that the insoluble product retained much of the expected acidity. The reducing power of the product, also as expected, was very small. The calcium acetate method assessed the residual acidity a t 46 millimoles per glucose.. unit , or 32 per cent of the theoretical amount, but the estimation with hydroxylamine hydrochloride was only 18 millimoles, perhaps because of inter ference by traces of potassium oxalate. Since the yield (86 per cent) or 100 g. was as high as those from more lightly oxidized samples, the larger amount of oxidant used in the preparation of V-5 had been expended in a more drastic oxidation of substantially the sarne accessible regions to soluble sub stances. Attention was focused on the 16 g. (14 per cent) o~ soluble products, which contained two thirds of the acidic groups in the adduct from V-5.

The water-soluble products were recovered as a dark brown syrup, and were combined with the residue from the water- alcohol-benzene washings of the largely unmodified, insoluble material. The whole was then reduced at reflux temperature for four hours with constant-boiling hydriodic acid and red phosphorus. Although most of the oxalic acid added to expel unreacted hydrogen cyanide from the cyanohydrin mixture, had been removed as sparingly soluble potassium hydrogen oxalate, sorne was present in the reduction with hydrogen iodide. Con- 66. trol experiments showed that the ratio of evolution of carbon dioxide from oxalic acid in hot hydriodic acid was still ap preciab1e even after three to four hours in the absence of red phosphorus. However, when 0.03 moles of oxalic acid and 0.06 moles of hydriodic acid were heated under reflux in the pres ence of 0.25 g. of red phosphorus, only a trace of carbon di oxide was noticeable after three and one-half hours. These resu1ts indicated that little, if any, oxa1ic acid would be

1eft after the reduction of the oxycel1ulose residues with the great excess of hydriodic acid and red phosphorus used. Af- ter hydrogenation in alkali with Raney nickel - aluminium powder to remove combined iodine, a pale yellow liquid was removed from a solution of the de-iodinated product by continuous ex traction with ether.

The yellow liquid from the extraction was taken up in barium hydroxide solution and re-extraction of this solution with ether yielded no neutral oils. None were espected, be cause any unmodified glucose ~~its should have yielded N-hexane, which would have vo1atilized during the evaporation of the ether. Perlin and Purves (5) isolated only a minute amount of neutral substances in a para11el reduction of unmodified lin ters, and suggested that these substances originated in impuri t Le s ,

The barium salts recovered from the aqueous layer of the above extraction were crystallized from aqueous acetone in two fractions and then recrystallized from water. The barium 67.

contents of the dried fractions were simiIar, and the carbon and hydrogen analyses on the first fraction suggested that the product was a mixture of the barium salts of hydroxy fatty acids of low molecular weight. After isolation by standard methods, the free acids (approximately 3 g. in weight) distilled over a wide range of temperatures at a pressure of 4 to 5 mm. Neither the original extract nor fractions of the distillate collected at various temperatures formed crystalline hydra zides of heptanoic or hydroxy-heptanoic acids such as were isolated from oxystarches by Purves and co-workers (6)(71).

The yields of the hi~her boiling fractions were also too small to permit of the removal of any impurities which might have prevented the crystallization.

Combined portions of fractions l and IV and a small amount of the trap contents obtained from the distillation were separated on paper chromatograms according to tile ascending method of Barker and Kennedy (79) in which the deve10ping li quid was ethanol, water, and concentrated ammonium hydroxide (volume ratio 95:5:1). The descending method was also used with ethanol or methanol, water and ~mnonium hydroxide as sol vents. parallel chromatograms were prepared of several simple acids in arder to have standard chromatograms for comparison. The separation of the combined fractions Figure 5 and Table XII indicated a mixture of acids. The spot of 1east mobility at the starting line was probably one or more dicarboxylic acids, since succinic acid by itself gave the sarne type of spot . . ------~------,

68.-

~ 5 ' 5 CM. SOLVENT FRONT

- - 14·0

t- - 12·0

,-\ , 1 ...,:, 1~1 t- '...... J - 10·0 ,., :..-:. \J e-- r: - 8 ·0 l ', 0 '...,:'' 1 l,. , 1 - .., 0 - a' 1 .. ' " 0 - ~ 0 - 4 ·0 ." f-- 1 . 1 - 2 '0 f""l\ 1 t-: 1 1IL. 1

nn o D G HA MHA CF S V B l A r 2·5 CM . l

FIGURE 5. Paper Chrornat ogrcm (one-half act.ua.L si ze) of simple fatt y acids by as cending rnet .hod

G Gl1l.te.r i c aci d T Trace HA 4- hyrl.roxyheptanoic acid V n-Valeric acId ~œA 2-methyl - 4-hyd roxyhexanoic aci d B n- Butyri.c acid CF Combined fracti on from oxyce llulose P n- Pro pi oni c acid S Succi ni c acid A Acet ic acid FT Faint trace 69.

'J.1ABLE XII

RF VALUES OF ACIDS BY THE ASCENDING ~ŒTHOD (See Figure 5)

Distance Trave11ed RF Acid as Ammonium Salt {cm. ) Value

G1utaric acid 3.7 0.12 4-Hydroxyheptanoic acid 16.5 0.53 22.8 0.74 2-Methyl-4-hydroxyhexanoic Acid 16.1 0.52 Combined fractions from oxyce11u1ose 1.8 0.06 4.8 0.15 10.1 0.33 15.6 0.50 19.2 0.62 Suecinic Acid 1.7 0.06 n-Va1erie Acid 17.7 0.57 n-Bubyr-tc Acid 15.7 0.51 n-Propionic Acid 11.8 0.38 Acetic Acid 9.4 0.30

Solvent Front 31.0 • • •• with a similar RF value, while that from glutaric acid was somewhat more extended. The second spot of the combined frac tions was very faint, but had a mobility close to that of gl u tarie aeid. However, the possibility of a three or four car bon hydroxy acid was not excluded, sinee a hydroxyl substitu ent deereased the RF value of an acid. Brown and Hall (76), for example, observed that propionie and lactic acids had RF values of 0.19 and 0.07 respectively, when butanol, saturated with 1.5 N ammonium hydroxide, was used as the developing solvent. The third and fourth spots from the combined frac tions were assumed to be a mixture of normal aeids from Cl to C4 with the possibility of 4-hydroxyheptanoic and 2-metllyl-4 hydroxyl1exanoic acids being present. The fifth spot had an RF value whieh was slightly gr ea t er than that for valerie aeid and was, perhaps, hexanoic acid. However, no similar spot ap peared when the deseending method was used.

The RF values for the acids by the descendine method, employing the same solvent, but a different chromatographie tank and smaller papers, were not much altered from those by the ascending method. The results for the descending method (Table XIII) were obtained by developing the two papers at the same time and under similar conditions.

When methanol was used instead of ethanol as the de veloping solvent, resolution of the aliphatic acids was not satisfactory, as ~le RF values for the C3 to C5 acids (Table XIV) 71.

