t h e e f f e c t o f i o n i z i n g r a d i a t i o n s o n c e r t a i n CARBOHYDRATES AND RELATED SUBSTANCES

Dissertation

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

ANDREW UICHAEL MICHELAKIS, B. Sc., M. Sc.

The Ohio State university 1959

Approved by

Adviser Department of Chemistry ACKNOWLEDGMENT

The author Is very grateful and Indebted to Professor M. L. Wolfram for his inspiration, encouragement and guidance In the course of this investigation. Th e association with all the members of the Sugar Alley has been very pleasant, acknowledgment Is expressed to all of them, and especially to Mr. Leo J. McCabe. The author wishes to express his sincere appreciation and thanks to Mrs. Mary Leonid&kls for typing this thesis. Acknowledgment is made to Hr. B. Schmidt and Professor Dudley Williams of the Department of Physics for their help in obtaining and interpreting the electron- spin resonance spectra. The financial help provided by the Quartermaster Food and Container Institute for the Armed Forces, Research and Development Command, Quartermaster Corps, U. S. Army, Is acknowledged. Acknowledgment is also made to the Department of Chemistry for support as a teaching assistant.

ii TABLE OF CONTENTS

IMS INTRODUCTION...... 1 Types of Radiation...... 2 I. Photolytic Radiation...... 2

II. Ionizing Radiation...... 5 1. Units and Terms Used In Ionizing Radiation ...... 3 2. Cathode Rays...... 4 3. Gamma Rays...... 5

4. Action of Radiation...... 6 STATEMENT OF PROBLEM...... 7

HISTORICAL...... 8

I. Photolytic Radiation..,...... 8 II. The Irradiation of Water...... 12 III. Direct and Indirect Action of Ionizing Radiation...... 16 IV. Distinction of Radiation Chemistry from Photochemistry...... 16 V. Irradiation of Polymers Other Than Carbohydrates...... 17 VI* Irradiation of Carbohydrates...... 18 1. Effect of Ionizing Radiation on Cellulose and Its Derivatives...... 18 2. Effect of Ionizing Radiation on Dextran...... SI 3. Effect of Ionizing Radiation on Inn 1 in...... 23

ill TABLE OF CONTENTS (Contd.)

£ass 4. Effect of Ionizing Radiation on Pectin...... 25 5. Effect of Ionizing Radiation on Starch...... 24

6 . Effect of Ionizing Radiation on Oligosaccharides...... 25 a. Rafflnose ...... 26 b. Maltose...... 26 c. Sucrose...... 26 d. Lactose...... 27 7* Effect of Ionizing Radiation on Monosaccharides...... 26

8 . Effect of Ionizing Radiation on 4-D-glucopyranoside...... 30 9. Effect of Ionizing Radiation on Polytaydric Alcohols...... 51 VII* Paramagnetic Resonance Study of Irradiated Carbohydrates...... 32 EXPERIMENTAL...... 54 I. Irradiation Sources...... 34 II. The Effect of Ionizing Radiation on Maltose. ••••...... 54 1. Irradiation of Maltose with Cathode Rays as Fifty Per Cent Aqueous Solutions at 0°C...... 34 a. Descending paper chromatography of maltose Irradiated as 50# aqueous solutions with cathode rays at 0°C...... 35

!▼ TABLE OF CONTENTS (Contd.)

Page 2* Irradiation of Maltose as Twenty Per Cent Aqueous Solutions with Cathode Beys...... 36 a. Descending paper chromatography of Irradiated 2 0 % aqueous maltose with cathode rays at ambient air, ice-water and ethanol-dry Ice temperatures.,**. 38 b. Determination of per cent hydrolysis of irradiated 20# aqueous maltose with cathode rays...... 39 c* Calculation of G values of irradiated 20 # aqueous maltose with cathode rays...... 40 d. Isolation of irradiation products from maltose irradiated with cathode rays 41 e. Acetylatlon of the slrupy monosaccharide fraction obtained from carbon column chromatography of maltose...... 42 f. The separation and Identification of the acetylated monosaccharide derivatives of irradiated 20# aqueous maltose at 0°C...... 43 III* The Effect of Ionizing Radiation on Celloblose...... 46 1* Irradiation of Celloblose with Cathode Rays as Fifty Per Cent Aqueous Solutions at 0°C...... 46 a* Descending paper chromatography of the Irradiated 50# aqueous celloblose samples*...... 46

v TABLE OF CONTENTS (Contd.)

£&&a 8 . Irradiation of Celloblose as Twenty Per Cent Aqueous Solutions with Cathode Bays at Q°C...... 47 a* Descending paper chromatography of Irradiated 2 0 % aqueous celloblose with cathode rays at 0°C...... 47 b. Determination of the per cent hydrolysis of 20% aqueous celloblose Irradiated with cathode rays at 0°C...... 46 c. Determination of 0 values of Irradiated 2 0% aqueous celloblose with cathode rays at 0°C...... 49 IV. The Effect of Ionising Radiation on Trehalose...... 49 1. Irradiation of Trehalose with Cathode Rays as Two Per Cent Aqueous Solutions at Ambient Air Temperature ••. 49 a. Chromatography of irradiated trehalose as 2% aqueous solutions with cathode rays 50 b. Determination of the per cent hydrolysis of trehalose Irradiated as 2$ aqueous solutions...... 50 c. Calculation of G values of irradiated trehalose...... 51 d. Isolation of Irradia tion products from irradiated trehalose. Dose received 80 megarads of cathode rays...... 51 e. taper chromatography of the monosaccharide fraction obtained from irradiated trehalose by separation on a carbon column ...... 58

vl TABLE OF CONTENTS (Contd.)

Eg&ft f# Acetylation of the obtained monosaccharide fraction..... 53 g„ The separation and Identification of the acetylated monosaccharide derivatives of Irradiated trehalose...... 54 V. The Effect of Ionising Radiation on Raff inose...... 55 1. The Irradiation of Rafflnose with Gamma Rays...... 55 a. Descending paper chromatography of Irradiated raff lnose as 8$ aqueous solutions with gamma rays...... 55 b. Ionophoresis of the raff lnose samples exposed to gamma radiation as 2% aqueous solutions...... 56 c. Determination of copper reducing sugar values of raff inose exposed to gamma radiation as 2 % aqueous solutions...... 57 2. The Irradiation of Two Percent Aqueous Raffinose with Cathode Rays at Ambient Air Temperature...... 56 a. Chromatography of the Irradiated 2$ aqueous raffinose solutions with cathode rays 58 b. Ionophoresis of rafflnose Irradiated with cathode rays as 2% aqueous solutions...... 59 c. Determination of the copper reducing sugar values of rafflnose irradiated as 2% aqueous solutions with cathode rays...... 60

vii TABLE OF CONTENTS (Contd.)

Page VI. The Effect of Ionising Radiation on Inulin...... 61 1. The Irradiation of Inulin as Two Per Cent Aqueous Solutions with Gamma Rays...... 61 a. Physical properties of inulin irradiated as 8$ aqueous solutions with Co^O gamma radiation at 24°C...... 61 b. Descending paper chromatography of inulin irradiated with gamma rays...... 62 c. Paper ionophoresis of inulin Irradiated with Co60 gamma radiation...... 62 d. Determination of the extent of inulin hydrolysis. Inulin exposed to gamma radiation as 2j6 aqueous solutions...... 63 e. Isolation of irradiation products from inulin irradiated with Co60 gamma radiation as 8% aqueous solutions...... 64 f. Paper chromatography of the slrupy fraction obtained from carbon column chromatographic separation of inulin irradiated with gamma rays...... 65 g. Acetylation of the slrupy monosaccharide fraction obtained from carbon column chromatography of Co60 gamma rays irradiated inulin...... 66 2. The Irradiation of Powdered Inulin with Cathode Rays...... 68 a. Effect of cathode ray radiation on the physical properties of powdered inulin...... 68 ▼ill TABLE OF CONTENTS (Contd.) Page b. Chromatography of powdered Inulin Irradiated with 400 megareps of cathode rays...... 68 S. The Irradiation of Two Per Cent Aqueous Inulin Solutions with Cathode Rays at Room Temperature...... 69 a. The effect of cathode radiation on the physical properties of 2 % dilute aqueous inulin solutions ...... 69 b. Chromatography of inulin irradiated with cathode rays as 2 % aqueous solutions...... 70 c. Ionophoresis of inulin irradiated with cathode rays as 2 % aqueous solutions...... 70 d. Extent of hydrolysis of inulin exposed to cathode radiation as 2 % aqueous solutions...... 71 VII. The Effect of Ionising Radiation on Amylose...... 72 1. The Irradiation of Two Per Cent Aqueous Mixture of Amylose with Gamma Radiation...... 72 a. Carbon column chromatography of amylose irradiated with gamma rays...... 72 b. Acetylation of the slrupy monosaccharide obtained from carbon column chromatography of irradiated amylose...... 73 c. The separation and identification of the acetylated monosaccharide derivatives of irradiated aqueous amylose mixture...... 74 VIII. The Effect of Ganna Radiation on D-Glucosamine Hydrochloride, D-Glucuronic acid and D-Galacturonlc Acid...... 76 lx TABLE OF CONTENTS (Contd.)

1 . Irradiation Procedure...... 76 £• The Collection of the Gases Produced from the Irradiated D- Glucosamine hydrochloride, D- Glucuronic Acid and D-Galacturonic Acid...... 77 3* Vapor Phase Chromatography of Gases Produced from D-Glucosamlne hydrochloride, D-Glucuronic Acid and D-Galacturonic Acid on Exposure to Guam* Radiation ...... 77 a. Apparatus...... 77 b. Analysis of the gaseous products.... 73 c. Tabulation of data...... 79 4, Paper Chromatography of Irradiated Aqueous Dilute D-Glucosamlne hydrochloride, D-Glucuronic Acid and D-Galacturonlc Acid...... 30 IX. Paramagnetic Resonance Studies of Irradiated Carbohydrates ...... 31 1. Irradiation of Certain Crystalline Carbohydrate Powders with Gamma Rays...... 81 2. Paramagnetic Resonance Spectra of the Irradiated Carbohydrate Powders...... 81 DISCUSSION OF RESULTS...... 84 I. The Effect of Cathode Radiation on Fifty Per Cent Aqueous Solutions of Maltose and Celloblose ...... 34 II. The Effect of Cathode Radiation on Twenty Per Cent Aqueous Maltose Irradiated at Ethanol-Dry Ice, Ice-Water and Ambient Air Temperatures...... 85 III. The Effect of Cathode Radiation on Twenty Per Cent Aqueous Celloblose Irradiated at Ice-Water Temperature...... 97 x TABLE OF CONTENTS (Contd.) Page IV. The Effect of Cathode Radiation on Two Per Cent Dilute Aqueous Solutions of Trehalose at Ambient Air Temperature.... 102 V. Effect of Ionising Radiations on Rafflnose...... 109 1. The Irradiation of Two Per Cent Dilute Aqueous Solutions of Rafflnose with Gamma Rays at Room Tempera ture.... •• 109 2. The Irradiation of Two Per Cent Dilute Aqueous Solutions of Rafflnose with Cathode Rays at Room Temperature.... 114 VI. Effect of Ionising Radiations on Inulin .... 118 1. The Irradiation of Dilute Aqueous Solutions of Inulin with Gamma Rays at Room Temperature...... 118 2. The Irradiation of Aqueous Dilute Solutions of Inulin with Cathode Rays at Room Temperature...... 124 3. Irradiation-Induced Depolymerisation of Powdered Inulin...... 127 VII. Irradiation-Induced Depolymerisation of Amylose...... 128 VIII. Ihvestlgatlon of Gases Produced an Irradiation of Carbohydrates...... 130 1. Effect of Gamma Radiation on D- Glucosamlne Hydrochloride...... 150 a. Gas chromatographic analysis...... 130 2. Effect of Gamma Radiation on Uronic Acids...... 132 a. Gas Chromatographic analysis...... 135

3 . Proposed Mechanism for the Formation of the G a s e s 138 xl TABLE OF CONTENTS (Contd.) Page a. D-Glucosamlne hydrochloride...... * 136 b. D-Glucuronic and D-galacturonlc acids...... 140 4. Carbohydrates and Genesis of Petroleum Hydrocarbons In Nature...... 141 IX. Paramagnetic Resonance Studies of Irradiated Crystalline Carbohydrate Powders...... 14S SUMMARY...... 156 CHRONOLOGICAL BIBLIOGRAPHY...... 162 AUTOBIOGRAPHY...... 168

xii LIST OF TABLES 2&ULA Page I* The Irradiation of 20# Aqueous Maltose with Cathode Bays...... 37 II, Extent of Hydrolysis of Maltose Irradiated as 20# Aqueous Solutions at Three Different Temperatures...... , 88 III, Extent of Hydrolysis of Celloblose Irradiated as 20# Aqueous Solutions with Cathode Rays at 0°C...... 99 IV, Reducing Sugar Values for Irradiated 2# Aqueous Trehalose...... 106 V. Reducing Sugar Values for Irradiated 2 % Aqueous Rafflnose with Gamma Rays...... 112 VI, Reducing Sugar Values of Aqueous Rafflnose Irradiated with Cathode Rays...... 117 VII, Reducing Sugar Values for Irradiated Two Per Cent Aqueous Inulin with Co60 Gamma Radiation...... 121 VIII, Reducing Sugar Values for Irradiated Two Per Cent Aqueous Inulin with Cathode Rays...... 126 IX, Gas Chromatography Peak Heights In cm, of the Gases Produced by Gamma Radiation of 2 % Dilute Aqueous Solutions of D- Glucosamine Hydrochloride, D-Glucuronic Acid and D-Galacturonic Acid, ...... 133 X, Micromoles of Gases Produced from D- GlucosamIne Hydrochloride, D-Glucuronic Aold and D-Galacturonic Acid...... 134

xlll LIST OF FIGURES Figure Page 1. The Irradiation of 2 0 % aqueous solutions of celloblose and maltose with cathode rays at 0°C. and at the rate of 5 z 10 reps/mln...... 90 £• Irradiation of 8 % aqueous solutions of trehalose with cathode rays at the rate of 5 z IQ6 rads/mia, and at ambient air temperature •... *...... 107 £a« Irradiation of 2 % aqueous solutions of rafflnose with cathode and gamma rays..... 113 3. Irradiation of 2 % aqueous solutions of Inulin with cathode and gamma rays...... 122 4. Electron spin resonance spectra of Irradiated D-glucuronic acid and D- galacturonic acid with gamma rays...... 144 5. Electron spin resonance spectra of irradiated maltose and celloblose with gamaa rays...... 146

6 . Electron spin resonance spectra of Irradiated trehalose and rafflnose with gamma rays ...... 147 7. Electron spin resonance spectrum of a mixture (1: 1 by wt.) of Irradiated maltose and Irradiated celloblose with gamma rays. Electron spin resonance spectrum of a mixture (1 : 1 by wt«) of irradiated D-glucuronic acid and D- galacturonlc acid with gamma rays...... 149

ziw INTRODUCTION

Carbohydrates, together with fats and proteins, comprise the three Important classes of food products * They are among the fundamental needs of the living organism. In all living cells the carbohydrates are the central pathway for the supply of energy needed for mechanical work and chemical reactions. In addition to these important functions, certain members of the carbohydrate family supply many of man*s industrial requirements. The entire cotton industry, the fermentation industries, foods and food processing, and numerous other commercial activities are dependent upon the availability of large supplies of carbohydrates. The presence of carbohydrates in foodstuffs, the increasing demand for food supplies and the development of new methods of preservation give special significance to the study of the effect of ionizing radiations on carbohydrates and related substances. The study of radiation chemistry began about fifty years ago and was primarily the subject of physical chemists. Consequently not much investigation has been directed towards the possible use of ionizing radiations for synthesis and other phases of organic chemistry. The ever-increasing availability and cheapness of sources of ionizing radiation has caused a new interest in the use of ionizing radiation. 1 2 Although the study of the Irradiation of carbohydrates is increasing rapidly, most of the reported work has been concerned with changes in physical properties with little emphasis placed on the chemical transformations that occur in carbohydrates as a result of being exposed to radiation. Very little emphasis was also placed on the processes which lead to the ultimate physical and chemical changes and on the Isolation and characterization of products due to irradiation. For these reasons and in recognition of the current interest, in the effect of radiations on carbon compounds, in the potential utility of these radiations for the sterilization of foods, deinfestation of grains and cereals, and for increasing the shelf life of foods and mediclnals, the effects of ionizing radiations on carbohydrates is studied in this work. Included in this study are the detection and determination of chemical changes, the study of free radicals formed and the isolation and characterization of products, including gases, produced from the carbohydrates due to irradiation.

Types of Radiation The radiation of matter, in general, could be divided into photolytic radiation and ionizing radiation.

I. Photolytic Radiation In photolytic radiation the molecule absorbs energy which causes the excitation of an electron to a high energy 3 state. This absorbed energy can be either lost by the electron returning to the ground state, or it can cause chemical reactions and decomposition of the irradiated substance•

II. Ionising Radiations The ionizing radiations can be divided into those caused by charged particles as alpha particles, beta particles, and protons, and that caused by neutral particles as with gamma rays, X-rays ana neutrons.

1 . Units and Terms Used la Ionizing Radiation The rate of absorption of radiation is often called the dose rate and when the absorbed radiation is integrated over the period of irradiation the term dose is used. The unit of X- and gamma ray dose (1) is the roentgen, r.,

(1) F. A. Bovey, "The Effects of Ionizing Radiation on Natural and Synthetic High Polymers" Interscience Publishers, Inc., New York, II. Y. 1058. which is defined as the Quantity of X- or gamma radiation which, on passing through air, produces ions carrying 1 electrostatic unit (e.s.u.) of electricity of either sign (but not both) per cubic centimeter at 0°C. and 760 mm. pressure. One r. corresponds to the absorption of 63 ergs/g. For particle radiation, the unit which is 4 commonly employed Is the rep. This is a roentgen equivalent physical and is defined as the quantity of radiation which upon absorption in 1 g. of body tissue or of water releases the same amount of energy as 1 r. of X- or gamma radiation. Since in tissue or water 1 r. of radiation releases 93 ergs/g., 1 rep is equivalent to 93 ergs of energy absorbed by a gram of Irradiated material. The International Congresses of Radiation in 1953 and 1956, officially adopted the unit rad which is a radiological unit equivalent to 100 ergs of energy absorbed by a gram of substance. In dealing with large doses of radiation, the common units are* the megarep, equal to 1 0 ®rep and the megarad, equal to lO^rad. The yields of rauiation-lnduced reactions can be expressed in two ways. These are the ionic yield, LI/H, and the energy yield, G. The ionic yield is the number of molecules changes, U, divided by the number, N, of ion pairs formed in the medium. The energy yield is the number of molecules changes per 100 ergs of energy absorbed, and is the more useful concept of the two as it can, in principle at any rate, be measured precisely, whereas in liquids and solids the number of ion pairs formed cannot be measured.

£• Cathode Rays Cathode rays or high-voltage electrons are artificially produced beta rays. Their penetration depends on the energy at which the electrons are produced. The first device for 5 producing this type of rays was the Van de Graaff generator (2,5). For the cathode Irradiation of

(S) R. J. Van do Graaff, Phys. Rev,, £Q, 1919 (1931), (3) F. S. Foster, D. R, Dewey and A. J. Gale, Nucleonics ii, No 10, pp. 14 (1953). carbohydrates the Resonant Transformer in conjunction with a cathode tube (4) was used. This source was located at

(4) J. A. Kn owl ton, R. R. Idahu, and J. V/. Rantfl, Nucleonics, 11. No 11, 64 (1954). the General Electric Company, Milwaukee, Wisconsin. Cathode rays are useful for surface irradiation and for irradiation of liquids. They give the greatest ionization per unit area and their penetration depends on the energy of the electrons.

3. fonnna ^ 7 ” Gamma rays are produced from fission waste products as wellas by placing in a nuclear reactor materials which are capable of becoming sources through activation. As an example, Co®^ which irradiated with neutrons produces Co®4"*

Co5SVn y Co60 p gamma rays which is very efficient source of gamma radiation. A 2000 curie Co^O source located at the Battelle Memorial Institute, 6 Columbus, Ohio and a 100,000 curies fuel elements source located at the Argonne National Laboratory, Lemont, Illinois, were used In this work. The dose rate of the first Is 400,000 reps/hour, and of the latter 20,000 rads/mln. Gamma rays are very penetrating and can be used for the Irradiation of large volumes of dense materials.

4. Action of Radiation When charged particles are used for the irradiation of carbohydrates, ionisation and exitation can occur by the interaction of the charged particles with the electrons in the atom. When gamma radiation is used, in order to have ionisation there still should take place interaction of charged particles. The gamma ray collides with an electron, scattering occurs and the recoil electron has sufficient energy to interact with electrons of other atoms and thus cause ionisation. One may expect to have, as a result of ionization, formation of free radicals, formation of new bonds, breakage of bonds, liberation of gases, polymerisation and depolymerisation. STATEMENT OF THE PROBLEM

"The Effect of Ionizing Radiation on Certain Carbohydrates and Related Substances"

The objective of this study was the Investigation of the changes occurred In carbohydrates exposed to Ionizing radiations as solids and water solutions. Compounds Investigated were: Ualtose, Celloblose, Trehalose, Rafflnose, Inulin, Amylose, D-Glucosamlne Hydrochloride, D-Glucuronic Acid and D-Galacturonic Acid.

7 HISTORICAL

I. Photolytic Radiation The first type of irradiation used for the irradiation of carbohydrates vas the photolytic radiation. The first work reported on the photolysis of carbohydrates was by Bierry, Ranc, and Henri (5)• They irradiated aqueous

(5) H. Bierry, A. Ranc, and V, Henri, Compt. rend., 151- 316 (lblO).

solutions of fructose with ultraviolet light. A measurable amount of carbon monoxide was evolved for a^24 hour exposure. The same solutions were exposed to ultraviolet radiation for several days. Formaldehyde, acetaldehyde and methanol were detected in the irradiated solutions. The reducing power and the optical rotation of the irradiated samples v/ere decreased. Glycerol was Irradiated with ultraviolet light and glycerose (6 ) was formed. The same workers irradiated

(6 ) H. Bierry, V, Henri and A, Ranc, Compt, rend., 152. 535 (1211). dilute aqueous sucrose solutions (7) for several days.

(7) H, Bierry, V. Henri, and A. Ranc, Compt. rend., 152. 162^ (1211).

6 9

An Inversion of sucrose was observed after 20 hours exposure to ultraviolet rays. Products identified were glucose, fructose and, after 46 hours of exposure, formaldehyde was detected together with the evolution of a gas containing carbon monoxide.

They thought that the reaction tahes place in two stages. In the first stage an acid is formed and in the second stage that acid causes the evolution of a gas.

