Poly(styrene peroxide) and Poly(methyl methacrylate
peroxide) for Grafting on Unsaturated Bacterial
Polyesters
Birten Cakmakli,1Baki Hazer,*1, 2Mehlika Borcakli2
1Zonguldak Karaelmas University, Department of Chemistry, 67100 Zonguldak, Turkey
2TUBITAK-Marmara Research Centre, Food Science and Technology Research Institute, Gebze 41470 Kocaeli, Turkey
E-mail: bhazer@karaelmas.edu.tr
Introduction
Natural aliphatic polyesters as intracellular energy and carbon storage materials produced by bacteria, (PHA)s, are considered to be truly biodegradable polymers.[1 – 3] Pseudomonas oleovorans has been investigated very intensively. This bacterium produces medium chain length PHA random copolymers of repeating units con-taining 6 to 12 carbon atoms. Pseudomonas oleovorans can produce polyester from a wide variety of carbon sub-strates, including alkanes, alcohols, alkanoic acid, alkenes and its derivatives.[4 – 9]Unsaturated PHAs were produced from substrates of edible oily acids obtained from hazel-nut, sesame, butter, olive, coconut and anchovy oil.[10, 11] The (PHA)s produced by Pseudomonas oleovorans are
considered to be thermoplastic elastomers.[12]
Bacterial polyester synthesized from soybean oily acids was sticky, waxy and soft material. To improve the mechanical and viscoelastic properties of biodegradable PHAs, several fine attempts have reported.[13 – 18]
Grafting is considered an important technique for modifying the physical and chemical properties of polymers.[19 – 23]Hazer has reported grafting reactions of poly(3-hydroxynonanoate) (PHN) with polystyrene and poly(methyl methacrylate) by per-oxidic initiators.[13] c-Irraddiation of a mixture of PHN and methyl methacrylate (MMA) also yielded PHN-g-PMMA graft copolymers.[15] Lenz et al.[16 – 18] obtained PHAs with double bonds from Pseudomonas oleovorans cofed with 10-undecenoic acid and nonanoic or octanoic
Full Paper: A new soluble terephthaloyl oligoperoxide
(OTP) was synthesized by the reaction of terephthaloyl peroxide and 2,5-dimethyl 2,5-dihydroperoxy hexane. Thermal polymerization of vinyl monomers (styrene, methyl methacrylate) with OTP yielded poly(styrene per-oxide) (PS-P) and poly(methyl methacrylate perper-oxide) (PMMA-P) which are used in the grafting reactions onto medium chain length unsaturated bacterial polyester obtained from soybean oily acids with Pseudomonas oleo-vorans poly(3-hydroxy alkanoate), (PHA). PS-g-PHA and PMMA-g-PHA graft copolymers isolated from related
homopolymers were characterizated by1H NMR
spectro-metry, FT-IR spectroscopy, thermal analysis and gel per-meation chromatographic (GPC) techniques. Swelling measurement of the crosslinked graft copolymers were also measured to calculate qvvalues.
Macromol. Biosci. 2001, 1, No. 8 iWILEY-VCH Verlag GmbH, D-69451 Weinheim 2001 1616-5187/2001/0811–0348$17.50+.50/0
Thermogravimetric trace of graft copolymers. (BC: 31-712-1).
acid. They performed the modification of the biopoly-esters, containing double bonds, via irradiation,[16] epoxi-dation[24] and chlorination.[14] Poly(ethylene glycol) was thermally grafted onto the PHAs containing double bonds by polyazoester.[25]Crosslinking properties of the unsatu-rated PHAs were also studied.[26]Free-radical polymeriza-tion of MMA initiated by benzoyl peroxide in the pre-sence of PHA-soybean also gives PHA-g-PMMA graft copolymers.[27] The first oligoperoxide was prepared by the reaction of phthaloyl dichloride with sodium perox-ide.[28, 29] Polymeric peroxides obtained by the polymeri-zation of vinyl monomers with an oligoperoxide can be successfully used in the block copolymerization.[30, 31]
In this work, a new soluble oligo(terephthaloyl per-oxide) (OTP) was used as initiator in the polymerization of styrene and methyl methacrylate to obtain active poly-mers which were thermally grafted on bacterial polyester containing unsaturated side chains.
