View, with References to the Chemical Literature, on the Action of Chemical Agents on Cellulose Nitrate

Home , Phenylhydrazine, Polyvinyl nitrate

THE CONTROLLED THERMAL DECOMPOSITION

OF CELLULOSE NITRATE: CARBONYL COMPOUNDS

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

GUY PIERRE ARSENAULT, B.A.Sc.

The Ohio State University 1958

Approved by:

Adviser Department of Chemistry ACKNOWLEDGMENT

The author wishea to extend his sincere apprecia

tion and thanks to professor M. L. Wolfrom for his

Interest In this work and for his guidance in technical

matters.

The spirit of sacrifice and unfailing assistance of

the writer’s wife, Anne, who made valiant efforts to

understand the field of chemistry, made this work possible.

The writer wishes to thank Messrs. H. R. Menapace

and V. G-. Wiley for the loan of their gas chromatographic

equipment and calibration charts, and for their spirit

of cooperation.

Mr. A. Chaney and Dr. F. Shafiaadeh of The Ohio State

University Research Foundation have given freely of their time at the writer’s request for aid. For this he is very thankful.

Appreciation is expressed to the Monsanto Chemical

Co. (1956-1957) and the Standard Oil Foundation inc.

(Indiana) (1957-1958) for fellowships provided by them.

This work was supported in part by the united States

Army Ordnance Department under contracts (unclassified;

DA3>0l9-ord-727, DA33-019-ord-14-76 and DA33-019-ord-2o42; supervising agency, The Ballistic Research Laboratories,

Aberdeen Proving Ground, Maryland) with The Ohio State

ii ill

University Research Foundation (Projects 496, 591 and

679). TABLE OF CONTENTS

Page

INTRODUCTION ...... 1

STATEMENT OF THE PROBLEM...... 3

HISTORICAL ...... 5

Decomposition of Cellulose Nitrate ..... 5

Preparation of 2 ,4-Dinitrophenyl- hydrazine Derivatives ...... 26

Chromatography of 2,4-Dinitrophenyl- hydrazine Derivatives ...... 37

Characterization and identification of 2,4-Dinltrophenylhydrazine Derivatives ...... 45

A. ultraviolet and visible spectrum 45

B. Infrared spectrum ...... '..... 50

C. Melting point ...... 52

D. Miscellaneous methods ...... 55

DISCUSSION OF RESULTS ...... 57

Thermal Decomposition of Cellulose Nitrate ...... 57

Fractionation, by Repeated Sublimation, of Some of the Major Organic Components of the Aqueous Solution of the Liquid Mixture of Cellulose Nitrate ignition Products ...... 61

preparation of 2,4-Dinitrophenyl- hydrazine Derivatives of Known Carbonyl Compounds ...... 69

A. Preparation of triose 2,4- dinitrophenylosazone ...... 7^

iv TABLE OF CONTENTS. Cont. Page

B. Preparation of the 2,4- dinitrophenylhydrazflmes of short carbon chain sugars ...... 77

0. Preparation of mesoxaldehyde tris(2,4-dinitrophenylhydrazone)... 79

D. Reaction of £>-glucose with 2 ,4-dinitrophenyThydrazlne ...... 81

E. Preparation of mesoxaldehyde l,2-bis{2 1 ^ ’-dinitrophenylhydrazone) ...... 83

F. Attempted preparation of mesoxaldehyde l,3-bis(2 ' ,4 '- dinitrophenylhydrazone) ...... 87

G-. Reaction of 2,4-dinitrophenylhydrazine with triose- reductone ...... 91

H. Miscellaneous data concerning 2 ,4-dinitrophenylhydrazine derivatives ...... 93

Chromatography of 2,4-Dinitrophenyl hydraz ine Derivatives of Known Carbonyl Compounds ...... 105

Fractionation, by Extraction with Alcohol (95?0, of the 2,4-Dinitro- phenylhydrazine Derivative of the Residue of the Repeatedly sublimed Condensate Aqueous Solution ...... 110

Investigation of the Alcohol-Soluble Fracti on ...... 114 investigation of the Alcohol- Insoluble Residue...... 12o

Attempted Isolation of Triose 2,4- Dinitrophenylhydrazone from the Condensate Aqueous Solution ...... 121

v TABLE OF CONTENTS. Pont. .rase Gas Absorption Chromatography of the Liquid. Mixture of Cellulose Nitrate ignition products ...... 124

Origin of Compounds Isolated from the Liquid Mixture of Cellulose Nitrate Ignition Products .... 135

EXPERIMENTAL...... 141

General Statements ...... 141

Chromatography of Derivatives of 2,4-Dinltrophenylhydrazine ...... 144

Reaction of Some Carbonyl Compounds with 2,4-Dinltrophenylhydrazine ...... 148

A. Preparation of the 2,4- dinitrophenylhydrazones of some oi -hydroxy carbonyl compounds .... 148

B. Preparation of D,L-glyceraldehyde 2,4-dlnItropTaefiylhydra- zone in the presence of a large excess of reagent ...... 151

C. Preparation of D,L-glyceraldehyde 2 ,4-dinitropMenylhydra- zone in the presence of an acid catalyst ...... 152

D. Preparation of triose 2,4- dlnitrophenylosazone ...... 152

E. Preparation of mesoxaldehyde tris(2,4-dinitrophenylhydrazone) .. 155

F. Reaction of D-glucose with 2 ,4-dinitrophenyTnydrazine 159

G. Preparation of mesoxaldehyde l,2-bist21,4'-dinitrophenyl hydrazone) ...... 16 0

vi TABLE OF CONTENTS, Pont.

Page

H. Conversion of mesoxaldehyde I ,2 -bis(2 ’,41-dlnitrophenylhydrazone) into mesoxaldehyde tris(2 ,4-dinitrophenylhydrazone).... 163

I. Attempted preparation of mesoxaldehyde l,3-bis(2 1,41 - dinitrophenylhydrazone) ...... 164

J. Reaction of 2,4-dinitrophenylhydrazine with triose- reductone ...... 165

Miscellaneous Data Concerning 2,4- Dinitrophenylhydrazine Derivatives ...... 170

A. Infrared spectra of 2,4- dinitrophenylhydrazine derivatives., I70

B. Sublimation of 2,4-dlnitrophenylhydrazine, 2,4-dinitroaniline and 2 ,4-dinitrophenylhydrazine derivatives ...... 170

C. Solubility of 2,4-dinitrophenylhydrazine derivatives in hot water ...... 172

D. Light absorption spectrum of mesoxaldehyde l,2 -bis(2 1,41 - dinitrophenylhydrazone) - Solvent effect ...... 172

Fractionation, by Repeated Sublimation, of Some of the Major Organic Components of the Aqueous Solution of the Liquid Mixture of Cellulose Nitrate Ignition products ...... 174-

Fractionation, by Extraction with Alcohol (95$)* of the 2,4-Dinitro- phenylhydrazine Derivative of the Residue of the Repeatedly Sublimed Condensate Aqueous Solution...... 178

vil TABLE OF' CONTENTS, Cont.

Pap;e

Investigation of the Alcohol- Soluble Fraction ...... 179

A. Chromatographic separation ... 179

B. Zone 1 ...... 179

C. Zone 2 ...... 182

D. Zone 3 ...... 184

E. Zone 4 ...... 185

F. Zone 5 ...... 186

G-, Zone 6 ...... 187

Investigation of the Alcohol- Insoluhle Residue ...... 189

Attempted Isolation of Trlose 2,4- Dinltrophenylhydrazone from the Condensate Aqueous Solution ...... 190

A, Reaction of the condensate aqueous solution with 2,4-dinitrophenylhydrazine in boiling ethanol.. 190

B. Reaction of the condensate aqueous solution with a super saturated solution of 2,4-dinitrophenylhydrazine in 2 ^ hydrochloric acid ...... 190

G-as Absorption Chromatography of the Liquid Mixture of Cellulose Nitrate Ignition Products ...... 193

SUMMARY ...... 199

BIBLIOGRAPHY ...... 203

AUTOBIOGRAPHY .... I...... 216

viii LIST OF TABLES

Table Page

I* Fractionation, by Repeated Sublimation, of Some of the Major Organic Components of the Aqueous Solution of the Liquid Mixture of Cellulose Nitrate Ignition products ...... 64

II. Melting Points of Some 2,4-Dinitro- phenylhydrazlne Derivatives ...... 70

III. Light Absorption Characteristics of Some Derivatives of 2,4- Dlnitrophenylhydrazine ...... 112

IV. Compounds Isolated, in Our Work, from the Liquid Mixture of Cellulose Nitrate ignition Products ...... 136

ix LIST OF FIG-URES

Figure Page

1. Product Relationships ...... 62

2. Light Absorption Spectrum of Mesoxaldehyde 1,2-Bis(2',4'-dinitro phenylhydrazone J j in Ethyl Acetate; - - - -, in Ethanol (95%) •••* 86

3- Infrared Absorption Spectrum of G-lycolaldehyde 2,4-Dinitrophenyl- hydrazone ...... 96

4. Infrared Absorption Spectrum of G-lyox- ylic Acid 2,4-Dinltrophenylhydrazone .. 97

5. Infrared Absorption Spectrum of Acetol 2,4-Dinitrophenylhydrazone ...... 98

6. Infrared Absorption Spectrum of Mesox- alic Acid 2 ,4-Dinitrophenylhydrazone .. 99

7. Infrared Absorption Spectrum of D,L- G-lyceraldehyde 2 ,4-Dinitrophenyl- “ hydrazone ...... 100

8. Infrared Absorption Spectrum of Di- hydroxyacetone 2,4-Dinitrophenyl- hydrazone ...... 101

9. Infrared Absorption Spectrum of Triose 2 ,4-Dinitrophenylosazone ...... 102

10. Infrared Absorption Spectrum of Mesox aldehyde Tris(2,4-dinitrophenylhydrazone) ...... 103

11. Infrared Absorption Spectrum of Mesoxaldehyde 1,2-Bis(2 1,4'-di nitrophenylhydrazone) ...... 104

12. Diagrammatic Representation of the Chromatogram of Extract I on Silicic Acid-Celite (5:1} 8% water) ...... 115

x LIST OF FIGURES. Qont.

Figure Page

13. Diagrammatic Representation of the Chromatographic Separation on Silicic Acid-Celite (2:1; 0% water) of the Residue of Evaporation of the Mother Liquors from the Recrystallizations of the Sublimate of the Material in Zone 1 117

14. Schematic Diagram of the Gas Absorp tion Chromatography Apparatus ...... 125

15. Recorded Gas Chromatogram of the Separation of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate) on a 1.22 m. (4‘) polyethyleneglycol- 400 Column ...... 127

16. infrared Absorption Spectrum of the Material in Zone 3 from the Separa tion, by Gas Chromatography on a 1.22 m. polyethyleneglycol-400 Column, of the Liquid Mixture of Cellulose Nitrate Ignition products (First Spiral Trap Condensate) ...... 129

17. Infrared Absorption Spectrum of the Material in Zone 3 from the Separa tion, by Gas Chromatography on a 4.88 m. Polyethyleneglycol-400 Column, of the Liquid Mixture of Cellulose Nitrate Ignition products (First Spiral Trap Condensate) ...... 130

18. Infrared Absorption Spectrum (in Carbon Disulfide; Compensated) of the Material in Zone 4 from the Separation, by Gas Chromatography, of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate) ...... 152

xi LIST OF FIGURES. Pont.

Figure ?age

19. Infrared Absorption Spectrum (Gas Phase) of the Material in Zone 4 from the separation, by Gas Chroma tography, of the Liquid Mixture of Cellulose Nitrate Ignition Products (FirBt Spiral Trap Condensate) .... 133

xli INTRODUCTION

Cellulose nitrate is the only Inorganic ester of cellulose which is produced commercially in large quantities.

The first preparation of cellulose nitrate was reported by Bracoimot (1) who Identified his product

(1) H. Braconnot, Ann. chim. et phys., /~"2_7 52, 290 (1833). with starch nitrate. A few years later Pelouze (2) alBO

(2) j. pelouze, Compt. rend., 713 (1838). prepared a nitrate ester of cellulose. Pelouze observed the combustion properties of the nitrate ester and suggested its possible use for artillery purposes. How ever, a clear distinction between cellulose nitrate and starch nitrate yet remained to be made. This was done by Sohbribein (3) who also reported the usefulness of

(3) Schonbein, Phil. Mag., 2b 7 (1847). cellulose nitrate as a propellant and an explosive.

Schonbein is generally credited with the discovery of cellulose nitrate.

The cellulose nitrate industry uses chiefly cotton linters and wood pulps as sources of cellulose. Mixtures of nitric acid, sulfuric acid and water, such as were introduced a century ago by Schonbein, are used commer cially as nitrating agents. This nitrating medium is seldom used in the laboratory.

The importance of cellulose nitrate has steadily increased since its discovery. Furthermore, this cellu lose ester is responsible for many changes in industrial arts and sciences. Cellulose nitrate has replaced black powder as the universal basis of all propellants.

Plastic compositions of practical purposes date from the discovery of celluloid, a mixture of camphor and cellu lose nitrate. The advent of cellulose nitrate lacquer after World War I marked a major advance in the field of protective surface coatings. STATEMENT OF THE PROBLEM

The controlled thermal decomposition of cellulose

nitrate, under ignition conditions at reduced pressure,

has been investigated in this Laboratory by Wolfrom and

co-workers (4-7). The nitrated oxycellulose of low

(4) M. L. Wolfrom, J. H. Frazer, L. P. Kuhn, E. E. Dickey, S. M. Olin, D. 0. Hoffman, R. S. Bower, A. Chaney, Eloise Carpenter and P. McWaln, J. Am. Chem. Soc., 77, 6573 (1955).

(5) M. L. Wolfrom, J. H. Frazer, L. P. Kuhn, E. E. Dickey, S. M. Olin, R. S. Bower, G-. G. Maher, J. D. Mur dock, A. Chaney and Eloise Carpenter, J. Am. Chem. Soc., 18, 4695 (1956).

(6) M. L. Wolfrom, A. Chaney and P. McWain, J. Am. Chem. Soc., in press.

(7) F. Shafizadeh and M. L. Wolfrom, J. Am. Chem. Soc., in press.

degree of polymerization which was obtained as a major

product In the pressure range of 2-5 mm. was succeeded by

a liquid mixture as the pressure was raised. This mixture

consisted mainly of water, formic acid, formaldehyde and

glyoxal. Th<=> results were Interpreted on the basis of

homolytic bond scission by analogy with the thermal decom

position of simple mononitrates. Thus, the initial step

of the ignition decomposition of cellulose nitrate was

postulated as the homolytic cleavage of the 0-N bond of

the nitrate groups with the formation of nitrogen dioxide

3 and alkoxyl radicals. Since alkoxyl radicals may lead by simple processes to the formation of carbonyl com pounds, a thorough knowledge of the identity of all carbonyl compounds formed as a result of the thermal decomposition of cellulose nitrate may shed some light on the decomposition mechanism.

The objective of this study was the continued investi gation of the carbonyl compounds present in the liquid product of the decomposition of cellulose nitrate and the development of methods for the isolation and character ization of these compounds. HISTORICAL

Decomposition of Cellulose Nitrate

In the decades which followed the initial prepara tion of cellulose nitrate by Braconnot (l), the manu facture and usage of this compound was a dangerous enterprise which led to several catastrophies. The nitrating agent used at that time, and still in use today, waB a mixture of nitric and sulfuric acids. Abel (8)

(8) F. Abel, Brit. Patent 1102 (1865). discovered that the stability of cellulose nitrate could be improved greatly by removing traces of the nitrating mixture retained by the product and invented a process to do so. The manufacture of cellulose nitrate was thus established on a safer basis.

The formation of sulfate esters of cellulose nitrate during nitration was recognized early in the twentieth century and soon this became understood to be a further cause of the instability of the nitrate ester of cellulose.

The preparation of cellulose nitrate on a commercial basis was firmly established in 1906 when Robertson (9) discovered

(9) R. Robertson, J. Soc. Ohem. Ind. (London), 2£, 624 (1906). that the unstable sulfate esters of cellulose nitrate are decomposed at least in part by steeping the product in 6 < boiling water while it still retains a small amount of mixed acid.

Thus cellulose nitrate undergoes a slow decomposi tion at room temperature. The hydrolysis of nitrate groups is believed to be caused by traceB of acid which have not been completely washed out or which result from the decom position of unstable sulfate ester groups, if the pro ducts formed are allowed to remain in contact with the cellulose nitrate, the decomposition reaction gains in momentum. Stabilizers are often added to cellulose ni trate to prevent this autooatalytic reaction. These stabilizers do not slow down the inherent, very slow rate of spontaneous degradation of the nitrate. They simply prevent the occurrence of a self-accelerating reaction by virtue of their ability to be nitrated, thus forming

Inactive products.

A number of simple and a few complex reaction pro ducts have been found as a result of the action on cellu lose nitrate of chemloals such as acids, bases and reducing agents (10,11). The photolytic decomposition of

(10) J. Barsha, in“Cellulose and Cellulose Deriva tives,1' "High Polymers," Vol. V, 2nd edition, E. Ott, H. M. Spurlin and Mildred W. Grafflin, Ed., interscience Publishers, inc., New York, N. Y., 1954, p. 751 gives a review, with references to the chemical literature, on the action of chemical agents on cellulose nitrate.

(11) The authors in reference 4 have made additions to the review given by BarBha in reference 10« 7 cellulose nitrate haa also been Investigated (12,13).

(12) D. Berthelot and H. G-audechon, Compt. rend., 154. 201 (1912).

(13) D. Berthelot and H. Gaudechon, Compt. rend., 154. 514 (1912).

Cellulose nitrate Is unique among the industrially significant high polymers because of its ability to de compose exothermlcally without the participation of oxygen.

The sole action of heat on cellulose nitrate has been the subject of a considerable amount of research because of the importance of this polymer and its thermal sensitivity.

Thermal decomposition of cellulose nitrate, of a high degree of nitration (ca. 11$ N or more), can be divided into three distinct classes. The first, slow thermal decomposition below the deflagration point, increases in rate with increasing temperature below 200°. The rate will decrease if the temperature is subsequently reduced by removal of the external heating source. The sample under study is usually heated at temperatures below 200° for long periods of time during which gases are evolved.

On prolonged heating, a point is reached when gases are no longer evolved from the sample and the decomposition residue is no longer explosive. The second class, ignition decomposition, occurs on Initiation by contact with sources of intense heat. This mode of decomposition, which is usually self-sustaining, is carried out under 8 conditions such that no pressure build-up is allowed in the ignition system. The last class, explosive decomposition, occurs when cellulose nitrate is subjected to an intense source of heat in a confined space. The products arising from each of these classifications are different.

The only products Isolated from the explosive decom position of cellulose nitrate are C0S » CO, Ns , CH4 , H* and Hs0.

The products obtained from the explosion of cellu lose nitrate were first investigated by Karolyl (14) who

(14) L. v. Karolyi, Ann. Phys. Chemie, 118, 544 (1863). detonated the substance in a sealed cast iron chamber placed inside a large, evacuated bomb. Upon decomposition of the sample by means of a heated platinum wire, the cast iron chamber was shattered and the gases formed were collected in the bomb. Under these conditions, GO, C08 ,

Ns , CH4 , Ha , Hs0 and a small carbonaceous residue were the only products of decomposition. The fact that no oxides of nitrogen are found in the product of the explosive decomposition of the nitrate ester of cellulose 9 was soon confirmed by Abel (15)•

(15) F. A. Abel, Proc. Roy. Soc. (London), 13, 2o4 (1864).

Sarrau and Vieille (16,17) made an extensive study

(16) Sarrau and Vieille, Bull. soc. chim., 22, 581 (1880).