Later on Berthelot ana Gaudechon (6 ) tested the effect of

(6 ) D. Berthelot and II. Gaudechon, Compt. rend.,

155. 401 (1 0 1 2 ).

both sunlight and ultraviolet radiation on dihyuroxyacetone, fructose and sorbose. The reaction was followed by the evolution of gases; carbon monoxide was the most abundant followed by carbon dioxide which in turn was followed by hydrogen and methane. The reaction in all cases was the same. The formation of an alcohol of one less carbon atom was noted. In ultraviolet light, however, accessory reactions were found to t&he place such as the photolysis of the products formed, decomposition of water into hydrogen and oxygen and partial combination of carbon 10 monoxide and hydrogen to form formaldehyde. The same authors (9) reported the decomposition of maltose and

(9) D. Berthelot and H. Gaudechon, Compt. rend., 156. 468 (1913). the decomposition and Inversion of sucrose by ultraviolet m radiation. As for the formation of gases, Berthelot and Gaudechon (9) disagreed with Ranc and Henri (7) concerning the mechanism of the gas formation. Berthelot stated that the gases produced from the irradiated materials were coming from a primary reaction caused directly by photolysis. This mechamism was based on the fact tiiat aqueous solutions of maltose remained neutral after irradiation, and yet, the evolution of carbon monoxide and hydrogen was observed. In 1924 Bierry and Rand (10), having conducted more

(10) H. Bierry and A. Ranc, Bull. Soc. Chim. Biol., 35. 771 (1924). experiments on photolysis of Retoses, obtained conclusive evidence for the formation of carbon monoxide and carbon dioxide and formaldehyde. BielecRi and Wurmser (11)

(11) J. Bielecki and R. Wurmser, Compt. rend., 154. 1429 (1912). 11 Initiated the decomposition of starch with ultraviolet light. Exposure of aqueous starch solutions to ultraviolet rays increased the conductance and the hydrogen ion concentration and decreased the optical rotation. The irradiation products were dextrins, reducing sugars, pentoses and formaldehyde. Exposure of aqueous solutions of sucrose to ultraviolet radiation (1 2 ) first caused inversion and as

(12) P. Beyrsdorfer and W. Iless, Ber., 5£, 1703 (1024).

exposure to light was continued, the glucose-fructose mixture was decomposed to simple aldehydes, ketoses and alcohols and finally into carbon dioxide, carbon monoxide, methane, and hydrogen. When D-glucose and D-fructose were

irradiated separately (1 2 ), each gave qualitative but not quantitavely the same products. Bernoulli and Cantlenl (13),

(13) A. L. Bernoulli and R. Cantlenl, Ilelv. Chlm. Acta, J&, 11D (1032).

investigating the photolysis of D-fructose and D-glucose solutions, found that the evolution of gas was proportional to the Intensity of the ultraviolet light. The addition of acids and bases to D-fructose solutions retarded the •volution of gases. However, the addition of acids to the 12 D-glucose solutions was found to accelerate the gas evolution. In 1036, Guilaume and Tonret (14) studied the

(14) A. Guilaume and G. Tanret, Bull, Soc. Chlm. Biol., 556 (1036). hydrolysis of glucosides by ultraviolet rays and found that sucrose was hydrolysed very slowly and maltose and trehalose were hydrolysed less than o0% as fast as sucrose.

II. The Irradiation of ffater Since tne irradiation of carbohydrates tabes place in most cases in water solution or in a moist atmosphere, it would be advisable to discuss briefly the effect of ionizing radiations on water. The effects of ionizing radiations on water could be divided into primary and secondary processes. Considering the action of beta particles in the primary process, they eject electrons from the water molecules leaving HgO ions. The ejected electrons will themselves cause more ionization or will be captured by other water molecules (15) to give %0*~ ions. On the other hand, beta

(15) D. £• Lea, nActions of Radiations on Living Cells," Cambridge, 1046 p. 47. particles might excite some water molecules to a higher electronic level, HgO*". In the secondary process one 13 ▼lew (15) Is that the following interaction occurs.

320*+HSO ______H3 O*+ • OH

Another view (16) is that the HfcO* ion might recapture a

(16) A. H. Samuel and J. L. magee, J. Chem. Phys., 21. 1080 (1053).

free electron and give an excited water molecule, probably of higher energy level than the previous one. Dainton (17)

(17) R. S. Dainton, J. Phys. and Colloid. Chem., £&, 400 (1048).

studied extensively the irradiation of water. According to him, the HgO*ion is formed during the irradiation of water. This formed ion can then interact as described belov/ with the resultant formation of other Ions and radicals, and the recombination of these ions and radicals.

H£ 0 > H20* + e'

Hg0+ ______p 0H*+

HgO*______^ 0H * + H*

HVH------, Hg 14

HgO + HgO* ______^ H50

2 OH------> HgOg

The primary radical formed is the OH* which is capable of causing oxidations. Guamon Barron investigated the effect of ionising radiation on aqueous solutions of sulfhydryl compounds and noted that when oxygen was dissolved in the solution it showed increased oxidations (Id) compared to

(Id) E. S. Gusmon Barron, Radiation Research, i, 109 (1954). the irradiations carried out in the absence of oxygen. He concluded that oxidations were caused by H0£ radicals and by the HgOg molecule in addition to the OH* radical. The HQg radical could be formed in the following manner,

H # + Og ______, HOg where the H* was formed as shown previously. H&rt and co-workers (19) noted that a negative ion is

(19) E. J. Hart, S. Gordon, and D. A. Hutchinson, J. Am. Chem. Soc., ££, 6165 (1953). produced by the capture of electrons when water is irradiated 15 with electrons.

HgO + e" ^ ^2^*"

It is postulated by Kowbotton (20) that the absorption of

(20) J. Rowbotton, Science, lib. 9U4 (lw54).

ionising radiation in water gives rise to two groups of radicals. The radicals of the first group are derived from ions while the radicals of the second group are derived from excited water molecules. If a solute is present it could react with one or the other of these two groups of radicals. Ghormley and Stewart (21) investigated the effect

(21) J. A. Ghormley and A. C. Stewart, J. Am. Chem. Soc., 28, 2934 (lw56).

of ionising radiation on ice and concluded that free radicals are much less mobile in solids than in liquids but on the other hand electrons may diffuse rapidly in solids and are of importance in reaction mechanisms. It is generally agreed (2 2 ) that the net result of the effect

(22) A. 0. Allen, Radiation Res., 1, 85 (1^54); E. J. Hart, ibid., 53; H. A. Dewharst, A. H. Samuel, and J. L. ..jagee, ibid., 82. 16 of Ionising radiation on water may be written as follows.

HoO y possible other species.

III. Direct and Indirect Action ot Ionising Radiation When a pure substance is exposed to ionizing radiation, the effects produced are (1) those caused by primary excitations and ionizationg originating in the substance itself, together with secondary reactions. When a substance is lrraaiated in solution, the effects produced (1) are due to direct hits on tne solute molecules and to radicals produced in the solvent which then react with the solute. The latter is the indirect action.

IV. Distinction of Radiation Chemistry fron Photochemistry The important features that distinguish (S3)

(23) E. Collinson and A. J. Swallow, Quart. Revs., b, 312 (1^55). radiation chemistry from photochemistry are: (1) that the energy absorption is not specific, that is, energy is absorbed by all components of a system, (11) that ions are formed in the primary act, and (ill) that the absorption of energy, being by collision of particles with molecules, tends to be localized along the tracks of the particles 17 and therefore, non-uniformily distributed throughout the system.

V. Irradiation of Polymers Other Than Carbohydrates Both cathode and gamma rays are useful In the radiation of organic compounds as well as In radiation sterilization (24).

(24) S. A. Goldblith and B. E. Proctor, Nucleonics, No. 2, 32 (1054).

Although these rays are fundamentally different, their respective merits are sufficient at present to prevent the exclusive use of one type. Two completely different changes occur when certain linear polymers are subjected to high speed electrons; they are synthesis and decomposition (25)•

(25) A. Charlesby, Nature, 171. 167 (1^53).

Polythene and polystyrene polymers became crosslinked while the methyl methacrylate resins disintegrated (26)•

(26) A. Charlesby and U. Boss, Nature, 171^ 1153 (1053). 18 Lawton and associates (27, 28) found that cathode rays

(27) E. J. Lawton, A. m. Beuche, and J. S. Balwlt, Nature, 172. 76 (1955). (28) A* A. Miller, E. J. Lawton, and J. S. Balwlt, J. Polymer Sci., 14, 503 (1554). caused crosslinking of the polymer chains leading to an Increase in molecular weight and scission of the chains resulting in a decrease in average molecular weight, Ihe nature of these reactions Is complicated considerably by the presence of air and moisture which introduce oxidative changes.

VI. Irradiation of Carbohydrates Carbohydrates are usually irradiated in air and in the presence of water varying from traces, in polymers, to dilute aqueous solutions for simple sugars. Polysaccharides have received more attention than monosaccharides and oligosaccharides.

1. Effect of Ipniffiipg Radiation on Cellulose and Its Derivatives Shoepfle and Connell (£2) exposed paper to cathode

(28) C. S. Shoepfle and L. H. Connell, Ind. Eng. Chem., gi, 529 (1929). 19 rays and observed the evolution of hydrogen, carbon monoxide and carbon dioxide, Wlnogradoff (30) observed that

(30) N. N. Wlnogradoff, Nature, 166. 123 (1950). cellulose acetate cracked and discolored on exposure to X-rays. Basswood became fermentable by rumen bacteria (31)

(31) E. J. Lawton, W. D. Bellamy, R. E. Hungate, i'. P. Bryant and E. Hall, Science, 113. 360 )1051). after exposure to 6.5 megareps of cathode rays. At 100 megareps the digestibility was comparable to that of hay, and the wood had become hygroscopic and friable, Volatlble acids, reducing sugars, and solubility Increased sharply beyond 10 megareps. It became clear that the cellulose underwent profound degradation, but the fate of the lignin portion of the wood was not so clear. It Is possible that lignin Is originally combined with the cellulose and that the developed fermentablllty Is due to the breakage of these bonds as well as to formation of soluble cellulose chain fragments. Cotton, wool, and cellulose derivatives were degraded (32) by high-speed

(32) K. Little, Nature, i70, 1075 (1052). £ 0 electrons. Lawton, Beuche, and Balwlt (£7) reported that cellulose underwent predominantly chain scission by cathode radiation. Sisman and Bopp (33) found that cellulose

(33) 0. Sisman and C. D. Bopp, ITuclear Sci. Abst., 8, No. 2792 (1951).

2, 6-dlproplonate (Forticel), cellulose dlacetate (Plastacele), and cellulose dinitrate (Pyralin) lost all tensile strength and elongation after exposure to pile radiation. They rated the /*-D-(l ■ ■ ■■ »4)-D-glucopyranosyl unit as among structures least stable to scission by ionizing radiation, exceeded only by polylsobutylene, polymethyl methacrylate, and polytetrafluorcethylene. Saeman, Uillett, and Lawton (34) irradiated cellulose

(34) J. F. Saeuan, 11. A. Mlllett, and E. J. Lawton, Ind. Dig. Chem., 44. 2348 (1952). from cotton linters with high-energy electrons and measured the rate of hydrolysis of the irradiated samples and the total glucose yield on complete hydrolysis. The rates of acid hydrolysis of the irradiated samples were much greater than those of the unirradiated samples. It was found that cotton linters cellulose irradiated with doses of over 500 megarep, showed only 54£ of the initial potential 2 1 sugar content, estimated by the reducing power of hydrolysates. Evidently the remainder had been destroyed by the radiation. At those high doses glucose was decomposed at about the same rate as cellulose. Aftereffects In the degradation of cellulose by gamma rays were studied by Glegg and Kertss (35). They measured

(35) R. E, Glegg and Z. I. Kertesz, Science, 124, 893 (1956), viscosities and noted that In the absence of water after-effects become Important and they occurred for as long as two weeks,

2, Effect of Ionising Radiation on Dextran The effect of ionizing radiation on dextran has received some attention because of the possibility of using radiation as a means of degrading the very high molecular weight "native* polymer to the 50-75,000 range necessary for use as a blood plasma extender. Ricketts and Rowe (36) exposed samples of native dextran In solid

(36) C. R. Ricketts and C, E. Rowe, Chen, and Ind., 189 (1954). form and in 1^ aqueous solution to Co 60 gamma radiation. 22 The Intrinsic viscosity was decreased more rapidly In solution than In the solid polymer. They further demonstrated that no significant crossllnklng occurred. A General Electric Company patent (37) disclosed the

(37) General Electric Co., Brit. Pat. 764,547 (1956). degradation of native dextran by high speed electrons to products In the 50-75,000 clinical range. Price, Bellamy, and Lawton (36) reported a detailed study of the degradation

(38) F. P. Price, W. D. Bellamy, and E. J. Lawton, J. Phys. Chem., £8, 621 (1954), of dry dextran by electron irradiation. The molecular weight was decreased from several millions to approximately 700,000 when it was exposed to 100 megarep. Irradiation In an oxygen-free atmosphere appeared to have less effect on dextran. Phillips and LLoody (39) observed the degradation

(39) G. 0. Phillips and G. J. Koody, J. Chem. Coe., 3534 (1958). of dextran when it was irradiated in aqueous solutions with gamma radiation. Their chromatographic examination revealed that the main irradiation products were glucose, 2 3 isomaltose, isomaltotriose, gluconic acid, glucuronic acid, glyoxal, erythrose, and glycolaldehyde.

3. Effect of Ionizing Radiation on Inulin Inulin vater solutions were irradiated with gamma radiation from a Co^° source (40, 41) and the ultraviolet

(40) tt. A. Khenokh, Do-lady Akad, Nauk. S.S.S.R., 104. 746 (1955). (41) X. A. Khenokh, J. Gen. Chem. Russ., 20. 1560 (1950). light absorption spectra were investigated but no definite products have been reported.

4. Effect of Ionising Radiation on Pectin After-effect in the irradiation of pectin wa3 observed by Glegg and Kertesx (35)• Viscosity measurements showed that the after-effect becomes more important when the samples are irradiated in the absence of water. Kertesz and eo-workers have reported (42) a more extensive study of the

(42) Z. I. Kertesx, B. H. Morgan, L. W. Tuttle and M. Lav in, Radiation Research, 372 (1956). effects of ionizing radiation on pectin in dry form, in solution, and in gel. Solid pectin is noticeably degraded 24 by 50,000 rep of electron or gamma radiation; In solution It Is even more sensitive. Sucrose, glucose, or fructose added to pectin solutions protect It. If the sugar concentration is high enough to allow jelly formation, the pectin is much more stable to Ionizing radiation.

5. Effect of Ionizing Radiation on Starch A brief report on the irradiation of starch was given by Brasch and associates (42)• Khenokh studied the effect

(42) A. Brasch, ... Huber and A. Waly, Arch. Biochem. and Biophys., 2451 (1£*52). of gamma-radlatlon (40) on starch solutions but no irradiation products have been reported. A rather extensive investigation on the amylose fraction of starch was performed by Stacey and co-workers (44). They exposed 0.1#

(44) E. J. Bourne, Stacey, and G. Vaugham, Chem. and Ind., 572 (1W56). aqueous solutions of amylose in vacuo and under nitrogen to doses of up to 0.2 megareps of gamma-radlation. There was observed an increase in reducing power of the solutions, as measured by copper reduction. The polymer became nonstaining to iodine when the reducing power reached 10# 25 of that obtainable on complete hydrolysis. When oxygen was admitted it caused marked inhibition both of the development of reducing power and of the loss of iodine-staining power. Paper chromatographic investigation of the irradiation products showed the presence of glucose, maltose, maltotriose, and one unidentified product evidently smaller than a pentose. The presence of carboxyl groups was noted. Irradiation products were not Isolated, It was concluded that the action of irradiation is not confined to hydrolytic scission of the glycosldic linkages, but includes also oxidation steps. Wolfrom and co-workers (45)

(45) il. L. Wolfrom, W. W. Blruiley and L. J. McCabe, Abstracts Papers, Am. Chem. Soc., 150. 16A (1956). found that cornstarch when irradiated with cathode rays becomes water-soluble with Increasing availability. Amylopectin was hydrolysed by the action of cathode rays and mono- , dl- , and tri-saccharide fractions were isolated.

6. Effect of Radiation on oligosaccharides The oligosaccharides (46) are a group of polymeric

(46) B. Helferich, E. Bohn and S. Winkler, Ber., 6ft. 089 (1930). £ 6 carbohydrates consisting of relatively Tew monosaccharide units. They are classified on the basis of the number of monosaccharide residues per mole, as disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, etc. a. Raffinose- Irradiation of aqueous raffinose solutions with gamma rays (40) yielded fructose and sucrose identified by paper chromatography. b. £4ftltqs§.. Khenokh (40) irradiated aqueous maltose solutions with gamma rays. The ultraviolet absorption spectra of the irradiated samples were studied but products were not identified. c. Sucrose. Aqueous sucrose solutions when irradiated with gamma radiation (40) yielded formaldehyde. Paper chromatography indicated fructose as one of the degradation products. X-rays colored crystalline sucrose to a reddish-brown color (47)• Irradiation in solution gave

(47) II. C. Reinhard and K. L. Tucker, Radiology, 12. 151 (1929).

some invert sugar, the amount of which was proportional to the time of exposure but was independent of the sucrose concentration. The extent of hydrolysis (based on the amount of invert sugar formed) of 50$ aqueous sucrose irradiated with cathode rays increased with increasing irradiation dosage (43). At 104 megareps the extent of 2 7

(48) M. L. Wolfrom, W. W. Binkley and L. J. LIcCabe, J. Am, Chem. Soc., 81, 1442 (1959), hydrolysis was 22,2, 27,0, and 37.8J6 while being cooled with ethanol-(solid carbon dioxide), ice and water, and ambient air, respectively. Identified products were fructose and glucose. Powdered sucrose irradiated with cathode rays was hydrolysed and the extent of hydrolysis as a function of dosage reached a maximum (48) at an intermediate dose. The extent of hydrolysis at the same irradiation doses was much greater when sucrose was irradiated as 50% aqueous solution than when irradiated as powder. d. Lactose. Action of gamma radiation on lactose 2ms been reported by Khenoch (41) with very little information. The sensitivity of milk to nonenxymlc browning is induced by ionizing radiation (49). Radiation-induced changes of

(49) J. H. Wortheim, Be. £. Proctor, and S. A. Goldblith, J. Dairy Science, §9, 1236 (1956). lactose were found to be largely responsible for this induced browning sensitivity as well as for the formation of certain products (49) wlUch can react with thiobarbituric acid to give red dyes (50, 51). The brown coloring of £8

(50) H. H. Streuli, Mitt. Lebseasm. u. Hyg., £§L, 225 (1957). (51) H. H. Streuli, ibid., 234.

Irradiated milk appeared to a certain degree during irradiation but mostly as an after-effect (50, 51). It was caused by an interaction of albumin and lactose.

7. Effect of Ionising Radiation on Monosaccharides D-Glucose has been irradiated in air as a dry powder and in dilute aqueous solutions previously acidified and boiled (34, 52)• The dry sugar after irradiation with

(52) B. E. Proctor and S. A. Goldbllth, Advances in Food Research, £, 119 (1951).

1 to 10 megareps showed up to 14# decrease of copper reduction value. The Irradiation of aqueous solutions of D-glucose (40) gave formaldehyde and this sugar was found to be attacked less vigorously than D-fructose. Phillips (53) found that D-glucose, D-galactose, and

(53) 0. 0. Phillips, Nature, 175. 1044 (1954).

D-mannose react at carbon atom 6 to give the corresponding uronic acid which was the only product he observed when 2 9 these sugars were irradiated as dilute aqueous solutions with X-rays, The reaction was accelerated by the presence of oxygen and the amount of uronlc acid formed was independent of the initial hexose concentration, Indicating indirect action of irradiation. It is lsnown that the hydroxyl radical is involved in Fenton's reaction (54, 55).

(54) H. J. H. Fenton, J. Chem. Soc., 399 (1354). (55) F. Haber and J. Vieiss, Proc. Royal Soc. (London), 1147. 333 (1934).

Phillips attempted to explain his results on the basis of the Fenton's reaction. The presence of substances (reducing and nonreducing sugars) other than D-glucose were found in Irradiated alkaline D-glucose solutions (56).

(56) Christine T. Bathner-Ejy and ftidre A. Balass, Radiation Research, £, 302 (1957)•

D-Glucose, in dilute aqueous solutions, on exposure to high-energy electrons from a 2 ifv. van de Graaff Generator, was degraded to a considerable extent (57)• Irradiation

(57) G. 0. Phillips, G. J. iloody and G. L. Mattoh, J. Chem. Soc., 3522 (1953). 30 products identified were glucuronic acid, gluconic acid, glyoxal, arablnose, erythrose, formaldehyde, saccharic acid, and dihydroxyacetone. The occurrence of post-irradiation reactions was shown by the liberation of gas for 24—30 hr. after the irradiation. Polymeric products have been reported to be formed from D-glucose when exposed to gamma radiation under vacuum or nitrogen atmosphere but not in the presence of oxygen (58).

(58) S. A. Barker, P. LI. Grant, LI. Stacey and R. B. Ward, Mature, 183. 376 (1959).

4-D-Glucose monohydrate and «<-D-fructose (as crystalline powders) were irradiated with cathode rays (59). The

(59) LI. L. Wolfrom, W. .7. Binhley, L. J. McCabe, 1. LI. Shen Han, and A. LI. Mlchelakls, Radiation Research, 10. 37 (1959). reducing values were measured and some physical and organoleptic changes were noted.

8. Effect of Ionising Radiation on flt-D-Glucorarranoside powdered and aqueous samples of methyl -D- glucopyranoside were exposed to cathode radiation at ice-water temperature and It was found (48) that the extent of hydrolysis (based on conversion to glucose) 3 1 was 3*3 and 6.3#, respectively. Glucose was Identified chromatographically as one of the irradiation products*

9. Effect of Ionising Kadlatlon on Poivhydria Alcohols Phillips (53) found that exposure of D-mannitol to electron irradiation gave mannose which was subsequently oxidised to mannuronic acid* Dilute aqueous solutions of the same alditol on exposure to gamma radiation were found (40) to yield some fructose, formaldehyde, and a substance with an absorption maximum of £65 zlja* Wolfrom and co-workers (59) investigated extensively the action of gamma and cathode rays on hexitols. D-Glucitol (sorbitol) aqueous solutions exposed to cathode radiation indicated by paper chromatography, the presence of D-glucltol (sorbitol), glucose, gulose, arablnose and xylose in the irradiation product. Although not established, the xylose and gulose should be of the L-3eries. Aqueous D-mannitol exposed to ionizing radiation gave mannose, arablnose and mannuronic acid (59). The D-mannose was Isolated and identified. An interpretation of these results was given on the bases of Fenton's reaction (54, 55) myo-Inositol, closely related to sugar alcohols, was altered chemically (60)

(60) A. F. Scott and A. H. Livermore, Abstract Papers Am. Chem. Soc., 5A (1954). 3 2 when its aqueous solutions were exposed to gamma radiation from a Co6^ source.

Paramagnetic resonance study of free radicals produced toy X-ray irradiation of nucleic acids, proteins, aminoacids, fatty acids, hormones and other compounds have been reported by Qordy and associates (72).

(72) u, Gordy and G. McCormick, Bull. Am. Phys. Soc., 1, 200 (1956); tf. Gordy and H. V«*. Shields, ibid., 267; H. Rexroad and \lm Gordy, ibid., 200.