TABL }:; XIII

Rp. V !'.1UI~S OF ACIDS BY THE DESCENDD!G ME'YrIOD Ethanol Water Amm on i um Hydroxide 95 : 5 1 (b y volume )

Distance Travelled RF Acid a s Ammonlum Sa lt --.lerll . ) Va lue p.eeti.c Ac i d ( a ) 9. 4 0 . 2 7

Succ i n i c Ac ie] 1 . 5 0. 0 (:

Bu tyrie Aeid(a ) 17.2 0.50

Pr opionie Aci d ( a ) 12.4 0 . 3 6

C- l uta r i c Ac i d 5.0 0.14

4-Eydroxyhen tanoic fi e l d 16.8 0.49

2 - M8 thyl -4-hydroxyhexanoic Acid 16.5 0.48

Combined F ractions f r om o xy eellulo se 1.5 0.04 4.7 0.14 9.4 0 . 2 7 16.5 0.48

( a) No. 2 Ch r oma togram. Solvent Front 3 4 . 6 cm. Others on No. l Chr oma t ogr am. Solvant Front 34. 6 cm. TABLE XIV

RF VALUES OF ACIDS BY THE DESCENDING METHOD

Methanol : Water Ammonium Hydroxide 95 5 l (by volume)

Distance Travelled RF Acid as Ammonium Salt ( cm. ) Value

Succinic Acid(a) 11.0 0.33 Combined Fractions(a) 12.0 0.34 18.5 0.53 to to 25.0 0.73(b) Acetic Acid 21.8 0.63 Propionic Acid 24.0 0.70 Butyric Acid 25.0 0.73 Valerie Acid 25.7 0.74

(a) Chromatogram No. 1. Solvent Front 34.8 cm. Others on Ghromatogram No. 2. Solvent Front 34.6 cm. (b) Continuous spot with limits shown. were not very different. The first spot of the combined frac tions from the oxycellulose reduction had an increased mobility which was closely paralleled by succinic acid. No distinct separation occurred with the remainder of the combined fractions, which produced a streak.

The results from the barium, carbon and hydrogen analyses, from the fractional distillation of the acids from the barium salts, and from the chromatographie experiments, aIl in

dicated that the products from ~le Kiliani reduction of cyano hydrated oxycellulose were a mixture of low molecular weight fatty acids and their hydroxy derivatives. The fact that poly carboxylic acids, such as succinic and glutaric acids, might also be present suggested that in the original oxycellulose there were glucose units which had been oxidized in more than one position and had suffered cleavage of their carbon chains. The great decrease in the total carboxyl contents of the oxy celluloses after cyanohydration, together with a relatively small loss in weight, also indicated that the oxidative attack was strongly localized. The oxidation of cellulose with oxalic acid and potassium dichromate in aqueous solution, even although the system swelled cellulose, was similar to that with chromium trioxide in a non-swelling acetic acid - acetic anhydride solu tion (5), the localized attack being restricted to 15 to 35 per cent of the swollen or unswollen cotton linters. The prod ucts, with their great alkali-sensitivity, were consequently unsuitable for structural studies by the Kiliani series of re-

actions. The fact that similar studies on chromic acid oxy- 74.

starches met with success (6)(71), was perhaps due ta the much greater dispersion of starch in aqueous solutions, resulting in a greater dispersal of oxidant through the starch. It ap pears therefore, that the clarification of the intensely 10 calized and complicated oxidations of cellulose with chromic acid will require new methods employing a neutral or acid, rather than an alkaline, medium. EXPERIMENTAL SECTION

Determination of Reducing Sugars by Condensation with Cyanide

Method A.

The glucose or fructose, 0.1 ta 0.2 g., was placed in a 125 ml. glass-stoppered Erlenmeyer flask, and 25 ml. of freshly prepared potassium cyanide solution of the appropriate concentration and buffered to the required pH, was added. Af- ter standing at a selected temperature for the desired length of time, the contents were washed into a 300 ml. standard taper, round-bottomed distilling flask, and were acidified with 7 ml. of 6 N hydrochloric acid in excess of the amount required to obtain neutrality toward methyl red. The flask was then connected to a bubbler system and nitrogen was passed through the contents for 30 minutes, or until no trace of hydro- cyanic acid cauld be detected. The solution was made more alkaline than pH Il.5 with 10 ml. of 20 per cent sodium hydroxide solution, the volume was made up to 100 ml. by addition of wa ter, and the flask was quickly attached to a one-piece still- head and condenser the outlet of which was in~ersed in 20 ml. of standard hydrochloric acid. The liquid was distilled at a rate which was strictly controlled by an electrical heating mantle operating at constant voltage. After exactly 25 minutes, when approximately 60 ml. of distillate had been collected, the contents of the receiver were titrated with standard alkali to 76.

a methyl purple end-point.

A reagent blank containing no sugar was similarly carried out, and the carbonyl content of the sample was obtained by subtraction of the blank from the observed amount of ammonia.

Method B. The glucose or fructose, 0.1 to 0.2 g., contained in a 125 ml. glass-stoppered Erlenmeyer flask, was dissolved in 25 ml. of potassium cyanide solution of the required pH and concentration. At the appropriate time the contents were transferred to a 300 ml. standard taper, round-bottomed dis tilling flask, and sufficient l N sodium hydroxide solution

(2 ml.) to adjust the aLka'It.nd ty to pH lL7 was a dded, Dis- tillation and titration of the distillate were similar to Method A.

A reagent blank was included with every set of sam pIes. If x represented the carbonyl content of the sample, then

y w(y z) x - z(w - x) or x - = w = (w - z )

where y = the ammonia recovered in the estimation, z = the ammonLa recovered in the blank, w = the original cyanide present.

By the use of tbese equations the correction for the blank was made to correspond to the ammonia evolved from the portion of 77. cyanide unused in the actual estimation.

It was necessary to use the highest grade of potas sium cyanide obtainable (97 per cent pure) for t his determination. Early work had shown that colorless aqueous solutions of sodium cyanide (95 per cent pure) and potassium cyanide (96 per cent pure) changed to a dark brown color when left at room temperature overnight. At longer times (48 to 96 hours) filterable material and dark oils were pr e s en t . The nature of these substances was not investigated.

Determination of Carboxyl Groups in Oxycellulose ___ The method of Yackel and Kenyon (26) as modified by Mees ook and Purves (38) was used. High-erade calcium acetate was dissolved in boilfng water in sufficient amount to make a 0.5 N solution. After boiling for several minutes, the solution was cooled to room temperature, filtered, and stored in a glass- stoppered bottle. Oxycellulose san~les, 0.2 to 0.4 g., which had been thorougnly de-ashed, were placed in 125 ml. glass- stoppered Erlenmeyer flasks and 60 ml. of the freshly prepared calcium acetate solution (pH 7.3 to 7.5) was added. Reagent blanks and unmodified cellulose blanks were run with every set of samples. After standing for 24 hours at room temperature, the mixtures were filtered into dry flasks through dry sintered- glass funnels, and 50 ml. aliquots of the filtrates from the blanks and samples were titrated to pH 8.3 with 0.01 N sodium hydroxide solution. A Beckmann pH meter, calibrated before- hand with buffer solutions of known pH, was employed. Since 78.

estimations tended to be low if the pH became more acid than 6.3 during the experiment, the weight of sample was chosen so that the pH never became less than 6.6. The carboxyl content (C) was expressed in millimoles per glucose unit as

60 M C • -go- x N x -w- x 162

where N = normality of alkali, M = ml. of alkali corrected for the cellulose blank,

W = weight of sample in grams. Base molecular weight of cellulose = 162.

Estimation of carbonyl Groups in Oxidized Cellulose

Hydroxylamine Hydrochloride Method In an effort to avoid high and variable reagent blanks caused by impure or contaminated samples, two lots of C.P. hy droxylamine hydrochloride, which did not give erratic results, were kept solely for these determinations.