(17) Sarrau and Vieille, Compt. rend., 8g, 165 (1879). of the effects of pressure on the products of explosion of cellulose nitrate. They exploded guncotton in a closed bomb. By varying the amount of this substance used in each case, they were able to produce pressures of up to 6000 atmospheres inside the bomb. Their findings may be summarized briefly as follows: as the pressure increased from 100 to 6000 atmospheres, the carbon dioxide and hydrogen contents of the gases increased, while their carbon monoxide and water contents decreased. The nitrogen content remained constant throughout the whole

range of pressures. Only C08, CO, Hs , Na and Ha0 were

found in the products, no CH,*, HCN or oxides of nitrogen having been detected. However, in some later work,

Sarrau and Vieille (18) reported the presence of small

(18) Sarrau and Vieille, Compt. rend., 105, 1222 (1887). 10 amounts of methane, at pressures of 5000 and 6000 atmospheres.

Presumably, oxides of nitrogen are formed in the course of the explosive decomposition of cellulose nitrate but they are reduced in secondary reactions which involve the conversion of carbon to its oxides.

Thus the only nitrogen-containing substance found is nitrogen gas.

The slow thermal decomposition of cellulose nitrate below its Ignition point affords a more complex product mixture than the explosive decomposition. The formation of C08 , CO, Ha0, Na , NO, N08 , NaO, Ha » CH*, HCHO, hydro carbons (unspecified) and renldual oxyoellulose nitrates has been reported. The gaBeous products have been the subject of considerable research. There is much contra diction in the work of different investigators. However, there are factors involved which permit a better under standing of these discrepancies. Minor changes in experi mental conditions may be critical in this field. Yet detailed descriptions of the experimental work are rare and a thorough examination of the products, rarer. To add to this difficulty, the original literature is not always available and, Instead, sketchy abstracts have to be consulted. Finally, much of the early work was done before suitable methods of stabilizing cellulose nitrate were known. The presence of traces of nitrating acids 11

would undoubtedly have some effect on the course of

the thermal decomposition of cellulose nitrate.

The earliest study on the thermal decomposition of

cellulose nitrate was reported by Abel (19). Abel

(19) F. A. Abel, J. Chem. Soc., 20, 505 (1867).

enclosed this substance in sealed tubes containing air

or nitrogen at atmospheric pressure. The tubeB were

heated at 100° for 21 to 28 hours. Nitric oxide was

formed in each case, pursuing his work, Abel (19) intro duced guncotton into a large bulb blown at the extremity

of a barometer tube. The barometer tube was placed with

its opening over mercury and exhausted. The bulb was

then heated to 65°. After the pressure inside the tube became equal to that of the atmosphere, the gases which

escaped from the tube were collected and analyzed. The

gases analyzed after a period of 11 weeks of heating were

reported to contain only C0a» NO and Ns* After a further

period of heating of nearly 12 months, the gases were

said to contain only C0a , Ha » NO, Na and traces of

hydrocarbons (unspecified), in addition, water was found

inside the bulb-tube.

Hoitaema (20) heated guncotton (13»8$ N) under

(2o) C. Hoitaema, Z. phya. Chem., 2£, 567 (1898) vacuum at temperatures "below 210°, until gases were no

longer evolved, A dark brown, peat-like, non-explosive,

solid residue was obtained which contained 7# N. The gases collected contained only C0S , NO, CO, Hs0, NO0 and N0. Will (21) soon confirmed part of Hoitsema's (2o)

(21) W. Will, Mitt. Centralstelle Wissensch. Techn. Untersuch., 5-40 (1902); J. Soo. Ohem. Ind. (London), 21, 1470, 1554, 1555 (1902). experimental results. Will (21) heated a sample of cellulose nitrate (13.1# N) at 135° in a current of carbon dioxide for 371 hours, after which time no further loss of weight took place on heating. A solid residue con taining 5.3# N was obtained. The weight of this residue was 33# of the weight of the starting material. Will con cluded that under the conditions of his experiment, the gaseous products consisted essentially of NO, H0O and CO.

Saposchnikoff and co-workers (22,23) reported that

(22) A. V. Saposchnikoff and M. Borisoff, J. Russ. Phys. Chem. Soc., .26, 836 (1904); J. Chem. Soc., 86-1. 799 (1904).

(23) A. Saposchnikoff, Mem. poudres salpetres, 42 (1907-1908); J. Soc. Chem. Ind. (London), 2J, 592 (1908). cellulose nitrate decomposes rapidly and completely on being heated at 150°. Thus for a sample containing 13.25# : N, 8 .9# of the original weight of cellulose nitrate 13 remained after the evolution of gases ceased. The gases evolved contained only NO, CO®, CO, N« and H80.

Saposchnikoff and co-workers (23-25) further reported

(24) A. V. Saposchnikoff and W. jaRellowitsch, J. Russ. Phys. Chem. Soc., 21* 822 (1905); J. Chem. Soc., A90-I. 68 (1906).

(25) A. V. Saposchnikoff, J. Russ. phys. Chem. Soc., £8, 1186 (1906); J. Chem. Soc., A92-I. 390 (1907); C. A., 1, 1323 (1907). comparative analyses of gases (C0S , CO, NO, N» and H20) formed when cellulose nitrates of 13.4$ N and 12$ N were heated at 150° and 120°. The results obtained with the two different samples of guncotton were quite similar.

The authors concluded that at the lower temperature less carbon dioxide, carbon monoxide and nitric oxide were formed than at 150°, while the nitrogen gas content was about the same in each case. At the higher temperature, however, the water content was approximately half of that at 120°. From his examination of the dark solid residue obtained at several temperatures below 160°, Saposchnikoff

(2 3 ^25) concluded that above 135°, the residue obtained in each case contained practically no nitrogen while below

135°» the residue contained a considerable amount of nitrogen but very little hydrogen. 14

Robertson and Napper (26) investigated the deoom-

(26) R. Robertson and S. S. Napper, J. Chem. Soc., 21, 764 (1907). position of cellulose nitrate (13# N) under conditions identical to those used by Will (21), and found that as much as 50# of the total nitrogen lost by the sample was in the form of nitrogen dioxide in the gaseous products.

Further studies allowed the authors (26) to conclude that the proportion of nitrogen as nitrogen dioxide to the total nitrogen lost by the sample decreased as the decom position gases remained in contact with the heated gun cotton for increased lengths df time. Robertson and

Napper (26) further observed that the ratio of nitrogen as nitrogen dioxide to the total nitrogen lost by the sample decreased steadily, when a sample of guncotton was heated in a stream of carbon dioxide for long periods of time (80 hours).

Koehler and Marqueyrol (2J) studied the composition

(27) Koehler and Marqueyrol, Mem. poudres, 18, 101 (1921); C.A., 16, 1866 (1922). of gases evolved on continuous heating of stabilized cellulose nitrate in a vacuum at 40°, 75° and 100°.

They concluded that the composition of the gases does not vary greatly with the temperature of heating. These 15 investigators reported the presence of NO, C0a» CO,

N80 and N8 in the gases, as well as traces of hydro carbons (unspecified). Koehler and Marqueyrol (28)

(28) Koehler and Marqueyrol, Mem. poudres, 18, 106 (1921); C. A., 16, 3398 (1922). have also reported that on heating a compressed dry sample of cellulose nitrate for 238 days at 75° in a dry vacuum, the only gases formed were C08 , N8 and 00, with traces of Na0 and CH*.

In 1931, Goujon (2 9 ) confirmed the work of Robertson

(29) F. Goujon, Mem. poudres, 24, 73 (1931); Brit. Ohem. Abstracts, 995B (1931); C. A., 2 5 , 5983 (1931). and Napper (26) concerning the large amount of nitrogen dioxide present in the gaseous products of decomposition.

Heating cellulose nitrate in a current of dry nitrogen for 8 hours at 135°, the author found that 60$ of the nitrogen was lost as nitric oxide and 30$ as nitrogen dioxide. The gaseous products contained N08 , NO, Na0, N8 ,

H3O, C08 and GO.

The gases evolved from a sample of cellulose nitrate heated at 109° were analyzed by Vandonl (30) and reported

(30) R. Vandonl, Compt. rend., 2ol. 674 (1935) 16 to contain only C08 , NO, Na0, CO, CH* and Na . No hydrogen gas was found.

Desmaroux and co-workers (31) heated samples of

^ (31) J. Desmaroux, R. Vandonl, L. Brlssaud, and Therese petltpas, Mem. poudres, 2£, 134 (1939). nitrated ramie fibers at 109°for periods of up to 2000 hours. The gases formed were collected over mercury.

Working with nitrated samples containing 14$ N, 12.78$ N and 11.88$ N, they were able to ascertain that, in all cases, a time is reached beyond which further heating yields no more gases. The decomposition residue reported for the starting material containing 12.78$ N made up

48$ of the original sample weight and contained 6.05$ N.

No further investigation of this residue was reported.

Incomplete gas analyses showed similar trends in all three samples. These trends may be summarized briefly: as time increased, the proportion of carbon dioxide in the gases also increased, whereas the proportion of nitric oxide decreased. The results for carbon monoxide were not conclusive, although it appeared as if the relative proportion of this gas remained appreciably constant throughout the time of evolution of gases. 17 Rideal and Robertson (32) studied the gaseous

(32) E. K. Rideal and A. J. B. Robertson, "Third Symposium on Combustion and Flame and Explosion Phenomena," The williams and Wilkins Co., Baltimore, Md., 194-9, p. 536. decomposition products of guncotton (10.3# N) heated in a high vacuum to 160° for 100 minutes. The gaseous pro ducts were collected in a liquid air trap and those gases which did not condense were collected with a Toepler pump.

The totality of the gaseous products was reported to be made up of N0b , NO, Ns0, Na » Ha , CO and C03. Some formalde hyde was detected by the formation of the dimedon derivative.

Of the gases found, the authors stated that only nitrogen dioxide disappeared quickly by secondary reaction at 200° and in general no nitrogen dioxide was found when the decomposition products were allowed to accumulate Instead of being continuously removed. This observation is similar to that made previously by Robertson and Napper

(2 6 ).

A new body of data on the kinetics of the thermal decomposition of cellulose nitrate has recently been published by Phillips, Orlick and Steinberger (33).

(33) R. W. Phillips, C. A. Orlick and R. Stein berger, j. Phys. Chem., 1034- (1955)» 18

Their findings clarify much of the earlier and some what contradictory reports in this field.

Under most conditions, the ignition of cellulose nitrate leaves little or no residue. The products of the ignition decomposition of cellulose nitrate most often reported are the following: N2 , 00, C08 and N0S •

Nitrous acid, cyanogen gas, Hs , NO, HON, acrolein, Hs0,

CH4 , a “cyanic compound'4 and a hydrocarbon have also been reported.

Fordos and G-ells (34) were the first workers to

(34) J. Fordos and A. G-elis, Compt. rend., 23, 982 (1846). report on the products of ignition decomposition of cellulose nitrate. They isolated silver cyanide by burning guncotton in a chamber closed at one end and leading at the other end to a solution of silver nitrate.

The authors stated that such a precipitate could be obtained only from 3 gases: cyanogen, hydrocyanic acid and ammonium cyanide. However, they did not state specifically which one of the three gases produced the silver cyanide precipitate. The authors reported that nitrogen dioxide was also present in the gaseous products. 19

Ransome (3 5 ) burned guncotton In an atmosphere of

(35) T. Ransome, Phil. Mag., C $ J -2Q> 1 (1847). carbon dioxide and collected the gases over water. No residue remained from the ignition. He showed that carbon monoxide was present in the gaseous products.

Porrett and Teschemacher (3 6 ) reported the presence

(36) R. porrett and E. P. Teschemacher, Mem. proc. Chem. Soc., 258 (1845-1848). of COj) 1 cyanogen gas, NO, 00, H80, carbon and crystalline oxalic acid among the products of ignition of cellulose nitrate. However, the authors use the term ^.nitrated lignin1' Interchangeably with cellulose nitrate in their paper and no mention is made of the method of preparing the sample they decomposed. For this reason, considerable discredit should be thrown on their work.

Karolyi (14) studied the products obtained from the ignition of guncotton placed in a barometric chamber.

The sample was ignited by heating a platinum wire in contact with the sample, under such conditions, the following products were reported*' CO, C08 , CH4 , N0a, Na,

Hg0 and a carbonaceous residue.

The combustion of guncotton in air or in other gases was studied by Abel (15 ), who also studied the "imperfect1' 20 combustion (the sample was ignited in an open tube and a current of hydrogen passed over the guncotton causing the combustion to cease) and the slow combustion

(ignition of guncotton in a highly rarefied atmosphere) of guncotton, in all cases, Abel reported the presence of nitrogen dioxide in the gaseous products, in cases of ‘'imperfect" and slow combustions, Abel detected the presence of cyanogen gas and nitrous acid among the products.

Sarrau and Viellle (3 7 ) have reported the presence

(37) Sarrau and Vleille, Compt. rend,, 90, 1112 (I8 8 0 ). of N0a , GO, 00s , Ha, Ns and a hydrocarbon (whose formula was given as OgH*) in the gases obtained by the Ignition of cellulose nitrate.

When cellulose nitrate was heated in the absence of air to the sudden evolution of gases and the resulting gases analyzed, Pfyl and Rasenaek (3 8 ) identified C0S , 00,

(38) B. Pfyl and Rasenaek, Arb. kals. G-esundheit- samt, 3 2 , 1 (1909)i C. A., 2* 2752 (1909).

Ns , NO, HGN and acrolein among the products. No hydro carbon, Ha or oxides of nitrogen other than NO were found in the gases. 21

In 1917, Trapani (39) reported the presence of

(39) E. Trapani, Attl accad. Lincel, 26, I, 332 (1917); C. A., 12, 368 (1918). formaldehyde in the products of ignition of cellulose nitrate. The author used Rimini's reagent (4o) to test

(4o) E. Rimini, Bull. eoc. chim., /"~3_7 2 0, 896 (1898). ~~ for formaldehyde.

The elucidation of a mechanism for the decomposition of cellulose nitrate, under conditions of a thermal nature, would be facilitated considerably if substances having carbon-carbon bonds could be isolated from the decomposition products. Yet, under most conditions, the decomposition of cellulose nitrate by the sole action of heat yields only gaseous products which provide little information as to the chemical nature of the intermediate products of decomposition. However, the conditions selected by

Wolfrom and co-workers (4-7) for the thermal decomposition of cellulose nitrate were such that substances having carbon-carbon bonds were found in the decomposition products, in the first investigation of a series,

Wolfrom and co-workers (4) dealt with the controlled thermal decomposition of cellulose nitrate, under Ignition

conditions, in the pressure range of 2-3 mm. propellant 22 cellulose nitrate (12.6$ N) was ignited at that pressure in order to remove the gaseous decomposition products rapidly from the high temperature zone. There was then left a solid residue which was characterized analytically and which on denitration and hydrolysis yielded cellobiose, ^-glucose, ]5-gluconic acid, D-erythrose and glyoxal. The results established the nature of the solid residue as a fragmented type of oxycellulose nitrate of an extremely low degree of polymerization.

Continuing their investigation, Wolfrom and co workers (5) found that increasing the pressure at which cellulose nitrate was ignited resulted in replacement of the solid residue by a liquid mixture. This mixture formed in maximum amount at ca. 30 mm. and decreased thereafter. It consisted mainly of water, formic acid, formaldehyde, hydrogen cyanide and glyoxal. The major organic constituents, formic acid, formaldehyde, hydrogen cyanide and glyoxal, were isolated as N-£-bromophenyl- formamide, formaldehyde dimethone, silver cyanide and glyoxal bis(phenylhydrazone), respectively. Quantitative assays were also devised for the four major organic products. By means of these methods of quantitative determination, Wolfrom and co-workers studied the formation of the major organic products as a function of increasing pressure. Thus, it was found that the amounts 23

of formic acid, formaldehyde and glyoxal attained a maximum below 60 mm. pressure and then decreased to

give a plateau in the pressure region of 200-500 mm.,

falling off rapidly thereafter with increasing pressure.

The fourth major organic product, hydrogen cyanide, was found in nearly equal amounts at all but the two

lowest pressures investigatddi.. The quantitative

assays for formic acid, formaldehyde and glyoxal were

also used to show that the amounts of these compounds

decreased inversely with the nitrogen content of the

cellulose nitrates employed.

The total free carbonyl content of the liquid

mixture was also Investigated (5)« The summation of

the amounts of carbonyl ascribed to formaldehyde and

glyoxal was lower, at all pressures, than the total

free carbonyl content as determined by the hydroxylamlne

hydrochloride method. For this reason, a .preliminary

investigation of the carbonyl compounds, other than

formaldehyde and glyoxal, was made, in this investi

gation the liquid mixture was derivatized with phenyl-

hydrazlne under conditions such that formaldehyde did

not form a derivative. The reaction product was

chromatographed on silicic acid. Several zones, aside

from that of glyoxal bis( phenylhydrazone), were present

in the chromatogram of the product and two of these

zones were identified as mesoxaldehyde 1,3-bis- 24

(phenylhydrazone) and triose phenylosazone. in their work, Wolfrom and co-workers (5) also Investigated the effect of the film casting solvent on the liquid mixture formed at 200 mm. pressure. The cellulose nitrate films used in their study had been cast from ethyl acetate or acetone, and it was estimated that ca.

5% of the casting solvent was retained by the films.

When acetone was used as the film casting solvent, acetone and acetic acid were found in the ignition products. When ethyl acetate was used, no acetone was found but a larger quantity of acetic acid was present and traces of ethanol were detected.

Wolfrom, Chaney and McWaln (6) later clarified, extended and correlated the previous work carried out in this Laboratory (4,5). Acetic acid and possibly ethanol were reported by these workers to be the only ignition products found which could be ascribed to traces of the residual ethyl acetate casting solvent in the cellulose nitrate sheets. Also, the previously reported (5) yield data for carbonyl compounds were shown to be slightly low as a result of o(-hydroxynitrlle formation. In addition, the overall carbon recovery from cellulose nitrate, taking into consideration all the products identified, was shown to be &9%» Finally,

Wolfrom, Chaney and MeWain (6) reignited, at 200 mm. 25 pressure, the solid oxycellulose nitrate which is afforded by the ignition of cellulose nitrate at

2 mm. pressure and obtained a liquid product mixture comparable in composition to that predicted for a liquid product mixture arising from the ignition of a cellulose nitrate of like nitrogen (9.5$) content.

In the most recent work in this series, Shafizadeh and wolfrom (7) reported on the radioassay of the products formed from the controlled ignition of cellulose-

C1^ nitrates predominantly labelled at Cl and C6 of the anhydro-D-glucose units. Their results indicated that

Cl gave mainly carbon dioxide and lesser amounts of

formic acid and glyoxal, and that the major product from

C6 was formaldehyde with lesser amounts of formic acid

and carbon dioxide. Preparation of 2,4-Dinltrophenvlhydrazlne Derivatives

Hydrazine and substituted hydrazines are frequently used in the characterization of carbonyl compounds, in this respect, 2 ,4-dinitrophenylhydrazine has received considerable attention since It forms derivatives which are highly insoluble and crystallize relatively easily.

Since there are as many methods of derivatizing carbonyl compounds with 2,4-dinitrophenylhydrazine as there are researchers who use this reagent, no attempt shall be made to list all modes of preparation. The terms ‘'hydrazone1' and *osazone* will be used for the sake of brevity and with the understanding that they refer to derivatives of

2,4-dinltrophenylhydrazine.

The discovery of 2 ,4-dinitrophenylhydrazine as a reagent for carbonyl compounds was made in 1894 by

Ourtius and Dedichen (4l) and by Purgotti (42).