Electron paramagnetic resonance studies on irradiated carbohydrates were reported by Combrisson and Ubersfeld (73)

(73) J. Combrisson and J. Ubersfeld Compt. rend., 23d. 1397 (1J54). and by O'Meara and Shaw (74). Several powdered carbohydrates

(74) J. P. O'Meara and T. Li. Shaw, Food Technology, 11. 132 (1957). have been Irradiated with X-rays and cathode rays (75). 33

(75) D. Williams, J. ii. Geusic, M. L. V.'olfrom, and L. J. McCabe, Proc. Nat. Acad. Sci., 44, 112Q (155t3),

The paramagnetic resonance spectra of the X-ray irradiated carbohydrates were round to be identical with the spectra of the cathode-ray irradiated samples (75). EXPERIMENTAL

The high-speed electrons (cathode rays) were supplied by a resonant transformer In conjunction with a cathode ray tube. The source utilised was a 1 Mev. peak, 500 /* beam-out unit (4) and was located at the General Electric Company, Milwaukee, Wisconsin; Its dose rate was 5 megareps/ min. Two gamma ray sources were used. One was a £000 curie Co 60 source located at the Battelle Memorial Institute, Columbus, Ohio which consisted of a number of Co®0 rodes placed In a water pool. The samples exposed to this source were Irradiated at the rate of 400,000 reps/hour. The other gamma ray source was a 100,000 curie fuel elements source located at the Argonne National Laboratory, Lemont, Illinois which consisted of a number of radioactive elements placed in a rack. The samples were placed In the rack for irradiation. The dose rate of this source was £0,000 rada/min. II. B a

i. I f j r t A f U w rf MfcltoH-rtja.Citiwflf a m m rtfiar n r ctat igmgm.fifliuiianfi at * Equal amounts of maltose (a) and distilled water were

(a) A product of the pfanstiehl Chemical Company, Waukegan, Illinois.

34 35 mixed In three open aluminum dishes (9.9 cm. in diameter) just prior to Irradiation and exposed to cathode rays In doses of 50, 200, and 400 megareps each at the rate of 5 megareps/mln. During the Irradiation the samples were oooled In an lce-water bath. After Irradiation, the samples were placed In amber-colored, screw-cap bottles and returned by air mall to this laboratory. Each of the Irradiated samples was diluted to about 40 ml. and lyophllised, and then stored In an evacuated desiccator over Drlerlte (b).

(b) Anydroua calcium sulfate, a product of the W. A. Hammond Drlerlte Company, Xenia, Ohio.

a. Duflgfflito piper gjggmtggmtor, gf mx % 9 s s toiaiattfl M W k MttfQltf W l „ hi 5°C. Two per cent aqueous solutions were prepared from the irradiated and lyophllised maltose samples. Additions of each of these solutions were made at the designated position Q cn. from the unpointed end of a 14 x 45 cm. (to tip of pointed end) sheet of Whatman No. 1 filter paper. The chromatogram was developed for SO hours with 1-butanol s ethanol s water (40sllsl9, volume ratio). The development took place In an all-glass chamber equipped for paper chromatography. The chromatogram was air-drled and then sprayed with l£ sodium metaperiodate, 1JC potassium 3 6 permanganate, and finally with bensidine reagent, according to the method of Wolfrom and Miller (61). All of the

(61) U. L. Wolfrom and J. B. Miller, Anal. Chem., 88. 1037 (1956).

irradiated maltose samples gave two spots, one of the same mobility as glucose and the other of the same mobility as maltose, Ohlrradiated maltose gave a single spot indicating chromatographic purity.

2. IrfdUtlnn nf ** Twantv Par C«nt Anii^oiut with Cittrtw flare Eighteen samples of 30# aqueous maltose (a) were prepared just prior to irradiation. They were placed in open aluminum containers (9.9 cm. in diameter) and exposed to cathode radiation in dose ranging from 20 to 100 megareps, at the rate of 10 megareps/mln. The irradiations were carried out at three temperatures, ambient air, lce-water, and ethanol-dry ice. Control samples of maltose were used and treated as the irradiated samples with the exception that they did not receive any dose. Bach of the irradiated samples was diluted to 60 ml. and lyophllised for 18 hours. The lyophllised product was kept in an evacuated deslocator over Drlerlte. Table I gives the doses and temperatures. 37

Table I

The IrredletIon of BQ% Aqueous Maltose With Cathode Bays

** B-^=-gg— BBBM--— -L_L - ! . I 1 I tJ n I J ■■■. ■ ■■ ^ Sample Dose* (megarep) Coolant

UL-1 (control) — ethanol-solid carbon dioxide ML-S (control) — Ice water ML-3 (control) — ambient air ML-4 SO ethanol-solid carbon dioxide ML-5 SO Ice water ML-6 so ambient air ML-7 40 ethanol-solld carbon dioxide ML-6 40 Ice water ML-9 40 ambient air ML-10 60 ethanol-solid carbon dioxide UL-U 60 Ice water ML-1S 60 ambient air ML-13 80 ethanol-solid carbon dioxide 1IL-14 80 Ice water HL-15 80 ambient air ML-16 100 ethanol-solid carbon dioxide ML-17 100 ice water ML-18 100 ambient air

•Delivered at the rate of & megareps/mln 38 a. Descend!ns paper chromatography of irradiated 20* aqueous maltose with oathode rave at ambient air. ice-water

and ••bhanoi^dry Ice temperatures . Two per cent aqueous solutions were made of the lyophilixed Irradiated maltose samples. A number of additions of each of these solutions mare applied at a specified position 3 cm. from the unpointed end of a 14 z 45 cm. sheet of Whatman 2fo. 1 filter paper. The chromatograms were developed for 30 hours with 1-butanol s ethanol s water (volume ratios 40sllil9), In an all-glass chamber equipped for chromatography. The chromatograms were air-dried. The air-dried chromatograms were sprayed with aniline-phthalate Indicator (1.66 g. of phthallc acid and 0.93 g. of aniline in 100 ml. of 1-butanol saturated with water) and then placed In a moist oven at 1Q5°C. for 5 minutes (62).

(62) S. M. Partridge, Nature, 164. 443 (1049).

In all the Irradiated samples the presence of maltose and glucose was observed. The Intensity of the glucose spots increased with increasing irradiation dosage and the maltose spots became less Intense with increasing dosage. No other substance than maltose was found to be present in the control maltose samples when chromatographed as described above. In a similar manner, two other sets of chromatograms were prepared and developed for 30 hours with 1-butanol t 3 9 ethanol i water t ammonium hydroxide, 40*40516!4 (vol. ratio)• The developed chromatograms were allowed to dry In air for several hours in order to remove the volatile aoetic acid. The one set of chromatograms was sprayed with O.SJC ninhydrin in absolute ethanol containing formic acid, and the other set wa3 sprayed with Bromocresol Green reagent. Both sets were placed in an oven for 5 minutes. No spots were observed for the irradiated samples. b. Determination of per cent hydrolysis of irradiated 20^ aqueous maltose with cathode rays. The per cent hydrolysis was found by determining the alkaline copper reducing values of the irradiated samples in accordance with the method of Somogyi (63)• An amount of

(63) M. Somogyi, J. Biol. Chem., 160. 61 (1945).

100 mg. each of the lyophilixed samples which has been irradiated at ambient air, ice-water, and ethanol-dry ice temperatures, was dissolved in distilled water and the volume adjusted to 100 ml. The resulting solutions were diluted 50 to 1 with distilled water. The copper-reducing values of the diluted solutions were determined by the Somogyi reagent. The mixtures were placed in a boiling water bath for 30 minutes. After cooling the solutions to room temperature, 1 ml. of 10JC aqueous potassium iodide was added to each without agitation. Allowing this, about 40 1.5 ml. of £ N sulfuric acid was added to each solution with vigorous mixing. The solutions were allowed to stand for 10 minutes, and then titrated with 0.005 N sodium thiosulfate solution. A 1% starch solution was used as indicator. The results are found in Table II. The per cent hydrolysis was estimated on the basis that 1 mg, of D-glucose reduces 7.4 ml. of Somogyi reagent and on the difference of reducing values of Irradiated and unirradiated maltose samples. The results are plotted in Figure 1. The Somogyi reagent was prepared as follows; £8 g. of anhydrous disodium phosphate and 40 g. of Rochelle salt were dissolved in about 700 ml. of water, 100 ml. of normal sodium hydroxide were added and then, with stirring, 80 ml. of lOjf copper sulfate solution was introduced. Finally, 180 g. of anhydrous sodium sulfate was added and, when dissolved, the solution was diluted to 1 liter and allowed to stand for £ days during which time impurities settled out. The solution was filtered through a good grade of filter paper. The reagent was alkalinised with a knife tip of sodium carbonate and kept for a long time without decomposition.

c. Calculation of G values of irradiated 20< aqueous maltose with cathode rays. In order to give more complete description of the effects of cathode ray irradiation on maltose, the G values were calculated. The calculation of these values is based on the apparent per cent sugar 4 1 hydrolysed. In the case of maltose, the per cent hydrolysis was based on conversion to glucose (copper reducing method of Somogyi ). The calculations were made as followsi Q s No. of molecules decomposed per 100 e.v. of energy absorbed. 1 rep * 93 ergs/g. 1 megarep s 56 i 1G10 ev./g. G ~ (fraction of sugar hydrolysed) (6.0g x IQ23) (100) (Mtf of Sugar) (megareps x 56 x 10^® ev./g,— megareps) The G values are shown in Table II.

d. Isolation of irradiation products from maltose irradiated with cathode rays. A slurry of Nuchar C— unground (c) was prepared with water and poured into a glass

(c) Onground Nuchar C. A product of the West Virginia Pulp and Paper Company, 35 E. Wacker Drive, Chicago, Illinois. chromatographic tube, in small portions, until a carbon column 300 x 40 mm. (diam.) was obtained (64). The column

(64) H. L. Whistler and D. R. Durso, J. Am. Chem. Soc., 2£, 677 (1950). was washed with 1000 ml. of 5J6 hydrochloric acid, followed with distilled water until the pH of the eluate was brought 48 to 6, The column was washed further with 1000 ml. of &% ammonium hydroxide and then with water until the eluate became neutral. At this point the carbon column was ready for use. An amount of 2 g. of lyophlllsed maltose sample, which had been Irradiated with cathode rays as S0£ aqueous solution at 0°C. and had received a dose of 100 megareps, was dissolved In 50 ml. of water. The resulting solution was chromatographed, according to the method of Whistler and Durso (64), on the prepared column. The chromatogram was developed with 3500 ml. of water to elute the monosaccharide fraction from the column. This fraction was concentrated under reduced pressure to sirup. The sirup was chromatographed on paper by applying three drops of It on designated positions of a 14 x 45 cm. sheet of Whatman No. 1 filter paper. After developing the chromatogram with 1-butanol i ethanol » water (40silt 19 volume ratios) and air drying, It was sprayed with aniline phthalate indioator (68). Only one intensive spot was obtained with the same mobility as glucose.

e. Acetylatlon of the slrupy monosaccharide fraction ghtatoA Xy«a w b ffl .gglwm gtattMitaraahy salVas. The slrupy monosaccharide fraction, obtained from the above carbon column, was dried ty addition of 5 portions of 20 ml. each of absolute methyl alcohol and evaporation to dryness under reduced pressure. A brownish white material was obtained, the acetylatlon of which was carried out by using 4 5 the sodium acetate method (65). An amount of 100 mg. of

(66) "Polarimetry, Saocbarlmetry and the Sugars," Circular 440, National Bureau of Standards, U. S. Government Printing Offioe, Washington, D. C, 1848, p. 488. freshly fused and grounded sodium acetate was placed In 50 ml, of distilled aoetlc anhydride. The mixture mas heated in a £50 ml, round-bottomed flask to about 100°C, The dry monosaccharide fraction mas added, stirring slovly, into the hot aoetlc anhydrlde-sodlum acetate mixture. When the addition mas completed, the mixture mas heated to about 100 C, for half an hour. After the completion of the reaction, the solution mas cooled to room temperature and then poured into 300 ml, of ice-water, This reaction mixture mas stirred for 1 hour and then let stand for S hours for the acetic anhydride to disintegrate. Extraction mlth three 8Q-ml, portions of chloroform follomed and the chloroform extracts mere combined. The combined chloroform extract mas mashed mlth mater and dried over anhydrous sodium sulfate for 84 hours. The dried chloroform solution mas concentrated to 10 ml, under reduoed pressure. f. The separation and identification of the acetylated monosacchi|Ttdg ^ffrf^t i v e s of Irradiated 804 a c u e ^ « at 0°G. The chromatographic separation of the acetylated monosaccharide fraction mas achieved on a column of 4 4 Magnesol (d) and Celite (•) (5/1 by wt.) aooording to the

(d) A synthetic hydrated magnesium acid silicate produced by the Yestvaco Chemical Division of the Food Machinery and Chemical Corp., South Charleston, V. Va. (e) No. 535, a dlatomaceous fllter-ald produced by the Johns-Uanville Co., New York, N. Y. method of UcNeely, Binkley, and Wolfram (66). A tapered,

(66) Y. H. ZfoNeely, V. W. Binkley and M. L. Yolfrom, J. Am. Chem. Soc., 67. 527 (1945). glass column was slowly and carefully filled with adsorbent mixture, using a partial vacuum from a water aspirator. Full vacuum was subsequently applied and the column was filled to a distance of 30 mm. from the top of the column. The adsorbent was tapped carefully to prevent channel formation. The dimensions of the prepared column were S00 x 40 mm. (dlam.). The column was prewetted with 30 ml. of benxene and the 10 ml. of chloroform solution containing the acetylated monosaccharide fraction was added Just before the level of bensene dropped to the surface of the column. It should be noted that at no time after the addition of the 30 ml. of bensene was the column allowed to run dry. The column was then developed with 1000 ml. of bensene x ethanol 4 6 mixture (500si by vol.). This developer was found to be suitable for the separation of model sugar acetates (66). The developed column was extruded and streaked with a freshly prepared solution of alkaline potassium permanganate (0*1 g. of potassium permanganate, 1.0 g. of sodium hydroxide, and 10 ml. of water). One main some was located at 90-100 mm. distance from the top of the column. This sone was separated from the rest of the adsorbent and eluted with 50 ml. of acetone. The acetone was filtered and the filtrate evaporated to dryness under reduced pressure. The material obtained was dissolved in 15 ml. of warm ethanol and decolorised with activated carbon. This substance was recrystallised from 95# ethanol; yield 90 mg., m. p. 130°- 133°C. uncor. in X-ray powder diffraction pattern of the crystalline derivative was obtained using filtered CuK^ radiation. The resulting pattern was compared and found to be Identical with that of an authentic sample of ^-D-glucopyranose pentaacetate (67) •

(67) U. L. Volfrom and H. B. Wood, J. Am. Cham. Soc., JX, 3175 (1949). 4 6 III. Ih« Eff.ct Of Tpnlting Radiation on Cellobiosetfili

1. Irradiation of Cellobiose with Cathode Rays as Fifty £er Cent Aqueous Solutions at Q°C. Three 50JC samples of aqueous cellobiose (f) were

(f) A product of the Pfanstiehl Chemical Company, Waukegan, Illinois. prepared just prior to irradiation and placed in open aluminum dishes (9.9 cm. in diameter). They were exposed to cathode ray Irradiation in doses of 50, 200 and 400 megareps at the rate of 5 megarep per minute while being cooled In an ice-water bath. The irradiated samples were diluted up to 50 ml., lyophillsed and stored in an evacuated desiccator over Drierite (b).

a. Descending paper chromatography of the Irradiated e samples. Two per cent aqueous solutions were prepared from the lyophillsed irradiated cellobiose samples. Additions of each of the solutions were made at a specified position, 8 cm. from the unpointed end of a 14 x 45 cm. sheet of Ho. 1 Whatman filter paper. The chromatogram was developed for 20 hours with 1-butanol i ethanol i water (volume ratios 40iUsl9), in an all-glass chamber equipped for chromatography. The air-dried chromatograms were sprayed with 1# sodium metaperiodate, 4 7 potassium permanganate, and finally with benaidine reagent (61)• No spots other than that corresponding to cellobiose were obtained.

S. Irradiation of Cejfobiose as Twenty Per Cent Aqueous Solutions with Cathode Rays at ▲ number of samples of 20% aqueous cellobiose (f) were prepared Just prior to irradiation. They mere placed in an open aluminum containers (9.9 cm. in diameter) and exposed to cathode radiation at the rate of 5 megareps/mln. while being cooled in an ice-water bath. The samples received £0, 40, 60, 80, and 100 megareps, respectively. A control sample was employed which received no radiation. Each of the Irradiated samples was diluted to 50 ml. and lyophillsed for 18 hours. The lyophillsed samples were kept in an evacuated desiccator over Drierite (b).

a. Descending paper chromatography of irradiated 20% ftflUSPWI CeUofelQM WCStfcgflg rtffl Two per cent aqueous solutions were prepared from each of the lyophillsed, irradiated, cellobiose samples which had received SO, 40, 60, 80, and 100 megarep, respectively. Several additions were applied at a specified position 8 cm. from the unpointed end of a 14 x 15 cm. sheet of Whatman No. 1 filter paper. The chromatograms were developed for 30 hours with 1-butanol t ethanol * water (volume ratio 40tllsl9). The air-dried chromatograms were sprayed with aniline phthalate indicator (6S) and placed in a moist oven for 5 minutes at 4 8 105°C. In all the Irradiated cellobiose samples the presence of cellobiose and glucose was indicated. The intensity of the glucose spot was increasing with increasing irradiation dosage. The reverse was observed with the Intensity of the cellobiose spot. In the control cellobiose sample only cellobiose was found to be present, indicating chromatographic purity.

b. nata^mlpation of tha par eant hvdrolvda of 20* aeneous cellobiose irradiated with cathode rays at 0°C. The per cent hydrolysis due to irradiation was measured by determining the alkaline copper-reducing values of the irradiated samples according to the method of Somogyi (65) • An amount of 100 mg. of each cellobiose sample was dissolved in distilled water and the volume was adjusted to 100 ml. The resulting solutions were diluted 50 to 1 with distilled water. An amount of 5 ml. of each aliquot was mixed thoroughly with 5 ml. of Somogyi reagent. The mixtures were placed In a boiling water bath for 50 minutes. After cooling the solutions to room temperature, 1 ml. of 10% aqueous potassium iodide was added to each without agitation. Addition of 1.6 ml. of 2 N sulfuric aoid to each solution was followed with vigorous agitation. The solutions were allowed to stand for 10 minutes and were then titrated with 0.005 N sodium thlosulfate solution. A l£ starch solution was vised as indicator. The results are found in Table III and are plotted in Figure 1. One mg. of D-glucose reduced 49 7.4 ml* of Somogyi reagent.

o. Determination of G values of Irradiated 20% aqueous cellobiose with cathode rays at 0°C. The calculation of the G values, for Irradiated cellobiose, was based on the apparent per cent sugar hydrolysed. In the case of cellobiose, the per cent hydrolysis was based on conversion to glucose (copper reducing method of Somogyi). The terms used were the same as In the case of maltose. Table III shows the G values of irradiated cellobiose.

IV. The Effect of Ionising Radiation on Trehalose

1. Irradiation of Trehalose with Cathode Rays as Two Per Cent Aqueous Solution at Ambient 41r Temperature The Irradiation was performed with cathode rays at the General Electric Company, Milwaukee, Wisconsin. Eight samples of crystallin trehalose (g), 2 g. each, were placed in

(g) Trehalose C. P., A product of the Pfanstlehl Chemical Company, Waukegan, Illinois. aluminum dishes (9.9 cm. diam.). One hundred grams of distilled water was placed in each of eight screw-cap, brown glass vials. These were shipped by air mall to the cathode ray source. Just before the irradiation, each trehalose sample was dissolved in the corresponding distilled water fraction, and the solutions were irradiated In doses of 50 0, £.5, 5, 7,5, 10, IS.5, 15, 17.5, and SO megarads at the rate of 5.0 x 106 rad/mln. The irradiated samples were returned to this laboratory where they were lyophillsed and placed In an evacuated desiccator over Drierite (b)•

Chromatography of Irradiated trehalose as £* aqueous solutions with cathode rays. Two per cent aqueous solutions were prepared and applied to a sheet of Whatman No, 1 filter paper (45 x 14 cm,) at positions 8 cm. from the unpointed end. The descending paper chromatograms were developed with 1-butanol ; ethanol : water, 40:11:19 (vol. ratio) for 30 hours. The developed chromatograms were air-dried, sprayed with aniline phthalate Indicator (68), and tere then placed in a moist oven at 105°C. for 5 minutes. In all of the irradiated samples, spots with the same mobility as D-glucose were obtained. The Intensity of the glucose spots Increased with Increasing irradiation dosage. The control sample showed no presence of glucose.

b. Determination of the per cent hydrolysis of trehalose irradiated as 2% aqueous solutions. An amount of 50 mg, from the irradiated samples was dissolved in distilled water and diluted to 50 ml. The method introduced by Somogyi was followed (63), From each solution, 5 ml. were taken and mixed with 5 ml, of Somogyi reagent. The mixtures were placed in a boiling water bath for 30 minutes. After cooling the solutions to room temperature, 1 ml. of 10# aqueous potassium iodide was added to each without agitation. An 51 addition of 1.5 ml. of 8 N sulfuric acid was made with vigorous mixing. The solutions were allowed to st.*md for 10 minutes and were then titrated with 0.005 N sodium thlosulfate solution. The results are found In Table IV. The apparent per cant hydrolysis was estimated on the basis that 1 mg. of D-glucose reduced 7.4 ml. of Somogyi reagent and that D-glucose was the product of hydrolysis. By plotting the per cent hydrolysis against dose, Figure 2 Is obtained.

c. (frlpn'Latl.op nf ft of ?gy.