The method of Gla.dding and Purves (50) was used , and since aqueous solutions of hydroxylamine hydrochloride were unstable after standing at room temperature for several hours, they were employed irr@ediately after preparation. Approximately 15 g. of the reagent was dissolved in 200 ml. of water and buf fered to pH 5 with 0.5 N sodium hydroxide solution (40 to 45 ml.). The volume was then made up to 300 ml., and 60 ml. aliquots were pipetted into 125 ml. dry, glass-stoppered Erlenmeyer flasks 79. containing 0.2 to 0.5 g. either of acidic oxycellulose or the calcium salts from the carboxyl determination. The weight of samples was chosen so that the pH did not become less than 4.5 during the ana l y s i s . After 1.5 hours at room temperature, the samples were recovered on separate dry sintered-glass funnels. Aliquots of the filtrates (50 ml.) were titrated electrometrical- ly to pH 3.2 with standard 0.1 N hydrochloric acid. The car bonyl content (x) was determined in millimoles per glucose unit as

x = (~g x N x : x 162) -c

where N = normality of acid, M = ml. of acid corrected for cellulose blank, W = weight of oxycellulose, C = millimoles of carboxyl per glucose unit found independently.

Tt was assumed that C = 0 when estimations were carried out on the calcium salts from the carb oxyl determination.

Cyanide Met h od The techniques used for glucose and fructose were applied with slight modifications to oxidized celluloses. To avoid transfer of the solid-liquid mixture from one container to another, the oxycellulose samples, 0.25 to 0. 5 g., were placed directly in 300 ml. standard taper round-bottomed dis tilling flasks together with 25 ml. of freshly prepared potassium cyanide solution of the required concentration and alkali- 80.

nity. This modirication of the technique was found to give

more reproducible results with oxycelluloses. At the appro-

priate time the samples were treated according to method A or

B as previously described for the analysis or simple sugars.

In the former method expulsion or unreacted cyanide was af-

fected by acidification with hydroehlorie .a c i d or an equivalent

amount of oxalic acid. After saponification or the oxycellu-

lose cyanohydrin and estimation or ammonia in the distillate as previously described, the 80lid residues were recovered on sin-

tered-glass runnels, steeped in oxalic acid, and then washed with distilled water until the filters gave no precipitatoowith

silver nitrate solution. Th e residues were then immersed for

15 minutes at a time in two la to 15 ml. volumes of anhydrous methanol. Final washing in similar volumes of thiophene-rree benzene was followed by drying i n high vacuum and storing over phosphorus pentoxide. Yields of the dried san~les were r eeorded and their earboxyl content was determined.

Estimation of the Aldehyde Content of Oxidized Cellulose

The me t h o d involved the re-oxidation of the oxycellulose with chlorous aeid, which was prepared by tile addition of

1.6 g. of sodium ehlorite (98 per cent p u r e ) to 29.2 ml. or wa

ter buffered with 13.2 ml. of glacial acetic acid to pH 2.5, as measured by a calibrated Be c kma nn pH meter.

In a typical e xperiment, two samples of an oxycellulose, prepared by the oxidation of unswollen linters i n 2 N oxalic acid and 0.5 N dichromate solution for 30 minutes, and each weighing

0.250 g. (dry weight), were placed in test tubes kept in a con

stant temperature bath at 25°C. Each sample was immersed in

6.6 ml. of the chlorous acid solution, which contained 0.25 g.

of sodium chlorite, and the oxidation was allowed to proceed

for one hour at 25°C. Af t er diluting the mixtures with ice water, the s amples were recovered on sintered-glass filters, washed with water, de-ashed by leaving in 10 ml. of 0.4 N oxalic

acid for 45 minutes, and again washed thorou&hly with distilled water until the filtrates were neutral and gave no precipitate with silver nitrate and baril~ nitrate solutions. After solvent

exchange through alcohol and benzene, the samples were dried lLD der vacuum overnight and stored over phosphorus pentoxide in an

evacuated desiccator. The weights of dried products were 0.248 and 0.246 g ., corresponding to an average recovery of 99 per

cent by weight. The carboxyl contents, determined by the calcium acetate method a s previously described, were 46 millimoles per

glucose unit in ea ch case. Subtraction of the original acid

content of 21 millimoles gave an average aldehyde content of

25 millimoles per glucose unit. The aldehyde groups d etermined in this way were assumed to be in the sixth positions of the glucose units, since no evidence of 2,3-glycol cleavage was ob served by Purves and colleagues (24) in chromic acid oxycelluloses.

The results obtained with a number of oxidized celluloses are shown in Table XV, and the origin al carboxyl contents are in

cluded for comparison. 82.

TABLE XV

RE-OXIDATION OF OXYCELLULOSE WITH CHLOROUS ACID

Carboxyl Content( a) % Oxy- Before After Al dehy de Yield Cellulose HC102 HC102 Content(b) of' Product

VI 10 35 25 99 10 35 VII 16 46 30 100 16 46 V-l 20 46 26 97 20 45 V-2 19 46 27 97 19 46 V-3 26 65 38 99 27 64 V-4 30 63 35 100 28 65

(a) IvIillimoles p er glucose un it; calcirun acetate me bh o d , (b) Mi l l i moles p er gl u cos e unit. 83.

Corper Reducing Power of Oxycelluloses

Three stock solutions required by the Reyes micro method (90) were pr epa r ed. Solution (a) contained 150 g. of apbydrous sodium carbonate and 50 g. of sodimn bicarbonate p er litre; solution (b) was prepared by dissolving sufficient crys talline cupric sulphate in water to make a 10 per cent solution; solution (c) contained 40 g. of ferric sulphate and 100 ml. of concentrated sulphuric acid per litre.

Approximately 0.25 g. of oxycellulose (corrected for mo isture content) was placed in a 17 cm. x 2 cm. reaction tube. A mixture containing 9.5 ml. of solution (a) and 0.5 ml. of solution (b) was heated to boiling and poured over the sample, the container being allowed to drain for 20 seconds. The re action tube was then immersed in a constant-level water bath to the desired depth and the bath temperature was maintained at 100°C. for three hours. The contents of the tube were stirred occasionally in order to distribute the oxycellulose and release gas bubbles. After exactly three hours, the tube was cooled in water, the oxycellulose and associated cuprous oxide were col lected quantitatively on a fine, sintered-glass funnel and were washed with distilled water t hree times. The funnel was then transferred to a clean 125 ml. filtering flask. The small amount of cuprous and cupric oxides deposited on the sides of the re action tube were dissolved in 1.5 ml. of solution (c) which was then poured over tb.e oxycellulose residue. Vfuen the dark colour

immediately caused by the oxidation of cuprous to cupric oxide 84.

had passed away, suction was applied and the solution drawn into the filter flask. Washing oE the reaction tube and irri- gation of the oxycellulose with solution (c) was repeated, and was followed by 4 extractions with 2 ml. volumes of cold distilled water, the oxycellulose being pressed with a flat-ended glass rod after each washing.

The filtrate and washings were then titrated with

0.0483 N potassium permanganate to a colorless end-point. The grams of copper reduced from the cupric ta the cuprous state by 100 g. of dry cellulose was calculated as

100 Capper number C x M x = 'N -B where C = 0.003071 the gr ams of copper equivalent to l.00 ml. of 0.0483 N potassium permanganate, M = ml. of potassium permanganate solution, W = weight of oxycellulose sample in gra.ms, B = blank correction.