(41) T. Curtius and G-. M. Dedichen, J. prakt. Chem., £ 2 J 5 0 , 24l (1894).

(42) A. Purgotti, G&zz. chim. ital., 24, 1554 (1894); J. Ohem. Soc., A68, 27 (1895).

They (41,42) described a few hydrazones of aliphatic and aromatic aldehydes and ketones. Several decades passed before the usefulness of the reagent became recognized. Bulow (43) suggested the detection of small

(43) C. Bulow, Science, 61, 344 (1925).

26 27 amounts of acetone by formation of its hydrazone in alcoholic hydrochloric acid. The preparation of 2,4- dinitrophenylhydrazones in boiling alcohol or in aqueous hydrochloric acid was reported by Brady and Elsmie (44).

(44) 0. L. Brady and Gladys V. Elsmie, Analyst, 5 1 , 77 (1926).

A large number of investigators published their work on the reaction of 2,4-dinitrophenylhydrazine with carbonyl compounds in the 1930's. Allen (45) selected alcoholic

(45) 0. F. H. Allen, J. Am. Chem. Soc., 5 2 , 2955 (1930). hydrochloric acid as the reaction medium for preparing many 2,4-dinitrophenylhydrazine derivatives. Later, Allen and Richmond (46) applied Allen's method (45) and prepared

(4$) C. F. H. Allen and j. H. Richmond, J. Org. Chem., 2, 223 (1937). several new hydrazones. The use of 2 ,4-dinitrophenylhydrazine dissolved in alcoholic sulfuric acid was intro duced by Brady (47) who thus prepared many derivatives,

(47) 0. L. Brady, J. Chem. Soc., 756 (1931) 28 three of which could not he prepared using Allen's method (45). Later, Ferrante and Bloom (48) suggested

(48) J. Ferrante and A. Bloom, Am. J. Pharm., 1 0 5 , 381 (1933). the replacement of ethanol by methanol in Brady's method

(47), Campbell (49), having observed literature melting

(49) N. R. Campbell, Analyst, 61, 391 (1936). point discrepancies of as much as 2o degrees for a single hydrazone, prepared by the method of Brady (47) a large number of known 2,4-dinitrophenylhydrazine derivatives and published a revised melting point table.

Some hydrazones were prepared by Collatz and

Neuberg (50) who used a supersaturated solution of 2,4-

(50) H. Collatz and Irene S. Neuberg, Blochem. Z., 255. 27 (1932). dlnitrophenylhydrazine in 2 N hydrochloric acid at 0° as reagent solution. The use of 2,4-dinitrophenylhydrazine dissolved in aqueous sulfuric acid was suggested by Torres and Brosa (51) and their reagent was used by Houghton (52),

(51) C. Torres and S. Brosa, Anales soc. espan. fis. y qufm., 5 1 , 34 (1933). (52) R. E. Houghton, Am. J. Pharm., 106, 62 (1934). 29

Perkins and Edwards (53) for the quantitative determi

(53) (j . W. Perkins and M. W. Edwards, Am. J. Pharm., loj, 2o8 (1935). nation of ketones and aldehydes. Some classical work concerning the quantitative, gravimetric determination of water-3oluble carbonyl compounds was done by Iddles and Jackson (54) who used a saturated 2 N hydrochloric

(54) H. A. Iddles and C. E. Jackson, Ind. Eng. Chem., Anal. Ed,, 6 , 454 (1934). acid solution of 2,4-dinitrophenylhydrazine. The quantl tative determination of water-insoluble carbonyl com pounds was investigated by iddles and co-workers (5 5 ).

(55) H. A. Iddles, A. W. Low, B. D. Rosen and R. T. Hart, Ind. Eng. Chem., Anal. Ed., 11, 102 (1939).

The above references merely give a glimpse of the enormous amount of research carried out in the 1 9 3 0 's on 2,4-dinitrophenylhydrazine. To these we might add the investigations of Cowley and Schuette (56), Strain (57)»

(56) M. A. Cowley and H. A. Schuette, J. Am. Chem. Soc., 5 5 , 3463 (1933).

(57) H. H. Strain, J. Am. Chem. Soc., 5£, 758 (1935). 30 and Roduta and Quibllan (58)*

(58) F. L. Roduta and G-. Quibllan, Rev. filiplna med. farm., 2£, 123 (1936); 0. A., £1, 985 (1937).

In the past decade, new methods of preparing deriva tives of 2,4-dinitrophenylhydrazine have been suggested.

Johnson (59) used an ethanol-phosphoric acid solution of

(59) G. D. Johnson, j. Am. Chem. Soc., 73, 5888 (1951). the reagent, instead, Thornton and Speck (6 0 ) preferred

(6 0 ) Barbara J. Thornton and J. C. Speck, Jr., Anal. Chem., 22, 899 (1950). a solution of 2,4-dinitrophenylhydrazine in aqueous per chloric acid and this new reagent solution was later

Introduced independently by others (6l).

(61) C. Neuberg, A. G-rauer and B. V. Pisha, Anal. Chlm. Acta, £, 238 (1952).

Some 2,4-dinltrophenylhydrazones were prepared in reflux ing pyridine by Braude and Timmons (62). The reaction

(62) E. A. Braude and C. J. Timmons, J. Chem. Soc., 3131 (1953). product (62) had to be purified by chromatography, a fact 31 which is not surprising in view of the instability of

2,4-dinitrophenylhydrazine in bases (57)• Recently,

Braddock and Willard (63) prepared hydrazine (H3NNH3)

(63) L. I. Braddock and M. L. Willard, J. Org. Chem., 18, 313 (1953). derivatives of aldehydes and ketones, and reacted these derivatives with 2,4-dinltro-chlorobenzene to form 2,4- dinitrophenylhydrazones.

The work which has been reviewed so far has dealt mostly with the reaction of 2,4-dinitrophenylhydrazine with relatively simple aldehydes and ketones. The 2,4- dinitrophenylhydrazones of more complex organic compounds have been prepared as well. Camphor and its derivatives are known to react with 2,4-dinitrophenylhydrazine (64,65).

(64) 0. Fernandez, L. Soc^as and C. Torres, Anales soc. espan. flTs. y qufm., ^0, 37 (1932).

(6 5 ) 0. Fernandez and Manuela Castillo, Anales soc. espan. ffs. y qu£m., jg, 81 (1935).

Steroid ketone 2,4-dinitrophenylhydrazones have been prepared (6 6 ,6 7 ).

(66) E. R. H. Jones, P. A. Wilkinson and R. H. Kerlogue, j. Chem. Soc., 391 (1942). (6 7 ) H. Reich, K. F. Crane and S. J. SanfHippo, J. Org. Chem., 18, 822 (1953). 32

In spite of reports to the contrary (45,4 7 ,5 7 ,6 8 ,

6 9 ), both hydrazones and osazones of "(-hydroxy carbonyl

(68) W. H. Linnell and I. Roushdi, Quart. J. Pharm. Pharmacol., 12, 252 (1939).

(6 9 ) T. Banks, 0. Vaughn and L. M. Marshall, Anal. Chem., 21, 1348 (1935).

compounds and sugars can be prepared provided the proper

2,4-dinitrophenylhydrazine reagent medium is UBed. The

preparation of sugar osazones, first reported by G-lazer

and Zuckermann (70), was investigated by Rabassa (7 1 ) and

(70) E. Glazer and N. Zuckermann, Z. physiol. Chem., 167. 37 (1927). (71) S. B. Rabassa, Rev. acad. cienc. Madrid, 51. 417 (1934); C. A., 2g, 28773 (1935).

by Neuberg and Strauss (72 ). The 2,4-dinitrophenylhydrazine

(72) C. Neuberg and E. Strauss, Arch. Biochem., 11, 457 (1946).

reagent solution suggested by Collatz and Neuberg (50) was

used by them and by others (7 3 ) in the preparation of

(73) D. B. Sprinson and E. Chargaff, j. Biol. Chem., 164, 417 (1946).

2,4-dinitrophenylbydrazones of low molecular weight 33 c< -hydroxy carbonyl compounds. The reaction of some sugars with 2,4-dinitrophenylhydrazine in boiling ethanol was shown by G-lazer and Zuckermann (70) to afford the hydrazone. The use of boiling ethanol in the preparation of 2,4-dinitrophenylhydrazones of hexoses and pentoses was further studied by Lloyd and Doherty (74). Reich and

(7^) E. A. Ll.ovd and D. G-. Doherty, J. Am. Chem. Soc., 24, 4214 (1952).

Samuels (7 5 ) have further shown that refluxing trioses

(75) H. Reich and Barbara K. Samuels, J. Org. Chem., 21, 68 (1956). with 2,4-dinitrophenylhydrazine in ethanol results in the formation of hydrazones.

Using equimolecular amounts of reagent and -hydroxy carbonyl compound in acidic solution, Reich and Samuels

(7 5 ) have reported that, on standing at room temperature, some amount of osazone is formed. Therefore, the prepara tion of the 2,4-dinitrophenylhydrazine derivative of

-hydroxy carbonyl compounds in perchloric acid solution, as suggested by Neuberg, G-rauer and Pisha (61), would undoubtedly lead to the formation of some osazone.

Occasionally, sugar 2,4-dinltrophenylhydrazones do not crystallize and form a gel-like mass instead (7^»7 6 ,7 7 ). 34

(76) L. M. White and Geraldine E. Secor, J. Am. Chem. soc., 6343 (1953).

(77) L. H. White and Geraldine E. Secor, Anal. Chem., 2£, 1016 (1955).

Solvates of hexose 2,4-dinitrophenylhydrazones have been reported (76,77).

Dicarbonyl compounds do not always yield a bis derivative of 2,4-dinitrophenylhydrazine. However, by a suitable choice of conditions, Reich and Hefle (78) were

(78) H. Reich and L. Hefle, jr., J. Org. Chem., 21. 708 (1956). able to prepare the mono 2,4-dinitrophenylhydrazones of some dicarbonyl compounds. -Dicarbonyl compounds usually form pyrazoles in the presence of 2,4-dinitro phenylhydrazine (47,79-81) although the bis derivative of

(79) J. W. Copenhaver, U. S. Patent 2,515,160.

(80) J. W. Copenhaver, U. S. patent 2,527,533.

(81) E. W. Malmberg, J. Am. Chem. Soc., J6, 980 (1954). malonaldehyde has been prepared (82),

(82) E. Rothstein, J. Chem. Soc., 1553 (194o). 35 Side reactions sometimes take place in the course of preparing 2,4-dinitrophenylhydrazine derivatives.

2 ,4-Dinltrophenylhydrazine monoacetate has been isolated as a by-product of the formation of 2,4-dinitrophenyl hydrazine derivatives in chloroform-acetic acid (57,67).

In some cases, the reaction medium chosen has resulted in esterification of hydroxyl groups (6 7 ) and acid groups

(46,57) present in the carbonyl compounds being derivatized.

The reaction of aldehydes and ketones with 2,4-di nitrophenylhydrazine at times leads to reaction products in which the carbonyl residue is different from the starting material. -Hydroxy carbonyl compounds tend to dehydrate easily in an acidic medium and afford -nnfeat urated 2,4-dinitrophenylhydrazones (46). 2-Kethoxycyclo- hexanone has been reported to afford a 1,2-bis(2,4-dinitrophenylhydrazone) (8 3 ). -Halo cyclic ketones may react

(8 3 ) H. Adkins and A. G-. Rossow, J. Am. Chem. Soc., II, 3836 (1949). to yield a 2 ,4-dinitrophenylhydrazone (84), a dehydrohalo-

(84) F. Ramirez and A. F. Kirby, J, Am. Chem. Soc., lit, 4331 (1952). genated 2,4-dinitrophenylhydrazone (84,85) or a 1,2-bis-

(8 5 ) C. Djerassl, J. Am. Chem. Soc., II, 1003 (1949). 36

(2,4-dinitrophenylhydrazone) (84).

The formation of hydrazides of 2,4-dinitrophenyl hydrazine has been studied by Cerezo and Olay (86).

(86) J. Cerezo and E. Olay, Anales soc. espan. ffs. y qufm., ^2, 1090 (1934).

There are methods available for the determination of total carbonyl by reaction with 2,4-dinitrophenyl hydrazine. These methods are based on the quantitative reaction of carbonyl compounds in the presence of excess

2,4-dinitrophenylhydrazine. The excess reagent is determined by azotometry (87)» by polarography (88)or by

(87) M. Yamagishi, M. Yokoo and S. Inoue, J. Pharm, Soc. japan, 25, 351 (1955); C. A., 4g, 9447® (1955).

(88) L. N. Petrova and E. N. Novikova, Khur. Priklad. Khim., 28, 219 (1955); C. A., 4£, 7448n (1955). titration with chloramine-T (8 9).

(89) A. Berka and.J. Zyka, Chem. Listy, ^0, 831 (1956); C. A., 50, 10111 (1956). Chromatogra-phv of 2.4-Dlnltror>henylhydrazine Derivatives

Studies on the chromatographic separation of 2,4- dinitrophenylhydrazine derivatives began soon after the reagent gained recognition. Strain (57) tested the suitability of several adsorbents with sets of known hydrazones: he reported that basic adsorbents such as magnesium oxide should not be used since they decompose

2 ,4-dlnitrophenylhydrazones.

Adsorption chromatography of some 2,4-dinitrophenyl hydrazine derivatives on magnesium sulfate was reported by

Stadtman (90). Wahhab separated hydrazones of furfural

(90) F. H. Stadtman, J. Am. Chem. Soc., 70, 3583 (1948). and its derivatives on the same adsorbent as well as on talc (91)* The 2,4-dinitrophenylhydrazine derivatives

(91) A. Wahhab, J. Am. Chem. Soc., £0, 3580 (1948). of carbonyl compounds present in rancid corn and avocado oils were separated on magnesium sulfate (9 2 ).

(92) J. Brekke and G-. Mackinney, J. Am. Oil Chemists' Soc., 2J, 238 (1950).

A large number of derivatives of 2 ,4-dinltrophenyl hydrazine was separated on bentonite (93»94).

37 38

(93) J. W. White, Jr., Anal. Chem., 2o, 726 (1948).

(94) J. A. Elvldge and Margaret Whalley, Chem. and Ind., 589 (1955).

The hydrazones of carbonyl compounds afforded by the nin- hydrin oxidation of amino acids were separated on zinc carbonate (95). The separation of 2,4-dinitrophenylhydra-

(95) F. Turba and E. v. Schrader-Beielstein, Naturwiss., J54, 57 (1 9 4 7 ). zones on calcium sulfate has also been reported (9 6 ).

(96) F. Sorm, M. Suchy and y. Herout, Chem. Llsty, 46 * 55 (1952); C. A., 46, 11134° (1952).

Alumina has frequently been used to separate 2,4- dinitrophenylhydrazine derivatives. The use of commercial

activated alumina is not recommended because of its alkali content (94). Chromatographic purification of

osazones on alumina (9 7 ) and of a steroidal hydrazone on

(97) J. R* Penney and s. S. Zilva, Blochem. J., JL> 403 (1943).

Florisll and alumina (9 8 ) have been reported. Hydrazones

(98) F. P. veitch and H. S. Mllone, J. Biol. Chem., 1^8 , 61 (1945). 39

of keto-steroids (99,100), aliphatic aldehydes and

(99) C. D. Johnston, Science, 106, 91 (1947).

(100) G. Zwingelstein, H. Pacheco and J. Jouanneteau, Compt. rend., 236. 1561 (1953).

ketones (101), and keto-aclds (102, 103) have been

(101) H. Adkins and G. Krsek, J. Am. Ghem. Soc., II, 3051 (1949).

(102) S. P. Datta, H. Harris, K. R. Rees, Biochem. J., 46, xxxvi (1950).

(103) K. Lohr, Biochem. Z., 52q . 115 (1950).

separated on alumina.

Silicic acid is a useful adsorbent for the separation

of derivatives of 2,4-dinitrophenylhydrazlne. The first

study in the use of silicic acid for this purpose was

reported by Roberts and Green (104). ozonization products,

(104) J. D. Roberts and Charlotte Green, Ind. Eng. Ghem., Anal. Ed., 18, 335 (1946).

after derivatlzation with 2,4-dinitrophenylhydrazine, were

separated on silicic acid by Carson (105). Reporting on

(105) J. F. Carson, J. Am. Chem. Soc., £5, 4652 (1951).

the analysis of mixtures of aldehydes and ketones, Gorden

and co-workers noted the difficulty of obtaining sharp

melting points from hydrazones Isolated by chromatography 4o on silicic acid (106). A study on the separation of

(106) B. E. Gorden, P. Wopat, jr., H. D. Burnham and L. C. Jones, Jr., Anal. Ohem., 2£, 1754 (1951).

2,4-dinitrophenylhydrazones of substituted benzaldehydes on silicic acid has been published (107). Silicic acid

(107) A. A. Rosen, K. V. Y. Sundstrom and W. F. Vogel, Anal. Ohem., 24, 412 (1952). was used by Malmberg to separate aliphatic aldehyde and ketone hydrazones and bis hydrazones (81). Malmberg also used hydrazones and bis hydrazones in his study on the effect of particle size on the chromatographic character istics of silicic acid (1 0 8 ).

(108) E. W. Malmberg, Anal. Ohem., 2£, 84o (1955).

Since several keto-acids are of biological importance, the separation of their hydrazones by paper chromatography, a method which is ideally suited to micro quantities of substances, has been the subject of much research.

Cavallini and co-workers have pioneered in this field

(1Q9-113). They are, however, by no means the only

(109) D. Cavallini, Nora Frontali and G. Toshl, Nature, 16^, 568 (1949). 41

(llo) D. Cavallini, Nora Frontali and G. Toshi, Boll, soc. ital. biol. sper., 2^, 286 (1949); C. A., 44, 8329 (1950).

(111) D. Cavallini, Nora Frontali and G. Toshi, Nature, 164, 792 (1949).

(112) D. Cavallini and Nora Frontali, Ricerca sci., 2^, 807 (1953); 0. A., 4£, 9863d (1953).

(113) D. Cavallini and Nora Frontali, Bioohem. Biophys. Acta, 1 5 , 439 (1954).

researchers in the field (114-118). Other workers have

(114) S. M. Altmann, E. M. Crook and 3 , p. Datta, Biochem. j., 42, lxiii (1951).

(115) D. Seligson and B. Shapiro, Anal, Chem., 24, 754 (1952).

(1 1 6 ) M. P. S. El Hawary and R. H. S. Thompson, Biochem. j., 340 (1953).

(117) A. I. Virtanen, J. K. Mlettinen and H. Kunttu, Acta Chem. Scand., J, 38 (1953).

(118) F. A. Isherwood and D. H. Crulckshank, Nature, 173, 121 (1954). reduced keto-acid 2,4-dinitrophenylhydrazones and chroma tographed the resulting amino acids on paper (119-121).

(119) G. H. N. Towers, J. F. Thompson and F. C. Steward, j. Am. Chem. Soc., J6t 2392 (1954).

(120) E. Kulonen, Scand. J. Clin. Lab. Invest., 5, 72 (1953); C. A., 41, 7573® (1953).

(121) A. Meister and Patricia A. Abendschein, Anal. Chem., 28, 171 (1956). 42

The hy&razone of a keto-acid often gives rise to two spots on a paper chromatogram and this phenom enon has been ascribed to the presence of geometrical isomers (112-115, 117-119). Towers and Mortimer (122)

(122) Gr. h . N. Towers and D. C. Mortimer, Nature, 174, 1189 (1954). have shown that this ready explanation is at fault in some caseB since l-hydroxy-6-nitro-l,2,3-benzotriazole may arise in the keto-acid hydrazone isolation procedure.

