Graft Copolymerization of Ethyl Acrylate Onto Gelatin Using Hydrogen Peroxide and Ascorbic Acid in Aqueous Medium

ANNE GEORGE, GANGA RADHAKRISHNAN, and K. THOMAS JOSEPH, Polymer Division, Central Leather Research Institute, Adyar,

Madras 600 020, India

Synopsis

Gelatin a derived protein from collagen can be modified at 60°C by allowing it to react with ethyl acrylate by use of a novel initiator technique. The polymeric ethyl acrylate side chain is chemically bonded to gelatin. The effects of synthetic variable in the graft copolymerization reactions have been discussed in the light of percent grafting, grafting efficiency, and the rate of polymerization.

INTRODUCTION Grafting of polymeric side chains offers an attractive techinque for al-

tering the properties, especially the surface characteristics, of the substrate. Many studies have dealt with grafting onto cellulose' and proteinaceous materials such as wool, silk, and c~ l l agen .~*~ Comparatively little infor- mation has been reported regarding the chemically initiated grafting of monomers onto gelatin. Grafting onto gelatin takes place by the formation of covalent bonds between gelatin and the polymeric side chain and this improves the physicochemical properties of the substrate. Furthermore, grafting procedures have indicated upgrading certain properties of leather, such as water penetration and abrasion

The initiator used in this system was hydrogen peroxide in combination with ascorbic acid. This novel initiator system was used for the first time in initiating grafting reactions. The aqueous ascorbic acid solution disso- ciates into ionic fragments and the monohydroascorbate ion is mainly re- sponsible for the strong reducing action of ascorbic acid in aqueous media.s

AH2+AH- + H +

(where AH2 and AH- represent ascorbic acid and ascorbate ion respectively). In this communication, grafting of poly(ethy1 acrylate) onto gelatin by use of hydrogen peroxide-ascorbic acid as initiator is reported as function of time, temperature, concentrations of monomer, initiator, and backbone.

EXPERIMENTAL

Materials Ethyl acrylate[EA] (Koch-Light Laboratories, Ltd.) was washed free of

inhibitor with 5% sodium hydroxide solution, distilled, and stored at 5°C. Hydrogen peroxide (HzO&, 6% (Sarabhai M. Chemicals, Ltd., G.R.) and As-

Journal of Polymer Science: Polymer Chemistry Edition, Vol. 23,2865-2874 (1985) @ 1985 John Wiley & Sons, Inc. CCC 03603676/85/112865-09$04.00

2866 GEORGE, RADHAKRISHNAN, AND JOSEPH

corbic acid (AHd (Sarabhai M. Chemicals, Ltd., G.R.) were used without further purification. Freshly prepared ascorbic acid solutions were used to prevent aerial oxidation, gelatin [GI (Riedel, Germany) was used as such in all investigations.

2.2 Method

Polymerizations were conducted at 60 L- 0.YC in Pyrex glass tubes with gas inlet and outlet arrangements. A 10% solution of gelatin was prepared in warm water and used in graft copolymerization reactions. Fresh solutions of gelatin were prepared to avoid bacterial growth. (10 mL of 10% Gelatin) was pipetted into the reaction tube. Nitrogen gas was passed through an alkaline pyrogallol solution to eliminate traces of oxygen. Required amounts of monomer and initiator were added. After a specific polymerization time (90 min), the reaction tube was immersed in a freezing mixture to arrest the reaction. The contents were then poured into ice-cold methanol. The polymer suspensions were then filtered in weighed sintered glass crucibles (IG-3) and dried in vacuum at 40-50°C overnight to a constant weight.

The percentage grafting was calculated as follows:

Weight of grafted poly(ethy1 acrylatel) Weight of backbone polymer Percent Grafting = x 100

The grafting efficiency (G.E.) was calculated using

Weight of grafted poly(ethy1 acrylate) Weight of grafted poly(ethy1 acrylate) % G.E. = x 100

+ weight of polyethyl acrylate

The rate of grafting (R,) was calculated from the weight of the total polymer formed and the weight of free homopolymer.