\Vhen highly reducing oxycellulose samples were used, it was necessary to increase the amounts of the reagent solutions employed. Reagent blanks were run simultaneously Witil every set of samples.

Table XVI compares the results by the Braidy method, employed by Clibbens and Ridge (34), and the above method for unswollen cellulose oxidized at 25°C. by a solution 0.04 N with respect to potassium dichromate and 0.20 N with respect to 85.

oxalic acid.

TABLE XVI

COMPARISON OF BRAIDY AND REYES COPPER NUNiliERS

Copper Number Time of Oxidation (minutes) Braidy Reyes

0 0.08 0.085 1 0.94 1.46 3 2.08 2.06 5 3.16 2.99

Moisture Content

Samples of de-waxed, air-dried linters, each weigb- ing approximately 1 g. were placed in an electrically heated oven at 105°C. for four hours. After cooling in a desiccator over phosphorus pentoxide for 30 minutes, the samples were then quickly weigbed and the 10ss in weigbt recorded as per cent moisture based on the air-dry weight.

Ash Content

Samples of moisture-free products, 0.05 to 0.10 g. were placed in weighed porcelain crucibles and ignited slowly before an electrical1y heated muffle furnace. The ashing was then completed by heating in the furnace overnight at 750°C.

The crucibles and contents were weighed after cooling for one hour in a desiccator over anhydrous calcium chloride. 86.

Pre-Treatments of Cellulose

Unswollen, high-grade cotton linters, kindly supplied by the Hercules Powder Company, were used in this research. The cotton was very thoroughly dewaxed by continuous extraction witl1 a solvent mixture consisting of one volume of ethanol and two volumes of thiophene-free benzene. After air-drying for ap proximately one week, the dewaxed linters were stored in closed containers in the dark until required for use. Two typical bat ches had moisture contents of 3.23 and 3.70 per cent.

Swollen linters were prepared according to the method of Gladding and Purves (50). ~uantities of air-dry linters from l to 30 g. were placed in an Erlenmeyer flask of suitable volume and were treated with 10 per cent sodium hydroxide solu tion pre-cooled to OOC. The volume of solution was 36 ml. per gram of cellulose. In order to lessen the possibility of degra dation by oxygen of the air, purified nitrogen was bubbled through the mixture for several minutes, and containers were ·then tightly c10sed and 1eft at OoC. for three hours. The swelling solution was then neutralized with 10 per cent acetic acid (previously cooled to OOC.) at such a rate that the temperature of the mix ture did not rise above 5°C. The slightly acidic contents were left for 30 minutes at OoC., and the cellulose collected on a sintered-glass funnel, care being taken to keep the swollen ma terial in a moist state. Further treatment with excess 10 per cent acetic acid was followed by thorough washing with dis tilled water at nearly freezing temperature until the filtrate 87. was no longer acid to methyl purple indicator and was neutral to litmus. The swollen cellulose was then stored in the moist state at 0°0. for use within 48 hours. In some experiments the swollen mass was treated with two volumes of anhydrous methanol for 15 minute periods (50 ml. per gram of cellulose) and then washed in two similar volumes of thiophene-free benzene. After drying under vacuum, the swollen cellulose was stored over phosphorus pentoxide in an evacuated desiccator until required for use.

In a typical r-un , the Los s from two sarnpLes of original dry weight 0.997 and 1.005 g. were 0.014 and 0.020 g. respectivelYl corresponding to an average loss of 1.7 per cent during swelling.

Oxidations with Sulphuric Acid - Dichromate

The ratio of cellulose to solution was 50 ml. per gram in aIl cases, and quantities of linters ranging from 1 to 5 g. were employed. In a typical experiment, 250 ml. of solu tion, which was 0.2 or 0.4 N with respect ta sulphuric acid and of various concentrations, 0.050 to 0.718 N, with respect to potassium dichromate, was placed in a 500 ml. Er1enmeyer f1ask kept in a constant temperature water bath at 20°0. lfuen the temperature of the solution was 20 t 0.2°, 5.00 g. of cellulose was added and evenly distributed by stirring with a glass rode Ohanges in the concentration of the dichromate in the solution were followed by adding potassium iodide to aliquots and ti- trating the liberated iodine with freshly prepared standard so- 88.

d i um t h:tosulphate, Ln p r-e s eri c e of starch a s ârid Lca t or-, Th e con-

sumption of o ~Jge n was calculated a R a t oms p e r gl u c o s e unit

(mol. wt , 1 62) of the cellulo s e wi t h t h e h e Lp of t h e fol l owi n e

equation:

Th e a sh contents of the dr i ed products from Exper i - ments 3 and 4 were 0.84 a n d 0 .86 per cent respect ively, whil e

t h e c opper number .o f' t h e oxidi zed c ellulose wit h t h e highe s t

con sumption of oxy gen p er glucose uni t (Experimen t 4) wa s 2 . 7 6.

Oxidat i on of Oxalic Ac id by Potassium Dichromate

Firth ml. of 0.200 N o xalic a cid , brought to t empera-

t ure in a 100 ml. flask kep t a t 30°0., wa s mixe d wit h 0.2452 g. of p u r e p o t a ss i um di chroma te. At the moment of mixi n g the concentration of dichromate was assumed to b e 0 . 100 e quivalents per litre. The concentration was measured at various times by tltration of 2 ml . aliquots with s t anda r d sodium t hiosulphate s olu tion i n the p resence of po t a s s ium i odide and added s ulph uric a c l d, The acidity was deternün ed by the titra tion of other aliquots with sta ndard s odium h ydroxide s olution, a correction being applied f or tile amount of dich~omate p res en t according to the followine; equation:

• 89.

This determinatlon was made on1y after 20 hours, when the rate of the reaction was sufficient1y slow to a110w an accurate comparison of dichromate and acid concentration at a given time.

In order to fo11ow the corresponding change in pH, the reactants were mixed in the same quantities as mentioned above, the pH of the solution was measured from the moment of mixing by means of a previous1y ca1ibrated Beckmann pH meter. The results are shown in Table XVII.

TABLE XVII

CHANGE IN pH OF AN OXALIC ACID - POTASSlillJ DICHRON~TE SOLUTION WITH TIME OF REACTION(a)

:Minutes ...EL Hours ~ 0 1.20 1.5 1.97 7 1.30 2.5 2.20 15 1.40 3.0 2.22 23 1.50 21.2 2.50 38 l.65 69.2 2.50 68 1.78

(a) Initial concentration: Oxalic aeid, 0.200 N; potassium dichromate, 0.100 N.

The carbon dioxide evolved in a 24-hour period was measured according to McCready, Swenson, and Ma cLay f s estimation (91) of uronic acid. In this case the apparatus was set up with the 100 ml. long-necked, side-armed reaction f1asks 90.

immersed in a constant temperature water bath at 30°C. instead of in the ail bath employed in the uranie acid determination.

In a typical experiment, 50 ml. of 0.200 N oxalic acid was added to each reaction flask, and the apparatus was

swept free of carbon dioxide. After 0.2452 g. of potassium di chromate had been quickly added ta the oxalic acid, carbon dioxide-free air was drawn at a controlled rate through the reaction flasks from tWo-foot towers containing ascarite.