Paper chromatographic separation of non keto-acid

2,4-dinitrophenylhydrazones and bis(2,4-dinitrophenyl hydrazones) have also been reported (123-127).

(123) r. G-. Rice, a. J. Keller and J. G-. Kirchner, Anal. Ghem., 2^, 194 (1951).

(124) D. F. Meigh, Nature, lJO, 579 (1952).

(125) v. Sykora and Z. Prochazka, ohem. Listy, 47. 1674 (1953); 0. A., 48* 3852® (1954).

(126) D. A. Forss, S. A. Dunstone and W. Stark, Chem. and Ind., 1292 (1954).

(127) w. J. Schmitt, E. J. Moriconl and W. F. O'Conner, Anal. Chem., 28, 249 (1956).

Kirchner and Keller have used silicic acid impreg nated paper strips for the separation of a few methyl ketone hydrazones (128). The partition chromatography of 43

(128) J. G-. Kirchner and G. J. Keller, J. Am. Chem. soc., J2, 1867 (1950).

2,4-dinitrophenylhydrazine derivatives of a few alde hydes, ketones, keto-acids and dicarbonyl compounds has been studied with acetylated filter paper as inert support for the stationary phase (1 2 9 ). 2,4-Dinitro-

(129) J. V. Kost£r and K. Slavfk, Collection Czechoslov. Chem. Communs., 1£, 17 (1 9 5 0 ). phenylhydrazones have been separated by Seligman and

Edmonds (130) on olive oil impregnated filter paper, by

(130) R. B. Seligman and M. D. Edmonds, Chem. and Ind., 1406 (1955).

Lynn, Steele and Staple (131) on paper impregnated with

(131) W. S. Lynn, jr., Lois A. Steele and E. Staple, Anal. Chem., 28, 132 (1956). phenoxyethanol, and by Klein and de Jong (132) on paraffin

(132) K. Klein and K. de Jong, Rec. trav. chlm., IS* 1285 (1956).

oil impregnated filter paper. Reverse phase partition

chromatography on paper was used by Hattori (133) to 44

(133) Y. Hattori, J • Sci. Research Inst. (Tokyo), 5Q, 51 (1956). separate the hydrazones of eight straight chain alde hydes and six methyl ketones.

Column partition chromatography of aliphatic 2,4- dinitrophenylhydrazones has been reported by Howard and

Tatchell (134) and by Kramer and van Duin (135)*

(134) G. A. Howard and A. R. Tatchell, Chem. and Ind., 219 (1954).

(135) Miss p. j. G. Kramer and H. van Duin, Rec. trav. chlm., 63 (1954).

2,4-Dinitrophenylhydrazones of <*(-keto-acids have been separated by paper electrophoresis and the spots quantitatively determined by colorimetry (136) or by

(136) H. Tauber, Anal. Chem., 2£, 287 (1955). polarimetry (137,138).

(137) W. J. P. Neish, Rec. trav. chlm., 72, 105 (1953). (138) W. J. P. Neish, Rec. trav. chlm., J2t 1098 (1953). Characterization and Identification of 2.4-plnltro- phenylhydrazlne Derivatives

A. Ultraviolet and visible spectrum

A comprehensive collection of light absorption data for derivatives of 2 ,4-dinitrophenylhydrazine is available. The first report in this field was published by Braude and Jones who recorded the ultraviolet and visible spectra data for some fifty derivatives of carbonyl compounds of widely different character (139).

(139) E. A. Braude and E. R. H. Jones, j. Chem. Soc., 498 (1945).

Specific studies concerning 2,4-dinitrophenylhydrazones of keto-sterolds (l4o), of keto-acids (141), of aromatic

(140) C. Djerassi and Elizabeth Ryan, J. Am. Chem. Soc., 21, 1000 (1949).

(141) D. Cavallini, Ricerca sci., 20, 803 (1950)j C. A., 45, 3295f (1951). ketones (142,143) and aldehydes (143), of flavanones (144),

(142) H. H. Szmant and H. J. Planinsek, J. Am. Chem. Soc., J2, 4o42 (1950).

(143) L. A. Jones, J. C. Holmes and R. B. Seligman, Anal. Chem., 28, 191 (1956).

(144) C. D. Douglass, Q. L. Morris and S. H. Wender, J. Am. Chem. Soc., 22> 4023 (1951).

45 of aliphatic aldehydes (106,14-3,14-5) and ketones (106,143),

(14-5) E. E. Lockhart, M. C. Merritt and c. D. Mead, J. Am. Chem. S o c , , . 858 (1951). of olefinic aldehydes (143) and ketones (143,146), of

(146) R. Heilmarm and R. G-lenat, Compt. rend., £2 4 , 1557 (1952). terpene aldehydes and ketones (147), and of furanic

(147) A. Duplessis-Kergomard and C. Sandris, Bull, soc. chlm. France, 1260 (1954). aldehydes and ketones (143) have been reported.

The light absorption spectrum of a derivative of

2 ,4-dinitrophenylhydrazine is important because it bears a direct relationship to the structure of the parent carbonyl compound, it has been pointed out by several authors that the wavelength position of the main maximum in the extinction curve varies significantly with the structure of the compound studied and that subsidiary bands show only slight correlation with the nature of the parent carbonyl compound (139,148). Thus, we shall be

(148) C. J. Timmons, j. Chem. Soc., 2613 (1957). 47 concerned only with the main absorption band, to illustrate the correlation mentioned above, the findings of Roberts and Green (149) may be summarized: the

(149) J. D. Roberts and Charlotte Green, J. Am. Chem. Soc., 68, 214 (1946). absorption maxima of 2,4-dinitrophenylhydrazones with no double bonds in conjugation with the imino linkage, with one double bond or unsubstituted aromatic ring in conju gation with the imino linkage, and with aromatic rings conjugated with the imino linkage fall in the range

349-363 mjJL , 377-379 mjx and 394-395 mjx » respectively.

A brief examination of Johnson's data is sufficient to show that the siibjeot is far more complex than the above example would lead one to believe (150).

(150) G. D. Johnson, J. Am. Chem. Soc., 2720 (1953).

The ultraviolet and visible spectra of 2,4-dinltrophenylhydrazine derivatives have been observed (139,140,

149) to show variations with a change of solvent. These variations are limited to small shifts in the wavelength

of maximum absorption and to slight changes in the molar

extinction coefficient. The subject has been studied at

length by Timmons (148).

Bis(2,4-dinitrophenylhydrazones) show normal 48

absorption (below ca. 360 mjx ) (139) except in the

case of o(-diketones (139,151) and conjugated alkene-

diones (152), where conjugation occurs between two hydra-

(151) A. L. Lehnlnger, J. Biol. Ohem., 149. 43 (1943).

(152) K. G. Lewis, J. Ohem. Soc., 1083 (1956).

zone systems and the position of the main maximum increases

to 400-450 m^x . Bis hydrazones of alicycllc -diketones

show a deviation from the above rule (153).

(153) F. Ramirez and R. J. Bellet, J. Am. Ghem. Soc., I6, 491 (1954).

The effect on the light absorption spectra of

derivatives of 2,4-dinltrophenylhydrazine of the addition

of alkali has been studied. In all cases, the wavelength

of the main maximum in neutral solution is shifted towards

the visible by adding alkali (143,149). The color change

in the presence of base has been shown to take place

only when an N-hydrogen is present (154-156), It is

(154) F. D. Ohattaway and a. R. Olemo, J. Chem. Soc., 122, 5041 (1923).

(155) F. Bohlmann, Ber., 84, 490 (1951).

(156) L. A. Jones, Abstracts papers Southwest Reg ional Meeting Am. Chem. Soc., Tulsa, Okla., Dec. 5-7, 1957, Paper no. 33. 49 therefore probable that 2,4-dinitrophenylhydrazones behave as weak acids in the presence of base and that the absorption observed in basic solution is due to the conjugate base of this acid (156),

2,4-Dlnitrophenylhydrazones and l,2-bis(2,4- dinitrophenylhydrazones) or 2,4-dinltrophenylosazones can be visually differentiated by dissolving them in alcoholic alkali. Hydrazones give a reddish-brown color with alkali, whereas osazones or 1,2-bis-hydrazones give a blue-violet color (157). Malmberg was thus able to

(157) C. Neuberg and E. Strauss, Arch. Biochem., X, 211 (1945). differentiate between monocarbonyl and «C,/3 -dicarbonyl compounds by streaking with base on the extruded chroma togram of a mixture of their 2,4-dlnltrophenylhydrazine derivatives (81)• Since flavanone 2,4-dinitrophenyl hydrazones give a violet color with alkali (144), the color differentiation discussed above should be limited to simple derivatives of 2,4-dinitrophenylhydrazine.

2,4-EJlinltrophenylhydrazine derivatives adhere strictly to the Lambert-Beer Law (106). They are there fore useful derivatives for the quantitative determination of carbonyl compounds (106,158,159).

(158) G. R. Lappin and L. C. Clark, Anal. Chem., 22, 541 (1951). 50

(159) A. S. Henick, M. P. Benoa and J. H. Mitchell, jr., J. Am. Oil Chemists' Soc., 31, 88, 447 (1954).

B. Infrared spectrum

Ross has suggested the use of the infrared spectra

of 2,4-dinitrophenylhydrazones for identification pur poses (160). He noted that the infrared spectra of

(16o) J. H. Ross, Anal. Chem., 2£, 1288 (1955). hydrazones of aldehydes and ketones are unique in that even closely related carbonyl compounds which differ

only by one methylene group exhibit markedly different

spectra. He further observed that a 2,4-dinitrophenylhydrazine derivative which contained as much as 10 per cent of another 2,4-dinitrophenylhydrazine derivative

could still be readily identified by infrared spectra.

The primary value of the infrared spectra of hydra

zones is in their use as “fingerprint'' references for

comparison with unknown spectra. However, some inform ation concerning the nature of the parent carbonyl

compound can be obtained from an examination of the

infrared spectrum of an unknown 2,4-dlnitrophenylhydrazine

derivative. For Instance, the spectra of crystalline

2 ,4-dinltrophenylhydrazine derivatives of aromatic

aldehydes and ketones exhibit four distinguishing bands

of nearly equal intensity in the region 13-15 microns 51 (16o). The position of the N-hydrogen stretching hand

has also been found to be indicative of the aldehydic

or ketonic nature of the parent carbonyl compound (143).

Olefinic and furanlc derivatives of 2,4-dinitrophenyl-

hydrazlne also have characteristic bands which facilitate

their identification (143).

Different ‘’forms" of acetaldehyde 2,4-dinitrophenyl

hydrazones have been shown to have different Infrared

spectra in Nujol (l6o). The syn and anti Isomers of 2-

furaldehyde 2 ,4-dinitrophenylhydrazone afforded dis

similar infrared spectra in potassium bromide (143).

A study of geometrical isomerism and absorption spectra

of some forty 2,4-dinitrophenylhydrazine derivatives has

been made and has revealed a method of assigning a syn or

anti configuration to the isomers (l6l). Isherwood and

(161) F. Ramirez and A. F. Kirby, J. Am. Chem. Soc., 26, 1037 (1954).

Jones have Interpreted the Infrared spectra of different

’‘forms*' of keto-acid 2,4-dinitrophenylhydrazones in terms

of syn-anti isomerism (162).

(162) f. A. isherwood and R. L. Jones, Nature, 175. 419 (1955). 52

C. Melting point

2 ,A—Dinitrophenylhydrazine derivatives are crys

talline compounds of high melting point, in general,

the hydrazones have sharp melting points below 225°,

whereas the bis hydrazones or the osazones melt with

decomposition above 225°. The melting point of a deriva

tive of 2,4-dinitrophenylhydrazine, as reported by diff

erent researchers, may vary by as much as twenty degrees

(49). The conflicting data recorded in the literature on

the hydrazones and osazones of 2 ,4-dinitrophenylhydrazine

may be ascribed to the extremely high melting point of

the osazones, to the possible occurrence of geometrical

isomerism, to polymorphism and to mixed crystal formation.

Brandstatter studied eleven aldehyde and ketone

hydrazones by means of Kofler's method of microthermal

analysis, and found that pight hydrazones showed complete

series of mixed crystals (163). Other cases of isomor-

(163) Maria Brandstatter, Mikrochemle ver Mikrochim. Acta, 3 2 , 33 (1944).

phism have been reported by Matthiessen (164).

(164)

As early as 1932, Bredereck presented evidence of the existence of two "forms" of the hydrazones of a 53 number of furfural derivatives (165). The two "forms"

(165) H. Bredereck, Ber., 65B. 1833 (1932). were red or yellow In color, had the same composition and molecular weight but dissimilar melting points which depressed each other's. The yellow "form” could be con verted to the more stable red "form" in alcoholic hydro chloric acid. Bredereck interpreted these results in terms of syn-antl isomerism. Braddock and associates

(166) were able to separate Bredereck's (1 6 5 ) isomers of t

(166) L. I. Braddock, K. Y. Garlow, L. I. Grim, A. F. Kirkpatrick, S. W. Pease, A. J. pollard, E. F. Price, T. L. Reissmann, H. A. Rose and M. L. Willard, Anal. Ohem., 2 5 , 301 (1953). furfuraldehyde by chromatography on bentonite-silica gel.

The number of geometrically isomeric hydrazones which have been reported is large (167-169) and it will

(167) W. M. D. Bryant, J. Am. Chem. Soc., 6 0 , 2815 (1938).

(168) W. Dirscherl and h . Nahm, Ber., 73B, 448 (1940).

(169) F. Ramirez and A. F. Kirby, J. Am. Chem. Soc., Z5, 6026 (1953). suffice to outline but one other case. Van Duin (170)

(170) H. van Duin, Rec. trav. chim., J8, 73 (1954). 54

reported the existence of two "forms*' of the 2,4-

dinitrophenylhydrazones of -keto-acid esters. These

isomers could be differentiated by melting point, ultra

violet spectra and retention volume in partition chro matography and they were interconvertible in the presence

of acid.

It is sometimes difficult to differentiate between polymorphism and isomerism of two "forms" of a 2,4- dinltrophenylhydrazone because of the limited physical

evidence presented (171). When two "forms" of a compound

(171) H. Brunner and E. H. Farmer, J. Chem. Soc., 1039 (1937). which cannot exist in isomeric modifications are reported, we are clearly faced with a case of polymorphism (166,172).

(172) G. L. Clark, W. I. Kaye and T. D. parks, Ind. Eng. Chem., Anal. Ed., 18, 310 (1946).

There are other cases which have been interpreted likewise

since more than 2 "forms" of some 2,4-dinitrophenylhydra

zones are known (163,173).

(173) L. I. Braddock and Mary L. Willard, Mikrochemie, 4o, 305 (1953). 55

D. Miscellaneous methods

The methods of optical crystallography have been

used by Bryant for the identification of aliphatic

aldehyde 2,4-dinitrophenylhydrazones (174). The appli-

(174) W. M. D. Bryant, J. Am. Ohem. Soc., 54. 3758 (1932).

cation of optical methods to the identification of 2,4- dinitrophenylhydrazine derivatives has also been dis

cussed by Mitchell (175).

(175) J. Mitchell, jr., Anal. Ohem., 21, 448 (1949).

The use of X-ray powder patterns for characterization

and identification of 2,4-dinitrophenylhydrazine deriva

tives was introduced in 1946 (172,1 76). The melting

(176) J. J. de Lange and J. P. W. Houtman, Rec. trav. chim., 6 5 , 891 (1946).

points of the 2,4-dinitrophenylhydrazones of a homologous

series of normal aliphatic aldehydes were found to be too

close for identification, whereas the long X-ray spaclngs

(powder patterns) were suitable for that purpose (177)•

(177) T. Malkin and T. 0. Trauter, J. Ghem. Soc., 1178 (1951). 56

X-ray powder patterns are particularly useful for the characterization of the several "forma” of some hydra zones (1 0 6 ,172). DISCUSSION OF RESULTS

Thermal Decomposition of Cellulose Nitrate

The controlled thermal decomposition of cellulose nitrate, under ignition conditions at reduced pressure, has been investigated by Wolfrom and co-workers (4-7).

These researchers found that the white solid (a nitrated oxycellulose of low degree of polymerization) obtained in the pressure range of 2-3 mm. (4) was succeeded by a liquid mixture as the pressure was raised (5)» This mixture consisted mainly of water, formic acid, formal dehyde and glyoxal. The aqueous solution of this liquid mixture shall hereafter be referred to as ''condensate aqueous solution."

The total free carbonyl content, determined by the hydroxylamine hydrochloride method, of the condensate aqueous solution was studied in this Laboratory as a function of the pressure at which cellulose nitrate was ignited (1 7 8 ). The pressure dependency of formaldehyde,

(178) See. reference 5 , Figure 5 . quantitatively assayed as formaldehyde dimethone, and of glyoxal, quantitatively assayed as glyoxal bis(phenylhydrazone), formation from cellulose nitrate was also investigated by Wolfrom and co-workers (1 7 9 ). At all

57 (179) See reference 5, Figure 3. pressures investigated, the total free carbonyl content of the condensate aqueous solution was larger than the sum of the amounts of carbonyl which could be ascribed to the presence of formaldehyde and glyoxal in the solution (1 7 8 ). Since it was the purpose of the present investigation to isolate and identify all carbonyl com pounds present in the condensate aqueous solution and since formaldehyde and glyoxal were already well-known components of this solution, it was desirable to carry out the cellulose nitrate ignition under conditions such that the largest possible proportion of unknown carbonyl compounds would be found in the condensate aqueous solution.

A primary consideration in this respect was the ignition pressure, since Wolfrom and co-workers (178) had shown that the formation, from ignited cellulose nitrate

(13.4$), of carbonyl compounds other than formaldehyde and glyoxal was nearly constant (within ca. 1C$) at all pressures investigated below 75 mm. but decreased steadily with increasing pressure above 75 ®nu Thus, In our work, cellulose nitrate was ignited at 75 mm. pressure.

Still another consideration in securing a condensate aqueous solution containing the largest possible 59 proportion of unknown carbonyl compounds was the nitrogen content of the cellulose nitrate ignited, since previous workers (180) had shown that the amounts

(180) See reference 5, Figure 4. of formaldehyde and glyoxal produced at a given ignition pressure decreased inversely with the nitrogen content of the cellulose nitrates employed, in the experience of workers in this Laboratory, the total free carbonyl con tent also decreased in a similar fashion, but the amounts of carbonyl compounds other than formaldehyde and glyoxal increased with a decrease in the nitrogen content of the cellulose nitrates ignited. It was preferable, therefore, to use in the present work the cellulose nitrate, among the nitrated celluloses at hand, containing the lowest percentage of nitrogen, namely, 12.6$ N.

Thus, cast sheets of cellulose nitrate of 12.6$ N content were prepared as described previously (4), employing ethyl acetate exclusively as the casting

solvent. Unless indicated otherwise, no attempt was made to remove the last residual traces of solvent from the

cellulose nitrate sheets. The apparatus employed for

the ignition, at 75 mm* pressure, of cellulose nitrate

has already been described (5)* The liquid decomposition 60 mixture was processed in the described manner (5) except that a much higher dilution with water was used, the final volume of the condensate aqueous solution being 50 ml. per g. of cellulose nitrate ignited. t

Fractionation, by Repeated Sublimation, of Some of the

Major Organic Components of the Aqueous Solution of the

Liquid Mixture of Cellulose Nitrate ignition Products

Even after a judicious selection of experimental conditions (ignition, at 75 mm., of 12.6% N cellulose nitrate) in order to obtain a condensate aqueous solution containing the largest possible proportion of unknown carbonyl compounds, the sum of the amounts of carbonyl ascribed to formaldehyde and glyoxal accounted for 74# of the total carbonyl content of the condensate aqueous solution (Table i). Since such a large proportion of formaldehyde and glyoxal would have interfered consider ably with the isolation of the unknown carbonyl compounds, the selective removal from the condensate aqueous solution of one or both of the major, known carbonyl constituents was desirable. For this purpose, the investigation described below was initiated.