The calculation of the G values for irradiated trehalose was based on the apparent per cent sugar hydrolysed. In this case, the per cent hydrolysis was based on conversion to glucose, measured In accordance with the copper reducing method of Somogyi (63). The terminology Is the same as In the case of maltose. These values are shown In Table IV.

d. Isolation of irradiation products from irradiated Dose received go maaarads of cathode rays. An amount of Nuchar C-unground (c) was mixed with distilled water to the extent of a slurry formation. This slurry was poured into a glass chromatographic tube In small portions until a carbon column of 300 x 4 mm. (dlam.) was obtained (64). The carbon column was washed with 1000 ml. of 5% hydrochloric acid, followed with distilled water until the pH of the eluate was brought to 6. The column was washed 62 further with 1000 ml. of 5% ammonium hydroxide and then with water until the eluate became neutral. An amount of 1.9 g. of trehalose sample which had received 20 megarads of cathode radiation was dissolved In 50 ml. of water, poured on top of the carbon column, and chromatographed in accordance with the method of Whistler and Durso (64)• The carbon column was developed with 3600 ml. of distilled water to elute the monosaccharide fraction. This fraction was concentrated under reduced pressure to a thin sirup.

e. Paper chromatography of the monosaccharide fraction from Irradiated trehalose by separation on a SfMVTl- The slrupy monosaccharide fraction was preliminarily paper chromatographed for an Indication of the sugars present. A descending paper chromatogram was prepared according to the method of Partridge (68). Three

(68) S. M. Partridge and R. G. Westhall, Blochem. J., 238 (1948); S. M. Partridge, Nature, 158, 270 (1946). additions of the slrupy fraction were made on a sheet of Whatman No. 1 filter paper (14 x 45 cm.) together with separate additions of known sample of the product to be identified. The chromatogram was developed for 20 hours with 1-butanol i ethanol t water (40sU*19, vol. ratio). The chromatogram was air-dried, sprayed with aniline phthalate reagent, and placed In a moist oven for 6 minutes 52 at K)5°C« (62). An intense spot corresponding to glucose was obtained.

f. Acetylatlon of tha st a i n e d monosaccharide fraction. The slrupy monosaccharide fraction obtained from the carbon column chromatography was dried by addition of 5 portions of 20 ml. each of absolute methyl alcohol and evaporation to dryness under reduced pressure. The material obtained was acetylated by using the sodium acetate method (65) • An amount of 100 mg. of freshly fused and grounded sodium acetate was mixed with 50 ml. of acetic anhydride in a 250 ml. round-bottomed flask and heated to about 100°C. The sugar was added slowly, with mixing, into the hot acetic anhydride-sodium acetate mixture. On the completion of the addition of the sugar, the reaction mixture was heated to about 100°C. for half an hour. After the completion of the reaction, the mixture was let to cool to room temperature and then poured Into 300 ml. of ice- water. The ice-water mixture was stirred for one hour and then let to stand for £ hours for the acetic anhydride to hydrolyse. Extraction with three 20-ml. portions of chloroform followed. The chloroform fractions were combined, washed with water, and dried for 24 hours over anhydrous sodium sulfate. After filtering the dried chloroform solution, it was concentrated to 10 ml. under reduced pressure# 54 g. The separation and Identification of the acetylated monosacoharlde derivatives of Irradiated trehalftsq. The separation of the acetylated monosaccharide was achieved on a Uagnesol (d) and Cellte (e) (5*1 by wt.) column (66), A tapered glass column was filled with absorbent mixture using a partial vacuum from a water aspirator. Full vacuum was subsequently applied and the column was filled to a distance of so mm. from the top of the column. The adsorbent was tapped carefully to prevent channel formation. The dimensions of the prepared column were S00 x 40 mm. (diam.). The column was prewetted with 30 ml. of bensene and then the 10 ml. of the chloroform solution containing the sugar aoetate were added to the column. It should be noted that at no time after the addition of the 30 ml. of benzene was the column allowed to run dry. The column was developed with 1000 ml. of bensene i ethanol solution (500*1, by vol.), (66). The developed chromatogram was extruded and streaked with a freshly prepared solution of alkaline potassium permanganate prepared by dissolving 1.0 g, sodium hydroxide and 0,1 g. of potassium permanganate In 10 ml. of distilled water. One main zone was found to be located at 94-101 mm. from the top of the column. This zone was eluted with 50 ml. of acetone. The acetone was filtered and evaporated to dryness under reduced pressure. The crude white solid obtained was dissolved in warm 96% ethanol and treated with a very small portion of activated carbon. 56 The mixture was filtered end on crystallisation a yield of 75 mg, of a crystalline material was obtained; m.p. 131- 133°C, uncor, An X-ray powder difractlon pattern of the crystalline derivative was obtained using filtered CuK^ radiation. The resulting pattern was compared and found to be identical with that of an authentic sample of ^-D-glucopyranose pentaacetate (67)•

V, The Effect of Ionising Radiation on Rafflnose 1, The Irradiation of Rafflnose witfr ffryg A Sfi solution of rafflnose was prepared ty dissolving £0 g, of rafflnose (h) In distilled water and diluted to

(h) Rafflnose pentahydrate, a product of the Pfanstlehl Chemical Company, Waujegan, Illinois,

1000 ml. This solution was divided equally In 4 tin cans of 20-ounce capacity, sealed, and exposed to gamma radiation an 6 from a Co source in doses ranging from 2,5 x 10 rep to 10 x 10® rep, at the rate of 400,000 rep per hour. The samples were irradiated at the temperature of the "swimming pool* unit. The irradiated products were lyophillsed and stored In an evacuated desiccator over Drierite (b)•

a, Pmifpdfrig paper chromatography of Irradiated glffhWffg tP S* P9lU.U-.gPg F U h f S m ? ffllg* Two per cent aqueous solutions of lyophillsed irradiated rafflnose 5® were prepared. Several additions of each of these solutions were made on a paper chromatogram of the usual dimensions. The chromatogram was developed with n-butanol s ethanol t water, 40x11*19 (vol. ratio) for 35 hours. The developed chromatogram was air-dried and then sprayed with aniline phthalate reagent (62)• The sprayed chromatogram was dried at room temperature and was then put in a moist oven for 5 min. at 105°C. The presence of meliblose, sucrose, galactose, fructose and glucose was detected in all the Irradiated rafflnose samples. The intensity of the spots Increased with Increasing irradiation dosage. Two chromatograms were prepared in similar manner and were developed for 35 hours with butanol-1 t ethanol x water x ammonium hydroxide (40x40*16x4, vol. ratio). One of the air-dried chromatograms (room temperature) was sprayed with Bromocresol Green reagent and the other with 0.2# nlnhydrln in absolute ethanol containing 5# formic acid. These spray reagents were for the detection of organic acids. No spots were produced by the rafflnose samples exposed to gamma radiation. D-Glucanic and D-galactonic acids were used as reference compounds.

b. Ionophoresis of the rafflnose samples exposed to radiation as 2* aqueous solutions. The solutions prepared for the chromatography of irradiated rafflnose were subsequently used for ionophoresis investigation. A number of additions of each of these solutions were 5? placed at specified positions on a sheet of Whatman No. 3 filter paper (55.9 z 14.0 cm.). The ionophoresis (69, 70) was run for 1.5 hours. The pattern vas dried in air and

(69) A. B. Foster, Chem. and Ind., 1050 (1952); Advances In Carbohydrate Chem., 81 (1957). (70) A. B. Foster and M. Stacey, J. of Applied Chem., £, 19 (1953). sprayed with aniline phth&late reagent (62)• The sprayed chromatograms were dried at room temperature and placed in an oven for 5 mln, at 105°C. The presence of mellbiose, sucrose, galactose, glucose, and fructose was observed. The intensity of the spots Increased with increasing irradiation dosage.

c. Determination of comer reducing sugar values of rafflnose exposed to gamm* radiation as aqueous solutions. An amount of 250 mg. of each of the irradiated rafflnose samples was dissolved in distilled water and the volume was adjusted to 100 ml. The resulting solutions were diluted 100 to 1 with distilled water. The copper-reducing values of the diluted solutions were determined by the method of Somogyi (63). An amount of 5.00 ml. of each of the diluted samples was mixed thoroughly with 5.00 ml. of Somogyi reagent. The mixtures were placed in a boiling water bath for 30 mln. After cooling the solutions at room temperature, 5ft 1*00 ml. of IQjC aqueous potassium iodide was added to each without agitation. Then, to each test tube was added 1.5ml. of £ N sulfuric acid. The test tubes were shaken vigorously during this addition. The solutions were allowed to stand for 10 mln. and were then titrated with 0.005 N sodium thlosulfate solution. A l£ starch solution was used as an Indicator. The results are found In Table V. By plotting the reducing values against dose Figure £a Is obtained.

£. The Irradiation of Two Per Cent Aqueous Rafflnose with Cathode Bay* al Ambient Air Temperature Just prior to Irradiation, 2% aqueous solutions of rafflnose pentahydrate (h) were prepared and placed In open aluminum dishes, which were used as irradiation containers. The samples were exposed to cathode rays In doses ranging from 0 to 10 megareps at the rate of 5 megareps/mln. These irradiations were done at ambient air temperature. The irradiated samples were lyophillsed and stored over Drlerlte (b) under reduced pressure.

«• Chromatography of the irradiated 2% aqueous rafflnose solutions with cathode rays. From the irradiated samples, 2% solutions were prepared and subjected to paper chromatography (88)* Whatman No. 1 filter paper was used for the preparation of three paper chromatograms. One chromatogram In which mellblose, sucrose, D-fructose, D-galactose and D-glucose were used as reference substances, 69 was developed with 1-butanol * ethanol i water (4b0 *11*19, vol. ratio), for 35 hours. After drying the developed chromatogram at room temperature, It was sprayed with aniline phthallc aoid indicator (62). The presence of mellblose, sucrose, galactose, fructose, and glucose was Indicated In all the Irradiated rafflnose samples. Two other chromatograms prepared In similar manner were developed for 35 hours with 1-butanol t ethanol : water s ammonium hydroxide (40*40:16*4, vol. ratio). The developed chromatograms were allowed to dry In air for several hours and then the one was sprayed with Bromocresol Green Indicator and the other with nlnhydrln reagent. Both chromatograms were placed In a moist oven for 5 minutes at 105°C, D-Gluconic and D-galactonic acids were used as reference substances. No acids were detected in the rafflnose samples exposed to cathode radiation,

b. Ionophoresis of rafflnose Irradiated with cathode rays as 2% aqueous solutions. Two per cent solutions of the Irradiated rafflnose samples were prepared and a number of additions were applied at specified positions on a sheet of Whatman No, 3 filter paper (55,9 x 14,0 cm,). The electrophoresis (69, 70) lasted 1,5 hours, Sucrose, mellblose, D-galactose, D-fructose, and D-glucose were used as reference substances. The ionophoretogram was dried at room temperature, sprayed with aniline phthalate reagent (62), and dried In the air. This Ionophoretogram was then placed 60 in a moist oven at 105 C. for 5 minutes. Mellblose, sucrose, glucose, galactose, and fructose were present In all the irradiated samples but not In the unirradiated rafflnose. The intensity of the spots increased with Increasing irradiation dosage.

C. termination of the contiar reducing sugar Values of raffinose irradiated as 2% aqueous solution with cathode ray, in amount of 185 mg. of each sample was dissolved in distilled water and the volume was adjusted to 50 ml. The resulting solutions were diluted 100 to 1. The Somagyi (65) method was followed. An amount of 5.00 ml. of each of the diluted samples was mixed with 5.00 ml* of Somagyi reagent. These mixtures were placed in a boiling water bath for 30 minutes. After cooling the solutions to room temperature, 1.00 ml. of 10% aqueous potassium iodide was added to each without agitation. Then 1.5 ml. of 2 N sulfuric acid was added to each with vigorous agitation. These solutions were allowed to stand for 10 minutes and were then titrated with 0.005 N sodium thiosulfate solution. A 1% starch solution was used as indicator. The values obtained are shown in Table VI. I$r plotting these values against dose, Figure 8a was obtained. 61

VI. The Effect of Ionlying Radiation on Inulln

1. The Irradiation of Inulln as Two Per Cent iflttfgm figlunw with ftiiTOi Rayt Twenty grams of inulln (1) were dissolved in 800 ml.

(i) Inulln C.P., A product of the Pfanstiehl Chemical Company, Waukegan, Illinois. of hot water. The volume was adjusted to 1000 ml. The solution was allowed to cool to room temperature and adjusted again to 1000 ml. This procedure was followed for the inulln to dissolve since it is insoluble in water at room temperature. Inulln, however, is soluble In hot water and does not precipitate out Immediately when the solution is brought to room temperature. The prepared Inulln solution was divided equally in 4 tin cans of SO ounce capacity. The sealed cans were lowered Into the "swlmlng pool* and exposed to Co60 gamma radiation at the following dosess £.5, 5, 7.5, and 10 megareps delivered at the rate of 400,000 rep/hr. The temperature of the samples during irradiation was that of the source (appr. S4°C.)• The irradiated products were lyophillsed and stored In an evacuated desiccator over Drlerite (b).

a. Physical properties of Inulln Irradiated as 2& Aft A aqueous solutions with C? fMBffl ft Ixmlln 62 samples which have been exposed to Co60 gamma radiation at doses of 10 megareps and 7.5 megareps, became completely soluble in water at room temperature• It should be noted that Inulin normally Is Insoluble In water at room temperature. The samples of inulin which were exposed to 2.5 and 5.0 megareps of gamma radiation, respectively, were Insoluble in water at room temperature. A brown coloration was developed in the Irradiated samples of Inulin which was more intense In the samples exposed to 10 and 7.5 megareps, respectively.

b. Descending paper chromatography of Inulin Irradiated with TflYf- Per cent aqueous solutions of the Irradiated samples were prepared and a number of additions were applied at specified positions of Whatman No. 1 filter paper (14 x 45 cm.). The chromatogram was developed for 35 hours with 1-butanol t ethanol t water (40*11*19, vol. ratio). The developed chromatogram was sprayed with aniline phthalate reagent (62) and placed In an oven at 105°C. for 5 minutes. Spots corresponding to fructose were obtained for samples exposed to 7.5 megareps and 10 megareps of gamma radiation. No fructose spots were observed for the samples exposed to lower dosages.

o. pager tagpfagrwlff

d, natai-pHiiatlon nt th« ortflnt of Inulin hvdralvfff.

Emlfal m m t i W W * 1* radiation as £* aqueous solutions. The extent of hydrolysis of inulin vas estimated by determining the copper reducing values of the irradiated samples • At first the copper reducing value of D-fructose vas determined. An amount of 0,100 grams of D-fructose vas dissolved in distilled vater and the volume vas adjusted to 100 ml, A 1,0 ml, aliquot of this solution vas adjusted to 100 ml, vlth vater. The Somogyi method vas folloved (63), An amount of 5 ml, of the sugar solution vas mixed vlth 5 ml, of Somogyi reagent. The mixtures vere placed in a boiling vater bath for £5 minutes, then cooled to room temperature. When cooled, 1,00 ml, of 64 10£ aqueous potassium Iodide vas added to each solution without agitation. In amount of 1.5 ml. of 2 N sulfuric acid vas subsequently added to each test tube with vigorous mixing. The solutions vere allowed to stand for 10 minutes and vere then titrated with 0.005 N sodium thlosulfate solution. A 1# starch solution vas used as an Indicator. It vas found that 1 mg. of D-fructose reduced 8.4 ml. of the alkaline copper solution. This figure vas used In determining the reducing pover of Irradiated inulin samples. An amount of 0.250 g. of each of the irradiated Inulin samples vas dissolved In hot vater, cooled, and the solutions adjusted to 100 ml. One ml. of this solution vas diluted to 50 ml. with distilled water. The alkaline copper reducing values of the Irradiated Inulin samples vere determined In the same exact manner as that of D-fructose. These values are shown In Table VII and their plot against dose Is shown in Figure 3.

e. Isolation of irradiation products from Inulin Irradiated with Co60 gamma radiation as aqueous solutions. An amount of Nuchar C-unground (c) vas mixed vlth distilled vater and a slurry wag obtained. This vas poured In portions Into a glass chromatographic tube and a carbon column, 300 x 30 mm. (dlam.), vas obtained. The column vas vashed vlth 1000 ml. of 5# hydrochloric acid and the hydrochloric a d d vas vashed out with distilled water 65 until the pH of the washings were about 6. The carbon column was subsequently washed with 1000 ml, of ammonium hydroxide and then with water until the eluate became almost neutral. The described washing procedure eliminates salts which, were they present In the carbon, would Interfere with the separation and purification of the sugars. At this point the carbon column was ready for use. Four grams vere taken from the inulin exposed to 10 megareps and 3 g. from the inulin exposed to 7,5 megareps of gamma radiation. They were mixed and dissolved In 100 ml. of distilled water. The resulting solution was chromatographed (64) on the prepared column. The column was developed with 3000 ml, of distilled water to remove the monosaccharides possibly present. The obtained fraction was concentrated under reduced pressure to a sirup,

f. fnptr Qf> fraction obtained from carbon column chTOTMrtffgrtHhi1 g gftTmriittftn gf

indication of the constituents of the monosaccharide fraction obtained from the carbon column. It was chromatographed on paper (66). Three drops from the thin sirup were applied to a sheet of Whatman Ho, 1 filter paper (14 x 45 cm.) and developed for 85 hours with 1-butanol i ethanol * water (40ill*19, vol. ratio). 66 The dried chromatogram vas sprayed vlth aniline phthalate (68) and vas then placed In a moist oven for 5 minutes at 1Q5°C. An Intense spot vas obtained corresponding to fructose.

g. Acetylatlon of the slrupy monosaccharide fraction 60 obtained froiq c&rban eolump chromatography "ft fa ffMTHi rays Irradiated Inulin, The slrupy monosaccharide fraction obtained from the above carbon column vas dried by addition of 4 portions, of 80 ml. each, of absolute methyl alcohol and evaporation to dryness under reduced pressure. A brovn sirup vas obtained, the acetylatlon of vhich vas carried out In accordance vlth Pacsu and Cramer (71) vlth very

(71) E. Pacsu and F. B. Cramer, J. Am. Chem. Soc., 59. 1148 (1937). small variations. An amount of 100 mg. of fused and quickly grounded sine chloride (j) vas mixed vlth 40 ml. (excess)

(j) A reagent grade product of Uatheson Company, Inc. Norvood (Cincinnati), Ohio. of acetic anhydride cooled In an ice-bath. This mixture vas poured Into a 850 ml. flask containing the dried slrupy monosaccharide fraction and the vhole mixture vas placed In an lce-bath. The reaction mixture vas stirred vigorously 67 with an electric stirrer at 0°C. for 4 hours. The temperature then was kept at £5°C. for 1 hour and finally at 50°C. for £ hours. The cooled solution was stirred with an equal volume of water for 1.5 hours, further diluted and neutralised with an excess of sodium bicarbonate. This solution was extracted with four SO-ml. portions of chloroform and the chloroform extracts were combined. The combined chloroform extract was dried for £4 hours over anhydrous sodium sulfate, filtered and evaporated under reduced pressure to a sirup. The slrupy acetylated material was dissolved In 10 ml. of absolute diethyl ether. After allowing to stand In the ice box for ten days, crystallisation occurred; yield 50 mg., m.p. 10£-106°C. uncor. An X-ray powder diffraction pattern of the crystalline derivative was obtained using filtered CuK^ radiation. The resulting pattern was compared and found to be Identical with that of an authentic sample of ^-D-fructose pentaacetate; X-ray powder diffraction data (84)s 11.33 m, 8.33 vw, 7.45 m (£), 6.68 vs (1),

(84) Interplanar spacing, A., CuK* radiation. Intensity of lines estimated visually: s, strong; m, medium; w, weak; v, very; parenthetic numerals indicate three strongest lines, 1 strongest.

5.34 m (3), 4.84 w, 4.34 w, 4.03 vw, 3.61 vw, 3.35 vw, 3.04 vw. 66 6. The Irradiation, of Powdered Inulin with Cathode Raya Two samples of Inulin (1), of 2 g. each, were placed In open aluminum dishes (9.9 cm., diam.) and exposed to cathode ray Irradiation In doses of 400 megareps at the rate of 5 megareps/min. During the Irradiation no temperature control was employed.

a. Effect of cathode ray radiation on the physical properties of powdered Inulin. Both powdered Inulin samples, each of which had been exposed to 400 megareps of cathode radiation, became reddish brown. It is known that inulin is insoluble in water at room temperature. The powdered inulin samples, however, after they were exposed to cathode radiation became very soluble in water at room temperature.

b. Chromatography of powdered inulin irradiated with 400 megarens of cathode rays. Two per cent aqueous solutions were prepared and applied to a sheet of Whatman No. 1 filter paper (45 x 14 cm.) at positions 8 cm. from the unpointed end. The descending chromatogram was developed with n-butanol i ethanol t water, 40x11*19 (vol. ratio) for 55 hours. The developed chromatogram was air dried, sprayed with aniline phthalate indicator, and then placed In a moist oven at 110°C. for 5 minutes (66). In both of the Irradiated samples, fructose appeared to be present. 69 Another chromatogram vas prepared and developed with 1-butanol i ethanol : vater : ammonium hydroxide, 40s40sl6:4, (vol. ratio) for 35 hours. The developed chromatogram vas air-dried, sprayed with Bromocresol Green reagent, and then placed in an oven at 110°C. for 5 minutes. No organic acids vere detected in the irradiated inulin samples.

^^Xathod^Ray^at^RooffleTemDSatSeSOlUtl°DS Five samples of inulin (i), of 1 g. each, were placed in open aluminum dishes (9.9 cm. dlam.). Fifty grams of distilled vater vere placed in each of five screv-cap brown glass vials. These vere shipped by air mail to the cathode ray source. Just before irradiation the distilled vater fractions vere boiled, the corresponding Inulin samples vere dissolved, and after being cooled, the samples were irradiated in doses of 0, 2.5, 5, 7.5, and 10 megareps at the rate of 5 megareps/min. The irradiated samples vere lyophiliaed and kept in an evacuated desiccator while being Investigated. A control sample of inulin vas used.

a. The effect of cathode radiation on the physical properties of 2* dilute aqueous inulin solutions. Inulin samples receiving 10 and 7.5 megareps of cathode radiation became soluble in vater. At room temperature, hovever, the samples exposed to 2.5 and 5 megareps vere insoluble in vater at the above temperature. It should be noted 70 that uairradiated inulin is Insoluble in vater at room temperature* A brown coloration was developed In the irradiated inulin samples which vas more pronounced in those exposed to 7*5 and 10 megareps of radiation*

b* Chromatography of inulin irradiated with cathode rays as 2* aqueous solutions. Two per cent aqueous solutions of the samples vhlch had received 2*5, 5, 7*5, and 10 megareps of cathode radiation, respectively, were prepared* The chromatography was performed in the same manner as described above for powdered inulin irradiated with 400 megareps of cathode rays* D-Fructose was used as reference substance. This experiment indicated that fructose was present in the samples exposed to 7*5 and 10 megareps cathode radiation* D-Fructose was not detected In the unirradiated Inulin and In the samples which had been exposed to 2*5 and 5 megareps*

o. lonophoresis of inulin irradiated with cathode rays as 2* aqueous solutions* From the solutions prepared for chromatography, a number of applications were made at designated positions of Whatman No. 3 filter paper

(55*9 m 14*0 cm.)* The usual electrophoresis procedure was followed ( 69, 70) and the lanophoretogram was developed for 1*5 hours, was air-dried and sprayed with aniline phthalic acid reagent (62) • It was placed in a moist oven for 5 minutes at 12jO°C*, after being dried in 71 the air. The presence of fructose vas indicated In the inulin samples exposed to 7*5 and 10 megareps of cathode rays. No fructose was detected in the control sample and in the samples exposed to 2.5 and 5 megareps.

d. of hydrolysis of inulin expose^ u at Muaon- The copper reduction method of Somogyi vas followed (63). An amount of 0.250 g. of each of the lyophiliaed irradiated inulin samples vas dissolved in hot distilled vater and after cooling, the solutions vere adjusted to 100 ml. From this solution, 1 ml. was taken and diluted to 50 ml. with distilled water, in amount of 5 ml. of Somogyi reagent vas mixed vlth 5 ml. of each solution in test tubes. The test tubes vere placed in a boiling wu^er bath for 25 minutes. After cooling to room temperature, 1 ml. of 1Q% aqueous potassium iodide vas added to each solution without agitation. An addition of 1.5 ml. of 2 N sulfuric acid vas followed by vigorous mixing. After standing for 10 minutes, the solutions vere titrated with 0.005 N sodium thlosulfate solution. A 1$ starch solution vas used as an Indicator. The results are Indicated in Table VIII. The D-fructose copper reducing value of 8.4 ml. determined previously was used in the calculations of inulin hydrolysis. Figure 3 gives a plot of the per cent hydrolysis of inulin against dose. 72 VII. The Effect of

1. The Irradiation of Two Per Cent Aqueous. iPxt-Ure of AfflZlffiM. .with QftdPft Radiation A 2$ suspension of 10 g. anylose (3) vas prepared

(J) A product of Stein Hall Co., New York, N, Y. with distilled vater and sealed in a tin can in the presence of air. This mixture vas exposed to gamma radiation at ambient air temperature. The gamma rays were provided by an 100,000 curies fuel element source of the Argonne National Laboratory, Lemont, Illinois. The amylose sample vas exposed to 15 megarads of gamma radiation at a rate of 20,000 rads/min. The Irradiated amylose sample (heterogeneous) vas lyophlllzed and then kept in an evacuated desiccator under reducing pressure over Drierite (b).

a. Carbon col^yn Qf t f v 1q s « ^rrfidiated Tilth KffP* riYP- A slurry of Huchar C-ung round (c) vas prepared vlth vater and was poured in portions into a glass chromatographic tube. A carbon column of 400 x 45 mm. (diam.) vas obtained. The column vas vashed vith 1000 ml. of b% hydrochloric acid followed by distilled vater to the extent that the pH of the eluate vas about 6. The vashlng of the column continued vith 1000 ml. of 5}6 ammonium 73 hydroxide and then with water until the eluate was about neutral. This washing procedure was necessary for the elimination of inorganic salts present in carbon which otherwise would contaminate the sugar fractions. At this point the column was ready and the Whistler and Durso procedure was followed (64), An amount of 5 g, of the lyophiliced irradiated amylose was mixed with 50 ml. of water and poured on the top of the carbon column. The column was eluted with 4,5 liters of distilled water to remove tit monosaccharide fraction, followed by elution of the disaccharide fraction with 5% ethanol by volume. Both fractions were concentrated to a thin sirup under reduced pressure. The sirups were chromatographed on paper (62)• Three drops of each were applied to designated positions of Whatman No, 1 filter paper. D-Glucose and maltose were used as reference substances. The paper chromatogram was developed for 30 hours with 1-butanol t ethanol t water (40:11*19, vol. ratio), dried In air, and sprayed with aniline phthalate reagent (62). The sprayed chromatogram was placed In an oven for 5 minutes at 110°C, Glucose was Indicated to be present In the monosaccharide fraction and maltose In the dissacharide fraction. b. Acetylatlon of the slrupy monosaccharide obtained from carbon column chromatography of Irradiated amylose.