The effluent gases passed into absorption towers con taining 0.2523 N sodium hydroxide. At the appropriate time, the sodium hydroxide was sucked from the absorption towers into Buchner flasks, and the towers were washed with distilled water. After the addition of 10 ml. of a 30 per cent solution of barium chloride solution, to precipitate carbonate, the contents of the flasks were titrated with 0.1035 N hydrochloric acid, phenol pl1thalein being used as indicator. Duplicate blanks, with the reaction flasks containing only oxalic acid, were rune

For six-hour runs, the carbon dioxide was determined by the method of Nanji, Paton, and Ling (92). In this case, carbon dioxide-free air was bubbled through the reacting mixture at a controlled rate and was swept fram the flask via a side-arm. This side-arm terminated in a parous porcelain disc placed at the bottom of a tower containing 100 ml. of 0.1492 N barium hy droxide solution. At the end of the run, when the precipitate of barium carbonate had settled, 10 ml. aliquots of the super natant liquid were titrated with standard hydrochloric acid to 91. a phenolphthalein end-point. The apparatus was checked by em ploying weighted amounts of anhydrous sodium carbonate, and the carbon dioxide recovered was 98.7 and 99.0 per cent of theory.

Oxidation of Cellulose by Potassium Dichromate - Oxalic Acid

Method l Fifty ml. of 0.200 N oxalic acid and 1.00 g. of air- dried cellulose, contained in a 100 ml. flask, were kept in a constant temperature water bath at 30°C.; 0.2452 g. of potassium dichromate was added and the oxidation allowed to proceed for six hours with occasional stirring. The oÀ~cellulose was then recovered in a sintered-glass funnel and washed with water until the filtrate was no longer acidic and gave no precipitate with bariurrl and silver nitrate solutions. After most of the water had been pressed from the product with a flat-ended glass rod, the material was allowed to dry in the air, and finally over phosphorus pentoxide under high vacuum for 24 hours. The yield of oxycellulose from duplicate samples of cellulose (dry weight 0.96 g.) was 0.95 g. in each case or 99 per cent of theory. The ash contents of the samples, after steeping in two 100 ml. volumes of 0.1 N hydrochloric acid for one hour, were 0.36 and 0.35 per cent.

In a similar experiment the evolution of carbon dioxide in the presence of cellulose was determined as described previously for a six-hour rune The oxidized cellulose was recovered, washed thoroughly with water, and the copper number determined

on portions of the air-dried material. The oxidizing solution

was left for approximately 96 hours, at which time the acidity

and concentration of dichromate were determined.

Wben swollen cellulose was oxidized by this method,

the reaction was allowed to proceed for 75 minutes, and analyses

were carried out on moist samples.

Method II

Fifty ml. of 1 N potassil~ dichromate and 1.00 g. of

air-dried cellulose containing 3.70 per cent moisture were mixed

together with a glass rod and were kept at 25°0. in a 250 ml.

flask. Then 50 ml. of a saturated solution of oxalic acid (ap

proximately 2.4 N) was added, and the oxidation permitted to

proceed for two hours, more acid being added as required in

order to keep the pH less than 2. After an additional hour

in the solution, the oxyeellulose was recovered, washecl, and

dried as previously deseribed. The yield of dried material was 0.96 g., or 100 per cent theory.

Method III

One g. of dewaxed linters was added ta 100 ml. of a

saturated solution of oxalic acid, the whole being contained

in a 2-litre Erlenmeyer flask immersed in a constant temgera

ture bath at 25°0. By means of a dropping funnel, 100 ml. of

1 N potas sium dlchromate solution was slowly added over a two hour period, and was followed by 50 ml. of saturated oxalie

acid solution. Finally 0.40 equivalents of finely powdered po- 93.

tassilnn dichromate was slowly added, with stirring, over a

period of six hours, and mor e oxalic acid was introduced as

required in order to maintain a pH less than 2. The oxidized

cellulose was left overnight in the solution, and was then re

covered on a sintered-glass filter, washed and dried. In a

typical experiment the yield of oxidized cellulose wa s 0.93 g,

or 97 peI' cent. The blue-green colour was removed by steeping

the product in 100 ml. of 0.5 N oxalic acid for 48 hours.

Me t h o d IV

The ratio of cellulose to oxidizing solution (1 g. to

80 ml.) was the same as that employed by Clibbens and Ridge (8),

but one tenth of the amounts were used. Samples of uns\Vollen

dewaxed linters, each 0.25 g., (corrected for moisture content),

were placed in 50 ml. test tubes immersed in a water-bath at

25°C. and 10.0 ml. of 0.4 N oxalic acid solution was added to

each tube. The mixtures were stirred to distribute the cellulose

a s uniformly as possible. Then 10.0 ml. volumes of 0.080 N po

t assium dichromate solution \Vere i ntroduced so tllat at the mo ment of mixing the solutions \Vere 0.2 N wi t h respect to o xalie

aeid and 0.040 N with respect to potassium dichromate. At the

end of the o xidation time set for each sample the s olution was

diluted with dis tilled wa t er a t 1 00e., the oxyceLl.ul.os e re

covered on a sintered-glass filter, and washed with five 30 ml.

volumes of distilled water.

A similar e xperiment was carried out with a series

of samnles of swollen linters to show the relationship between 94.

the copper number of swollen and unswollen linters, and the time of oxidation.

Change in Carbonyl and carboxyl Content during Oxidation

Potassium dichromate, 24.5 g. was dissolved in one litre of water at 25°C. and unswollen dewaxed cotton linters of moisture-free weight 10.0 g. was then uniformly distributed throughout the solution by stirring with a glass rode Follow ing the addition of 126 g. of oxalic acid dihydrate the mixture was stirred vigorously every three or four minutes. At each of four 30-minute intervals portions of the oxidized product weighing 2 to 2.5 g. were taken from the solution, recovered on a sintered-glass filter, and washed thoroughly with 500 ml. of distilled water near OoC. until the filtrate gave no precipitate with silver or barium nitrate solutions. After being steeped in three volumes of 0.4 N oxaltc acid (50 ml. per gram oE material) for 45 to 60 minutes, the oxycellulose was again thorougb- ly washed in distilled water until the filtrate was non-acidic and gave no precipitate with barium nitrate. Excess water was then sucked from the oxycellulose, and the remainder was extracted by solvent-exchange through two volumes of anhydrous metilanol (50 ml. per g. of product) and through similar volumes of thiophene-free benzene for 30 minutes. Final drying was carried out under higb vacuwn and the series of products, oxycelluloses V, from unswollen linters, was stored over phosphorus pentoxide in an evacuated desiccator. 95.

The yield from the combined wet portions was 9.9 g. based on the fact that 1.0 g. of unswollen linters contained

2.6 g. of water under similar conditions of treatment.

A similar procedure was employed for the preparation of oxycelluloses V from dried or moist swollen linters in batches ranging from 2 to 10 g. The times of oxidation were 30,

60, 90 and 120 minutes. In some cases the oxidized material was divided into two portions, one being stored in the moist state at ooe., while the other was dried as described above.

A product of a lower degree of oxidation (oxycellu lose VI) was obtained by treatment of dried swollen cellulose with one-fifth the amounts of reagents per g. as was employed for o~Jcelluloses V. In a typical experiment, 2.0 g. of lin ters was oxidized for 120 minutes in 200 ml. of solution con taining 0.98 g. of potassium dichromate and 5.04 g. of oxalic acid dihydrate. The product was recovered as described for oxyceL'Lu'Lo e e s V.

Oxycellulose VII was prepared from dried swollen lin ters by a similar procedure, except that the amounts of oxalic acid and dichromate were 0.02 and 0.01 equivalents, respectively, per g. of cellulose. In a typical experiment 3.00 g. of swollen linters were oxidized for two hours in 300 ml. of solution con taining 1.47 g. of potassium dichromate and 3.78 g. of oxalic acid dihydrate. The yield of dried product was 2.95 g. Moist swollen linters were similarly treated, except that the time of oxidation was 75 minutes. 96.