A condensate aqueous solution was frozen and sublimed under freeze-drying conditions. The sublimation residue was dissolved in water and sublimed as above. This opera tion was repeated once again, after which there was then left the final residue of sublimation, Residue ill (See

Figure 1, a product relationships diagram which outlines our investigation of the controlled thermal decomposition

61 62

Cellulose nitrate (12.6$ N) Ignition at 75 mm, ] First spiral trap Condensate aqueous condensate solution

Gas absorption Repeated low* c hromat ographlc temperature separation sublimation

Minor Major zones zones (Zones 1 to 4)

Sublimates Residue III I, II and III 2,4-Dinltro* phenylbydra- zine

2,4-Dinitro- phenylhydrazine derivative

Extraction with alcohol (95$) I Extract Extract Alcohol- II insoluble residue

Chromat ographio separation on Recrystalli silicic acid zation

Zones 1 to 6 Glyoxal bls(2,4-dinitrophenylhydrazone)

Figure 1. product Relationships 65 of cellulose nitrate). The sublimates were numbered, namely, Sublimates I, II and ill, in the order in which they were obtained. The results of formaldehyde and glyoxal analyses on the condensate aqueous solution, on

Residue H I and on the three sublimates are shown in

Table I. Thus, it has been demonstrated that repeated sublimation of the condensate aqueous solution has the effect of removing formaldehyde in almost quantitative amounts from the solution, whereas glyoxal remains in the solution. The reason for using, in our work, a higher dilution of the condensate aqueous solution

(50 ml. per g. of cellulose nitrate ignited) than in previous work (20 ml. per g. of cellulose nitrate ignited) (5) is now apparent (Table I, footnote h) since the higher dilution favored the removal of formaldehyde from the solution, by sublimation.

Incidental to our study of the removal of formalde hyde from the condensate aqueous solution by repeated sublimation, analyses for formic acid, a known ignition product of cellulose nitrate (5 ), were carried out on the condensate aqueous solution, on Residue III and on the three sublimates (Table i). Thus, it was shown that formic acid was quantitatively and readily removed from the condensate aqueous solution, by sublimation. This finding, as well as the separation of glyoxal from formaldehyde by sublimation of the condensate aqueous Table I

Fractionation, by Repeated Sublimation, of Some of the Ma;jor Organic Components of

the Aqueous Solution of the Liquid Mixture of Cellulose Nitrate Ignition Products^

. Product,- mmoles/mmole ignited^

Fractions of Formaldehyde- Glyoxal— Formic Acid— Total Free Unidentified the Condensate , Carbonyl Carbonyl Aqueous Solution— Content^ Content^

Condensate aqueous 0.255 0.255 0.208 1 .0*f 0.275 solution

Sublimate I 0 .188- 0.002 0.187 ------

Sublimate II 0.025 0.002 0.008 ------

Sublimate III 0.008 none none ------

Residue III 0.012 0.222 0.008 0.599 O.1V 3

Sumi 0.233 0.226 0.203 ------

- Cellulose nitrate, 12.6$ N, cast from ethyl acetate solution and ignited at 75 mm. pressure, with the exception made in footnote f. Table I. Cont.

" The quantitative assays for formaldehyde, glyoxal, formic acid and total free carbonyl content are described in the Experimental part.

q — The znmoles of cellulose nitrate were calculated by division of the grams decomposed by the sum of the millimolecular weight of one anhydro-D-glucose unit (0 .1 6 2 ) and the increase in millimolecular weight caused by the nitrate ester groups CO.OM-j times the degree of substitution).

— See Experimental part for details of their preparation.

— The formaldehyde, glyoxal and total free carbonyl analyses were carried out on the various fractions immediately after the fractions were obtained, with the exception of the condensate aqueous solution which was analyzed prior to freezing and subliming. — The formic acid investigation was carried out on an aged (2 weeks old) condensate aqueous solution (final volume, 10 ml. per g. of cellulose nitrate ignited) from the ignition of 1 3 .2$ IT cellulose nitrate at 200 mm. pressure. The sublimations were spaced at 6b hour intervals and all formic acid analyses were performed several weeks later.

^ Calculated by subtracting, from the total free carbonyl content of a fraction, the amount of formaldehyde and twice the amount of glyoxal present in the same fraction. ~ Separate experiments showed that ca. 50$ less formaldehyde is found in Sublimate I when a more concentrated (5 fold; 10 ml. per g* of cellulose nitrate ignited) condensate aqueous solution is sublimed.

— Calculated by adding the amounts of formaldehyde, glyoxal or formic acid in the three sublimates and Residue III. 66 solution, was useful in the investigation carried out by

Shafizadeh and Wolfrom (7) on the ignition of cellulose- 1 h. nitrates.

In the course of the sublimation experiments which have been described above, it was found that the formalde hyde content of a condensate aqueous solution decreased when the solution stood at room temperature. Thus, the

formaldehyde content was 0.256, 0.242, 0.220 and 0.215 mmoles per mmole of cellulose nitrate ignited (181) after

(181) The mmoles of cellulose nitrate were calculated by division of the gramB decomposed by the sum of the millimolecular weight of one anbydro-D-glucoBe unit (0.162) and the increase in millimolecular wefght caused by the nitrate ester groups (0.045 times the degree of substitu tion) . a condensate aqueous solution had stood at room tempera

ture for 16, 63, 240 and 333 hours, respectively. It is

apparent, then, that the formaldehyde content decreases

more rapidly at the beginning of aging than at the later

stages of aging. These results may be accounted for by

the very recent work of Wolfrom, Chaney and McWaln (6).

These authors have made an extensive study of the changes

which take place in a condensate aqueous solution during

the first 72 hours of aging and have shown that the aging

effects which they observed could be ascribed primarily 67 to the known interaction of cyanide ions with carbonyl compounds. These researchers also believed that other aging processes caused by the small amounts of nitrogen oxides and acids present in the condensate aqueous solu tion probably occurred but were of less Importance than the oyanobydrin addition reaction. Thus, the cyano- hydrin condensation reaction which is almost complete in the first 72 hours of aging (6) is undoubtedly responsible for the relatively rapid decrease in formaldehyde content which was observed, in our work, at the beginning of aging, whereas the relatively slow decrease in formalde hyde content which occurred in the later stages of aging is probably due to oxidation processes caused by nitrogen oxides and acids.

An examination of Table I (last column) shows that there is a difference of 0.132 mmoles of carbonyl per mmole of cellulose nitrate Ignited (181) between the unidentified carbonyl content of the condensate aqueous solution and that of Residue III. This difference may be largely due to aging processes, since Wolfrom, Chaney and McWain (182) have reported that the total free

(182) See reference 6, Table I. carbonyl content of a condensate aqueous solution 68 decreased by 0.07 mmoles per mmole of cellulose nitrate ignited (181), during the first 72 hours after ignition.

In addition, a small part of the difference between the unidentified carbonyl content of the condensate aqueous solution and that of Residue h i may be caused by the removal of volatile carbonyl compounds other than formal dehyde, on subliming the condensate aqueous solution.

The presence, in the condensate aqueous solution, of volatile carbonyl compounds other than formaldehyde is not in doubt since three such compounds, namely, acetone, acetaldehyde and acrolein, were isolated in the course of our work. Preparation of 2.4-Dlnitrophenylhvdrazlne Derivatives of Known Carbonyl Compounds

Since it was intended to use 2,4-dinitrophenylhydra zine in derivatlzing the carbonyl compounds present in the residue of the repeatedly sublimed condensate aqueous solution, it was necessary to have at hand a number of

2.4-dinitrophenylhydrazine derivatives of known carbonyl compounds to serve as model derivatives. Such model derivatives were required in order to devise methods for the separation of complex mixtures of 2,4-dinltrophenylhydrazine derivatives by chromatography. Furthermore, model derivatives were required for comparison with and identification of the compounds arising from the reaction of 2,4-dinitrophenylhydrazine with Residue III (Figure l).

The melting pointB and recrystallization solvents of

2.4-dinitrophenylhydrazine derivatives, some of which were well-known, prepared by us are given in Table II. The literature melting points, where available, are also listed in Table II. in our work, we were especially Interested in model 2,4-dinitrophenylhydrazine derivatives of short carbon chain (two and three carbon atoms) sugars and oxidation products thereof, without carbon fragmentation.

These model derivatives, and in many cases the parent carbonyl compounds as well, were little or not known.

69 Table II

Melting Points of Some 2,1f-Dinitrophanylhydrazine Derivatives

Our WorkS. Recorded in Literature

2, *f-Dinitro- Meltingh Recrystallization Melting Reference phenylhydrazine Point, - Solvent Point, Derivative °c. °c.

A. Hydra zone

Glyoxylie acid 193-19**.5 Water 190 dec. M-7 19b 183 203 121

Pyruvic acid 218.5-220.5 Acetic acid 213 216 121 218 57

Mesoxalic acid 203.5-205.5 2 N HC1 202 l8*f 205 121

Glycolaldehyde 159-161 Abs. ethanol 155-156 50

Acetol 137.5-138.5 Abs. ethanol 127.5-129.5 57 13^.5-136.5 75

D,L-Glyceral- 165-168 Ethanol (95$) 167 185,186 dehyde 170 75 Table IIT Cont.

Our Work- Recorded in Literature

2',^-Binitro- Melting, Recrystallization Melting Reference phenylhydrazine Point, - Solvent Point, Derivative °C. c.

Dihydroxy- 159-167 Abs. ethanol, 161 187 acetone ethyl acetate^ 1 6 3 -16^ 185 168-169 75

Acetone 127-128.5 Ethanol (95%) 126 150 128 ^5

Formaldehyde 167.0-167.5 Ethanol (95$) 155 b5 16**-165 150 167 106

Acetaldehyde 166-168 Abs. ethanol l*t8 167 156-157 167,172 168-170 167

B. Bis hydrazone or osazone

Triose 267-268 dec. Nitrobenzene 265 dec. 185 278 75 301-306 dec. 188

D-Glucose 263-267 dec. Nitrobenzene 2*f0 dec. 189 256-257 dec. 70 288-289 dec. 16b Table II. Cont.

Our Work- Recorded in Literature

2 ,!+-Dinitr o- Melting Recrystallization Melting Reference phenylhydrazine Point ,22 Solvent Point, Derivative °C. °c.

Mesoxaldehyde 262-269 dec.i (1 ,2)2 Methylglyoxal 30^.5-305.5 dec. Nitrobenzene 295 dec. 82 299-300 310 75

Glyoxal 336-338 dec. Nitrobenzene 326-328 57 330 190

C. Others

Mesoxaldehyde 306-308 dec. Nitrobenzene tris(2 ,*f-dinitrophenylhydra- zone)H

— See Experimental part for the methods of preparing the 2 , dinitrophenylhydrazine derivatives. Table II. Cont. i. — All compounds were recrystallized to constant melting point from a single solvent with the exception of mesoxaldehyde l,2-bis(2’,1f*-dinitrophenylhydrazone) (see footnote e) and dihydroxyacetone 2,4— dinitrophehylhydrazone (see footnote c); all melting points were determined with a Kofler micro hot stage and are corrected.

£ Recrystallized to constant melting point from abs. ethanol and from ethyl acetate.

— New compound.

— See Experimental part for method of purification.

(I8 3 ) J. E. Cadotte, G. G. S. Dutton, I. J. Goldstein, Bertha A. Lewis, F. Smith and J. W. Van Cleve, J. Am. Chem. Soc., 22, 691 (1957)*

(l8»+) F. P. Clift and R. P. Cook, Biochem. J., 26, 1800 (1932).

(185) C. Neuberg and H. Collatz, Biochem. Z., 223. *f9*+ (1930).

(186) B. C. Pressman, L. Anderson and H. A. Lardy, J. Am. Chem. Soc., 2k0b (1950). (1 8 7 ) R. P. Bell and E. C. Baughan, J. Chem. Soc., 19^7 (1937). (188) G. Matthiessen and H. Hagedorn, Mikrochimie ver. Mikrochim. Acta, 22, 55 (19^1). (1 8 9 ) E. C. Noyons, Chem. Weekblad, 32> ^ 9 (19^2).

(190) S. Glasstone and A. Hickling, J. Chem. Soc., 820 (1936). 74

In addition, where some literature was available on

2,4-dinitrophenylhydrazine derivatives of low mole cular weight sugars and oxidation products thereof, it was often contradictory. For instance, Collatz and

Neuberg (50) were the sole researchers ever to report on the preparation of glycolaldehyde 2,4-dinitrophenylhydrazone. in contrast, Banks, Vaughn and Marshall (6 9 ) were unable to prepare this derivative. In the course of our work, the 2,4-dinitrophenylhydrazine derivatives of several low molecular weight sugars and oxidation products thereof were prepared, and their preparation is discussed below.

A. Preparation of triose 2,4-dinitrophenylosazone

Dihydroxyacetone and glyceraldehyde are known to rearrange to methylglyoxal in the presence of an acid catalyst (191). For this reason, reports of the prep-

(191) C. L. Bernier and W. L. Evans, J. Am. Chem. Soc., 6 0 , 1381 (1938), have reviewed the action of acids and bases on dihydroxyacetone and glyceraldehyde. aration, starting from either of the trioses mentioned above, of pure triose 2 ,4-dinitrophenylosazone should be viewed with suspicion unless some evidence is presented 75 to establish the absence of methylglyoxal bis(2 ,4-dinit rophenylhydra zone) in the product. For instance, the reaction, at room temperature, of dihydroxyacetone with 2,4-dinitrophenylhydrazine in 2 ij hydrochloric acid

(saturated solution of 2,4-dinitrophenylhydrazine) was reported by Reich and Samuels (75) to afford pure triose

2 ,4-dlnitrophenylosazone. These researchers based their claim on a nitrogen analysis of their product and on its formation of an acetate. This evidence is certainly not sufficient to exclude the possibility of the presence of methylglyoxal bis(2,4-dinitrophenylhydrazone) in their product since triose 2,4-dinltrophenylosazone and methyl glyoxal bis(2,4-dinitrophenylhydrazone) differ but little in nitrogen content ^ 25.00 and 2 5 .92# N for the triose and the methylglyoxal derivatives, respectively), and since the presence of some amount of methylglyoxal bis(2,-

4-dinitrophenylhydrazone) would not have interfered with the formation and precipitation of the acetate derivative

(no yield reported), under the conditions which were used.

In our hands, the preparation of pure triose 2,4-dlnitrophenylosazone, as reported by Reich and Samuels (75) >

could not be repeated, methylglyoxal bis(2,4-dinitrophenylhydrazone) being found as a contaminant in the product. The possibility that methylglyoxal, which is

known to be a product of aging of dihydroxyacetone (192), 76

(192) P. A. Levene and A. Walti, J. Biol. Chem., 18, 23 (1928). was present in our sample of dihydroxyacetone was elimi nated. This was done by reacting dihydroxyacetone with

2.4-dinitrophenylhydrazine in boiling alcohol and chroma tographing, on silicic acid, the crude dihydroxyacetone

2.4-dinitrophenylhydrazone obtained therefrom under conditions such that methylglyoxal bis(2,4-dinitrophenylhydrazone) would have been found on the chromatogram had it been present in the crude reaction mixture. Several recrystallizations from nitrobenzene, of the crude triose

2.4-dinitrophenylosazone prepared following the procedure of Reich and Samuels (75) afforded a material of melting point 280-284° dec., melting point Intermediate between those of pure triose 2,4-dlnitrophenylosazone and methyl glyoxal bis(2,4-dinitrophenylhydrazone)(Table II). The recrystallized material still contained methylglyoxal bis(2,4-dinitrophenylhydrazone), as shown by its chroma togram on Biliclc acid, pure triose 2,4-dinitrophenylosazone was finally obtained by chromatography, on silicic acid, of the products obtained following the procedure of Reich and Samuels (75)• 77 B. Preparation of the 2,4—dinitrophenylhydrazones of short carbon chain sugars

The reaction of low molecular weight sugars (2 and

3 carbon atoms) with 2,4-dinitrophenylhydrazine in refluxing ethanol (75) or in 2 N hydrochloric acid at 0°

(supersaturated, solution with respect to 2,4-dinitro phenylhydrazine) (50) has been reported to afford 2,4- dinitrophenylhydrazonee. These reports were confirmed in the course of our investigation.

In addition, we showed, in the following way, that the reaction of low molecular weight sugars (2 and 3 carbon atoms) with 2,4-dinitrophenylhydrazine, in refluxing ethanol, takes place without any oxidation of the hydroxyl group adjacent to the carbonyl group and in the case of glyceraldehyde and dihydroxyacetone without rearrangement (191) as well. Glycolaldehyde, acetol and dihydroxyacetone were reacted with 2,4-dinitrophenyl hydrazine in refluxing alcohol. A 1($ excess of the carbonyl compound was used in each case and the prepara tions were refluxed for three hours. Exploratory chro matograms (silicic acid) of the crude reaction products showed that the reaction took place, in each case, with

out formation of any 2,4-dinitrophenylosazone. In addition, no methylglyoxal bis(2,4-dinltrophenylhydra-

zone) or glyceraldehyde 2,4-dlnltrophenylhydrazone was 79 found, by chromatography, in the dihydroxyacetone reaction product. Our investigation of the preparation of 2,4-dinitrophenylhydrazine derivatives in refluxing ethanol was then extended to D,L-glyceraldehyde, but the conditions used were more severe than those used with glycolaldehyde, dihydroxyacetone and acetol. The prep aration was refluxed for 16 hours and a threefold excess of reagent was used. Nevertheless, an exploratory chromatogram indicated the absence of 2,4-dinitroaniline, triose 2,4-dinitrophenylosazone, dihydroxyacetone 2,4- dinitrophenylhydrazone and methylglyoxal bis(2,4-dlnitrophenylhydrazone) in the crude reaction product.

We have also shown, in the following manner, that the derivatization of D,L-glyceraldehyde with 2,4-dinitro- 3 ■ X X phenylhydrazine in 2 g hydrochloric acid at 0° (super saturated solution with respect to 2,4-dinitrophenyl hydrazine ; 17 mg. of reagent per ml. of solution) takes place without oxidation and without rearrangement. A

10% excess of the triose was used and the reaction was allowed to take place for six hours. The crude reaction product (92$ yield) was shown by silicic acid chroma tography to contain only D,L-glyceraldehyde 2,4-dinitrophenylhydrazone. In contrast, Reich and Samuels (75)* using a saturated solution of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid at room temperature (4 mg. of reagent per ml. of solution), found it necessary, in the 79 derivatlzatlon of trloses, to limit the reaction time to lesa than one hour and to use a twofold excess of the trioses in order to avoid the formation of 2,4-dinitro- phenylosazones.

C. Preparation of mesoxaldehyde tris(2,4-dinitrophenylhydrazone)

Harries and Turk (193) have reported that the reaction

(193) G. Harries and H. 0. Turk, Ann., 374. 350 (1910). of mesoxaldehyde with an excess of_p-nitrophenylhydrazine in boiling aqueous hydrochloric acid resulted in the formation of mesoxaldehyde tris(j3-nltrophenylhydrazone).

A similar procedure was used, in our work, to derivatize mesoxaldehyde with 2,4-dinitrophenylhydrazine. An exploratory chromatogram, on silicic acid, of the deriva tive obtained therefrom showed the presence of three constituents in the reaction product. Recrystallizations of the reaction product from nitrobenzene failed to afford a chromatographically pure compound. Mesoxalde hyde tris(2,4-dinltrophenylhydrazone), the desired com pound, was finally obtained by chromatographing the crude reaction product on silicic acid. This compound proved to be the largest constituent of the reaction 80 mixture, and the least adsorbed of its three constit uents, on silicic acid.