The slrupy monosaccharide fraction was dried by addition of 74 4 portions, or SO ml. each, of absolute methanol and evaporation to dryness under reduced pressure. The sodium acetate method vas used for the acetylatlon of the material obtained (65). An amount of 100 mg. of freshly fused and grounded sodium acetate was mixed vlth 50 ml. of acetic anhydride In a £50 ml. round-bottomed flask. The mixture vas heated to 100°C. The dried sirup vas added vlth mixing Into the hot acetic anhydride-sodium acetate mixture. On the completion of the addition, the reaction mixture vas heated to about 100°C. for 0.5 hour. After completion of the reaction, the mixture vas allowed to cool and vas then poured Into 300 ml. of ice-water. The aqueous mixture was stirred for 1 hour and allowed to stand for S hours for the acetic anhydride to hydrolyse, extracted vlth chloroform, the chloroform vashed vith vater and dried over anhydrous sodium sulfate. This chloroform extract was concentrated to 10 ml. under reduced pressure*

c. The separation and Identification of the acetylated monosaccharide derivatives of irradiated aeneous amvlose _ The separation of the acetylated monosaccharide vas achieved on a Magnesol-Celite column (66)• A mixture of Uagnesol i Celite (5x1, by vt.) vas prepared. A tapered glass column vas filled vlth absorbent mixture using a partial vacuum from a vater aspirator. Full vacuum vas subsequently applied and the column vas filled to a distance of 30 mm. from the top. The adsorbent vas tapped carefully 75 carefully to prevent channel formation. The dimensions of the prepared column vere £00 x 40 mm. (dlam.). The column vas prewetted vlth 30 ml. of benzene, and then the chloroform-sugar acetate solution vas added to the column. The column vas developed vlth 1200 ml. of bensene : ethanol solution (500:1, by vol.) (66). It should be noted that at no time after the addition of the 30 ml. of benzene vas the column allowed to run dry. After the development, the column vas extruded and streaked vlth a freshly prepared solution of alkaline potassium permanganate. This solution vas prepared by dissolving 1.0 g sodium hydroxide and 0.1 g. of potassium permanganate In 10 ml. of vater. A zone located at 96*105 mm. from the column top was obtained. This zone vas sectioned from the rest of the Llagnesol-Celite column and eluted with 50 ml. of acetone. The acetone adsorbent mixture was filtered and evaporated to dryness under reduced pressure. The obtained crude white solid vas dissolved In warm 95# ethanol and treated vlth a very small portion of activated carbon. This mixture vas filtered and on standing In the ice box overnight, an amount of 70 mg. of a crystalline substance vas obtained; m.p. 131-134°C. uncor. An X-ray powder diffraction pattern of the crystalline derivative vas obtained using filtered CuK^ radiation. The obtained pattern vas compared and found to be identical with that of an authentic sample of y9-D-glucopyranose pentaacetate. 76 VIII. Tfty Effect of flaimna Radiation on Ine Hrtrochlorlde.iwmm ,wr. D-Galactur onlc Acid

1. Irradiation Procedure Amounts of 8 g • each of D-glucosamine hydrochloride (k),

(k) D-Glucosamlne hydrochloride C.P., a product of Pfanstlehl Chemical Co., Waukegan, Illinois.

D-glucuronic acid (1) and D-galacturonic acid (1), vere

(1) Products of The Nutritional Biochemical Corp., Cleveland, Ohio. dissolved in vater and the volume adjusted to 400 cc. for each sample. Each solution vas sealed In a plain tin can of 500 cc. capacity and in the presence of air. The samples vere exposed at room temperature (about 27°C.) to gamma radiation provided by a fuel element source of 100,000 curies. The dose rate of the source vas SO,000 rads/min. and the radiations received by each sample were: D-glucosamlne hydrochloride, 15 megarads; D-glucuronic acid, 5 megarads; and D-galacturonlc acid, 5 megarads. The dose rate of the source vas 20,000 rads/min. By the end of the exposure the cans vere bulged due to gas formation. The irradiated sample-containing cans vere kept for several veeks in the 77 deep frees* before Investigation, s. Mat CoUegtlsa gfthe taft Oases Produced from the osamlne Hydrochloride. > ,tfi4 P-Qft34gtxff9ffiLg A s M The samples were gradually brought to room temperature and the gas from each can was allowed to escape into the corresponding evacuated weather balloon (m), Before use, the weather balloons were placed for 30 minutes in warm

(m) Product of Dewey and Almy Chemical Company, Cambridge, Massachusetts• water and dried at room temperature. The volume of the gas collected in the balloon was measured by displacement of the corresponding volume of water at 27°C. and 740 mm, pressure of mercury. Each can initially contained 100 cc. of air. The volume of gases (air initially present and gas produced) of each of irradiated D-glucosamIne hydrochloride, D-glucuronic acid and D-galacturonlc acid, measured at the above conditions, were 800, 175, and 190 cc,, respectively.

3, ptoflfftptor

(76) J. V. Kraus, Ph.D. Thesis, Department of Chemistry, Ohio State University, Columbus, Ohio (1957)•

of a helium gas source, a pressure regulator, a manometer, a surge tank, a thermal conductivity cell, a sample introduction system, a gas chromatography column, a spiral glass trap, a flow meter and an electronic strip chart recorder. The helium pressure of the column was measured by a mercury manometer with the flow rate of helium through the apparatus measured by a moving bubble flow meter. The components of the thermal conductivity apparatus (76) were a Gow-Mac cell, a 100 ohm and a 2 ohm potentiometer, a 250 milliampere D. C. meter, a 6 volt storage battery, one six-prong plug and jack, the connecting wiring and a Brown electronic recorder. The output signal was transmitted to the recorder through the Wheatstone bridge circuit. One of the gas chromatography columns was 16 ft. long and was packed with Molecular Sieve, the other was 8 ft. long and was packed with silica gel. Each column was suspended Inside a glass tube (6.0 cm. dlam.), wrapped with flexible heating tape and the entire tube covered with a Magnesite jacket for thermal stability.

b. Anal vat* of the gaseous products. The helium flow

through the gas chromatography by-pass was started and the filament current to the thermoconductivity cell was turned 79 on and adjusted to 155 milllamperes• The flow of helium was established by the regulator valve and measured by the flow meter. When equilibrium was obtained, 2 cc. of the unknown gaseous products were Injected, ty the use of a syringe, Into the gas chromatography trap and the helium was re-routed through this trap. To Identify each component and measure its characteristic elution time, a pure compound was put through the gas column and the peaks obtained were compared directly. By this gas chromatographic investigation, it was found that carbon monoxide, carbon dioxide, hydrogen, methane, ethane, and an unidentified gas were produced from the aqueous D-glucosamlne hydrochloride, D-glucuronic acid, and D-galacturonlc acid exposed to gamma radiation. An amount of air was placed In a weather balloon under the same conditions and length of time as the gases produced from the Irradiated dilute aqueous carbohydrate solutions. An air sample of 2 cc. was chromatographed under the same conditions and neither methane nor ethane were found to be present.

c. Tabulation of data. The gas chromatographic peak heights of the characteristic peaks of the gaseous irradiation products are shown in Table IX. These peak heights represent the distance of the highest point of the peak from the base line. The peak heights of known 8 cc. samples, under the same experimental conditions, were measured. For 2 cc. 80 samples, 1 cm, peak heights of carbon monoxide, carbon dioxide, hydrogen, methane and ethane were 2,85, 2.5, 10,9, 1,95, and 2,20 micromoles, respectively. Using these standard values, the quantity of each gas, produced from the Irradiated sugar, could be calculated. It should be noted that hydrogen can be produced not only from the sugar molecule but also from water on Irradiation (17), 1 sample calculation is given below for the carbon monoxide produced from the Irradiated D-glucosamlne hydrochloride. Since 1 cm, peak height of carbon monoxide corresponds to 2,85 micromoles In 2 cc. of known sample, the 0,3 cm. peak height of the unknown will correspond to 0.3 x 2,85 = 0,855 micromoles In 2 cc, of sample. Therefore, the 200 cc. of collected gases contain 0,855 x 200 * 171 micromoles of carbon monoxide which was produced from the Irradiated 8 g, (3,78 x 10* micromoles) of D-glucosamlne hydrochloride. The calculated gaseous quantities are shown In Table X,

4 , Piper OtfgBEtamghy Agufpm Act

After the collection of the gases, the Irradiated as 2^ samples were chromatographed on paper. It should be noted that all the samples were reddish-brown after the irradiation. This color was more pronounced In the case of D-glucosamlne hydrochloride and the solution was acidic. Several drops of each sample were applied to a Whatman No, 1 filter paper and subjected to descending 81 development for 30 hours vlth 1-butanol : ethanol s water (40*11*19 vol. ratio), In an all-glass chamber equipped for chromatography. The alr-drled chromatograms vere sprayed vlth aniline phth&llc acid reagent (68) and placed in an oven at 105°C. for 5 minutes. This experiment Indicated the presence of four Irradiation products In the Irradiated D-glucuronic and D-galacturonic acid and three in the irradiated D-glucosamine hydrochloride samples.

IX. RftSQjpfflce Studies of Irradiated Carbohydrates

1. Irradiation, of Certain Crystalline Carbohydrate f^nyrffl Rjjyy

Powdered samples of maltose, cellobiose, trehalose, raffinose pentahydrate, inulin, D-glucuronic acid and D-galacturonlc acid vere sealed in the presence of air, in tin cans and exposed to gamma radiation In doses of 5 megarads. The radiation vas provided by the 100,000 curies fuel elements gamma ray source located at the Argonne National Laboratory, Lemont, Illinois. The temperature during the irradiation vas that of the room (about 87°). s . PflripmrpflU? Rtfrfflftasa Ss.gfitr* Irradiated Carbohydrate Powders The spectrograph employed in this Investigation consisted of an X-band Klystron operating at constant frequency (vs$317 Uc/sec.), controlled by a secondary as frequency standard monitored by WWV. Small amounts of each Irradiated powdered samples of maltose, cellobiose, trehalose, rafflnose pentahydrate, Inulin, D-glucuronic acid and D-galacturonlc acid were placed In melting point capillary tubes (100 x 2.0 mm.). The samples were mounted in a reflection type quarts resonant cavity, situated between the six-inch diameter poles of an electromagnet supplying a strong magnetic field. This magnetic field could be varied by means of a clock drive attached to a potentiometer In the magnet power supply (75)• The magnetic field modulation at 2000 cycles/sec. was provided by means of two small colls mounted on the pole faces of the magnet. A lock-in amplifier was employed, so that the trace displayed on the recorded chart was the first derivative of the absorption signal. The magnetic field was homogeneous to within 1 gauss over the sample sise used. Irradiated inulin gave no absorption signal. D-Galacturonlc and D-glucuronic acids gave signals which were ten times stronger than those obtained from the other carbohydrates• The results are shown in Figures 4, 5, 6, In order to obtain more Information about the nature of the radicals formed from carbohydrate molecules having little difference In structure, the following experiment was performed. Irradiated maltose and cellobiose were thoroughly mixed ltl by weight and the electran-spln resonance spectrum of the obtained mixture was taken. 83 The eleotron-spln resonance spectra of mixture (1*1, by weight) of Irradiated D-glucuronic a d d — irradiated D-galacturonlc acid, was also taken. The results are shown in Figure 7 (I and II). DISCUSSION OF RESULTS

I. The. .Effect of Cathode Radiation on Fifty Per Cent Aqueous Solutions pt Maltose and Cellobiose Maltose and Cellobiose are tvo disaccharides, each composed of tvo glucopyranose units. Maltose Is a 4-0-4-D-glucopyranosyl-D-glucose and cellobiose Is 4-0-^-D-gluc opyranosy1-D-glucose. Investigations concerned vith the action of ionizing radiations on sucrose and methyl 4-D-glucopyranoside have indicated (66) that the glycosldlc bond is preferentially attached. In order to determine the relative ease in cleavage of «(-D andfi-D glycosldlc linkages, an investigation of the action of ionizing radiations on maltose and cellobiose vas conducted. This investigation should indicate also the comparative susceptibilities of sucrose, maltose, and cellobiose to attack by ionizing radiations. Maltose and Cellobiose, as 50% dilute aqueous solutions, at 0 C., vere exposed in aluminum dishes to cathode ray irradiation in doses ranging from 50 to 400 megareps at the rate of 5 megareps/min. Descending paper chromatography of the aqueous maltose exposed to cathode radiation indicated the presence of glucose. In all cases the concentration of glucose appeared to be very small but increased vlth increasing radiation dosage. Samples of aqueous cellobiose, vhlch had been Irradiated under the 64 85 same conditions, shoved no glucose present when subjected to paper chromatography. Thus, paper chromatographic data suggest that the4-D glycosldlc linkage Is more readily attacked than Is the /J-D glycosldlc linkage, by Ionizing radiations, but neither glycosldlc bond Is attacked to any large extent. This Is In accord with the relative hydrolysis of these dlsaccharldes by acids. The results, along with preliminary analytical data, suggest also that maltose and cellobiose are hydrolyzed to a much lesser extent than sucrose when these disaccharides are exposed to Ionising radiations.

II. The Effect of Cathode .Radiation on Twenty Per Cent Aqueous Maltose Irradiated at Ethanol-Dry Ice. Ice-Water and. Ambient Air Temperatures Maltose, a synonym for 4-0-*-D-Glucopyranosyl-D- glucose, is a reducing dlsaccharlde• Among the most Important changes In carbohydrates directly attributable to high energy electron bombardments are the polymerisation and depolymerization reactions. The hydrolytic splitting of glycosldlc bonds of carbohydrate polymers is susceptible to detection and measurement. Such radiation depolymerization can well be Investigated through the preliminary investigation of model compounds, such as maltose, which serve as polymerizing units for these substances. It was known that carbohydrates exposed to Ionising radiations in the solid state were degraded but not to a large extent (59). When, however, the same 86 substances vere Irradiated In aqueous solutions, they exhibited more extensive depolymerisation changes. The effect of high energy electron irradiation on dilute aqueous maltose solutions and at different temperatures vas Investigated. The concentration of the solutions chosen vas 20J6. The vater vas added to the sugar Just prior to Irradiation and the resulting solutions, previously cooled vith ambient air (first series), lce-vater (second series), and ethyl alcohol-solid carbon dioxide (third series), vere exposed in open aluminum containers to cathode rays at a rate of 5 megareps/min. The lots in each series received from 20 to 100 megareps as is shovn in Table I. High irradiation doses vere employed in order to magnify the chemical changes so that they could be clearly recognised and readily measured. Maltose solutions of the same concentrations, treated in exactly the same manner as the irradiated samples but receiving no radiation, vere employed as control samples. The maltose samples, after being lyophllised, vere subjected to descending paper chromatography (62, 68) and glucose vas found to be present in all the irradiated samples. The intensity of the glucose spot increased vlth increasing radiation dosage and decreased vith increasing temperature. No glucose vas present in the control samples vhieh indicates that the formation of glucose is due to the cathode radiation. The possibility of acids formed from 07 the sugar was explored chromatographically but no adds ware detected. This indicates that under the Irradiation conditions employed either no adds vere formed or they were present In no detectable quantities. The extent of decomposition of maltose was estimated on the basis of the amount of copper reduced (63) by the Irradiated and unirradiated samples. These results are shorn in Table II. The apparent per cent maltose hydrolysed was calculated on the basis that glucose was the only reducing substance formed. The maximum amount of copper reducing substances was present in the products from the Irradiation of maltose cooled by ethanol and solid carbon dioxide (see Table II); the value corresponding to 1Q0 megareps represents a 16.21# hydrolysis of maltose. The maltose solution was a frosen mass during this irradiation; thus, any thermal change was markedly reduced. The extent of hydrolysis of maltose cooled by ambient air during the irradiation treatment was less; the value of the sample exposed to 100 megareps was 1S.16£. The corresponding figures for the irradiation series cooled with ice-water were Intermediate, 14.86)6 hydrolysis at 100 million reps (see Table II and Figure 1). Similar results have been reported previously (48) for the Irradiation of sucrose. To Interpret these results a probably thermal effect Is Involved. Normally, one would expect the maximum amount of copper-reducing substances to be present in the samples 88 Table II Extent of Hydrolysis of Maltose Irradiated as ZQ% Aqueous Solutions at Three Different Temperatures with Cathode Hays

Ml. of copper Ml* of copper solution re­ solution re­ duced per 5 ml* duced per mg. Apparent aliquot of of non-lrrad* Per non-lrrad* sample minus Cent Radiation sample minus ml. of copper Hydro- G received ml* of copper solution re­ lysis* solution re­ duced per mg. duced per 5 ml. of Irrad. aliquot of sample irrad. sample

Cooled with ethanol - solid carbon dioxide 0.0 MREP 0.00 ml. 0.00 ml* 0.00

£0.0 ■ 0.05 IT 0.30 r 4*05 6.12 40.0 N 0.05 If 0.50 R 6.75 5.30 60.0 ■ 0.08 II 0.80 If 10.54 5.42 80.0 « 0.11 n 1.10 If 14.86 5.64 100.0 N 0.1£ « l.£0 If 16.21 5.09

•Based on hydrolysis to glucose* 89

Table II (contd.)