3. RESULTS AND DISCUSSIONS Various experiments were conducted in order to understand the nature

of graft copolymerization onto this biopolymer. Effect of time, temperature, monomer concentration, initiator concentration, and gelatin concentration were studied.

3.1 Effect of Reaction Time Table I presents the dependence of percentage grafting, grafting effi-

ciency, and rate of grafting on the reaction time. These were found to increase gradually as the reaction time become longer. This suggests that a considerable number of active centers were formed as expected and hence the availability as grafting sites. The rate of grafting was found to be con- stant after 90 min. This may be due to a shortage of available grafting sites as the reaction proceeds.

GRAM' COPOLYMERIZATION OF ETHYL ACRYLATE 2867

TABLE I Effect of Time on Grafting.[EA] = 1.8 x lo-' mol L-l; [Gelatin] = 3.3 X lo-' mol L-1; H,Oz = 10 x 10-2 mol L-l; Temperature = 60'C; [AA] = 10 X

mol L-1; Total volume = 50 mL.

Grafting Percent Time R, x 106 (mid mol lit-l sec-l efficiency grafting

30 60 90 120 180 210

16.22 17.46 25.56 28.32 28.82 29.37

61.66 64.50 71.80 78.28 81.36 82.00

23.00 28.45 41.10 46.72 45.24 48.18

3.2 Effect of Temperature

The effect of polymerization temperature affects the radical formation rate, diffusion of radicals, growth, and termination of the polymer radicals. From Table I1 it is seen that lower reaction temperature favored higher percent grafting. At higher temperatures the primary radicals initiate homo-polymerization thereby showing a decrease in percent grafting, graft- ing efficiency, and rate of grafting.

3.3 Effect of Monomer Concentration

An increase in monomer concentration was found to inrease percent grafting, grafting efficiency, and the rate of graft copolymerization of ethyl acrylate onto gelatin (Table I11 and Fig. 1). The percent grafting value depends on the molecular weight of the grafts as well as on the number of grafts; since molecular weight increases with increase in monomer concen- tration the increase in percent grafting may be understandable. A similar trend was obtained when poly(ethy1 acrylate) was graft copolymerized onto wool in the presence of ceric ammonium nitrate as redox initiator? The homopolymer formed was less when hydrogen peroxide-ascorbic acid was used as the initiator, as compared with persulphate.

TABLE I1 Effect of Temperature on Grafting

[EA] = 1.8 x 10-1 mol L-l; [Gelatin] = 3.3 x mol L-l; H20z = 10 x mol L-l; total volume = 50 mL; [AA] = 10 x mol L-I; time = 90 min.

Temperature R, x 106 Grafting Grafting ("0 mol L-1 sec-' efficiency (%)

30 24.77 97.85 68.33 40 19.73 94.33 52.79 50 18.36 92.74 50.39 60 13.85 84.20 41.24 70 11.24 80.12 35.74 80 8.42 76.45 27.52

2868 GEORGE, RADHAKRISHNAN, AND JOSEPH

TABLE I11 Effect of Monomer Concentration on Grafting.

temperature = WC; [Gelatin] = 3.3 x lo-' mol L-l; time = 90 min. HzOz = 10 x mol L-l; total volume = 50 mL; [AA] = 10 x mol L-l;

[EA] x 10' R, x 106 Grafting Grafting mol L-l mol L-lsec-l efficiency (%)

1.846 2.769 3.692 4.615 5.538 .461

2.27 20.95 35.27 56.65 80.45

118.03

78.24 81.02 85.68 88.37 90.19 92.28

31.50 52.47 95.22

152.99 180.34 217.21

3.4 Effect of Initiator Concentration

mol. L - I to 30 x mol L-1 and ascorbic acid was varied from 6 x to 30 x mol L-I. The ratio of hydrogen peroxide concentration to ascorbic acid concentration was 101.