The two constituents, other than mesoxaldehyde tris(2,4-dinitrophenylhydrazone), present in the product of reaction of mesoxaldehyde with an excess of 2,4-di nitrophenylhydrazine in hot aqueous hydrochloric acid have not been characterized, although they were isolated, separately, as by-products of the chromatographic puri fication of mesoxaldehyde tris(2,4-dinitrophenylhydrazone).

The experimental evidence which was gathered allows us, however, to speculate as to the nature of these constit uents. The infrared spectra of both constituents were consistent with their being derivatives of 2,4-dlnltro- phenylhydrazlne. Furthermore, the infrared spectra of both constituents were found to contain no carbonyl absorption band, thus eliminating the possibility that either one was a mesoxaldehyde bis(2,4-dlnltrophenylhydrazone). 3oth constituents gave a blue-violet color when treated with alkali. This fact may be taken as an indication of the presence of 2,4-dinitrophenylhydrazine residues /~(N03 )8CsH3NHNf7 adjacent positions (157)* in each of the two constituents. On the basis of the evidence which has been presented, it is suggested that both constituents are 2,4-dinitrophenylhydrazine deriva tives of polycarbonyl compounds, possibly derivatives of polymeric nature. 81

D. Reaction of saD-glucose with 2,4-dinitrophenylhydrazine The derivatization, at room temperature, of dihydroxj- aoetone, glyceraldehyde, acetol and glycolaldehyde with

2.4-dinitrophenylhydrazine, in 3C$ perchloric acid solu tion, has been reported by Neuberg, Grauer and Risha (6l)f

These authors, however, did not state whether or not monosaccharides with five and six carbon atoms reacted with 2,4-dinitrophenylhydrazine under the conditions which they used in derivatizing the monosaccharides with two and three carbon atoms. Such being the case, we attempted to prepare a 2,4-dinitrophenylhydrazine deriva tive, in 30% perchloric acid, of g-glucose. On prolonged standing of the reaction mixture at room temperature, essentially no derivative was formed. The conclusions which may be drawn from this single experiment are necessarily tentative. However, further investigation may show that a sharp difference in reactivity towards

2.4-dinitrophenylhydrazine in 30^ perchloric acid exists between monosaccharides of low and high molecular weights.

The preparation of D-glucose 2,4-dlnltrophenyl- osazone ha3 been reported on several occasions (70,72,

151,164,189). Among these methods of preparing D-glucose

2.4-dinitrophenylosazone, that which was reported by

Neuberg and Strauss (72) was the most attractive, in spit© of the fact that no elemental analysis or melting point was shown, since they reported a quantitative yield of crystalline D-glucose 2,4-dinltrophenylosazone.

In our hands, however, the preparation of D-glucose

2,4-dinltrophenylosazone using the method described by

Neuberg and Strauss (72) afforded a quantitative yield, calculated as jD-glucose 2,4-dinitrophenylosazone, of a semi-crystalline material containing a considerable amount of tars and melting over a very wide range in temperature. Since we found that 2,4-dinitrophenyl hydrazine tars are very soluble in nitrobenzene and that D-glucose 2 ,4-dinitrophenylosazone is only very slightly soluble In that solvent, the semi-crystalline material was readily purified by extraction with nitro benzene. The progress of the purification by extraction could be followed visually since 2,4-dlnitrophenylhydrazine tarB form dark reddish-black solutions in nitrobenzene. Thus, on repeated extraction, the color

of the nitrobenzene extracts changed from black to

orange, pure D-glucose 2,4-dinitrophenylosazone was

obtained in 28% yield (overall) by recrystallization

from nitrobenzene of the nitrobenzene extraction residue. 83

E. Preparation of mesoxaldehyde l,2-bis(21,4’-dlnltrophenylhydrazone)

Glucose phenylosazone has successfully heen con verted to mesoxaldehyde 1,2-bis(phenylhydrazone) hy periodate oxidation (194,195) and a similar approach,

(194) E. Chargaff and B. Magaeanik, J. Am. Chem. Soc., 62, 1459 (1947).

(195) C. F. Huebner and K. P. Link, J. Am. Chem. Soc., J2, 4812 (1950).

starting from D-glucose 2 ,4-dlnitrophenylosazone, was

used in our preparation of mesoxaldehyde l,2-bis(2',4'- dinitrophenylhydrazone).

H»N-Dimethylformamide-water was found to be a good

solvent medium for the periodate oxidation of D-glucose

2,4-dinitrophenylosazone since the reactants were far more soluble in that medium than the reaction product.

However, a small scale exploratory chromatogram (silicic

acid) revealed that the reaction product could be

separated into two constituents. It was not possible to

obtain a chromatographically pure compound by recrystalli

zation of the reaction product from nitrobenzene since

the reaction product was somewhat sensitive to heat.

Mesoxaldehyde l,2-bis(2’,4’-dinitrophenylhydrazone), the

leBs adsorbed of the two constituents of the reaotion

product, was finally obtained in a pure form by 84 chromatography, on silicic acid, of the periodate oxidation product of D-glucose 2,4-dinitrophenylosazone.

There were two constituents in the periodate oxi dation product of D-glucose 2,4-dinitrophenylosazone and only one has been accounted for. The other con stituent, of greater adsorption affinity on silicic acid than mesoxaldehyde l,2-bis(2’,4'-dinitrophenylhydrazone), gave, as in the case of the mesoxaldehyde derivative, a blue-violet color with alkali (157). This unknown constituent, which was not investigated, differed quite markedly from the mesoxaldehyde derivative in its chroma tographic properties, and this fact lends to the belief that it was not isomeric v,ith the mesoxaldehyde deriva tive. it is suggested that the constituent of the second zone is of polymeric nature, or that it is a product of secondary oxidation since it is known that l,2-bis(phenylhydrazones) react with periodate (196).

(196) J. E. Gourtois, A. Wickstrom and p. Le Dizet, Bull. soc. chim. France, 1006 (1952).

Mesoxaldehyde l,2-bis(2’ ,4 ’-dinitropheny lhydrazone) was prepared by still another method. The prolonged room temperature reaction of equimolar amounts of mesoxaldehyde and 2,4-dinitrophenylhydrazine in ethanol afforded a substance which, after purification by chromatography, was identified as mesoxaldehyde 85

1.2-bis(21,4'-dinitrophenylhydrazone). i * Mesoxaldehyde l,2-bis(2 ' ,4'-dinitrophenylhydrazone) was converted to mesoxaldehyde tris(2,4-dlnitrophenylhydrazone) by reaction with 2,4-dinitrophenylhydrazine in dimethyIsulfoxide. This conversion provided the remaining evidence necessary to show that the substance which has been, thus far, referred to as mesoxaldehyde

1.2-bis(2',4'-dinitrophenylhydrazone) has indeed the structure which has been assigned to it. The infrared spectrum (carbonyl absorption band at 6.0/1 ), elemental analysis and reaction with 2,4-dinitrophenylhydrazine are conclusive proofs that the substance is a bis 2,4- dinitrophenylhydrazone of mesoxaldehyde. The location of the 2,4-dinitrophenylhydrazine residues

N0S JfcCgHsNHN-/ in 1,2-posltions rather than in the 1,3-positions of mesoxaldehyde is expected since the substance was obtained from D-glucose 2,4-dinitro phenylosazone, and confirmed by measurement of the light absorption spectrum in 95% ethanol (maximum at

445 m/l ).

The light absorption spectra of mesoxaldehyde

1.2-bis(2 ' ,4'-dinitrophenylhydrazone) in ethanol (95%) and in ethyl acetate are shown in Figure 2. The difference between these two spectra is very marked and Optical density. 0.8 h*0.8 0,6 0.4 0.2 i ty ctt; i tao (95$)* in Ethanol -- - t Acetate; in Ethyl Mesoxaldehyde 1,2-Bis 1,2-Bis Mesoxaldehyde 9(2 *-dinitrophenylhydrazone) s 320 Figure 2, Light Absorption Spectrum of Spectrum Absorption Light 2, Figure 6 0 440 400 360 86 X , m/i. 8 520 480

no solvent effects of thiB kind have heretofore been

reported In the case of derivatives of 2,4-dinitro

phenylhydrazine. It is suggested that this difference

may be due to hydrogen bonding, since the hydrogen

bonded species I may predominate in ethyl acetate,

whereas the hydrogen bonded species II may predominate

in ethanol (95$)*

H H ) I 0HsCH*O-H 0=CHC- -GH II II N N .N NH I I NH NH

■NO. 0SN

F. Attempted preparation of mesoxaldehyde l,3-bis(21,4'- i • dinitrophenylhydrazone)

In addition to the preparation of mesoxaldehyde tris(2,4-dinitrophenylhydrazone) and mesoxaldehyde 1,2- bis(2',4'-dinitrophenylhydrazone) which have already been discussed, several attempts, all of which were

unsuccessful, were made to prepare mesoxaldehyde

l,j5-bis(2 1,4'-dinitrophenylhydrazone). All of these

attempts were made by reacting mesoxaldehyde with 88

2 ,4-dinitrophenylhydrazlne, in different solvent media.

It was believed that a suitable choice of solvent medium might lead to the preferential formation of mesoxaldehyde l,3-bls(2',4'-dinitrophenylhydrazone)•

First, an attempt was made to react, at room temperature, mesoxaldehyde with 2,4-dinitrophenylhydrazine in acetic acid, in tetrahydrofuran and In pyridine.

When these reaction mixtures stood for one hour, no precipitate formed in any of them. These reaotion mixtures were then heated, and all of them afforded pre cipitates. Exploratory silicic acid chromatograms of these precipitates indicated the presence, in each pre cipitate, of several constituents. The least adsorbed constituent of each precipitate had chromatographic properties identical with those of authentic mesoxalde hyde tris(2 ,4-dinltrophenylhydrazone). This was con sidered to be significant since authentic mesoxaldehyde

1,2-bls(2'-4’-dinitrophenylhydrazone), a compound which should have chromatographic properties very similar to those of mesoxaldehyde l,3-bis(2',4'-dinitrophenyl hydrazone), is less adsorbed on silicic acid than authentic mesoxaldehyde tris(2 ,4-dinitrophenylhydrazone).

Since no constituent less adsorbed, on silicic acid, than mesoxaldehyde tris(2,4-dlnltrophenylhydrazone) was found in the precipitates mentioned above, the investigation of the precipitates was not pursued further. 89

Our last attempts to prepare mesoxaldehyde 1,3-

bls(21,4’-dlnitrophenylhydrazone) were made by reacting, at room temperature, mesoxaldehyde with 2,4-dinitrophenyl-

hydrazine in 2 N hydrochloric acid and in acetic acid-N,N-

dlmethylformamide. In both cases, a precipitate was

obtained, the exploratory chromatogram (silicic acid)

of which showed the presence of a single constituent below that which had chromatographic properties Identical

with those of authentic mesoxaldehyde tris(2,4~dlnitro-

phenylhydrazone). in each case, the chromatographic

properties of this single constituent were identical with

those of authentic mesoxaldehyde l,2-bls(21,4'-dinltro-

phenylhydrazone), and in one case (preparation in 2

hydrochloric acid) this single constituent was shown to

have a light absorption spectrum identical with that

of mesoxaldehyde l,2-bis(21,41-dinitrophenylhydrazone).

Although the chromatograms of the products of our

attempts to prepare mesoxaldehyde l,3-bis(2*,4'-dinltro-

phenylhydrazone) contained one or more zones which were

above that of mesoxaldehyde trls(2,4-dinitrophenylhydra-

zone), it is unlikely that any of these zones contained

mesoxaldehyde l,3-bis(2',4'-dinitrophenylhydrazone).

This assertion is supported by the fact that mesoxal

dehyde l,2-bis(2',4'-dinitrophenylhydrazone), a compound

which should have chromatographic properties very similar to those of mesoxaldehyde 1,3-bis(2’-4'-dinitrophenylhydrazone), is less adsorbed on sillclo acid than mesoxal dehyde tris(2,4-dinitrophenylhydrazone)♦ Furthermore, two unidentified zones were also found above that of mesoxaldehyde tris(2,4-dinltrophenylhydrazone) in the chromatogram of the product of reaction of mesoxaldehyde with 2,4-dinitrophenylhydrazine in hot 2 N hydrochloric acid /"see under ''preparation of mesoxaldehyde tri8(2,4- dlnitrophenylhydrazone)|[7. No carbonyl absorption band was found in the infrared spectra of the constituents of these two unidentified zones, showing that neither zone contained mesoxaldehyde l,3-bis(2 1,4'-dlnltrophenylhydrazone).

The foregoing discussion, further strengthened by the preparation of mesoxaldehyde l,2-bis(2',4'-dinltrophenylhydrazone) in ethanol /"see under " Preparation of mesoxaldehyde l,2-bls(21,4’-dinit rophenylhydrazone)27, tends to indicate that mesoxaldehyde l,3-bis(21,4'- dinitrophenylhydrazone) may be extremely difficult to prepare directly, by reacting mesoxaldehyde with 2,4- dinit rophenylhydrazine. 91

G-. Reaction of 2,4-dinitrophenylhydrazlne with triose- reductone

The term * reductone" will be used, other usage notwithstanding, as a generic name for compounds having structural features I in common. Triose-reductone,11, is the simplest of all reductones.

\ H\ s ; c = 0 h n c= o

HO >=< OH HO >=

I II

No investigation of the reaction of triose-reductone with 2 ,4-dinitrophenylhydrazine has been reported in the literature. In our work, attempts were made to prepare trlose-reductone 2,4-dinitrophenylhydrazone in boiling ethanol and in ethanol at room temperature, to prepare triose-reductone bis(2,4-dinitrophenylhydrazone)

in 2 N hydrochloric acid, and to prepare triose-reductone tris(2,4-dinitrophenylhydrazone) in acetic acid. A darkening of the hot and of the cold ethanol preparations was observed and was ascribed to polymerization of the

reaction product. Although the attempts to prepare

2.4-dinitrophenylhydrazine derivatives of triose-reduc

tone by the four methods listed above were unsuccessful,

products were obtained in each case. These products,

most of which had unusual colors for derivatives of

2.4-dinitrophenylhydrazIne, were chromatographed and 92 separated into many zones. In most cases, two zones, the constituents of which had chromatographic proper ties identical with those of authentic mesoxaldehyde l,2-bls(2',4'-dinitrophenylhydrazone) and mesoxaldehyde tris(2,4-dinitrophenylhydrazone), respectively, were present in the chromatograms of the products. The investi gation of these products was not pursued, since it was apparent that they contained, in varying proportions,

2,4-dinitrophenylhydrazine derivatives of mesoxaldehyde.

Triose-reductone was also reacted at room temperature with an excess of 2,4-dinitrophenylhydrazine in 30$ per chloric acid. The product thus obtained was purified by chromatography and was shown to be identical with authentic mesoxaldehyde tris(2,4-dinitrophenylhydrazone).

The ease with which triose-reductone was oxidized to mesoxaldehyde by 2,4-dinitrophenylhydrazine was not unexpected since reductones are strong reducing agents

(197). In addition, triose-reductone had previously

(197) H. v. Euler and H. Hasselqulst, "Reduktone. Ihre chemischen Eigenschaften und blochemischen Wirkungen," F. Enke, Stuttgart, Germany, 1950, p. 1. been shown to afford a mesoxaldehyde derivative when reacted with phenylhydrazine at room temperature (198). 93

(198) H. v. Euler, H. Hasselqulst and U. Loov, Arklv Kemi, Mineral. Geol., 26A . No. 17 (194-8).

H. Miscellaneous data concerning 2,4— dinitrophenylhydra-

zlne derivatives

In the course of our work, in addition to the preparation of rare and unknown derivatives of 2,4— dinitrophenylhydrazlne, some data were gathered concerning the

sublimation properties of derivatives of 2,4-dinitrophenyl hydrazine, and a collection was made of the infrared

absorption spectra of rare 2,4-dinitrophenylhydrazine derivatives.

The sublimation properties of some derivatives of

2,4-dinitrophenylhydrazine have been studied by Matthiessen

and Hagedorn (188) who carried out their study with a

Kofler micro melting point apparatus. These authors

reported that aliphatic hydrazones, keto-acld hydrazones,

osazones and bis hydrazones of 2,4-dinitrophenylhydrazine

sublimed (undecomposed) on heating in a high vacuum.

Our study of the sublimation properties of derivatives

of 2,4-dinitrophenylhydrazine was limited to a single

temperature (110°) and pressure (0.5 mm.). Under these

conditions, it was possible to separate a complex mixture

of 2,4-dinitrophenylhydrazine derivatives into two

fractions, since keto-acid and aliphatic 94

2 .4-dlnitrophenylhydrazones sublimed whereas other derivatives of 2,4-dinitrophenylhydrazine did not,

2 .4-Dinitrophenylhydrazine itself and 2,4-dinitroaniline also sublimed at 110° and 0.5 mm. pressure.

The infrared absorption spectra of a large number of derivatives of 2,4-dinitrophenylhydrazine are available in the literature (143,160-162). However, the infrared absorption spectra of the rare 2,4-dinitrophenylhydrazine derivatives which we prepared were not reported in the literature. These infrared absorption spectra were taken with a Perkin-Elmer spectrophotometer Model Ho. 21, using the potassium bromide technique, and are shown in Figures

3 to 11, in the following order: glycolaldehyde 2,4- dinitrophenylhydrazone, glyoxylic acid 2,4-dinitrophenyl hydrazone, acetol 2,4-dinitrophenylhydrazone, mesoxalic acid 2,4-dinitrophenylhydrazone, D,L-glyceraldehyde 2,4- S 5 S I dinitrophenylhydrazone, dihydroxyacetone 2,4-dinitrophenyl

hydrazone, triose 2,4-dinitrophenylosazone, mesoxaldehyde.

tris(2,4-dinitrophenylhydrazone) and mesoxaldehyde 1,2- bis(2’,4’-dinitrophenylhydrazone). The instrument

calibration (theoretical water absorption band at 2.66

microns) is also shown in the lower left-hand corner of

Figures 3 to 11. The calibration is the right-hand

absorption band, of the two bands taken with the instrument

on single beam. The infrared absorption spectra shown in Figures 3 to 11 have many points of comparison with each other, but they are nevertheless dissimilar.

For this reason they are of great value as "fingerprints" in the positive identification of unknown derivatives of

2,4-dinitrophenylhydraz ine. Figure 3. Infrared Absorption Spectrum of Glycolaldehyde 2, trophenylhydrazona. Figure V . Infrared Absorption Spectrum of Glyoxylic Acid 2 ,’h—i>initrophenylhydrazone. Figure 5 . Infrared Absorption Spectrum of Acstol 2,^-Dinitrophenylhydrazone• Figure 6m Infrared Absorption Spectrum of Mesoxalic Acid 2, V-Blnitrophenylhyarazone. *

Figure 7 . Infrarad Absorption Spectrum of D,L-Glyceraldehyde 2,V-Dinitrophenylhydrazone. \go_ io6 J22:

9 0 BO JSO_

so ■2£ M.

60 60 60 101 52. 52.

130 30 30 30

20 20 20

Figure 8. Infrared Absorption Spectrum of Bdhydroxyacetone 2 ,*f-Dinitrophenylhydrazone. 8930

Figure 9« Infrared Absorption Spectrum of Triose 2,**-Dinitrophenylosazone* m

jffl Mil;

rtt m Mi

i±L1

Figure 10. Infrared Absorption Spectrum of Mesoxaldehyde Tris(2,U— dinitrophenylhydrazone). Figure 11* Infrarad Absorption Spectrum of Mesoxaldehyde 1,2-313(2* jM-dinitrophenylhydrazona). Chromatography of 2,4-Plnlt rophenylhydraz Ine Derivatives of Known Carbonyl Compounds

Our preparation of 2,4-dinitrophenylhydrazine deriva tives of some carbonyl compounds has Just been described.