HI. of copper III. of copper solution re­ solution re­ duced per 5 ml. duced per mg. aliquot of of non-lrrad. Apparent non-lrrad, sample minus Per sample minus ml. of copper Cent Radiation ml. of copper solution re­ Hydro- G received solution re­ duced per mg. lysis* duced per 5 ml. of irrad. aliquot of sample irrad. sample

0 .0 IIREP 0.0 0 ml. 0 .0 0 ml. 0.0 0

2 0 .0 n 0 .0 2 n 0 .2 0 it 2.7 0 4 .4 4

40 .0 « 0 .0 3 n 0.3 0 it 4 .0 5 3 .1 7

60.0 n 0 .0 7 n 0 .7 0 tr 9.46 4 ,9 2

60 .0 n 0 .0 9 TJ 0 .9 0 n 1 2 .1 6 4 .7 7

100.0 u 0 . 1 1 It 1 .1 0 it 14.86 4 .6 6 Cooled with ambient air

0 .0 LIREP 0 .0 0 ml. 0 .0 0 ml. 0.0 0

20 .0 n 0 .0 2 II 0 .2 0 it 2.7 0 4 .2 4

4 0 .0 ti 0.0 25 n 0 .2 5 tt 3,3 8 2.66

60.0 n 0 .0 6 tt 0 .6 0 it 8 .1 1 4 .2 5

8 0 .0 n 0 .0 8 it 0 .8 0 it 1 0 .54 4 .1 5

100.0 n 0 ,0 9 it 0 .9 0 n 1 2 .1 6 5 .8 2

* Based, on hydrolysis to glucose. Gel lob lose and maltose hydrolyzed^ % 0 i. . h irdain f 0 qeu sltos f oeilobiose of solutions aqueous 20% of irradiation The I. Fig. n mloe ih ahd ry a 0G n a te rate the at and 0*G at rays cathode with maltose and f x 0 rp/i. A, ats; celiobiose). , O maltose; , (A reps/min. 10s x 5 of 20 oe megareps) (m Dose 0 4 90 60 0 6 00

91 Irradiated at higher temperature (ambient air), since the rate of hydrolysis of maltose should be faster at the higher temperature* In the case of sucrose, It was suggested (46) that the rate of alteration of the copper-reducing substances to nonreducing products was fastest at the highest temperature (ambient air), and this change may be caused by a thermal effect. The physical appearance of the Irradiated dilute maltose samples Indicated that a brown color was developed most In the samples cooled with ambient air. The least coloration was developed In the samples Irradiated at ethanol and solid carbon dioxide temperature. The brown coloration of the maltose samples, Irradiated while being cooled with Ice and water, was intermediate. If the brown color Is related to thermal reaction then the thermal effect Is in agreement with the results obtained by the determination of the amount of reducing products. Another view could be considered. It Is known that cathode rays attack not only the solute but also the solvent (££), Free radicals, like H* and OH*, given off by the water, during the Irradiation, attack the solute and this Is the so-called Indirect action of radiation. Since the solutions of maltose at ethanol-solid carbon dioxide temperature were a solid mass during the bombardment, the energetic free radicals formed could be trapped at Imperfections In the solid. These radicals being In high 98 concentration after the irradiation and while the sample Is gradually brought to room temperature, may oausa a graater attack on tha sugar present. Another intarpratatlan nay ba given to explain tha prasanca of smallar amounts of raduolng substanoas formed on cathode radiation of dilute maltose solutions at ambient air temperature. Tha D-glucose formed as a result of glyoosldlc cleavage could ba more readily damaged by Irradiation at ambient air temperature than at lover temperatures. This is quite likely since D-glucose, which la the probable product In the Irradiation of maltose, is radiation sensitive, in analogy to this can be found in the work by Wolfrom and his associates (48) who found that the percentage hydrolysis of irradiated aqueous sucrose was less at higher temperature and suggested it is due to the destruction of the irradiation products $ glucose and especially fructose. Similar observation vas made by Saernan (77) vho found that a peak in the acid hydrolysis of

(77) J. F. Saeman, Ind. Efcg. Chexa., 43 (1945). cellulose was due to the destruction of glucose. Ghormley and Stewart (81) Investigated the action of radiation on ice and found that free radicals are certainly much less mobile, but on the other hand electrons may diffuse rapidly. Perhaps this could partially explain my results in the 93 irradiation of maltose at ethanol-solid carbon dioxide temperature with high speed electrons* The results obtained from the oopper reduction values of irradiated maltose at lce-water temperature are plotted in Figure 1 and are extrapolated to sero* In this graph the extent of hydrolysis against dose is delineated* The apparent per cent hydrolysis was found to Increase with increasing irradiation dosage In a linear fashion, probably owing to the fact that only one bond was being hydrolysed* For a better description of the effect of cathode radiation on maltose dilute solutions, the G (glucose) values (34, 78) were calculated* These values are shown in

(78) U. Burton, J. Phys. Colloid Chem., & , 611 (1947)*

Table II and their calculation, as it is described in all the experimental section, was based on the apparent per cent sugar hydrolysed* In the case of maltose the per cent hydrolysis was based on conversion to D-gluoose (copper reduction method)* In calculating the G values of Irradiated maltose, no allowance was made for the nonreducing substances formed, therefore, the actual G values are probably somewhat higher than the calculated ones* The G (glucose) calculated values of irradiated maltose, at ethanol-solid carbon dioxide temperature, are somewhat greater than those of the samples irradiated at ambient air temperature, and those of the samples irradiated at lce-water temperature are intermediate. 94 In the first of the above three series of Irradiated samples (ethanol-solid carbon dioxide temperature) the values range between 5*09 and 6.18; in the second series (lce-water temperature) they range between 4.44 and 4.92 and In the third series between 2*66 and 4.85. The higher 0 values at lower temperatures could be expected since the extent of maltose hydrolysis is somewhat greater at those temperatures. Although this Is the case, one can see that these values are considerably close together, especially for samples exposed to Ionising radiation at the same temperature. The G values obtained for Irradiated maltose are less than those reported (48) for irradiated sucrose. This is very reasonable, since the glycosidlc bond in sucrose is more labile than is the glycosldic bond in maltose. Some important features have been reported to distinguish ionising radiation from photolytic radiation (S3), however, the data obtained from the irradiation of maltose clearly indioate that these two types of radiation have some common effects on carbohydrates. Guillaume and his associates (14,79)

(79) A. Guillaume and G. Tanret, Compt. rend., 201- 1057 (1935). studied the effect of ultraviolet radiation on dilute aqueous solutions of maltose and sucrose and found that maltose was hydrolysed less than 30% as fast as sucrose. This is In 95 close agreement with the results obtained from the Irradiation of maltose and those reported (48) on the Irradiation of sucrose. Is a consequence, the glycosidic bond In sucrose Is more labile than the glycosidic bond of maltose. Heidt (80), In order to explain the results of

(80) L. J. Heidt, J. Framtlin Inst., £54. 475 (1942). ultraviolet radiation on some glycosides, suggested that energy absorbed by a part of the molecule travels intramolecularly along the chain of atoms and eventually ruptures the acetal oxygen bridge. A similar phenomenon could be postulated for the splitting of the labile glycosidle bond of maltose. In order to confirm the results obtained from paper chromatography of irradiated maltose, one of the Irradiated samples with a high concentration of D-glucose was subjected to carbon column chromatography. The irradiated maltose sample chosen was the one exposed to 100 megareps of cathode radiation at ioe-water temperature. A carbon column was prepared and washed, first with dilute hydrochloric acid, then with dilute ammonium hydroxide and finally with water. This washing was found very useful for the separation and purification of sugar fractions, since the inorganic salts present In the carbon were eliminated before the introduction of the sugar under investigation. The flowing chromatogram 96 was developed with water according to the method of Whistler and Durso (64)• This developer removed the monosaccharide fraction from the adsorbent column. The recovered monosaccharide fraction was paper chromatographed (66) and the presence of D-glucose was clearly Indicated. The sirupy monosaccharide fraction was acetylated by the sodium acetate method (65) which Is one of the most satisfactory methods for preparing acetyl sugars, because the catalyst is easily removed. The acetylated product was chromatographed on a hydrated magnesium acid silicate column employing bensene : ethyl alcohol (500 si, volume ratio) as the developer (66). Elution of the principal zone and subsequent recrystallixatlon of the product from ethanol, yielded a crystalline substance which was identified asyff-D- glucopyranose pentaacetate by its melting point and X-ray powder diffraction pattern. The yield of y?-D-glucose pentaacetate was probably reduced by the nature of the acetylatlon and the efficiency of adsorbate recovery from the columns. The Isolation and identification of D-glucose as its )9-D-glucopyranose pentaacetate definitely proves the degradation of maltose to D-glucose by cathode radiation. 97 III. The ^Effect of Cathode_Radiation,on Twenty Per Cent Aqueous Celloblose Istdftrttr I«BBWft.tart Celloblose is a reducing disaccharide which consists of two glucopyranose units. It closely resembles maltose, however, as these two disaccharides differ only In the stereochemical nature of their glycosidic linkages. The products of hydrolysis of both dlssacharides are Identical but maltose Is a A-O-el-D-glucopyranosyl-D-glucose and celloblose Is a 4-0-yP-D-glucopyranosyl-D-glucose. To study the effect of ionizing radiation on celloblose In more dilute solutions and for more complete comparison of the susceptibility of^-D and^-D glycosidic linkages to radiation, 20% aqueous celloblose solutions were exposed to cathode rays at lce-water temperature. The dose rate was & megareps/mln., and the samples received from £0 to 100 megareps of radiation. A control sample was used and treated In the same manner as the rest of the samples but received no radiation. Descending paper chromatography of the lyophllised samples showed the presence of glucose in all the irradiated ones but not in the control sample. The intensity of each glucose spot was Increased with Increasing radiation dose. Chromatographic Investigation for the detection of acids In all the irradiated samples gave negative results. This would Indicate that either no acids were formed or if they were formed they were present in such small quantities as 96 to not be detected. This is in reasonable agreement with the results obtained from the irradiation of maltose at the same temperature, dilution and doses. The extent of celloblose hydrolysis was estimated by determining the copper reduction values according to the method of Gomogyi (63). The results obtained are shown in Table III. The sample exposed to SO megareps gave the same amount of glucose as that of maltose exposed to the same dose. However, the percentage of hydrolysis in celloblose samples exposed to higher doses was less than that of the corresponding maltose samples treated in a similar manner. The difference obtained is not very great but becomes greater at higher doses. These results show that at lower doses there is not much difference in the susceptibility of 4-D and ft-D glycosidic linkages but at higher doses the 4-D glycosidic linkage in maltose is much more labile to cathode radiation and is in accordance with the relative hydrolysis of these disaccharides by acids. Ey plotting the per cent of celloblose hydrolysed versus dose and extrapolating to sero, a straight line was obtained as shown in Figure 1. The increase of hydrolysis with increasing irradiation dosage in a linear fashion, probably comes from the fact that only one bond was being hydrolysed. Guillaume and Tanret (14) reported that y9-D glycosides were hydrolysed by ultraviolet radiation very slowly and were more resistant than 4-D glycosides. This is in 99

Table III

Qctent of Hydrolysis of Celloblose Irradiated as 20% Aqueous Solutions with Cathode Hays at 0°C*

Ml, of copper ill. of copper solution re­ solution re­ duced per 5 ml. duced per mg. aliquot of of non-lrrad. Per non-lrrad• sample minus cent Radiation sample minus ml. of copper hydro­ received ml. of copper solution re­ lysis* solution re­ duced per mg. duced per 5 ml. of irrad. aliquot of sample Irrad. sample

0 MREP 0 .0 0 ml. 0 ,0 0 ml. 0.0 0

20 ti 0 .0 2 n 0.2 0 tt 2.7 0 4 .2 4

40 it 0 .0 3 it 0.30 n 4.0 0 3.7 0

60 it 0 .0 5 it 0 .5 0 tt 6.80 5 ,5 6

60 it 0 .0 6 tt 0 .6 0 it 8 .1 1 3 .1 9

1 0 0 n 0 .0 8 tt 0 .0 0 tt 10 .54 5 .2 1

♦Based on hydrolysis to glucose 100 agreement with the results obtained from the study of celloblose and maltose. Furthermore, by comparison of the results obtained from the maltose and celloblose Investigation with those obtained from sucrose by Wolfrom and his associates (48), one can note that the extent of hydrolysis decreased In the order sucrose, maltose, celloblose. The effect of dilution Is of Importance In this Investigation. It was found that exposure of 50£ aqueous solutions of celloblose to cathode radiation resulted to no detectable degradation of the sugar. However, Irradiation of J20J6 solutions of the same sugar effected degradation to a reasonable extent. The results obtained undoubtedly prove that Indirect action Is very common In radiation chemistry (S3). It has been postulated that on irradiation of dilute aqueous solutions, radicals such as H*, OH* and possibly other species are formed by the direct action on water (17)• Free radicals are also formed In the solute as we proved by the paramagnetic resonance study of celloblose irradiated In the absence of water. The creation of active free radicals from the solvent shows that the solute can be attacked not only directly by the radiation but also by these radicals. The celloblose Irradiation at more dilute solutions could be considered as an example of Indirect action. Assuming a non-selective absorption of radiation energy and no marked transfer of energy before the Ions and excited molecules of water break down, then the actual 101 number of free radicals and other species formed from the water must be greater than the number formed from the solute (23). If one makes a rough estimation, the number of radicals may be In the ratio by weight, of the two substances present,. Consequently, at higher dilutions the number of radicals formed Is greater and since hydroxyl radicals and hydrogen atoms are highly reactive, the sugar Is far more likely to be affected by these than directly by the radiation. In the Irradiation of 20% dilute aqueous solutions of celloblose the actual number of free radicals and other products formed from the water would be very roughly twice the same number of the Irradiated 50% celloblose. This difference Is probably enough to give us the results obtained. The energy yields G, that is, the number of molecules changed per 100 ev, of energy absorbed, were calculated for the Irradiated celloblose samples. The calculation of these values (78) was based on the apparent per cent sugar hydrolysed and the latter was based on conversion to glucose (copper reduction method)• These values are shown In Table III. They range from 3,1b to 4.24 for the samples exposed from 20 to 100 megareps. They are rather constant and smaller than those obtained from maltose samples of the same dilution, exposed to cathode radiation at the same dose and temperature. This is not unexpected, since the glycosidic bond In maltose is more labile than the 108 glycosidic bond In celloblose.

IV. Tha. Effect.of Cathode Radiation on Two Per Cent Dilute Aoueous Solutions of Trehalose at Ambient Air Temperature Natural trehalose Is a nonreducing dlsaccharlde which on hydrolysis yields two moles of D-glucose. This of-D- glucopyranosyl 4 —D-glucopyranoside was chosen, among the nonreducing dlsaccharldes of D-glucose, to study the radiation effects on carbohydrates at considerably more dilute solution. A number of trehalose 8% dilute aqueous solutions were prepared just prior to irradiation and exposed to cathode radiation at the rate of 5 megarads/min. The irradiation was performed at ambient air temperature and the doses received were 2.5, 5, 7.5, 10, 12.5, 15, 17.5, and 80 megarads. A control sample was employed and was treated just as the rest of the samples but received no dose. All the samples after radiation treatment were lyophlliaed and chromatographed according to the method of Partridge (68, 66). The paper chromatographic investigation showed the presence of glucose in all the Irradiated samples but not in the control sample. This evidence suggests that trehalose was hydrolysed by the high energy electron bombardment. At higher dosages the degradation of trehalose was greater since the Intensity of the glucose spot was more pronounced. No acids were found to be present. In any of the Irradiated trehalose samples, with the chromatographic 103 procedure employed. The results obtained are in agreement with, those reported fcy Guillaume and Tanret (14) on the effect of ultraviolet radiation on aqueous solutions of trehalose. These workers found that trehalose was hydrolysed but not to any great extent. A further proof of the degradation of trehalose to D-glucose was the separation and isolation of D-glucose from irradiated trehalose and its identification as ^-D-glucopyranose pentaacetate. For the isolation of the reducing sugar fraction from the irradiated trehalose a carbon column was prepared. Before the development of the chromatogram, the carbon column was washed very carefully to eliminate Inorganic salts present in the carbon which certainly would interfere with the separation and purity of the sugar fractions. At first dilute hydrochloric acid was employed, then water and dilute ammonium hydroxide. The ammonium hydroxide was finally washed off with a considerable amount of water until the column was neutral. The trehalose sample exposed to £0 megarads was chromatographed since analytical data Indicated that it gave more hydrolysis products than any other sample. The flowing chromatogram was developed with water (64)• The eluted monosaccharide fraction was recovered and chromatographed on paper (66), in order to obtain some preliminary evidence of its nature. The only oompound shown to be present in this fraction was D-glucose. 104 The recovered and dried sirupy fraction was acetylated by employing the sodium acetate method (65). The acetylated product was chromatographed on Hagnesol-Cellte column employing as developer a mixture of benzene and ethyl alcohol* The zones were located with alkaline potassium permanganate solution* Elution of the principal zone and subsequent r©crystallization of the product from ethanol, yielded a crystalline substance which was Identified by its melting point and X-ray powder diffraction pattern as ^-D-glucopyranose pentaacetate* The detection of stable free radicals, in Irradiated trehalose, by the electron-spln resonance study and the hydrolysis observed in dilute samples, obviously Indicate the formation of stable free radicals or activated molecules, which, on coming In contact with free radicals from water or activated water molecules, result In the split of the most labile bond. In this case the most label bond Is the glyconsldlc bond* A number of workers (81, 88, AS,) to explain the cleavage of peptides at the peptide bond,

(81) D* C* Carpenter, J* Franklin Inst*, £38* 76 (1941). (88) D* C* Carpenter, J. Am. Chem. Soc*, 68- 889 (1940)* (83) Inez Uandl and A* D* McLaren, Nature, 164. 749 (1949),

suggested that the energy absorbed travels along the chain resulting in the rupture of the molecule at the most labile 105 bonds. A similar phenomenon probably takes place in the trehalose molecule. The apparent per cent trehalose hydrolysed was calculated by the copper reduction method (63). It was found that the extent of hydrolysis of %% aqueous solutions of trehalose Increased with Increased irradiation dosage. The determined per cent hydrolysis was plotted against irradiation dosage and, by extrapolation to zero, Figure 2 was obtained. At lower doses (0 to 7.5 megarads), the per cent hydrolysis was found to Increase in a linear fashion with increasing irradiation dosage. At higher doses (7.5 to 20 megarads), a deviation from the straight line was observed; however, the per cent hydrolysis was still Increasing but at a smaller rate than at the lower dosages. This would not mean that trehalose is attacked by radiation to less extent at higher doses. Since D-glucose was the product of hydrolysis, apparently it was degraded by irradiation at a higher rate in higher doses than in lower ones. At higher irradiation dosages, the difference between the rate of formation and the rate of disappearance of D- gluoose gradually decreased. This probably was caused by the Increased degradation of D-glucose formed. This phenomenon was not observed in the case of maltose and celloblose; however, one must keep in mind that these sugars were irradiated at much higher concentrations than trehalose. Apparently D-glucose is considerably more labile to radiation 106

Table IV

Reducing Sugar Values for Irradiated 2% Aqueous Trehalose

No. of ml. No. of ml. copper copper Dose solution solution Hydrolysis# (megarads) reduced reduced per cent G per 5 ml. per mg. of aliquot sample

0.0 0.00 0.00 0.00 2.5 0.53 0.11 1.48 17.4 5.0 1.00 0.20 2.70 16.2 7.5 1.50 0.30 4.05 16,2 10.0 1.85 0.36 4.87 14.6 12.5 2.03 0.41 5.54 13.3 15.0 2.86 0.45 6.08 12.2 17.5 2.40 0.48 6.48 11.1 20.0 2.50 0.50 6.76 10.2

♦Based on hydrolysis to glucose* Trehalose hydrolyzed, % 0 Fig Z

cathode rays at the rate of 5 x id* rods/min and and rods/min id* x 5 of rate the at rays cathode Irradiation of of Irradiation min ar temperature. air ambient 5 oe me go rodsDose, 2% 107

qeu sltos f rhls with trehalose of solutions aqueous 10 15 20 at

108 at higher dilutions and this may be due to the greater Indirect action. The radiation yields G (78), that is the number of molecules changed per 100 ev. of energy absorbed, were calculated for the irradiated trehalose samples. The calculation of the G (glucose) values was based on the apparent per cent trehalose hydrolysed to O-glucose by irradiation. The results, tabulated in Table IV, show that the G values range from 10.2 to 17.4. These values are greater than those obtained in the irradiation of maltose and celloblose. This is not unexpected, since trehalose was exposed to cathode radiation at considerably greater dilutions than maltose and celloblose. Such a difference could be due to greater indirect effect in more dilute solutions (23)• Table IV shows that the G (D-glucose) values for irradiated aqueous trehalose are greater at lower dosages. This could be due to an oxygen effect or to some indirect effect (48). It has been reported (14) that trehalose was hydrolysed less than 3QJC as fast as sucrose when exposed to ultraviolet rays. This is in analogy to the results obtained from the cathode irradiation of trehalose when compared to the results reported on sucrose (48)• 109 V, rt IgTl^FiVnfl Relations on Rafflnose

1. Tha Irradiation of Two Par Cant Dilute Aqueous Solutions of Rafflnose with Gamma Rays at Room Temperature Rafflnose Is a trisaccharide which on complete acid hydrolysis gives one mole each of D-glucose, D-fructose, and D-galactose. This 0-4-D-galactopyranosyl-(l' — ■» 6)- 0-4-.D-glucopyranosyl-(l ■■■ >2 )-y^D-fructofuranoside was chosen as the substrate for the study of the effect of radiation on trlsaccharldes and, In addition, to compare the effect of cathode radiation versus gamma Irradiation on carbohydrates* It was considered that since rafflnose contains two linkages, one of which is thert-D- galactopyranosldlc linkage present In mellbiose, and the other is the «(-D-glycopyranos idle one present In sucrose, the irradiation of this sugar should give considerable Information on the relative resistance of these linkages to Ionising radiation. A number of &£ solutions of rafflnose were exposed, 60 In sealed tin cans, to gamma radiation from a Co source In doses of 2*5, 5, 7*5, and 10 megareps at the rate of 400,000 reps/hour* The irradiation temperature was about £4°C* A control sample was employed but received no dose* Paper chromatographic Investigation (62) of the lyophilixed samples indicated the presence of mellbiose, sucrose, galactose, glucose and fructose, In all the Irradiated 110 wunple* but not In the control sample. To obtain more evidence on the effect of gamma radiation on rafflnose, the samples were subjected to ionophoresis (69, 70) and the results obtained were In complete agreement with those obtained from paper chromatography. In both cases the spot Intensity of the Irradiation products Increased with Increasing radiation dosage; however, the reverse was observed with the rafflnose spot. The evidence from paper chromatography and Ionophoresis indicated the presence of substantial amounts of fructose and mellbiose, which would mean that the rt-D-glycosidle bond in the sucrose part of the molecule was very labile to gamma radiation; however, the 4% -D-galactosldic bond In the mellbiose part was more resistant. This can be seen more pronouncedly in the samples exposed to lower radiation dosages. In these samples just detectable amounts of sucrose, galactose and glucose were present. This is in analogy with evidence obtained from the literature on acid hydrolysis of rafflnose (85)•

(85) C. Schelbler and H. Mlttelmeler, £er«, 22. 1680, 3120 (1869).

Schelbler and Ulttelmeier found that mild acid hydrolysis of rafflnose affected only one linkage with the formation of mellbiose and D-fructose. Khenokh (40) reported the presence only of sucrose and fructose In irradiated aqueous solutions Ill of rafflnose. In tnis work not only fructose and sucrose but also mellbiose, galactose and glucose were detected. No organic acids were found to be present In the Irradiated rafflnose by chromatographic analysis. In order to obtain further information on the extent of the hydrolytic processes that occurred in the rafflnose samples exposed to gamma radiation, the copper reducing values were determined (63)• Rafflnose is a nonreducing sugar but on hydrolysis all the products are reducing. On irradiation, rafflnose was degraded to the intermediate hydrolysis products mellbiose and sucrose, besides the three monosaccharides. Moreover, these irradiation products were formed to a different extent. Therefore, there was no single substance or mixture of substances of lcnown concentration to be used as reference for the calculation of the per cent hydrolysis. However, examination of the copper reducing values shown in Table V will give information on the extent of the overall hydrolysis due to irradiation. The copper reduction values increase with increasing radiation dccage which indicates that at higher dosages more reducing products are formed. The extent of hydrolysis as a function of dosage was nonlinear since two different bonds were being hydrolyzed (Figure 8a)• A relative comparison of the irradiated rafflnose reducing values with the reducing values of other irradiated oligosaccharides, Indicates that rafflnose exhibits greater susceptibility to ionizing radiation than 112

Table V

Reducing Sugar Values for Irradiated 2% Aqueous R&ffinose with Gamma Rays

Dose* Ml. of Cu** solution Ml. of Cu* solution (megareps) reduced per 5 ml. reduced per mg. of aliquot sample

0,0 0.0 0.0 2.5 0.08 0.64 5.0 0.17 1.36 7.5 0.22 1.76 10.0 0.26 2.08

•Delivered at the rate of 400,000 reps/hr Copper reduction values 2.5h 0 2 - 5 Q i.2. raito o 2 qeu sltos f raffinose of solutions aqueous 2% of Irradiation 2o. Fig. - A , cathode cathode , A ih ahd ad am ry (o, gma rays; gamma , o ( rays gamma and cathode with 25 oe (megoreps) Dose 113 rays). 5 7

114 maltose, cellobioso or trehalose. This Is not unexpected since rafflnose contains the same glycosidic linkage as sucrose.