An initial increase in the initiator concentration was found to increase percent grafting, grafting efficiency, and rate of grafting of ethyl acrylate onto gelatin (Table IV). The higher percent grafting obtained upon using relatively lower concentration of hydrogen peroxide suggests that hydrogen

The concentration of hydrogen peroxide was varied from 6 x

0.1 0.2 0.3 0.4 0 . 5 0.6 0.7 - [ € A ] M -

Fig. 1. Effect of ~ o n o m e r ] on grafting. (A) 4 vs FA]; (B) percent grafting vs FA].

GRAFT COPOLYMERIZATION OF ETHYL ACRYLATE 2869

TABLE IV Effect of Initiator Concentration on Grafting.

[EA] = 1.8 x 10-l mol L-l; total volume = 50 mL; [Gelatin] = 3.3 X lo-' mol L-l; temperature = 60°C; time = 90 min.

HzOz x 102 [AAJ x 103 R, x 106 Grafting (%) mol L-I mol L-I mol L-l sec-'

Efficiency Grafting

6 10 16 20 24 30

6 6.03 96.12 34.35 10 10.00 94.32 38.22 16 15.85 90.60 40.03 20 11.93 84.57 34.77 24 9.43 78.29 25.46 30 5.74 70.31 15.50

peroxide forms a redox system with ascorbic acid. The decomposition of hydrogen peroxide is most probably as shown by eq. (1)

The .OH radical abstracts hydrogen atom from the gelatin backbone to yield gelatin macroradical, which is capable of initiating grafting as shown by the mechanism (2) - (4),

Gel - OH +.OH -+ Gel - 0. + H20 Gel - 0. + M -+ Gel - 0 - M.

Gel - 0 - M. + nM -, Grafted polymer

(2) (3)

(4)

Where M is the vinyl monomer. The reaction suggested by eq. (1)-(4) seems to proceed favorably up to hydrogen peroxide concentration of 16 x mol. L-l. Beyond this, the .OH radicals may largely participate in termi- nation processes with the growing polymer chain. The .OH radicals may also further decompose hydrogen peroxide as shown by eqs. (5) and (6),

The ultimate effect of participation of .OH radicals in termination, com- bination, and decomposition of hydrogen peroxide certainly lower percent grafting, grafting efficiency, and rate of grafting. Similar results were ob- tained in the graft copolymerization of vinyl monomers onto modified cotton with use of hydrogen peroxide as initiator."

The plot of log R, vs log [ I ] gave a slope value of unity for ethyl acrylate monomer and the plot of R, vs [ I ] was a straight line passing through the origin showing first-order dependence of initiator concentration on the rate (Fig. 2). ( [ I ] represents concentration of hydrogen peroxide.) This suggests that the termination step is first order in the growing radical concentration. This is similar to the results obtained in wool grafting.12

2870 GEORGE, RADHAKRISHNAN, AND JOSEPH

0 4 8 1 2 16 2 0 2 4 - A

0.6 0.8 1 0 1.2 1.4 1.6 1.8 - B

A : I x 1 0 ~ t . 1 - B : L o g I t 3

Fig. 2. Effect of [Initiator] on rate of grafting. (A) Rg vs [I]; (B) Log Rg vs Log [I].

3.5 Effect of Backbone Concentration

From Table V it is seen that an increase in gelatin concentration increases percent grafting, grafting efficiency, and rate of grafting. This shows that the higher the gelatin concentration, the more grafting sites formed. Similar results were obtained when methyl methacrylate was graft copolymerized onto bl00d.l~

The plot of log Rg vs log [Gelatin] gave a slope value of unity (Fig.3). This may be due to the heterogeneous nature of the system as the grafted polymer precipitates out during the course of polymerization.