These derivatives were required in order to devise methods for the separation of complex mixtures of known 2,4-dinitro phenylhydrazine derivatives by chromatography. The necessity of being able to separate complex mixtures of known 2,4-dinitrophenylhydrazine derivatives by chroma tography is obvious since the product of the derivatization of Residue ill (Figure l) with 2,4-dinitrophenylhydrazine was expected to contain a large number of 2,4-dinitrophenyl hydrazine derivatives which would have to be separated.

The chromatographic separation of 2,4-dinitrophenyl hydrazine derivatives on silicic acid has often been reported (81,104-107). As a case in point, the procedure described by Malmberg (81) was used in the course of this work to separate the 2,4-dinitrophenylhydrazones of acetone, acetaldehyde and formaldehyde, in general, however, the work published on the separation of 2,4-dinitrophenyl hydrazine derivatives on silicic acid was of limited value for our purpose since it was concerned mainly with the derivatives of saturated and unsaturated aliphatic carbonyl compounds and with the derivatives of aromatic carbonyl

105 107 This relationship was adhered to by the 2,4-dinitro phenylhydrazine derivatives which we separated on silicic acld-Celite (5^1» 8$ water). To give but one example, both l),L-glyceraldehyde 2,4-dlnitrophenylhydrazone and dibydroxyacetone 2,4-dinitrophenylhydrazone had greater affinity for the adsorbent than triose 2,4- dlnitrophenylosazone which, in turn, had greater affinity for the adsorbent than mesoxaldehyde tris(2,4-dinitrophenylhydrazone). Thus, the influence of polarity is evident since these compounds decreased in adsorption affinity in spite of their increasing molecular weights. The preparation of some 2,4-dinitrophenylhydrazine derivatives has already been discussed. Occasionally, these derivatives could not be obtained in a chromatographically pure form by recrystallizations of their crude preparations. Although these derivatives could have been obtained in a pure form by chromatographing their crude preparations on silicic acid-Celite (5*lj 8$ water), this method of purification would have been quite tedious because of the extreme insolubility of the derivatives. In addition, this method of purification was much too elaborate to use for the purification of these crude preparations since the crude preparation of any one of these derivatives contained at the most three constituents, since the desired constituent was usually present in amounts much larger than the other one or two constituents, and since the constituents of 106 compounds. It became necessary, therefore, to develop a method of separating, on silicic acid, 2,4-dinltro- phenylhydrazlne derivatives of interest to us.

A silicic acid-Celite (5:1) mixture containing adsorbed water proved to be very satisfactory for our purpose. The chromatographic separations were carried out under reduced pressure (200 mm.). The solute, dissolved in nltrobenzene-benzene (1:4 by volume), was adsorbed on the column and the chromatogram was developed with benzene. Where necessary, benzene-ether mixtures of increasing developing strength were used in order to develop the chromatogram. Thus, it was possible to separate the following mixture of known 2,4-dinitrophenyl hydrazine derivatives on silicic acid-Oelite (5:lj 8$ water). These derivatives are listed in the order of their decreasing adsorption affinity: D,L-glyceraldehyde

2,4-dinitrophenylhydrazone, dihydroxyacetone 2,4-dinitro phenylhydrazone, triose 2,4-dinltrophenylosazone, mesox aldehyde trls(2,4-dinitrophenylhydrazone), mesoxaldehyde l,2-bis(21,4'-dlnitrophenylhydrazone), glyoxal bis(2,4- dinitrophenylhydrazone) and methylglyoxal bls(2,4-dinitrophenylhydrazone).

A relationship exists between the polarity of sub stances and their behavior on a chromatographic column. 108 any one of these crude preparations differed markedly in their chromatographic properties. These considerations led us to develop the method of purification which is described below. This method bears some resemblance to the method of frontal analysis of Tiselius, Claesson and collaborators (199)•

(199) E. Lederer and M. Lederer, "Chromatography. A Review of principles and Applications,'* Elsevier Publishing Co., New York, N. Y., 1953, p. 3-

The mixture of slightly soluble substances was dissolved in warm nitrobenzene (1 volume) and the solution was diluted with benzene (4 volumes), a non-solvent.

The highly dilute, supersaturated solution was then placed on the silicic acid-Celite (5:1; Qffo water) column top, and this addition was maintained continuously.

After the adsorbent was washed, the material in the colored zones on the column or the material in the column effluent was recovered, depending on the component which was desired. This procedure was applied to the purifi

cation of crude preparations of triose 2,4-dinitrophenyl-

osazone, mesoxaldehyde l,2-bls(2*,4’-dinitrophenylhydrazone) and mesoxaldehyde trie(2,4idinitrophenylhydrazone). As much as one gram of the crude preparations of these

substances was thus purified at one time, on a column

of 54 mm. diameter. The elemental analyses of the 109 substances thus obtained were in excellent agreement with the calculated analyses. The recovery of the desired component of a crude preparation was facilitated

in some cases since the column effluent was a super

saturated solution. Cooling of this effluent at 4° afforded in separate instances mesoxaldehyde l,2«ble(21,41 dinitrophenylhydrazone) and mesoxaldehyde tris(2,4-dinitro phenylhydrazone) (from reductone) in 57% and 30$ yield,

respectively (based on the amount of the mixture chroma

tographed) . Fractionation, by Extraction with Alcohol (95#)« of the

2 .4-Dlnitrophenvlhvdrazlne Derivative of the Residue of

the Repeatedly Sublimed Condensate Aqueous Solution

Our investigation of the condensate aqueous solution

(Figure 1) was continued. Residue m , arising from the

repeated low-temperature sublimation of the condensate

aqueous solution, was derivatized with 2,4-dinitrophenyl-

hydrazlne in 30$ perchloric acid. Since glyoxal

accounted for 75$ of the total free carbonyl content of

Residue h i (Table I), the 2,4-dinitrophenylhydrazine derivative of Residue m was expected to contain mostly glyoxal bis(2,4-dinitrophenylhydrazone). Such a large proportion of glyoxal bis(2,4-dinitrophenylhydrazone) would have Interfered greatly with the isolation of other derivatives of 2,4-dinitrophenylhydrazine present in the

same mixture. For this reason, and with the knowledge

that glyoxal bis(2,4-dinitrophenylhydrazone) is less

soluble in most organic solvents than 2,4-dinitrophenyl-

hydrazones, the 2,4-dinitrophenylhydrazine derivative of

Residue ill was fractionated by extraction.

The 2,4-dinitrophenylhydrazine derivative of Residue

III was extracted with ethanol (95$)« Extract I was thus

obtained, in 7.8$ yield. The residue of extraction was

re-extracted with 95$ ethanol, affording Extract II in

3*4$ yield. There was then left an alcohol-insoluble

10)0 11$ residue (Figure 1). There is some evidence which indicates that the extraction of the 2 ,4-dlnitrophenylhydrazine derivative of Residue ill with 95$ ethanol resulted in the fractionation of the components of that derivative. This evidence is based mainly on light absorption measurements.

Light absorption studies of known derivatives of

2,4-dinitrophenylhydrazine have been made by others

(159,156) and have shown that l,2-bis(2,4-dlnitrophenylhydrazones) absorb at longer wavelengths than 2,4-dlnitrophenylhydrazones which have neither ethylenic double bonds nor aromatic rings in conjugation with the imlno linkage. Table ill shows the wavelength position of the main maximum ( X max.) in the extinction curves of formaldehyde 2,4-dinitrophenylhydrazone and glyoxal bis(2,4-dinitrophenylhydrazone) which are used to illus trate the generalization made above and which have light absorption spectra typical of those of the 2,4-dinitro phenylhydrazine derivatives in which we were interested.

Table ill also shows the value "R" for the above two known derivatives of 2,4-dinitrophenylhydrazine, "R" being defined, for a given substance in solution, as the quotient resulting from the division of the numerical value of its optical density at 44l mjK by the numerical value of its optical density at 5^9 m/t • Thus " R'* is smaller for formaldehyde 2,4-dinitrophenylhydrazone than Table III

Light Absorption Characteristics of Some Derivatives of 2,^-Dinitrophenylhydrazine^

Product” X max. f^mjJL R3

F ormaldehyde 2 , dinitrophenylhydrazone 3**9 0.070

Glyoxal bis(2, ^f-dinitrophenylhydrazone) kkl 3.15 2,lf-Dinitrophenylhydrazine derivative of --- 2.1k Residue III

Extract I --- 0.50

Extract II --- 1.0

Alcohol-insoluble residue --- 2.35

— The measurements of the light absorption characteristics were taken in dioxane.

— See Experimental part for details of their preparation.

— Wavelength of the main maximum in the extinction curve; concentration, 1.00 mg. per 100 ml.

j — For a given substance in solution, R is the quotient resulting from the division of the numerical value of its optical density at 4^1 m^t by the numerical value of its optical density at 3^9 mu. ; thus, R is independent of concentration. ' 113 for glyoxal bis(2,4-dlnitrophenylhydrazone).

Extending our discussion to a hypothetical mixture

of 2 ,4-dlnitrophenylhydrazones and l,2-bis(2,4-dinitrophenylhydrazones), *' R" is then a measure of the amount

of 2,4-dinitrophenylhydrazones In the mixture. Thus, a

small value of "R" would Indicate the presence of a large amount of 2,4-dinitrophenylhydrazones in the mixture. The

concept of **R" was applied to the 2,4-dinitrophenylhydra

zine derivative of Residue m , to Extracts I and II, and

to the alcohol-insoluble residue (Figure 1). The results

are shown in Table ill. These results clearly show that

there is a larger concentration of 2,4-dinitrophenylhydra

zones in Extracts I and II than in the 2,4-dinitrophenyl

hydrazine derivative of Residue III. This conclusion is

supported by the fact that the "R" value of the alcohol-

insoluble residue is larger than that of the 2,4-dinitro

phenylhydrazine derivative of Residue III, as expected if

2,4-dinitrophenylhydrazones were removed from the 2,4-

dinitrophenylhydrazine derivative of Residue III. The

sharp increase, on successive extraction, of the *'R" value

of the extracts, accompanied by a decrease in the

percentage of material extracted (Extract I, 7«8j£j

Extract II, 3.4$), also Indicates that some components

of the 2,4-dinitrophenylhydrazine derivative of Residue

III are selectively extracted with alcohol. Investigation of the Alcohol-Soluble Fraction

It Is reasonable to assume that both Extracts I and

II (Figure 1) would have qualitatively the same compo

sition. Thus, our investigation of the alcohol-soluble

fraction of the 2,4-dinitrophenylhydrazlne derivative of

Residue III was limited to Extract I. Extract I was

chromatographed on silicic acid-Celite (5:1» 8# water)

following the method discussed under "Chromatography of

2,4-Dinitrophenylhydrazine Derivatives of Known Carbonyl

Compounds" and was separated into six zones. A diagram

matic representation of the chromatogram, after full develop

ment, is shown in Figure 12. The progress of the separation

was followed with ease since the zones are colored. On

elution from the column, the materials in Zones 1 and 2

were collected separately, by means of a fraction collector.

The materials in Zones 3 to 6 were obtained by extrusion

of the column and elution of the desired sections of the

column with 95$ ethanol. The percentage of material found

in each zone, based on the total amount of Extract I which

was chromatographed, was as follows*. Zone 1, 22#; Zone 2,

22#j Zone 3, 11$ i Zone 4, 3$i Zone 5, 4#j Zone 6, 43$.

» The components of each zone were identified by comparison

of their chromatographic properties and infrared spectra

with those of authentic specimens. In some cases, the

114 115

VM Zone 6

m Zone 5

Zone 4

Zone 3

Zones I and 2to receiver

Figure 12. Diagrammatic Representation of the Chromatogram of Extract I on Silicic Acid- Celite (5*1? 8# water). 116 identity of the components of each zone was further established by melting point, mixed melting point or visible absorption spectrum.

The material in Zone 1 was purified by sublimation at 80° and ca. 0.5 mm. pressure, several recrystalllza- tions of the sublimate afforded acetone 2,4-dinitrophenylhydrazone, the major component of Zone 1. The residue of evaporation of the mother liquors was chromatographed on silicic acld-Celite (2:1} 0$ water), following the method of Malmberg (81), and was separated into four zones.

A diagrammatic representation of the chromatogram, after full development, is shown in Figure 13* Zones 1-D, 1-0 and 1-B afforded the 2,4-dinitrophenylhydrazones of formaldehyde, acetaldehyde and acetone, respectively.

There was too little material (ca. 1 mg.) in Zone 1-A to allow us to pursue our investigation of this zone which appeared to contain several components.

The sublimation, at 80° and ca. 0.5 mm. pressure, of

Zone 2 resulted in its fractionation into a sublimate, identified as 2,4-dinitroaniline, and a residue, identi fied as methylglyoxal bis(2,4-dinitrophenylhydrazone).

G-lyoxal bis(2,4-dinitrophenylhydrazone), 2,4-dinitrophenylhydrazine and the material in Zone 3 had similar chromatographic properties on silicic acid-Cellte (5:1;

8$ water). The material in Zone 3 was shown to contain 117

Zone l-D

Zone l- C

Zone l- B

Zone I-A to receiver

Figure 13* Diagrammatic Representation of the Chromatographic Separation on Silicic Acid-Celite (2*1; 0% water) of the Residue of Evaporation of the Mother Liquors from the Recrystallizations of the Sublimate of the Material in Zone 1. 118 no 2,4-dinltrophenylhydrazine since no part of it sublimed at 110° and ca. 0.5 mm. pressure. G-lyoxal bis(2,4-dinitrophenylhydrazone) was the only component of this zone.

The material in Zone 4 was further separated into three zones by chromatography on silicic acid-Celite

(5:1» 8# water). These zones were numbered 4-A, 4-B and

4-0 in the order in which they appeared from the bottom of the column. An insufficient amount of the material in each one of these zones prohibited a full investi gation of their nature. It was established, however, that none of the known 2 ,4-dlnltrophenylhydrazine deriva tives at hand had properties (infrared spectrum, ultra violet and visible spectrum, and chromatographic behavior) identical with those of the materials in Zones 4-A, 4 -b and 4-0. It is suggested on the basis of the infrared spectra, ultraviolet and visible spectra, and chromato graphic properties of the materials in these zones that they were 2 ,4-dinltrophenylhydrazine derivatives of polymeric nature.

The material in Zone 5> after rechromatography, yielded mesoxaldehyde tris(2,4-dinltrophenylhydrazone).

Triose 2,4-dinitrophenylosazone was found to be a minor component of Zone 6, the zone which contained more material than any one of the other five zones. 119

Investigation of the material in Zone 6 by chromatography on silicic acM-Cellte (5tl* 8# water) yielded several

zones. None of the materials in these zones had chroma tographic properties identical with those of any of the authentic substances available except triose 2,4-dinltrophenylosazone. The materials in all of the unidentified

zones gave a blue-violet color with alkali, which may be

taken as an indication of the presence of~2,4-dinitrophenylhydrazine residues i.r08 )aG6H3NHN=7 in adjacent positions (157) in these materials. The materials in

the unidentified zones are probably polymeric in nature.

This explanation is supported by the black, tar-like

appearance of the material in Zone 6. Investigation of the Alcohol-Insoluble Residue

The alcohol-Insoluble residue (Figure 1) was re crystallized twice from nitrobenzene to afford glyoxal bis(2,4-dinitrophenylhydrazone) in 22% yield. The low yield of glyoxal bis(2,4-dinitrophenylhydrazone) was expected because the mother liquor of the first recrys tallization was pitch black, indicating that the alcohol- insoluble residue contained a considerable amount of tars.

G-lyoxal bis (2,4-dinit rophenylhydraz one) was by no means the only component, aside from tars, of the alcohol- insoluble residue. This fact was shown by a comparison of the chromatographic properties of the alcohol-soluble

(Extract l) and alcohol-insoluble (alcohol-insoluble residue) fractions. The chromatograms were qualitatively the same with the exception of the absence of Zones 1 and

4 (Figure 12) in the chromatogram of the alcohol-insoluble fraction. Since methylglyoxal bls(2,4-dinitrophenylhydrazone), mesoxaldehyde tris(2,4-dinitrophenylhydrazone) and triose 2 ,4-dlnitrophenylosazone are quite insoluble in alcohol and were present in Zones 2, 5 and 6, respec tively, of the alcohol-soluble fraction (Extract i), it is reasonable to assume that these substances are also present in the alcohol-insoluble residue as is

indicated by its chromatogram.

120 Attempted Isolation of Triose 2,4-Plnitrophenylh.ydrazone from the Condensate Aqueous Solution

The isolation of 2,4-dlnitroanlline from Zone 2

(Figure 12) of the chromatogram of Extract I suggests osazone formation in the derivatization, with 2,4- dinltrophenylhydrazine, of Residue ill (Figure 1). in addition, methylglyoxal bis(2,4-dinitrophenylhydrazone) and triose 2,4-dinitrophenylosazone were also isolated from Extract I. Since the above three compounds could have arisen (75) from either glyceraldehyde or dlhydroxy- acetone, an attempt was made to Isolate either of the trioses (as the 2,4-dinltrophenylhydrazones) from the condensate aqueous solution.

The method of preparation of the 2,4-dinitrophenylhydrazine derivative of trioses can be selected so that the reaction takes place without oxidation of the hydroxyl group adjacent to the carbonyl group and without rearrange ment (191) of the trioses (see discussion under "Prepara tion of the 2,4-dinitrophenylhydrazones of short carbon chain sugars"). In addition, the solubility in hot water of D,L-glyceraldehyde 2,4-dinitrophenylhydrazone and dihydroxyacetone 2,4-dinitrophenylhydrazone was found to be much greater than that of any other 2,4-dinitrophenylhydrazine derivative previously isolated from the conden sate aqueous solution (derivatized with

121 122

2,4-dinitrophenylhydrazine). Finally, D,£-glyceraldehyde 2 ,4-dlnitrophenylhydrazone was shown to be

stable in high vacuum at 110° for over 24 hours. Under

these conditions, dihydroxyacetone 2,4-dinitrophenyl-

hydrazone showed signs of charring. The knowledge of all the facts listed above was combined in our attempts

to isolate either of the trioses (as the 2,4-dinitro-

phenylhydrazones) from the condensate aqueous solution.

The first attempt was made by preparing the 2,4- dlnitrophenylhydrazine derivative of a neutralized,

concentrated condensate aqueous solution, in boiling

ethanol. The reaction resulted in a brown to black product which undoubtedly contained a large proportion

of tars which would have greatly interfered with the

isolation of either of the triose 2,4-dinltrophenylhydra-

zones by chromatography. Since another method of pre

paring the 2,4-dinitrophenylhydrazone of trioses was

known, this attempt was abandoned.

In our second attempt, the preparation of the 2,4-

dinitrophenylhydrazine derivative, using a supersaturated

solution of 2,4-dinitrophenylhydrazine in 2 hydrochloric

acid at 0°, of a condensate aqueous solution resulted

in a product which was extracted with hot water. The

residue after evaporation of the solvent was sublimed

at 110° and 0.5 mm. pressure. Under these conditions,

authentic triose 2,4-dinltrophenylhydrazones do not sublime. A charred sublimation residue resulted from the sublimation of the water extract. This residue was shown by chromatography on silicic acid-Celite

(5:1; 8% water) to contain no triose 2,4-dinitrophenylhydrazones. feaa Absorption Ohromatography of the Liquid Mixture of

Cellulose Nitrate ignition Products

Our Isolation of minute amounts of acetaldehyde, as the 2,4-dinitrophenylhydrazone (ca. 5 mg.), from the liquid mixture arising from the ignition of 26 g. of cellulose nitrate suggested that the ignition products might contain many organic compounds which could be isolated via long, multi-step routes only with great difficulty because of their instability, volatility or minute quantity. Hence, a search for these organio compounds by gas absorption chromatography (Figure 1) was initiated since this mode of separation provided a rapid method of analysis for the freshly prepared liquid mixture of ignition products.