J2. The Irradiation of Two Per Cent Dilute Aqueous Solutions of Raffinose with Cathode Rays at Room Temperature In order to study the relative effects of gamma rays and cathode rays on 2^ aqueous solutions of raffinose, a number of rafflnose samples were prepared and exposed to cathode radiation at the rate of 5 megareps/mln. Aluminum containers were used and the samples received 2.5, 5, 7.5, and 10 megareps of radiation. The control sample used was treated In the same manner as the rest of the samples but received no radiation. Paper chromatography (63) and ionophoresls (69, 70), performed In the same manner as In the case of rafflnose exposed to gamma radiation, Indicated the presence of mellblose, sucrose, galactose, glucose, and fructose In all the Irradiated samples but not In the control sample. These results substantiate those obtained In the gamma ray Irradiated rafflnose samples. Chromatography and electrophoresis evidence demonstrated that the extent of hydrolysis Increased with Increasing radiation dosage. The rafflnose residue decreased with Increased radiation. Substantial amounts of fructose and mellblose were Indicated to be present In all the irradiated samples. As In the case of gamma radiation, the fructose and mellblose spots were quite Intenseve, In comparison to the other products, at the 115 lower dosages. This would Indicate that the *-D- glycopyranosldic bond and not the o(-D-galactopyranosIdic bond, Is preferentially attacked. By an electron-spin resonance study of Irradiated rafflnose powder, the presence of stable free radicals was found. These undoubtedly resulted from the primary act of radiation on the sugar molecule. In dilute solutions, one has to consider also the Indirect effect (23)• It might be expected that the free radicals produced from the irradiated water (hydrogen atoms and hydroxyl radicals) would be so reactive that little specificity of attack could occur when raffinose solutions were irradiated. Lloreover, since the energy of radiation is so great it would be expected to cause fission at the site of the primary act, according to the statistical principle (86).

(86) h. Burton, J. Chem. Educ., £8, 404 (1951).

However, the results obtained from the irradiation of raffinose indicated that tills sugar is an important exception. This apparently is partly due to the fact that ionisation and excitation energy may be transferred from the site of the primary act to the site at which reaction occurs. In raffinose the most labile reaction site was the e(-D-glycopyranosidic bond. Ho detectable amounts of organic acids were observed 116 by paper chromatography of the raffinose samples exposed to cathode radiation. For better information on the cathode ray action on raffinose in comparison to that of gamma radiation, the reducing values were measured (63) and are shown in Table VI. These values increase with irradiation dosage nonlinearly, as shown in Figure 2a, probably due to the fission of two different bonds by irradiation. The increase of reducing values is in agreement with the chromatographic and ionophoresis results. No copper reduction was exhibited by the control sample. Comparison of the copper reduction values (Tables V and VI) from the two kinds of radiation shows that at lower doses (2.5 megareps), they are the same. At higher dosages, the values for the gamma-irradiated rafflnose are slightly greater. This would Indicate that the gamma rays were more effective. Another factor that could be considered is the longer exposure to gamma radiation since the dose rate was only 400,000 reps/min. 117

Table VI

Reducing Sugar Values of Aqueous Rafflnose Irradiated with Cathode Rays

Doses Ml. of copper Ml. of copper (meg streps) solution reduced solution reduced per 5 ml. aliquot per mg. of sample

0.0 0.00 0.00 2.5 0.08 0.64 5.0 0.16 1,28 7.5 0.21 1.68 10.0 0.23 l.«4

♦Delivered at the rate of 5 megareps/min 118 VI. Effect of Ionizing Radiations on Inulln

1. ?he Irradiation of Dilute Aqueous Solutions gfc Iflaj-lfl-JLlth Gfunffft p^yg f.t.-Ropni lemperjLtjjrg Several materials of high molecular weight are degraded on Irradiation and others exhibit an Increase in molecular weight. The effects of radiation on a large number of synthetic polymers have now been investigated (86) and a

(86) A. Charlesby, nucleonics, 18 (1054). number of interesting changes, like cross-linking and degradation, were observed, which would lead to a different physical behavior of the polymers. The present investigation was concerned with a study of the ionising radiation effects on the carbohydrate polymer inulln, which is a polyfructosefructofuranoside with linkages between Cl and C£. Since the degradation of polymers can be brought about by irradiation in dilute solutions, due to Indirect action, it appeared advisable to investigate the effect of gamma radiation on 2 % aqueous inulin and to attempt the isolation and identification of the irradiation products. Two per cent aqueous solutions of inulln were exposed to Co60 radiation, at the rate of 400,000 reps/hour, to receive £.5, 5, 7.5 and 10 megareps of gamma rays, A control sample was employed. Xt is known that inulin is soluble in hot water and not directly so at room temperature. However, 1X9 the recovered solids exposed to 7.5 and 10 megareps of radiation became brown and very soluble in the same solvent at room temperature. The control sample and the inulin receiving only 2.5 and 5 megareps were not soluble in water. This change in solubility at higher doses apparently is due to the modification of inulln and degradation to smaller fractions which are soluble in water at the usual temperature. These results are In agreement with those obtained by VYolfrom and associates (45) on cathode radiation of corn starch. Degradation might have been caused by the indirect effect of the action of hydrogen atoms and hydroxyl radicals (25) on inulin. The degradation in the presence of oxygen could also be caused by the HOg radical (18) as was observed by Alexander and his associates (80) in the degradation of poly (methyl acrylic acid) by radiation.

(89) P. Alexander and Z, Fox, Trans. Faraday Soc., 50. 605 (1954).

The lyophiliwed, irradiated samples were subjected to paper chromatography (62) and paper ionophoresis (69, 70) for the detection of reducing sugars. Fructose was detected only in the samples exposed to 7.5 and 10 megareps. Fructose was not detected in the samples exposed to lower doses. The proof of the presence of fructose in the irradiation products is significant in that, aw observed with other sugars, there 180 is * cleavage of the glycosidlc linkages as a result of ionizing radiation. In order to study the extent of inulin degradation, the per cent hydrolysis was estimated from the determined copper reduction values, based on conversion to D-fructose. The results obtained are shown in Table VII. These values were plotted against radiation dose and extrapolated to zero. Figure 3 shows that the extent of hydrolysis increased nonlinearly with increasing radiation dose. This figure suggests that, in doses between 0 and 5 megareps, Inulln underwent radiation hydrolysis at a slow rate apparently due to the small amount of radiation energy absorbed at these low doses. Another explanation could be that the radical yield in the solvent is small at lower doses so that no extensive indirect action could take place in this region. At about 7 megareps irradiation doses, the extent of hydrolysis increased rapidly. Apparently in this region the ionization and excitation energy, acquired by the inulin molecule, increased to such an extent that it could be transferred along the chain of the polymer causing fission at the glycosidlc linkages. In this region the increased radical yield in water undoubtedly played a very important role. At higher doses, as can be seen in Figure 3, the increase in fructose content becomes smaller. This probably is a consequence of an increased rate of fructose degradation due to the increased radiation dose (46). 121

Table VII

Reducing Sugar Values for Irradiated Tvg-Per Cent Aqueous Inulin, with Co60 Gamma Radiation

No. of ml. No. of ml. Dose* Cu** reduced of Cu** re­ Per cent (megareps) per 5 ml. of duced per mg. hydrolysis** aliquot of sample

0.0 0.00 0.00 0.00 2.5 0.01 0.04 0.47 5.0 0.04 0.16 1.90 7.5 0.10 0.40 4.76 10.0 0.15 0.52 6.18

*The rate of gamma Irradiation vas 400,000 reps/hr. **Eased on conversion to D-fructose. One mg. of D-fructose reduces 3,4 ml. of Cu++ solution. Reducing products, i 3 roito of irrodiotion 3 Fig and gamma rays ( Q» gamma gamma Q» ( rays gamma and 25 2 % qeu sltos f nln ih cathode with inulin of solutions aqueous 122 rays* ahd rays). cathode ^ 7.5

123 A further Investigation of gamma radiation of Inulin was concerned with the isolation and the identification of Irradiation products. A carbon column was prepared and washed very carefully as described in the experimental section. Since the concentration of radiation products was not very high, lots from two Irradiated inulin samples weremixed and subjected to carbon column chromatographic separation. The elution of the chromatogram was performed according to the method of Whistler and Durso (64). The recovered monosaccharide fraction was chromatographed on paper (62) before acetylation and fructose was indicated to be present. The acetylation of the dried, sirupy monosaccharide fraction was performed according to the improved method of Pacsu and Cramer (71), with very small variations. The sirupy acetylated material crystallised from absolute diethyl ether in ten days. The crystalline product was identified by melting point and X-ray powder diffraction pattern as ^-D-fructo3e pentaacetate. X-ray powder diffraction data (84) are recorded in the experimental part of this work. Although Pacsu and Cramer (71) used their method for the preparation of penta-O-acetyl-keto-D- fructose, the yj-D-fructose pentaacetate was obtained. The latter has been reported as present In the mother liquors from the crystallization of keto-D-fructose pentaacetate (97)•

(97) II. L. Wolfrom and A. Thompson, J. Am. Soc., 56. 880 (1934). 124 The isolation of fructose from the irradiation products, and its acetate identification, is a clear proof that •inulln is depolymerised when exposed to gamma radiation*

2. The Irradiation of Aqueous. Dilute Solutions of Inulin with Cathode ftays Room Temperature In order to study the effect of cathode rays on inulin and to compare it to the effect of gamma radiation on this carbohydrate polymer, a number of 2 % aqueous solutions of inulin were exposed at room temperature to cathode radiation in doses of £.5, 5, 7.5 and lo megareps at the rate of 5 megareps/min. A sample treated in the same manner but receiving no radiation was used as control. The recovered material from samples exposed to 7.5 and 10 megareps attained complete water solubility but those exposed to 2.5 and 5 megareps were insoluble. The solubility results are in agreement with those obtained from inulin irradiated with gamma rays and indicate that at higher doses extensive modification and fragmentation of the polymer had occurred. Fructose was identified by paper chromatography and ionophoresis in the samples which received 7.5 and 10 megareps, indicating that at higher doses degradation of inulin to the single polymer units takes place. The fact that fructose was not found to be present in the samples exposed to the lower dosages Indicated that inulin is not attacked to any reasonable extent by small quantities 125 of radiation energy. Similar results were obtained from the gamma ray investigation. Evidence from paper chromatography indicated that no detectable quantities of acids were formed from inulin by irradiation. For further Investigation of the depolymerization of inulin, the copper reducing values were measured (63) and the extent of hydrolysis was estimated on conversion to fructose. The values obtained are shown in Table VIII and delineated against dose in Figure 3. The extent of hydrolysis increased with increasing irradiation. However, at doses lower than 5 megareps this increase was quite small. This would indicate that in this region the glycosidlc linkage present in inulin is rather resistant to the ionization and exitation energy absorbed by this polymer, moreover the radical yield in the solvent, to cause indirect action on the 1 ■■■+ £ linkage, perhaps was small. Figure 3 shows a rather sharp increase of hydrolytic action at doses around 7 megareps. At this point the glycosidlc linkage present in Inulin seems to be more labile to the gradually Increasing radical yield in water and to the absorbed higher ionization and ex1tation energy, as well. At doses close to 10 megareps the rate of Increase in the reducing sugar content appears to be somewhat smaller than in the previous region. Such a behavior would indicate not only that inulin is depolymerized but also that the fructose formed begins to become labile to radiation and undergoes degradation. 126

Table VIII

Reducing Sugar Values for Irradiated Two Per Cent Aqueous Inulln, with Cathode Rays

La. of Cu** ia. of cu** solution solution Per cent** Dose* reduced per reduced per hydrolysis (megareps) 5 ml. of mg. of aliquot sample

0.0 0.000 0.00 0.00 £.5 0.015 0.06 0.70 5.0 0.030 0.14 1.62 7.5 0.070 0.28 3.33 10.0 0.120 0.46 5.52

♦Rate of cathode irradiation, 5 negareps/min. ♦♦Based on conversion to fructose. One mg. of D-fructose reduces 6.4 ml. of Cu+* solution. 127 The overall cathode radiation effects on inulin polymer are very similar to the gamma radiation effects on the same substance. There are, however, two exceptions. As can be seen in Tables VII and VIII, the extent of hydrolysis of the sample exposed to 2.5 megareps of cathode radiation was greater than that of the sample exposed to the same dose of gamma radiation. This might be caused by the higher dose rate, in the case of cathode radiation. At higher doses hydrolysis appears to be greater in the ca3e of gamma radiation although the dose rate was 400,000 reps/hr. This is not surprising since the samples were in contact longer with the radiation source and gamma rays are very penetrating.

3. Irradiation-Induced Deoolymerlaation of Powdered Jrmi in Two samples of inulin were exposed to 400 megareps of cathode rays at tne rate of 5 megareps/min. At these high doses Inulin became brown and completely soluble in water. Examination of the products cleaved from the polymer chain revealed fructose to be present. Modification and depolymerixation of inulin occurred by the direct action of radiation and its structure Is apparently very sensitive to those high dosages. Since fructose was present in the irradiated samples, the special weak points which can be severed by direct action are the glycosidlc linkages. One may suggest that at least part of the energy released by ionisation and excitation within the inulln molecule became localized at the junction points causing the fission of inulin. 128 VII. Irradiation-Induced Depolarization of Aftyjggy Amylose Is a linear carbohydrate polymer composed of ol-D-glucopyranose units which are uniformly linked (l >4). The amylose investigation was concerned with the isolation and Identification of any monosaccharides or oligosaccharides resulting from the degradation of amylose by gamma radiation. A 2% aqueous mixture of amylose was exposed to 15 megarads (see units of radiation) of fuel element gamma radiation at the rate of 2 x 104 rads/min. Amylose dissolved partially in water at this dosage. This would lnuicate modification (45) and possibly fragmentation of the polymer. The irradiated amylose sample was lyophilized and subjected to column chromatography. The most effective method of separating the low molecular weight fractions of irradiated amylose was by column chromatography on carbon. The column was prepared and washed as described in the experimental section. Elution of such columns according to Whistler and Durso (64) gave the monosaccharide fraction and with 5% aqueous ethanol, the dissacharide fraction. Descending paper chromatography of these fractions indicated glucose and maltose (44) which apparently were irradiation products. The free sugar from the monosaccharide fraction was further separated by acetylation (65) and chromatographic techniques (66)• Conclusive evidence of D-glucose as being one of the irradiation products was obtained by the melting point and X-ray powder diffraction pattern identification (67) 1£9 of its y&-D-glucopyranose pentaacetate. The amount of D-glucose was not large, relative to the amount of amylose Irradiated and certainly it was not the major part (44) of the degradation products. Although D-glucose was not the only irradiation product its presence was significant in that, as observed with maltose, there is a cleavage of the glycosidlc linkage as a result of irradiation. The separation and Isolation of products from irradiated amylose were also valuable in obtaining an evaluation of the fractionation procedure used. Stacey and his associates (44) studied the irradiation effects on Q ml% amylose solutions. Glucose, maltose, maltotriose, and maltotetraose were found, by paper chromatographic techniques, to be present; however, they did not isolate any of these products. Our isolation of D-glucose supports their evidence and proves the depolymerisation of amylose by ionizing radiation. 7/olfrom and his associates (45) found that amylopectin was hydrolyzed to smaller fragments by cathode radiation. All this evidence supports the radiation-induced degradation of carbohydrate polymers. Since amylose was irradiated in high dilution, the major depolymerization action was apparently caused by tne attack of reactive free radicals produced from the solvent (17). One should not, however, exclude the direct action (S3) of gamma radiation on the degradation of amylose since ionization and excitation ISO could simultaneously take place.

VIII. Investigation of Gases Produced on Irradiation of Carbohydrates

1. Rffaet nf Gamma Rarilat^ on D-Gluon.qflni jne Hydrochloride 2-Amino-2-deoxy-^-D-glucopyranose (D-glucosamine) hydrochloride, in 2% aqueous solution, was exposed to fuel element monochromatic gamma radiation source at the rate of 2 x 10^ rad/mln. The sample was irradiated in a tin can at room temperature and received a total dose of 15 megarads. The produced gases were collected as described in the experimental section. The irradiated dilute solution of D-glucosamine hydrochloride was chromatographed on paper according to Partridge (62) and three unidentified irradiation products were found to be present. The chromatographic evidence and the deep reddish-brown color present, would indicate irradiation damage of the D-glucosamlne hydrochloride sample. This damage could be caused mainly by the indirect effect (17); however, the direct effect should not be Ignored since the presence of free radicals in irradiated D- glucosamlne hydrochloride has been established (75).

a. Gas chromatographic analysis. The collected gases were chromatographed (76) as described in the experimental section. Carbon monoxide, carbon dioxide, hydrogen, methane, ethane, and an unknown gas were identified among the gaseous irradiation products. This identification was performed on 131 the basis of known samples of the above gases. No higher hydrocarbons were detected. Apparently all these gases were produced from the sugar solution since their production from the collection container, and the presence of hydrocarbons in the air, were excluded by control samples. The irradiation-inauced evolution of carbon monoxide, carbon dioxide, and hydrogen were not unexpected and they are in agreement with literature evidence (5,10, 13,91) on the production of gaes from carbohydrates and

(91) C. R. Maxwell, D. C. Petersen, and N. E. Sharpless, Radiation Research, 1, 530 (1954); IT. E. Sharpiess, A. E. Blair, and C. R. :.Iaxwell, ibid., 2, 135 (1955). amino acids by photolysis and ionising radiation. The production of hydrocarbons from carbohydrates by ionising radiation has never been reported. The methane evolution from D-glucosamine hydrochloride suggests a similarity between ionising radiation and ultraviolet irradiation effects on carbohydrates. Berthelot and Gaudechon (8) detected the evolution of methane from D-fructose and Beyersdorfer and Hess (12) from sucrose solutions; however, ethane formation has not been reported. Hydrogen gas apparently came not only from the sugar but also from the solvent (17)• 132 The gas chromatography peak heights were measured (see Table IX) ana the quantity of each gas produced was estimated on the basis of known concentration samples. Table X shows the gases produced In terms of micromoles from 3,78 x 104 micromoles of D-glucosamine hydrochloride. Uydrogen was found to be produced in the largest quantity obviously due to its evolution from the solute and solvent. Carbon dioxide was second in the series and carbon monoxide was third. Uethane and ethane were produced in smaller quantities. The production of carbon dioxide from D-glucosamine, as shown in Table X, was greater than from the uronic acids irradiated at almost the same molar concentration. The carbon dioxide excess probably is due to the fact that the D-glucosamine hydrochloride sample received three times as much dose as the acids. Undoubtedly ammonia was one of the products from D-glucosamlne.

2. Effect of Kadlarion on Uronic Acids In the present investigation, the objective was to determine the effects of ionizing radiations on the decomposition of uronic acids to gaseous products. By the determination of the nature and quantity of the produced gases, it was possible to examine, more closely and critically, the radiation induced changes in carbohyurates and related substances. 123

Table IX

Gas Chromatography Peak Heights* in cm. of the Gases Produced by Gemma Radiation of 2% Dilute Aqueous Solutions of D-Glucosamine Hydrochloride, D-Glucuranic Acid, and D-gal&cturonic Acid

Gases D-Glucosamlne D-Glucuronic D-Galacturonic hydrochloride acid acid

CO 0.3 0.5 0.5 COg** 0.6 0.4 0.4 %*** 0.2 0.2 0.3 C ^ 0.2 0.3 0.3 trace 0.05 0.05 unknown 0.05 0.1 0.1

■•Measured at 27°C. and 740 mm. pressure of mercury. **The peak height due to the 0.05 cc, of carbon dioxide present in 100 cc. of air is not included. ***The peak height due to the 0.01 cc. of hydrogen present in 100 cc. of air is not included. 134

Table X

4 Micromoles of Gases Produced from 3.78 x 10 /tmoles of D-Glucosamine Hydrochloride, 4.13 z 104 moles of D-Glucuronic and 4.13 z 104 ^moles of D-Galacturonic Acid Exposed to Gamma Radiation as Aqueous Solutions

D-Glucosamine D-Glucuronic D-Galacturonic Gases hydrochloride* acid** acid**

CO 171 249 270 °o2 300 175 190

% 436 481 414 78 98 111 ^ 2 % trace 19 19

*E*posed to 15 megarads of gamma radiation. ♦♦Exposed to 5 megarads of gamma radiation. The dose rate was 80,000 rads/mln. 135 Two per cent solutions of D-glucuronic acid and D- galacturonic acid, in tin cans, were exposed to a fuel element source of monochromatic gamma radiation at the A rate of 2 x 10 rads/min. Each sample received 5 megarads of radiation at room temperature. The gases produced were collected in weather balloons as described in the experimental section. Chromatographic investigation (62) of the irradiated solutions indicated the presence of four unidentified radiation products in each of the D- glucuronic acid and D-galactaronic acid samples. Apparently irradiation damage of the uronic acids was the cause of the appearance of those products. In the presence of water the radiation damage probably was caused by the reaction of these substances with free radicals formed from the solvent (17). It would not be correct, hovrever, to exclude the direct action of radiation, since we found the presence of trapped-free radicals in irradiated D-glucuronic and D-galacturonic acids.

a. Gas chromatographic analysis. Vapor phase chromatography of the collected gases identified the presence of carbon monoxide, carbon dioxide, hydrogen, methane, ethane, and an uihoiown gas in the D-glucuronic and D-galacturonic acid samples. These gases were produced from the sugar solutions by irradiation and were identified and measured with standard samples. Table IX shows the chromatography peak heights obtained and Table X gives the number of 126 4 micromoles of each gas produced from 4,12 x 10 micromoles each of D-glucuronic acid and D-galacturonic acid. Hydrogen was evolved in larger quantities from the solutions of uronic acids. Apparently it was produced not only from the solute but also from the solvent (17, 22). The close agreement in the number of hydrogen micromoles produced from the samples suggests relative accuracy of the obtained quantitative results and indicates that the major fraction of hydrogen gas comes from the irradiated solvent. The second place in number of micromoles is held by the carbon monoxide and the values obtained from each uronic acid are rather close together. This would indicate that the rate of rauiation-induced decurbonylation is faster than the rate of the rauiation-induced decarboxylation. Moreover, the radiation-inuuced decarbonylation rates are very close together. The same holds for the rauiation-induced decarboxylation rates of these two uronic acids. This is not surprising, since the structures of the two investigated acids differ only in the configuration at C4. Methane and ethane were found to be radiation-induced products from the two acids. This is a new consequence of ionizing rauiation effects on carbohydrate substances. Berthelot ana Gaudechon (8) reported the evolution of methaie from D-fructose, caused by ultraviolet light irradiation of its aqueous solutions. However, they did not report the production of ethane. The transformation of carbohydrates 137 to hydrocarbons by Ionizing radiation is a phenomenon we have observed which may be of importance in nature. The number of ethane micromoles produced is the same in both uronic acid samples and much less than the methane micromoles present. A consequence of this would be that ethane came mainly from methane. In the case of methane, the carbon-hydrogen bond breaks on irradiation (92) and

(92) S. C. Lind, nTlie Chemical Effects of Alpha Particles and Electrons," 2nd Ed., The Chemical Catalogue Co. Inc., Hew York, (1928). the methyl radicals combine to give ethane. The experimental evidence, supported by the reported methane irradiation, would indicate that ethane was mostly an indirect product produced from the evolved methane, since the gaseous products were kept in the containers during the exposure. However, one must not exclude the possibility of the production of ethane directly from the sugar. The paper chromatography findings indicated that the irradiation products are of smaller molecular weight than the irradiated compounds. These findings in conjunction with the results obtained from the gas analysis, prove the degradation of carbohydrate substances to fragments as small as metiiane molecules. 158 3. Proposed Mechanism for the Formation fiQSOftSiS The Investigated compounds were Irradiated in water solution. It has been reported (16, 19,22) that excited molecules, free radicals, ions, and other species are produced in irradiated water. The most important species are the H and HO radicals. Similar species and other free radicals are produced from the solute on irradiation. Undoubtedly the reactions that took place in our investigations were free radical reactions.