TABLE V Effect of Gelatin Concentration.

x mol. L-l; time = 90 min. [EA] = 1.8 x 10-I mol L-l; H202 = 10 X mol L-l; total volume = 50 mL; [AA] = 10

[Gelatin] x 10' R, x 106 Grafting Grafting (mol lit) mol L-I sec-' efficiency (%)

2.2 16.2 74.61 27.64 2.5 19.1 77.27 34.19 3.2 24.0 80.42 41.56 3.8 29.9 83.58 49.24 4.8 36.0 87.12 58.31 6.3 48.0- 88.32 62.

GRAFT COPOLYMERIZATION OF ETHYL ACRYLATE 2871

A

0 2 4 6 B I0 _1 A

0.2 0.4 0.6 0.8 1.0 1.2 - B

A : [ G ] x 1 0 2 M - ; 8 : l op [ G ] + 2

Fig. 3. Effect of [Gelatin] on rate of grafting. (A) 4 vs [GI; (B) Log I$ vs Log [GI.

4. Kinetics and Rate Law From the foregoing discussion, the rate of graft copolymerization Rg may

be expressed as

R, a [EAI2.O [I]'.O [G]'.O

5. Characterization of Graft Copolymers

One of the most important problems in the characterization of gelatin ethyl acrylate graft copolymers in common with the other graft copolymers is the fundamental question of whether true grafting has really occurred or not. In the present investigation the following techniques have been used for characterization.

5.1 Selective Solvent Extraction

The simplest evidence for graft copolymerization is based on differences in solubilities between the graft copolymer and unbound homopolymer. The ungrafted homopolymer of ethyl acrylate could be readily removed from gelatin by extraction with acetone. Such evidence was claimed as a proof of grafting in the preparation of starch graft copolymer^.^*

5.2 Treatment of Isolated Grafts with Ninhydrin Reagent

Proof of grafting can be ascertained by detection of amino acid end groups in the grafts isolated by acid hydrolysis of the graft copolymers. The isolated

2872 GEORGE, RADHAKRISHNAN, AND JOSEPH

grafts were treated with ninhydrin reagent, which gave the characteristic blue color normally associated with the presence of amino acids.

In the case of physical mixture obtained by intimately mixing backbone with homopolymer, no blue color was noticed. These results indicate that true grafting of the polymer to the amino acid residues in gelatin has occurred.

5.3 IR Spectra Infrared spectroscopy has been found to be a valuable tool in the study

of graft copolymerization reactions. This technique has been extensively used (a) to establish the proof of grafting, (b) to ensure complete removal of nongrafted homopolymer from the grafted products and, (c) to determine the structure of the grafted products.

In the present investigation the IR spectra of gelatin, gelatin graft co- polymer, and ethyl acrylate homopolymer have shown that actual grafting took place (Fig.4). The spectra showed the characteristic amide absorption at 1550 and 1660 cm-' in addition to carbonyl peaks at 1730 cm-l.

5.4 Amino Acid Analysis of the Graft Copolymers Graft copolymers were hydrolyzed by use of 6N HC1 for 22 h at 110°C

and evaporated under vacuum at 40-50°C. The residue was dissolved in sodium citrate buffer at pH 2.2 and then analyzed.

The data from amino acid analysis can be used to determine exactly the number of residues of each amino acid in the protein molecule. Table VI shows the results of amino acid analysis of control and samples. Samples with different percent grafting were chosen for analysis. The control ex- periment contained no monomer.

W 0 Z a m

m

LT 0 Ln

Q

LOO0 3000 2000 1200 800 600 400

F RE W E NC Y CM-' Fig. 4. IR Spectra. (A) IR spectra of pure gelatin; (B) IR spectra of gelatin-g-polfiethy1

acrylate); (C) IR spectra of poly(ethy1 acrylate).