In a control experiment involving the oxidation of dried swollen linters, 1.90 g., to oxycellulose V-4, the yield of the dried product was 1.93 g., or 101 per cent of theory. The ash content, after de-ashing in 0.4 N oxalic acid, was 0.17 per cent.

Large-Scale Oxidation of Cellulose ~ Oxycellulose V-5)

Unswollen linters, 31.8 g . of moisture content 3.70 was known to yield 30 g. of dry product after swelling in cau.s- tic soda. This amount was swollen in 1080 ml. of 10 per cent sodium hydroxide for three hours at OoC. and was recovered in water at OoC. as previously described. Without being dried, the wet mass, containing approximately 80 per cent water, was oxidized for 60 minutes according to the procedure for oxycelluloses V. The blue green product was washed with 4 litres of distilled water and then steeped in a total of 1500 ml. of 0.4 N oxalic acid for two hours with occasional shaking. Final washing with water, until the filtrate was no longer acidic and gave no precipitation with barium nitrate solution, yielded a pure white p r-oduc t ,

One g. portions from each of four separate preparations were combined and mixed thoroughly by stirring in water. The wet mass Was dried through alcohol and benzene and was used for carbonyl and carboxyl determinations. The carbonyl content, as determined by the cyanide method, was 89 and 90 millimoles per glu- 97. cose unit in duplicate samples; the corresponding values by the hydroxylamine method were 65 and 69 millimoles per glucose unit. The carboxyl content (calcium acetate metb.od) and the aldehyde content (chlorous acid method), as estimated on duplicate samples, were 44 and 44, and 46 and 47 millimoles per glucose unit, re spectively. The apparent ketone content, based on the difference between cyanide carbonyl and the aldehyde content, was therefore 43 millimoles per glucose unit.

Nitration of Oxycellulose V-5 A modification by Segall (93) of Berl's method (94), based upon nitration without degradation, was used. The nitra tion mixture consisted of 78 per cent by weight of fuming nitric acid and 22 per cent of phosphorus pentoxide.

Fuming nitric acid, 39 g. (26 ml.) was placed in a 100 ml. glass-stoppered flask in a constant temperature bath at - 100e. and Il g. of phosphorus pentoxide was slowly added over a period of 20 minutes. The mixture was then stored at ooe. for 48 hours, when the phosphorus pentoxide was completely dissolved. Dried oxycellulose V-5, 0.6 g. was added to the homogeneous medium and the nitration was allowed to proceed for four hours at ooe. The nitrated oxycellulose was recovered on a sintered-glass filter, and as much as possible of the spent acid was pressed out. The product was immersed in excess 50 per cent alcohol at -lOoe., and was washed with 3 volumes of this solution. After boiling for five minutes in 3 changes of 96 per cent ethanol and squeezing out the product after each boil- 98.

ing, the stabilized nitrated oxycellulose was washed with peroxide-free ether, dried under vacuum at room temperature, and stored over phosphorus p en t oxi de in an evacuated desicca- tore

The nitrogen content was determined by a modification of the micro-Kjeldah1 method (95). Since the method gave a hydroxyl substitution of 2.91 per gl ucos e unit with unmodified cellulose, and a nitrogen content of 13.90 p er cent, the theoretical nitrate substitution of the oxycel1ulose was assumed to be 2.91 minus the carboxyl and carbony1 substitutions found byexperiment. The carboxyl and carbony1contents of oxyce11u- lose V-5 were 44 and 99 millimoles per glucose unit respective1y, based on the moist product. Therefore t he t heoretical n i t r a t e substitution was 2.77 per gl u cos e unit, corresponding to a nitrogen content of 13.50 per cent and a mo1ecular weight of 287.0 per basic unit. The nitrogen content observed was 13.33 and 13.37 per cent corresponding to a nitrate substitution of 2.72 and a molecu1ar weight of 284.7. The yield of nitrated oxycellu1ose was therefore 99.2 par cent.

Estimation of the Average Degree of PolYŒerization Acetone solutions of the nitrated oxyce11u1ose were prepared to have concentrations ranging from 0.025 to 0.250 per cent. The specifie viscosities (rj sp.) of these solutions were determined at 25°C. with an ostwald viscometer.

In a typical case, a 0. 0312 per cent solution gave 99.

a time of f10w of 114.5 seconds, based on the average of three

values varying by not more ~lan one-tenth of a second. The average time of f10w for pure acetone was 105.3 seconds. The r elative and specifie viscosities were therefore 1.087 and 0.087 respective1y. Emp10ying the concentration equation,

1 + K 11sp

independent1y arrived at by Huggins (96) and Schu1z and Blaschke (97), and accepting the value of K as 0.31 for cellulose trinitrate in acetone (98), the intrinsic viscosity, [11 J %, was

0.087/0.031 or 2.71 1 + 0.31 x 0.087

1~is value was in close agreement with that determined graphica1- ly (Figure 6). The average degree of polymerization was ca1- culated from the relationship emp 10yed by Mi t chel l (99):

D.P. = 100[11J % = 271.

The average degree of polymerization of the unmodified linters was 1675, the data being kind1y supp1ied by Dr. T.E. Time11.

Methy1ation of Oxy cel l ul ose V-5 with Diazomethane Oxycellulose V-5, 0.7 g., was pl a ce d in 70 ml. of anhydrous 1,4-dioxane, and sufficient water- was added to make the moisture content 0. 25 per cent (55). Af ter degassing the oxy-

cellulose suspension under vacuum, the mixture was saturated Oi i i i 1 o r-- 10 6 o o 0/ 10 l() o

o o o 10 6 o

4°Cl o --1 o o 10 V /0 6 ,~ ,.

o o o ~I 1 1 1 1 o 0 -100 0·200 0-300 CONCENTRATION IN GRAfwlS PER 100fwlL. ACETONE

FIGURE 6. Det.ernti r.at i.on of the intrinsic viscosity of nitrated oxycellulose V-5 in acetone at 25°C.

Log l Tl~p]:: 0.429 [Tl]% = 2.69 8 c-'o • 101.

with diazomethane until the solution had assumed a p er manen t yel Low t i n t . The suspens ion was l eft at room temp erature for

24 hours, after which time the excess diazomethane was removed.

The product was recovered, washed with 100 ml. of anhydrous

1,4-dioxane, and finally with 200 ml. of thiophene-free ben-

zene. Af t er drying in vacuo, the material was stored over phosphorus pentoxide in a n evacuated desiccator.

In order to determine the total carbonyl plus carboxyl content, duplicate samples of the methylated product, each weighing 0.25 g., were treated with an aqueous solution of hydroxylamine hydrochloride, as previously described. The amount of carbonyl plus carboxyl groups was found in each case to be 62 millimoles p er glucose unit, as compared to a value of III millimoles per base unit for the original oxycellulose

V-5. The reducing power of the methylated product was comparable to that of oxycellulose V-5, when tested with Schiff's fuchsin- sulphurous acid reagent.

Preparation and Reduction of the Cyanohydrin of oxycellu1ose V-5

Over a period of 20 minutes, 29 g. of oxycellulose V-5 was a dded to 2850 ml. of 0 . 30 M potassium cyanide solution at pH 10, care being taken that the temperature did not rise above

30°C. After a reaction period of 24 hours, 240 ml. of a solu tion of 1 N potassium hydroxide and 760 ml. of water were added.