a. D-Glucosanine hydrochloride. Sharpless and his associates (95) reported the deamination of alanine on

(95) N. E. Sharpless, A. E. Blelr, and C. R. Maxwell, Radiation Research, &, 417 (1955). irradiation to give propionic acid, lactic acid and pyruvic acid.

a%-CHNHg-C02H — ■» CH5CHgC02H CH3-CH0H-C0gH +CH3-C0-C0gH

A similar mechanism may take place with D-glucosamlne on irradiation. It has been also reported (55,59) that 139

HC=0 IIC=0 HC=0 HC=0 HC=0 t I T I f HCHHg CHOH C-0 CHg HCHHg i i t i i HOCH KOCH + HOCH HOCH +. HOCH t JLr-n'" * l i f t HCOH HCOH HCOH HCOH HCOH t i i i t HCOH HCOH HCOH HCOH HCOH i t » i t CHgOH CHgOH CHgOH CHgOH COgH

D-Glucosamine I II III IV selective Irradiation-Induced oxidations take place at C6 and uronic acids are obtained from aldohexoses. The produced ammonia would form ammonium chloride. Compound II is an osone. Osones are knovm to be produced by the action of Fenton*s reagent on carbohydrates (94). Decarbonylatlon

(94) H. S. Morrell and J. M. Crafts, J. Chem. Soc., 75. 786 (1899). of this osone would give carbon monoxide and an aldopentose (59). Decarboxylation of IV would give carbon dioxide. Carbon dioxide could also be produced by radiation-induced oxidation of carbon monoxide in the presence of oxygen. Etydrogen atoms are formed from water by irradiation (17) and possibly from the sugar molecule. The combination of these atoms would give hydrogen gas. One may assume decarbonylatlon of III and subsequent split of a methyl radical which in the presence of hydrogen atoms would give methane. Ethane might 1 40 have been produced from D-glucosamine by some unknown mechanism. A greater possibility for the ethane production would be by irradiation of the evolved methane (9£).

sch4------► C g H e + %

The above postulated mechanisms ty no means represent the only processes that may take place. We suggest the Irradiation of isotope-labeled D-glucosamine hydrochloride to obtain definite Information concerning the mechanisms Involved.

b. D-Glucuronlc and D-galacturonlc acids. Since their structures differ only in the relative configuration about 04, similar processes leading to the identified gases may have taken place. The carbon monoxide could be produced from the radiation-induced decarbonylatlon and could be proven by introducing labeled carbon in position one. Irradiation of carboxylic acids gives rise to decarboxylation (95)• A similar reaction may explain the formation of

(95) C. S. Sheppard and V. L. Burton, J. An. Chem. Soc., §0, 1636 (1946). carbon dioxlue from uronic acids and could be proven by introducing labeled carbon in position six. Carbon dioxide could also be produced from the evloved carbon monoxide in the presence of oxygen, hydrogen gas was probably formed 141 from hydrogen atoms which could be produced from the water present and from the acids. Methane apparently was produced from the acids by some sort of free radical mechanism, proof of which could be accomplished by the use of isotope- labeled uronic acids. One may suggest that ethane was produced either directly from the acids or from the evolved methane. The second possibility is in accord with work reported by Lind (92) who reported the formation of ethane by irraaiation of methane.

4. Carbohydrates and Genesis of Petroleum Hydrocarbons in Nature During the last 25 years considerable progress has been made in the development of an adequate theory on the genesis of petroleum. The biogenesis theory alone would not explain the formation of natural gas and oil. Although not proven, it has been suggested (u6) that bacteria, radiation and

(9 6 ) K . Van lies and li. A. Van Wes ten, "Aspects of the Constitution of Mineral Oils," Elsevier Publishing Co., II. Y., (1951). catalytic cracking co-operated in the formation of petroleum hydrocarbons. The transformation of carbohydrates to hydrocarbons reported in our wont, gives experimental evidence for the genesis of natural gas from organic matter by irradiation. 142 IX. Paramagnetic liesonance Studies of Irradiated Crystalline Carbohydrate Powders When organic compounds in the solid state are exposed to ionizing radiation, the organic molecule may lose an electron and give an ionized molecule with an unpaired electron in one of its orbitals. The removed electron may be trapped in the crystal lattice, or become attached to a neighboring molecule. Ionizing radiation may produce either positive or negative ions. These Ions may become stable, uncharged free radicals with unpaired electrons. If the concentration of these stable free radicals is high, a detectable electron-spin resonance would result. Paramagnetic resonance studies on carbohydrates exposed to X-ray ana catnoue radiations were reported by hTolfrom and his associates (75). In the present investigation maltose, cellobioce, trehalose, raffinose, D-glucuronic acid, D-galacturonic acid and inulin were exposed, as solids, to fuel element gamma radiation at room temperature. The radiation rate was 20,00j raas/min. and each sample received a dose of 5 megarads. All the irradiated samples were stored in deep freeze before investigation. The spectrograph employed in the present study is described briefly in the experimental section. In the figures to be presented, the first derivatives of the absorption spectra recorded on the chart are shown. The hiperfine structure for D-galacturonic acid is centered on g - 1.^06 £ 0.002. For D-glucuronic acid, maltose, cellobiose, 143 trehalose and raffinose, the hyperfine structure is centered on g = 1.0U6 £ Q.0U3. The signal intensity from the free radicals in the irradiated D-galacturonic and D-glucuronic acids are approximately ten times those from the other irradiated carbohydrates and this is more than can be accounted for, by the narrower line. Irradiated D-galacturonic acid gave a spectrum with a strong single peai: caused by an unpaired electron of a g value equal to 1.096 £ 0.002 (Figure 4). The small peaks at the sides appear too small to be a part of a hyperfine structure and probably indicate tiie presence of another free radical. The width of the line is 13 gauss t2 and the obtained spectrum shows that the unpaireu electron is v/ell removed from any proton. D-Glucuronic acid exposed to gamma radiation at the same dose as D-galacturonic acid, gave a spectrum with a strong single peai: (Figure 4) caused by an unpaired electron with a g = 1.006 £ 0.003. The presence of small peaks as in the case of D-galacturonic acid was not indicated. The width of tue line is the same as in the case of D-galacturonic acid and the spectrum shows the unpaired electron to be renoveu from any proton. The only difference in structure of D-glucuronic and D-galacturonic acid is tne relative configuration about C4. This fact may explain the similarity of their paramagnetic resonance spectra produced from the main free radical present. V ■ 9317 Mc/eec. V * 9317 Me/tee.

3959 39)5 3395 9355 Magnetic Field (g o u tt) Magnetic Field tgoues) H C - 0 C - 0 H -f-O H H-C-OH HO-C-Ht HO-C-H H-C-OH HO-C-H H-^-O H H -^ -O H COjH go2 h O-Glucuronic acid D-Golacturonic acid g. 4. Electron spin resonance spectro o* irradiated 0 -glucuronic acid ond O-galacturonic acid with gamma rays. The curves represent first derivatives of the actual absorption lines. 145 The Irradiated trehalose spectrum consists of two absorption peaks which are clearly resolved (Figure 6). If these peaks are associated with a hyperfine-structure doublet, then, for this pair of lines, (A/gfl) is 10 gauss and g is l.bB6 t, O.OQd. This would indicate an interaction of the magnetic moment of the unpaired electron with the magnetic moment of the nearby single proton in the radical. Perhaps the unpaired electron is on a carbon atom attached to hydrogen. The signal obtained from irradiated trehalose was about ten times less intense than that of irradiated D-galacturonic and D-glucuronic acids. The paramagnetic resonance spectra obtained from irradiated maltose, cellobiose and raffinose are very similar (Figures 5,6). In each one of them there seems to be a tendency toward splitting into a hyperflne-structure doublet. This splitting is just barely resolved. It is not possible to arrive at a value for {k/g/f) but it appears to be less than 5 gauss. Again this would indicate interaction of tne magnetic moment of the unpaired electron with that of a single proton, but the proton is somewhat more removed from the impaired electron than in the case of irradiated trehalose. Because of the 1/r3 dependence of tne interacting field, this distance would not be much greater than in tne case of trehalose. The electron-spin resonance spectrum of a mixture

(111 by weight), of irradiated maltose-irradiated cellobiose V - 9317 Mc/sec

3315 Magnetic Field (gou*) Mognetic Field (go je t)

HOH OH HOH HO OH OH

Cellobiose ig 5 Electron spin resonance spectra of irradiated maltose and cellobiose with gamma rays. The curves represent first derivatives of the actual absorption lines. V = 9317 Mc/ttC V * 931 7 Mc/sec-

9315

Magnetic Field (gauss) Magnetic Field (gauss)

OH

loh HO H OH H CHjOH H OH OH H Treftotoee Ruff inose

Fig 6. Electron spin resonance spectra of irradiated trehalose and raffinose with gamma rays. The curves represent first derivatives of the actual absorption lines 148 was taken under the same conditions* The hyperfine structure obtained is centered at g » 1.996 t 0*003. The separation of the lines is within about 7 gauss (Figure 7)• From the obtained spectrum it is suggested that the radicals in Irradiated maltose and cellobiose are probably similar. Irradiated D-galacturonic acid and irradiated D- glucuronic a d d were mixed in 1:1 (ratio by weight) and the electron-spln resonance spectrum of the obtained mixture shows that the main radicals are probably similar in both acids (Figure 7). Irradiated inulln (a D-fructose polymer) gave no signals* This is due to the fact that the inulln is an amorphous solid and contains no crystal lattice to trap the fragments* iJy experiments suggest that paramagnetic studies could be used for the determination of the degree of crystallinlty of cellulose preparations* In general, in all the samples, except those of the acids, the additional broadening of the lines might be caused by the superposition of fields from nearby electrons* There is a strong possibility that the unpaired electron present in the molecules of the Irradiated cellobiose, maltose, trehalose, and raffinose is on a carbon atom located close to an electron-rich center of the molecule* However, in the case of Irradiated D-galacturonic and D-glucuronic acids, since there is no broadening of the lines, the unpaired electron of the stable free radical is V * 9317 Mc / m C. 9 4 1

3355 5335 3315 5 3 5 5 5315 Mognetic Field (goues) Mognetic Field (gouet) I 3L

Fig. 7 1 = Electron spin resononce spectrum of a mixture (|:| by wt.) of irradiated maltose and irradiated cellobiose with gamma rays. I = Electron spin resonance spectrum of a mixture (I I by wt.) of irrodiated D-glucuronic acid and D-galocturonic acid with gamma rays. The curves represent first derivatives of the actual absorption lines 150 possibly on a carbon atom located not close to an electron-rich center of the molecule. The main free radical present in the irradiated uronlc acids is suggested to be formed by the elimination of a hydrogen atom from a carblnol-carbon atom. This is in agreement with results obtained by McDonell and Newton (87)

(87) W, R. McDonell and A, S. Newton, J. Amer. Chem, Soc., 4651 (1054)• who reported that the bonds, linking the carbinol-carbon atom to hydrogens, in primary and secondary alcohols are preferentially broken by irradiation. The fission of these hydrogen bonds is probably due to their being weakened by the polarisation induced in them by the hydroxyl group. Consideration of the electron-spin resonance spectra of irradiated D-glucuronic acid, D-galacturonic acid, maltose, cellobiose, trehalose and raffinose suggest the existence of stable free radicals trapped after gamma irradiation and are in agreement with results, obtained In other carbohydrates, already found in the literature (73,75,90).

(90) P. M. Grant, R. B, Ward and D, H, Whlffen,

i J, Chem, Soc., 4635 (1958), 151 The presented experimental evidence obtained from the study of the effect of ionising radiations on the glycosidic linkages, along with the results from the electron-spin resonance study, suggest that the D-glucopyranosyl free radical I vas trapped after gamma irradiation of maltose, cellobiose and trehalose. In the case of D-glucuronic acid the paramagnetic resonance study suggests a stable free

CHgOH COgH

HOH OH OH HO HO i t r t H OH H OH

II radical of type II to be present where the unpaired electron is located at C5. The location of the unpaired electron at C5 is more probable since tertiary free radicals are more stable than secondary and primary, and at this position there is more resonance stabilisation with the carboxyl group and the oxygen. The main stable free radical trapped after gamma irradiation of D-galacturonic acid could be of type II with opposite configuration about C4. Formula I fulfills tne experimental requirement that the unpaired electron is attached to a carbon which carries a proton and Formula il is attached to a carbon which carries no proton. 152 A comparison of the spectrum obtained from Irradiated D-glucuronic acid with that obtained from irradiated 4-D- glucopyranose (75) could be made. The«(-D-glucopyranose spectrum consists of two absorption peaks which are clearly resolved; this would indicate an Interaction of the magnetic moment of the unpaired electron with the magnetic moment of the nearby single proton In the radical. In the case of D-glucuronic acid this is not observed, since a strong single absorption peak was obtained. These two compounds differ in C6. The C6 in D-glucose is a carbinol-carbon and in D-glucuronic acid the C6 is a carboxyl-carbon atom. Since this is the only difference between them, the substitution of the -CHgOH group by -COgH could be associated with the elimination of the extra absorption peak from the D-glucuronic acid spectrum. This evidence contributes to the possibility of the unpaired electron being at C6 of the irradiated D-glucose. A comparison between the electron-spin resonance obtained from the Irradiated D-galacturonic acid could also be made. In the D-galactopyranose spectrum there is a tendency toward splitting into a hyperfine-structure doublet as in the case of D-glucose. This again would indicate an interaction of the magnetic moment of the unpaired electron with the magnetic moment of the nearby single proton in the radical. In the spectrum of the irradiated D-galacturonic acid in the single peak due to the trapped, main free radical, a tendency toward 153 splitting is entirely eliminated. These two compounds differ only in C6. The substitution of the -CHgOH in O-galactose by -COgH in D-galacturonlc acid could be associated with the difference in their spectra. This evidence, as in the case of D-glucose and D-glucuronic acid, contributes to the possibility of the unpaired electron being at C6 of the irradiated O-galactose. A correlation of our results with those obtained by Wolfrom and associates (75) and other information already in the literature (87,90) would suggest that 111 could be the main free radical trapped in the irradiated o( -D-glucose and IV in the Irradiated -O-galactose.

CHOH 4hoh

H HO i i CC i t OH H

III IV

The developed approach of interpreting the electron-spin resonance spectra obtained from the gamma ray irradiated carbohydrates could be applied in the interpretation of that obtained (75) from the X-ray irradiated D-glucosamine hydrochloride. Their experimental evidence in correlation with my results would suggest that one of the stable free 154 radio*l present in the X-ray Irradiated D-glucosamine hydrochloride could be V* Salts of this nature have been

V reported in the literature (98) and are the so-called Wuster's

(98) W. A. Waters, "The Chemistry of Free Radicals,n Oxford (1948), p. 75. salts. On the basis of arguments presented in the present work, an Interpretation of the spectra obtained from Irradiated 4-L-rhamnose (6-deoxy-oC-L-mannopyranose) monohydrate and «t-D-mannopyranose (75) could be made. It could be suggested that the electron-spin resonance spectra of these two carbohydrates are very similar and not markedly different as it has been reported (75)• This perhaps could be explained on the basis of their related structures. Besides enantiomorphlsm and hydration, they differ only in having a 155 methyl group on C6 of the rhamnose entity where a hydrorymethyl group is present in the mannose structure. By looking at the published spectra of these two compounds one may consider that they are different whereas they are not. The lines are mlrror-lmages to each other and the location of the peaks In either left or right of the base line Is caused by the oscillation of the pointer which can swing to either side on recording the same signal. The probably main radicals present are shown below (VI, VII)•

HOCH

H H H H

VI VII

The use of deuterium on C6 of D-glucose, D-galactose, 4-L-rhamnose, ©l-D-mannopyranose, and on the nitrogen of the D-glucosamine hydrochloride is suggested for furtner proof of the stable free radicals formed by irradiation of these compounds• SU1UARY The effects of high-speed electrons (cathode rays) on 50% aqueous solutions of the disaccharides maltose and cellobiose were studied. An investigation of the relative susceptibilities of 4-D and y0-D glycosldic linkages to ionizing radiation was made. Paper chromatographic evidence suggests that the «f-D linkage in these dlssacharides (as with acidic hydrolysis) is more readily attacked than is the yF-D linkage, but neither glycosldic bond is attacked to any large extent. D-Glucose was detected in the irradiated samples of maltose by paper chromatography. The extent of hydrolysis of maltose and cellobiose was studied by determining their copper reducing values. A series of 20% aqueous maltose solutions were Irradiated with cathode rays at ambient air, ice-water and ethanol- (solid carbon dioxide) temperatures and the changes caused by irradiation were investigated. It was found, by chromatography and copper reduction, that the extent of Irradiation damage is greater at lower temperatures and is increased by Increasing dosage. Glucose was detected chromatographically in all the irradiated maltose samples. The extent of hydrolysis of maltose solutions, exposed to radiation at ice-water temperature, Increased In a linear fashion. The G values were determined on the basis of the per cent of maltose hydrolyzed. These values were found to be greater at lower temperatures ana were very close together in each series of irradiated samples. D-Glucose was isolated by carbon column 156 157 chromatography, acetylated, chromatographed on Llagnesol- Cellte and Identified as crystalline yB-D-glucopyranose pentaacetate• Samples of cellobiose exposed to high energy electron Irradiation, at ice-water temperature and as 20% aqueous solutions were damaged to such an extent that glucose was detected by chromatography In all the Irradiated samples. The extent of hydrolysis was measured by copper reduction methods and was found to increase linearly with the radiation dose* A comparison of the indirect effect at the concentrations of 20% and 50j£ was made and was found to be greater at higher dilutions* The G values and the extent of hydrolysis of cellobiose were found to be less than in the case of maltose irradiated under the same conditions* These results indicated that the glycosldic linkage of cellobiose is more resistant to ionizing raalatlon than is theo(-D glycosldic linkage of maltose. The effect of ionizing radiation on aqueous solutions of the disaccharide trehalose was studied by chromatography and reduction procedures. The G values were determined* The amount of reducing products present increased with radiation* The increase was linear at low doses but not at higher doses. Irradiated trehalose was subjected to carbon column chromatography for the isolation of radiation products* A monosaccharide sirupy fraction was obtained, chromatographed, acetylated, Isolated by column chromatography and Identified 158 as crystalline ^-D-glucopyranose pentaacetate. The effects of gamma and cathode radiations on dilute raffinose aqueous solutions were studied. Llelibiose, sucrose, glucose, galactose and fructose were detected chromatographically in all the irradiated raffinose samples. Fructose and mellbiose were the main radiation products which indicates that the o(-D glycosldic linkage is more susceptible than is the 4-D-galactosidic linkage of the raffinose molecule. The extent of hydrolysis was followed by reduction methods and was found to increase nonlinearly with increasing radiation. Gamma rays and cathode rays had similar action on raffinose. Irradiation of the disaccharides maltose, cellobiose, and trehalose, and the trisaccharide raffinose, demonstrated that the glycosldic bond is especially sensitive to the action of ionizing radiations. The polysaccharide inulln was subjected to gamma as well as to cathode irradiation at several doses. D-Fructose was detected by paper chromatography and ionophoresis in samples irradiated at higher doses. Determination of the reducing values and the extent of hydrolysis indicated that the content of reducing products Increased with increasing radiation. Gamma and cathode rays caused similar effects on inulln but to a somewhat different extent. Column isolatlve studies were made, as in the case of trehalose, and the monosaccharide fraction obtained was acetylated and 159 Identified as crystalline yB-D-fructose pentaacetate. Inulln exposed to higher doses became soluble In water at room temperature• Irradiated powdered Inulln with high doses of cathode radiation was damaged to such an extent that It became soluble In water and fructose was one of the products. For the Isolation of products due to ionizing radiation, an aqueous amylose suspension was Irradiated with gamma rays and subjected to carbon column chromatography• To obtain better results from our separation procedure, the carbon column was washed with dilute hydrochloric acid and ammonium hydroxide. A monosaccharide and a disaccharide fraction were obtained. Maltose and D-glucose were found to be among the irradiation products by paper chromatography. The D- Glucose was identified as its crystalline ^0-D-glucopyranose pentaacetate. A study of the gases produced by irradiation of dilute aqueous solutions of D-glucosamine hydrochloride, D-glucuronic acid and D-galacturonic acid was made. These compounds were exposed to gamma radiation. From all these compounds the following gases were obtained: carbon dioxide, carbon monoxide, hydrogen, methane, ethane and an unidentified gas. The amount of each gas produced from each irradiated compound was determined and methane and ethane were found to be present in lesser quantities than the other gases. 1 6 0

Radiation caused the transformation of carbohydrate substances to hydrocarbons. This experimental evidence may partially explain the formation of petroleum hydrocarbons In nature. A mechanism of the gas formation was postulated. Free radical studies were made on the following carbohydrates Irradiated as solids with gamma rays: maltose, cellobiose, trehalose, raffinose, D-glucuronic acid, D-galacturonic acid and inulln. Electron-spin resonance spectra were obtained for all the Investigated compounds except the amorphous inulln. The paramagnetic resonance spectra study shows the creation of free radicals from the carbohydrate molecules. The free radicals were present in much higher concentration in D-glucuronic and D-galacturonic acids. Two different stable radicals were present in the irradiated D-galacturonic acid. Electron-spin resonance spectra of mixed irradiated carbohydrates, identified the radicals present in cellobiose and maltose to be similar. Similarly the main stable free radicals in Irradiated D-glucuronic and D-galacturonic acids were similar. Possible structures for the free radicals trapped after gamma Irradiation of maltose, cellobiose, trehalose, D- glucuronlc acid and D-galacturonic acid have been postulated on the basis of the experimental data obtained. 161 An interpretation of paramagnetic resonance spectra, already reported in the literature lay other workers, was made in correlation of the results obtained from the present work and possible structures for the stable free radical present in irradiated D-glucosamine hydrochloride, 4-D- glucose, c^-D-galactose, «<-L-rhamnose, and cf-D-mannopyranose have been postulated. CHRONOLOGICAL BIBLIOGRAPHY

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I, Andrew ;.r. Llichelakis, was b o m In Kassanous, Crete, Greece, on August 12, 1927. After having completed my primary education, I entered the Gymnasium of Science of Herakleian where I received university preparatory training. In 1946 I entered che College of Agriculture, and In 1947 the School of Medicine of Athens university, Greece, after succeeding in the entrance examinations of these schools. I enrolled concurrently In these two schools and in 1952 received ny first university degree from the College of Agriculture. In 1955 I interrupted my medical education on being awarded a Fulbright grant for advanced studies in the United States. I attended the university of Kansas and received the 21. Sc. degree in chemistry in 1956. In the autumn of 1955 I entered the Ohio State University Graduate School to work for the Doctor of Philosophy degree in chemistry. Since then, I have attended the Ohio State University and worked part of the time as a teaching assistant and part of the time as a research fellow.

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