GRAFI' COPOLYMERIZATION OF ETHYL ACRYLATE 2873

TABLE VI Amino Acid Composition of Gelatin and Gelatin Graft Copolymers

Amino acids Control Sample I Sample I1

Hydroxyproline Aspartic Acid Threonine Serine Glutanic acid Proline G 1 y c i n e Alanine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Hydroxylyse Lysine Histidine Arginine

103.7 50.7 14.0 36.2 73.5 150.3 335.0 100.3 17.7 0.5 12.6 21.0 1.2 9.7 6.8 23.9 3.4

8840.0 1000.5 -

150.2 41.2 10.2 21.2 68.2 200.1 333.1 74.5 17.2

12.2 18.2 0.9 8.5 5.0 15.5 1.2 32.1

-

160.0 47.6 12.2 18.3 63.2 195.4 330.5 76.2 16.1

10.6 16.0 0.9 8.0 5.8 10.4 1.0 28.2

-

999.5 1000.7

Condition

Reaction time Temperature Gelatin

[WI Amount of monomer

Sample Sample I I1

90 min. 90 min. 60°C 60°C

3.2 x 10-4

10 x 10-3

3.2 x 10-4

10 x 10-3 mol L-l mol L-'

1 mL 2 mL

A comparison of the amino acid composition of grafted and ungrafted gelatins (Table VI) showed a significant decrease in serine, histidine, ty- rosine, lysine, threonine, alanine, and arginine. These results, therefore, indicate that these groups may be involved as grafting sites. It is not clear whether the increase in the proline and hydroxy-proline content is caused by the transformation of other amino acids by the action of oxidizing agent used in the present study.

That proline and hydroxyproline residues in proteins are susceptible to oxidation has been demonstrated by earlier workers.15J6

The primary radicals SO;- produced would initiate the graft copoly- merization reaction. Further, these primary radicals may form a redox system with some reducing sites in gelatin to form free radicals on the backbone itself. Similar results has been obtained by Ikada, Nishizaki, and Sakurada.17

References 1. Norman G. Gaylord, J. Polym. Sci. C, 37, 153 (1972). 2. H. L. Needles, J. Polym. Sci. B, 5 , 595 (1967). 3. H. L. Needles, J. Appl. Polym. Scz., 12, 1557 (1968).

2874 GEORGE, RADHAKRISHNAN, AND JOSEPH

4. A. H. Korn, S. M. Feairheller, and E. M. Filachione, J. Am. Leathar Chem., 68, 111

5. A. H. Korn, S. M. Feairheller, and E. M. Filachione, Leather Munufi, 89, 33 (1972). 6. V. Svobada, Kozarstvi, 20, 158 (1970). 7. W. R. Dyson, M. A. Knight, and R. L. Sykea, J. Soc. Leather Technol. Chem, 57, 31

8. R. R. Geinstead, J. Am. Chem Soc., 82, 3464 (1960). 9. B. N. Misra, Inderjeet K. Mehta, and M e s h Dogra, J. Macrvmol. Sci. Chem A,

(1972).

(1973).

12(10), 1513 (1978). 10. N. K. Boardmon and M. Lipson, J. Soc. @em Colour., 67,271 (1951). 11. S. Kamenskaya and S. Medveder, Acta. Phys. Chem USSR, 13, 565 (1956). 12. A. J. Keknnow, J. Appl. Polym. Sci., 14, 3033 (1970). 13. G. F. Fanta, R. C. BUR, C. R. Russell, and C. E. Rist, J. Appl. PoZym. Sci., 10, 929

14. C. E. Brockway and P. A. Seaberg, 3: Polym Sci. A-l,51, 1313 (1967). 15. N. R. Moudgal, V. Srinivasan, and P. S. Sarma, J. Sci Ind Res. C, 1 4 6 7 (1956). 16. E. Bradbury and C. Martin, Proc. Roy. Soc. (zondon) Ser. A, 214, 183 (1952). 17. Y. Ikada, Y. Nishizaki, and I. Sakurada, J. Polym. Sci. Polym. Chem Ed, 12, 1829

(1966).

(1974).

Received May 15, 1984 Accepted February 26, 1985