The gas abscrption chromatography apparatus is shown in Figure 14 (2oo). A column of polyethyleneglycol-400

(200) This apparatus waB kindly lent by Messrs. H. R. Menapace and V. G-. Wiley. was selected for our purpose since the following components of the condensate aqueous solution were known to react with the adsorbent and to be retained permanently by the column (2ol): formic acid, acetic acid (5), formaldehyde,

(201) Private communication from Messrs. H. R. Menapace and V. G-. Wiley.

324, 125

Hoke needle valve & Manometer

Regulator yD

Thermal conductivity Helium cell

Serum cap

4 ' column in Flowmeter —0 - insulated jacket

Condensation traps

Figure l*f, Schematic Diagram of the Gas Absorption Chromatography Apparatus, 126 glyoxal and methylglyoxal. Solvent sheets (202), cast

(2o2) Private communication from Dr. L. P. Kuhn. from ethyl acetate, of cellulose nitrate were ignited.

The condensate (liquid mixture of ignition products) found in the first spiral trap (nearer to the combustion tube) (5) was used in this study since a large amount of water would have interfered with the chromatography had the condensate aqueous solution been used. Identification of the products obtained by gas chromatography was made by infrared spectrum and elution time.

Figure 15 shows the recorded gas chromatogram of the first spiral trap condensate separated on a 1.22 m. (4*) column. This recorded chromatogram shows, apart from the large zone ascribed to volatile gases, four major zones and some minor peaks. The materials in Zones 1 and 2 were identified, respectively, as acetaldehyde and ethyl acetate.

The presence of ethyl acetate in the ignition products shows that the "solvent-free" sheets (202) ignited still contained residual traces of the sheet casting solvent, ethyl acetate.

The gas chromatographic study was continued on a

4.88 m. column. The use of a longer column resulted in a better separation of the components of the first spiral trap condensate. Thus, on the longer column, the region Recorder response, mv. a 1.22 m* Of*) Polyethyleneglycol-wO Column. Of*) Polyethyleneglycol-wO m* 1.22 a Separation of the Liquid Mixture of Cellulose Nitrate ofCellulose Mixture theLiquid of Separation giinPout (is Sia rpCnest) on Condensate) Trap (First Spiral Products Ignition Zone Figure 15. Recorded Gas Chromatogram of the of Chromatogram Gas Recorded 15. Figure < o — — o ! * oe 2 Zone UJ . > 040 4 30 ? 2 1 ie min. Time,

50 60 oe 3 Zone oe 4 Zone 5 T3I k O I 5 Q- 5 I" c _ c >> □ 0 8

0 9

128 prior to Zone 1 (Figure 15) was found to be complex

and the small peaks between Zone 1 and Zone 2 were well

separated, it was thus possible to isolate the material

in the small peak located immediately before Zone 2

(Figure 15). This material was identified as acrolein.

The material in the zone prior to the acrolein peak had an elution time identical with that of acetone.

The infrared spectra (in carbon disulfide) of the material in Zone 3 from the 1,22 m. and the 4.88 m.

columns are shown in Figures 16 and 17, respectively.

These spectra are very similar, each showing only one major spectral absorption band at 10.5 microns. These

spectra allow us to conclude that the material in Zone 3

has not previously been isolated from the condensate aqueous solution. The material in Zone 3 is still uniden

tlfied. However, on the assumption that the material in

Zone 3 is a single compound, it is possible to conclude

that the material in Zone 3 is not a carbonyl compound

since its infrared absorption spectra (Figures 16 and 17)

show no strong spectral absorption band in the region

5 .5-6.0 microns (203).

(203) L. J. Bellamy, "The infrared Spectra of Complex Molecules," John Wiley and Sons, Inc., Hew York, N.' Y., 1954, p. 114. ‘ 1 62

Figure 16. Infrared Absorption Spectrum of the Material in Zone 3 from the Separation, by Gas Chromatography on a 1,22 m. Polyethyl enaglycol-J+OO Column* of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate). 90

BO 130

50 6 0 80

40 40

Figure 17* Infrared Absorption Spectrum of the Material in 2one 3 from the Separation, by Gas Chromatography on a *+,88 m. Polyethyleneglycol-1+00 Column, of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate), 131 The infrared spectra of the material in Zone 4, in carbon disulfide and in the gas phase, are shown in

Figures 18 and 19, respectively. As in the case of the material in Zone 4, hydrogen cyanide was found to have, in its infrared spectrum in carton disulfide, a broad spectral absorption band at 13*9 microns. This band was displaced, as in the case of the material in Zone 4, to a sharp peak at 14.1 microns, when the Infrared spectrum of hydrogen cyanide was taken in the gas phase.

Furthermore, hydrogen cyanide waB found to have an elution time Identical with that of the material in Zone 4 and showed no infrared spectral absorption bands which were absent in the spectra of Zone 4. It is suggested, there fore, that hydrogen cyanide is present in Zone 4.

However, the infrared spectra of Zone 4 leave no doubt that this zone contains another constituent, beside hydrogen cyanide. This other constituent may be a car bonyl compound since the absorption band at 5.8 microns

(203) ('Figures 18 and 19) is absent in the infrared spectra of hydrogen cyanide.

The amount of acetaldehyde and ethyl acetate present in the condensate aqueous solution was estimated from the recorded gas chromatogram (Figure 15) to be 0.02 and

0.01 mmoles per mmole of cellulose nitrate ignited (181), respectively. For the purpose of calculating this

estimate, it was necessary to assume that all of the 20

Figure 18, Infrared Absorption Spectrum (in Carbon Disulfide; Compensated) of the Material In Sane if from the Separation, by Gas Chromatography, of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate), lo £ $SSL 223.

80 SSL J -

70 70 JO 03: 2SL

jfii 60-

-S£ »b

4 0 '

30 30 30 30

20 20 23 JO_

Figure 19* Infrared Absorption Spectrum (Gas Phase) of the Material in Zone from the Separation, by Gas Chromatography, of the Liquid Mixture of Cellulose Nitrate Ignition Products (First Spiral Trap Condensate)* 134 ethyl aoetate and acetaldehyde resulting from the ignition of cellulose nitrate would be found in the first spiral trap condensate.

It was necessary to heat the sample of the first spiral trap condensate at the point of its insertion on the gas chromatographic column In order to volatilize it.

This resulted in charring of a seemingly large, porous, non-volatile residue. The gas chromatographic results may thus have been affected.

Ethanol has an elution time a little longer than that of the material in Zone 4 (Figure 15) and was absent in the first spiral trap condensate studied by gas chromatography. However, traces of ethanol have previously been found in the condensate aqueous solution

(5). In order to explain these different results, it is suggested that the traces of ethanol which have previously been reported (5) arose from the slow acid hydrolysis of the ethyl acetate present in the condensate aqueous solution. Origin of Compounds isolated, from the Liquid Mixture of

Cellulose Nitrate ignition Products

The oompounds which have been isolated, In the course of our work, from the liquid mixture of cellulose nitrate ignition products are shown in Table IV. This mixture has previously been reported to contain several substances, among which were formaldehyde, glyoxal and hydrogen cyanide (5,6). The presence of these three compounds in the liquid mixture of ignition products has been confirmed during the present work. Their modes of formation or positions of origin, in the anhydro-J)-glucose units, have alBo previously been studied (5-7).

in our work, "solvent-free’’ sheets (2o2) of cellulose nitrate were found, on ignition, to afford a liquid mix ture which contained ethyl acetate, the sheet casting solvent. This was not unexpected in view of a study which was made elsewhere of casting solvent retention by cellu lose nitrate films (2o4). The presence of residual casting

(204) F. D. Miles, "Cellulose Nitrate," Interscience Publishers, Inc., New York, N. Y . , 1955, PP. 203-205* solvent in the cellulose nitrate ignited is undoubtedly responsible for several of the compounds isolated in the complex mixture of ignition products.

135 Table IV

Compounds Isolated, in Our Work, from the Liquid Mixture of Cellulose Nitrate

Ignition Products^

As Derivatives of 2,*f-Dinitrophenylhydrazine5 By Gas Absorption Chromatography^

Acetone 2 ,if-dinitrophenylhydrazone Acetaldehyde

Acetaldehyde 2 , lf-dinitrophenylhydrazone Acetone^

Formaldehyde 2 , lf-dinitrophenylhydrazone Acrolein

Methylglyoxal bis(2 , lf-dinitrophenylhydrazone) Ethyl acetate

Glyoxal bis(2 , lf-dinitrophenylhydrazone) Unidentified compound

Mesoxaldehyde tris(2 ,*f-dinitrophenylhydrazone) Hydrogen cyanide—

Triose 2 ,1f-dinitrophenylosazone

— Cellulose nitrate, 1 2 ,6% N, cast from ethyl acetate solution and ignited at 75 run* pressure. — Details of the isolation procedures are given in the Experimental part.

— Identified by elution time only.

— The zone containing hydrogen cyanide also contained some other material as well. 137 Acetone had heen reported to he absent In the product of ignition of cellulose nitrate films cast

from ethyl acetate (5 ). By the use of more sensitive

techniques, it has now been shown that acetone is a product of Ignition. Acetone may have originated from

the pyrolysis of ethyl acetate (205)* However, this is

(205) R. D. Obolentsev and Y. N* Usov, Doklady Akad. Nauk. S.S.S.R., 11, 489 (1950)j 0. A., 45, 547 (1951). by no means certain since acetone has been reported

among the products of pyrolysis of cotton cellulose (2o6).

(206) P. Klaeon, G-. v. Heidenstam and E. Norlin, Z. angew. Ghem., 22, 1205 (1909)*

Cellulose nitrate is stored in a moist atmosphere

and, before films are prepared, is dehydrated with

absolute alcohol. The dehydrated material is freed of

most of the alcohol by filtration and then dissolved in

ethyl acetate (4). Since the presence of ethyl acetate

in "solvent-free" sheets (2o2) has been demonstrated,

the presence of ethanol cannot be in doubt. However,

unlike ethyl acetate, ethanol was not found in the freshly

prepared ignition products. It is suggested, therefore,

that ethanol pyrolyzes to acetaldehyde (207) or is 138

(207) C. D. Hurd, “The Pyrolysis of Carbon Compounds," The Chemical Catalog Company, Inc., New York, N. Y., 1929, p. 149. oxidized to the same compound by nitrogen dioxide.

The compounds isolated from the decomposition products of cellulose nitrate are not necessarily the original frag ments of the residual casting solvent or of the anbydro-|)- glucose units. Some of the compounds in the ignition mixture are undoubtedly derived from secondary reactions.

Crotonaldehyde is found among the products of pyrolysis of acetaldehyde and is believed to originate from an aldol condensation of two molecules of acetaldehyde, followed by dehydration (2o8). A similar reaction between formaldehyde

(2o8) See reference 2 0 7 i P« 2 3 7 * and acetaldehyde would yield acrolein which was isolated from the cellulose nitrate ignition products and which had previously been reported as a product of ignition (38).

However, since compounds, other than acrolein, containing three carbon atoms have been Isolated from the condensate aqueous solution, it is also possible that acrolein origi nated from the anhydro-D-glucose units in the cellulose nitrate ignited. 139 The reaction of the ignition products with substi

tuted hydrazines has afforded a mesoxaldehyde derivative both in this work and in a prior instance (5)• Since

the mesoxaldehyde derivatives isolated could have been

obtained from either triose-reductone or mesoxaldehyde, an attempt was made to determine whether or not triose- reductone was present in the ignition products (2o9). A

(209) M. L. Wolfrom and E. E. Dickey, unpublished results.

standard triose-reductone solution was quantitatively

oxidized by Tillman's reagent (2,6-dichloro-phenol-indo- phenol) or by a standard iodine solution. The results were inconclusive, however, when these methods were applied to the product of ignition, at 20 mm. pressure,

of 12.6$ N cellulose nitrate. The iodometric titration and the titration with Tillman's reagent gave widely divergent values, 6 x 10 mmoles and 9 x 10 ^ mmoles of triose-reductone, respectively, per mmole of cellulose

nitrate ignited (181).

The presence of methylglyoxal bls(2,4-dinitrophenyl-

hydrazone), trlose 2 ,4-dinitrophenylosazone and 2,4-

dinitroanlline in Extract I (Figure l) led this worker

to believe that these compounds had originated from a

triose (75). Two unsuccessful attempts were made to l4o isolate a triose as Its 2 ,4-dinltrophenylhydrazone.

The failure to isolate a triose does not rule out the possibility of its presence in the ignition products.

However, the interpretation which leads one to the belief that a triose was present in the condensate aqueous solution may have been at fault. There is qualitative evidence to the effect that methylglyoxal Is present in the products of thermal decomposition of polyvinyl nitrate and its isolation is being pursued (2lo). Since either

(2lo) M. L. Wolfrom, A. Chaney and K. S. Ennor, unpublished results. ------of the trloses could hardly have been formed from polyvinyl nitrate, it is suggested that the methylglyoxal found in the present work was formed by some recombination process.

Malmberg (81) has reported the Isolation of "synthetic products," such as methylglyoxal, from a methane-air flame.

Formyl and methyl radicals are the only chemical entities necessary for the "synthesis" of methylglyoxal, and these radicals are undoubtedly formed in the course of the ignition of cellulose nitrate which is known to contain residual traces of the casting solvent. EXPERIMENTAL

General Statements

Cast sheets of cellulose nitrate containing 12.6# N were prepared as described previously (4), employing ethyl acetate as the solvent. The controlled thermal decomposition of cellulose nitrate was carried out at

75 mm. pressure in the apparatus described by Wolfrom and associates (5). The condensate obtained as the product of ignition was removed from the ignition tube and spiral traps (allowed to stand at 0° for one hour prior to removal) by dissolving it in water. After filtration, the solution was diluted to the desired volume, namely,

50 ml. per g. of cellulose nitrate ignited, with water.

This aqueous solution shall be referred to hereafter as the "condensate aqueous solution." Formaldehyde analyses of the condensate aqueous solution from three separate ignitions agreed within + 3#. Thus, the ability of this operator to obtain reproducible results has been demon strated.

Unless otherwise indicated, no attempt was made to remove the last residual traces of the film casting

Bolvent. When such an attempt was made, cellulose nitrate sheets were cut into strips (2 x 10 cm.). The strips were submerged in water, at room temperature, for one week

141 142 with occasional changes of water. The strips were then submerged in water, at 90°, for 48 hours. The water was removed by decantation, and the cellulose nitrate strips were dried to constant weight .in vacuo over phosphorus pentoxlde. The strips obtained in this fashion were considered to be essentially free of removable ethyl acetate (2o2).

All melting points were determined with a Kofler micro hot stage and are corrected. The melting point of substances which decompose on melting was taken by placing the samples on this stage five to ten degrees below the melting point and, then, raising the temperature at a rate of oa. four degrees per minute.

The elemental analyses were carried out by Galbraith

Kicroanalytieal Laboratory, Knoxville, Tenn.

The light absorption spectra covering the range of wavelength from 320 to 600 mp were determined with a

Beckman model DU spectrophotometer, using 1 cm. silica cells. The solvent used in each case is shown in parentheses.

The infrared spectra were taken in a perkin-Elmer spectrophotometer Model 21 equipped with sodium chloride optics. The phase in which these spectra were obtained, varied. Where the spectra were measured using the potassium bromide technique, they are identified as

"(KBr)." The spectra taken in carbon disulfide 143

(compensated) In a 3 mm. cell are shown as ‘'(CSg)."

Spectra labelled "(gas phase)" were measured In a 10

cm. gas cell with sodium chloride windows. Chromatography of Derivatives of 2 .A—Dlnltrophenvlhydra- zlne

The chromatographic separations desoribed herein were performed on silicic acid (Analytical Reagent,

Mallinckrodt Chemical Works, St. Louis, Mo.) and Celite

(No. 535, Johns-Manvilie Co., New York, N. Y.) mixtures.

Specifically, three such mixtures were used.

The preparation of a silicic acid-Celite (2*1) mix ture free from water removable by heating at 200° was described by Trueblood and Malmberg (211,212). The silicic

(211) K. N. Trueblood and E. W. Malmberg, Anal. Chem., 21, 1055 (1949).

(212) K; N. Trueblood and E. W. Malmberg, J. Am. Chem. Soc., J2, 4112 (1950).' ' acid used in this case was ground before mixing with

Celite (108). This adsorbent, identified as "(2:1),*' was used for the separation of the 2,4-dinitrophenylhydrazine derivatives of formaldehyde, acetaldehyde and acetone, following Malmberg's method (81).

Silicic acid and Celite were mixed in the ratio of

5:1 and the mixture was dried overnight at 200° (211,212).

This adsorbent, which is free from adsorbed water and is identified as "(5:1; 0^),° was used for the chromatographic purification of new derivatives of 2,4-dinitrophenylhydrazlne.

144 145

The separation of unknown, complex mixtures of

2,4-dinitrophenylhydrazine derivatives was achieved on a silicic acid-Celite (5:1) mixture containing 8% of adsorbed water. This adsorbent, referred to as "(5:1;

8^)," was prepared by either of two equally satisfactory methods. In both cases, silicic acid-Celite (5:1* 0%) was used. One method consisted in mixing the adsorbent of 0$ water content with enough silicic acid-Celite (5:1), of water content higher than 8$, to give the desired results. The other method consisted in adding a calcu lated amount of water to the adsorbent (5:1;

In all cases, the chromatographic columns were packed and developed at 200 mm. pressure. Before the solute was added to the column, the adsorbent was prewashed, unless mentioned otherwise, with a sufficient amount of benzene to wet the column completely. Mixtures of two solvents were often used as developers. Their proportions, referred to in terms of ratios or percentages, were by volume. Zones which, on development of a chromatogram, moved off the column were collected separately by means of a fraction collector.

Comparative chromatograms and chromatographic tests of purity were carried out on silicic acid-Celite 146

(5:1; 8$). A representative sample of the substance

under investigation was dissolved in nitrobenzene. A

0.2 ml. aliquot of the nitrobenzene solution, containing

1 mg. or less of the substance, was diluted with 0.8 ml.

of benzene and added immediately to a column (8 mm., diam., x 2o cm.) of adsorbent. The chromatogram was developed with benzene and, when necessary, with benzene-ether mixtures of increasing developing strength. For compara

tive purposes, both known and unknown substances were

chromatographed simultaneously on separate columns and, after full development, the Inside core position of the

zones in the cut columns was measured. Since it is known

that sodium hydroxide gives a blue to violet color with

2,4-dinltrophenylosazones or l,2-bls(2,4-dinitrophenyl-

hydrazones) and a red to brown color with 2,4-dlnitro-

phenylhydrazones (157), additional information was obtained by streaking the zones with 1 N sodium hydroxide (81).

The following known substances are listed in the

order of their adsorptive affinity on silicic acid-Celite

(5:1; 8$), beginning with the least adsorbed: formalde hyde 2,4-dinltrophenylhydrazone, methylglyoxal bis(2,4-

dinit rophenylhydrazone) and 2,4-dinitroaniline, glyoxal

bis(2,4-dlnitrophenylhydrazone) and 2,4-dlnitrophenyl-