The ammonia was expelled by heating the mixture at 65 to 70°C. under reduced pressure until 2000 ml. of distillate had been collected. Purified nitrogen was bubbled through the solution 102.

durin g the distillation in order to decrease the possibility of degradation by alkali in the presence of atmospheric oxygene

After the addition of 2 litres of water to the contents of the distilling flask, the mixture was cooled and acidified to pH l to 2 by oxalic acid. Expulsion of unreacted hydrogen cyan ide was then effected by heating the suspension on a steam bath under reduced pressure for approximately six hours, during which time 2 litres of distillate were removed. After cooling the solution, the cellulose was collected on a sintered-glass filter and washed with 500 ml. of distilled water, the washings being combined with the supernatant liquors. The combined in soluble residues obtained from a total of 116 g. of oxidized cellulose were furtber washed thoroughly with about 2 litres of water, then with 2 volumes of methanol totalling 800 cc., and finally twice steeped in 400 ml. of thiophene-free benzene.

After drying under vacuum over phosphorus pentoxide, the yield of the pale amber, non-fibrous product was 100 g. The dried powdery material, which had an almost negligible reducing power, had an ash content of 0.50 per cent. Portions of the product were Dnmersed in calcium acetate solution and the carboxyl con tent was determined. The total carboxyl and carbonyl content was also estimated by the hydroxylamine hydrochloride method.

The water-alcohol-benzene washings were evaporated to dryness, and 4 g. of a white residue were recovered. The solid, which tested strongly for oxalate ion, was set aside (see below).

The supernatant liquor and first water washing from each of the four preparations were separately concentrated 103.

in vacuo to approximately 400 ml. and then cooled, the white,

crystalline potassium oxalate being recovered on a filter and

washed with 200 ml. of water. Subsequent r eduction in volume

interrupted by the removal of further amounts of oxalate, re

sulted in 50 ml. of a dark brown, syrupy concentrate. The con

centrates from the four experinlents were reduced to dryness

yielding, 47.3 g. of brown, brittle residue which still tested

strongly for the o xalate ion.

This residue, together with that from the alcohol ben

zene washings, was added slowly to a suspension of 330 ml. of

constant-boiling hydrogen iodide and 12 g. of red phosphorus,

an amount ca1cu1ated to be twice that required both for the re

duction of the a1kali-soluble portion of the saponified oxycel

lulose cyanobydrin and for the decomposition of the oxalic ac1d

or potassium oxalates present. After the mixture had been

gently heated in order to dissolve the carbohydrates without

charring, the flask was connected to a condenser having a short distilling coluwn, and with regulated heating a slow distillation was carried out until approximately 40 ml. of distillate had been collected at 126-127°0., the temperature of constant-boiling hydriodic acid. The flask was rapidly transferred to a reflux

condenser and the contents were gen t l y h eated under r eflux for

four hours. After three hours 40 ml. of hydriodic acid were added to replace the amount which had been disti11ed. At the

end of the reflux period the distil1ate, was combined with the

reaction mixture and the whole cooled to room temperature. The products were then continuously extracted with ether for 12 hours 104.

a nd the extract reduced ta a dark brown syrup by evaporation of

the ether at atmospheric p r e ssur e . The residual syrup was hy

drogenated accord Lng to the method of Schwenk and others (73)

by solution in 400 ml. of 2 lIT potassium hydroxide solution and

by the addition of 12 g. of Raney nickel-almninium powder, in

small amolUlts over a 30-minute periode Af ter the foaming had

subsided, the reaction mixture was heated on a steam bath for

15 minutes, using a watch-glass to cover the beaker, and then

heated at 90-95°C. for one hour, occasional stirring being re

quired to prevent the catalyst from "caking". The catalyst

was removed by filtration, washed with 500 ml. of distilled

water, and the pale yellow filtrate was slowly acidified to

pH 1 with 50 per cent sulphuric acid. The i odide ions formed

during the reduction were removed from solution by precipita

tion wi th s I Lver a cetate and hydrogenation was as sume d to be

complete when the ftltrate ga v e a negative result for combined

iodine with the sodium fusion test. Free iodine was absent

as indicated by the starch test. Af t e r a continuous extraction

of the filtrate with ether for twelve h ou r s , the ether was

evaporated at atmospheric pressure, and the concent r-ate , light

yellow in color and at pH 2.5, was made up to a volume of 50

ml. with distilled wa ter. A saturated solution of bar-Lum hy

droxide, 350 ml. was added and the mixture now pH 9, was gently h eated under reflux for on e hour on a s team bath. 1Nhile the

solution was still hot, carbon dioxide gas wa s bubbled bhr-ough,

and the precipitate of barium carbonate was removed by filtra

tion. This p r o c e s s was repeated until the hot filtrate no 105.

longer ga v e a p r e c ip i t a t e with carbon dioxide gas. The f i l trate was cooled and extracted with half its volume of ether and ~)e colorless extract was dried with anhydrous sodium sul-

ph a t e at 5°C. No neutral o i l s were obtained on evaporation of the ether at atmospheric pressure.

The aqueous solution of barium salts, pH 8.0, was heated on a steam bath at r educed pressure and concentrated to a volume of 30 ml. Two v olumes of fr e shly distilled a c e t on e were added to the cooled s o l u t i on and the mixture was left overnight at room temperature. The white p r e c i p i t a te which f ormed was r ecovered, washed with 70 per cent aqueous acetone, and dried over calcium chloride in an evacuated desiccator.

Recrystallization from water yielded 2.56 g. of crystalline solide (Fraction A). The c ombined mother liquor and washings were concentrated a l mo s t to dryness at r educed pressure; nine volumes of acetone were added, and the solution 1eft overnight at room temperature. The resulting precipitate was recrystallized from water, yielding an additional 1.93 g. of crystalline prod uct (Fraction B) •

.Analysis Fraction A. Found , C, 14.4, 14.6; H, 2.94, 3.02;

Ba, 50.6, 50.7 per cent. Fraction B; Ba, 50.7,

51 . 7 p er cent.

Both f r a ct i on s were considered similar and the analysis of r'z-act ton A corresp onde d to the empirical formula C3. 3 H8.2

05.4 Ba , This resul t sugges ted bhat the cr-ys tals were d erived f r om a m:ixture of hydroxy fatty acids of low mol ecular weight. 106.

Th e mixture of bar ittm s alts, together with t h e residues from tile combined mother liquor s and wa shin gs, were d i s - s ol v e d i n wa t e r and a cidi f i ed t o pH l by t he addi t i on of s ul - ph u r i c a c i d . AI' ter a conti n u ous extraction with e ther f or four h o urs , t he e x t r act wa s dried wi th a nhydrous sodium sul- ph a t e o ver n i gh t a t 5°C. Distillation of the e ther at atmo s- ph e r i c pres sure 1eft a pp r oximatel y 3 g . of a pale y el 10w li- quld wbich, when distilled at r e duced pre ssure, ga v e the wide r-ange of' fraction s shown in Ta ble AvIlI. Th e h i ghe r boiling f ractions ( III an d IV) repre s ented approximately 1 0 per cent of t h e e xtract. Attemp t s ta prepare c r y stalline h y drazides, by treatment of ea ch f r a c t i on with hydraz i n e h y drate (100), were uns ucces sful. Portions of Fraction l and IV were comb i n e d with a small amount of the tra p-contents, and the mi xt u re was furth er studie d by t he me thod of pap e r chromatography.

TABLE XVIII

FRACTIONAL DISTILLATION OFACI DS FROM TH E BA RIUM SALTS

Distillation Refractive Appr ox imat e 25 Yield pH Fraction Te mp. Pre ssure Index l1D (z-) lin wate r )

l 46-60° 5 mm. 1.371 0. 520 4 II 60-70° 5 mm. 1.376 0.161 4 III 70-75° 5 mm. 1.407 0.034 5-6 >98 0 4 mm. 1.454 0 . 2 70 5-6 Tra~Ya) <46 ta 75° 5 mm . 1.368 1.37 4

(a) Coo1ed with so1id carbon dioxide between r e ceiver a n d vacuum pump, 107.

Ascending Method

Barker and Kennedy1s method (79) for the separation

of volatile aliphatic acids, employing iVhatman No. l filter

paper was a dopt ed, The strips of p a p e r were 8.pproximately 55

cm. in 1ength and 45 cm. in width.

The ammonium salts of 4-hydroxyheptanoic and 2-methyl

4-hydroxyhexanoic acids were preparGd by heating the crystalltne hydrazides for severa1 minutes with di1ute su1phuric acid, cool

ing and adding ammoniQm hydroxidc until the solutions were al kaline (pH 8-9). The solutions were then diluted so tllat one ml . contained about 4 mg. of acid. Other known acids were s1 milar1y treated, and also a mixture containing 10 mg. of Frac

tion l, 7 mg. of Fraction IV and 20 mg. of the contents of the

trap (b.p. < 46 to 75° at 5 mm.) p er ml.

The ammonium salts of the acids were applied to the starting 1ine, 5 cm. from the bottom of the paper, in amounts which had been found appropriate in preliminary e xp er-Iment.s,

After a~plication of the spots, the paper was ralled inta a

cylinder and pinneà together in such a way that no averlapping accurred. The roll was then placed in an air-tight chromato graphie tank filled ta a denth of 2.5 cm. with approximately

2 litres of a solution c ontaining ethanol, water, and cancentra

ted ffiillnonium hydroxide in the ratio 95:5:1 by volume. After de velopment of the chromatogram for eight hours at room tempera ture the location of the solvent front was noted, and the paper was dried in an aven at 105°0. for six minutes. The chromato- 108.

gram was then sprayed with a solution containing 50 mg. of bromo

phenol blue and 200 mg. of citric acid in 100 ml. of water. The

spots were easily observed by the intense blue color r esulting

from the buffering effect of the a cid a nions, the background

being yellow, the acid color of the indicator.

De s cending Method

The strips of Vfuatman No. l paper used in this method were 45 cm. in length and 10 cm. in width, and the ammon Lum aaLts

of the acids were applied to a starting line 10 cm. from the top

of the pap e r . The p a p e r s were then suspended from a stainless

s t e el trough in an air-tight chromatographie tank, after a re

servoir, filled with the appropriate solvent, had been placed

at the bottom of tha tank. After one hour, when the atmosphere was saturated witb tbe solvent vapor, the trough was filled with

the solvent via a small open~ng at the top of the tank. The

opening was then closed a nd the chromatogram developed. Wi t h

ethanol, water, and mnmonium hydroxide in the volume ratio

95:5:1, the solvent front moved 35 cm. in approximately six hours. When the Ethanol was replaced by methanol, the chromato

gram was developed in about four hours. Sm~~ARY AND CLAIMS TO ORIGINAL RESEARGH

1. Confirmation was obtained for previous reports

that the rate of oxidation of cellulose, by potassium di

chromate, as judged by increase in copper number, was much

greater in oxalic than in sulphuric acid.

2. Vfuen oxalic acid was estlmated by decrease in acidity and chromate ion by an iodometric method, 1 mole of potas

sium dichromate was equivalent to 7 moles of oxalic acid, but

the evolution of carbon dioxide ceased at 5 moles. It was assumed that 2 moles of the oxalic acid formed a colored com plex with the mole of dichromate, although the possibility was not examined in detail~ Cellulose, when present, reduced 15 per cent or less of the oxldant, and the oxidation of cellulose was tentatively attributed to some unstable intermediate dur ing the reaction, since in the brief period of time the direct oxidation of the cellulose by dichromate was negligible. The period when the oxalic acid was rapidly oxidized was also the period when the cellulose was rapidly oxidized.

3. Although the estimation of aldoses by the amount of ammonia liberated by saponification of the cyanohydrin was twice reported to give quantitative results, those with fruc tose were very high. It was confirmed that the estimation gave correct results with fructose when 0.3 M potassium cyanide was used at a pH not less than Il, or by using 0.03 M potassium 110.

cyanide at any pH more alkaline than 8. The presence of unmo dified cellulose increased the stability of the solution of po tas sium cyanide used as a blank.

Total carbonyl groups in dichromate - oxalic acid o~Jcelluloses were estimated by the cyanohydrin method with results that agreed weIl with the standard hydroxylamine hy drochloride method unless the latter was rendered inaccurate by prior drying the oxycellulose bhr-cugh me t h a n ol and benzene or by the 9 r e s en ce of much chromium containing ash.

4. Unswollen and highly swollen linters were oxidized by p o t a s s i um dichromate and oxalic acid until they acquired

50 to 190 milliatoms of oxygen per gl u co s e unit. The oxycel luloses were obtained in nearly quantitative yields, the most highly oxidized product still retaining a degree of polymeriza tion of about 270, as determined by the nitration method, and possessing a low ash content. The accessibility of swollen cellulose was greater than that of unswollen linters, in agree ment with the results previously found with other chromic acid oxidations of cellulose.

5. An increase i n t h e ratio of oxalic acid to dichromate increased the proportion of total carb onyl to carboxyl g r oup s .

In lightly oxidized products the carbonyl content was almost en t i r el y aldehydic in nature, as estimated by the chlorous acid metilo d. ~~en gr e a t er quantities of r eactants were em~loyed, the ketone gr oup s increased to approximately 50 per cent of the total carbonyl content. The ketone content passed through Ill.

a maximum a t approximately one hour of oxidation. Methylation

of the most highly oxidized product with diazomethane did not

abol i sh the reducing power, and presumably esterified s orne of

the carboxylic acid gr oup s , a s evidence in the literature sug

gested.

6. In the condensation of hydrogen cyanide more of

the oxidized cellulose passed into solution at pH 8, than at pH 10, perhaps because cyanohydration was more rapid than al

kaline cleavage at the latter pH.

A procedure involving the cyanohydrin reaction at pH

10 was applied to 116 g. of an oxycellulose averaging 47 milli moles of aldehyde, 43 of k etone, and 44 of carboxyl, per glu

cose unit. Only one third of the expected number of carboxyl groups remained in the 100 g . (86 per cent) of the non-reducing, insoluble product which was not further examined. The soluble products (16 g .) recovered from the liquors was reduced wit h boiling hydriodic a c i d and r ed phosphorus to a con~lex mixture of fatty acids. Paper p a r t i t i on chromatograpby made it p r ob a b l e

that they ranged from acetic to butyric and included succinic acid. 2-Methyl hexanoic acid or lactone, the product expected from 2-keto glucose units in the o~Tcellulose, could not be definitely identifie d. The r esults clearly showed that the oxidation even of highly swollen cellulose, with p o t a s s i um di

chromate was bath local and intense. 112.

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