Experimental Part
Materials2,5-Dimethyl-2,5-dihydroperoxyhexane (Luperox 2.5–2.5) was a product of Cincinatti Milacron Chem. Corp. It was
recrystallized from CCl4before use.
Terephthaloyl chloride was a product of Fluka and was used without further purification.
Styrene and methyl methacrylate were supplied from Aldrich and were freed from inhibitor by vacuum distillation over CaH2.
All other chemicals were reagent grade and used as received.
PHA Biosynthesis
Stock cultures of P. oleovorans (ATCC 29347) were used in all growth and polymer production experiments. P. oleovor-ans was grown on soybean oily acid substrate and the result-ing polymer was extracted by usresult-ing methods in the litera-ture.[27]
Substrates
Soybean oil was extracted from the related agricultural prod-ucts grown in Turkey. The acid obtained from the hydrolysis of soybean oil included both saturated and unsaturated acids in different weight percentages. Unsaturated acids, including oleic, linoleic and palmitoleic acids, comprised of the acids
from soybean.[32] Saturated acids, including both palmitic
and stearic acids, were approximately 18% ens, diens and tri-ens as unsaturated acids were about 18–26, 50–55 and 7– 10 wt.-%, respectively.
Synthesis of Oligoperoxide (OTP)
For the synthesis of
oligo(terephthaloyl-5-peroxy-2,5-dimethyl hexyl peroxide), 2.909 g of Luperox 2.5–2.5 was dissolved in 35 ml of 10% KOH with stirring at 0 8C. After the dissolution of the dihydroperoxide, a cooled solution of terephthaloyl chloride in ether (3.318 g terephthaloyl chlor-ide in 33.2 ml ether) was slowly added dropwise over 0.5 h. Vigorous stirring was continued at 0–5 8C for 1 h. The organic phase was washed with water and dried over
anhy-drous Na2SO4. The solvent was evaporated and dried in a
vacuum oven at room temperature for a day. The yield was 3.92 g. The product was purified and peroxygen amount was found to be 18 wt.-% by iodometric analysis. It was soluble in chloroform, chlorinated solvents and the vinyl monomers used.
Synthesis of Active Polystyrene (Active PS)
In a pyrex tube, a given amount of styrene and the
oligoper-oxide in CHCl3 were charged separately. Argon was
intro-duced through a needle into the tube for about 3 min to expel the air. The tightly capped tube was then put in an oil bath at 68 8C. After the required time, the contents of the tube were coagulated in methanol. The active PS sample was dried overnight under vacuum at 30 8C. Table 1 show the charac-teristic data of the styrene polymerization.
Table 1. Results and conditions of the synthesis of active PS and active PMMA.
Run No. OTP
g Monomer Yield g 1O1O1 wt:-% Mw 610ÿ4 MWDa) Styrene ml MMA ml 31-61b) 0.252 5.5 – 0.24 0.41 3.9 1.54 31-62b) 0.502 5.5 – 0.76 0.36 3.6 1.42 31-64b) 0.125 5.5 – 0.34 0.26 4.2 1.51 31-65b) 0.054 5.1 – 0.40 0.11 5.7 1.62 31-66b) 0.066 12 – 0.76 0.13 5.6 1.67 30-41c, d) 0.243 – 5 0.38 0.42 7.6 1.65 31-712c, d) 1.020 – 5 3.01 0.51 5.2 1.63
a) MWD: molecular weight distribution. b) At 68 8C.
c) Polymerization was carried out in 5 ml of CHCl 3. d) At 80 8C.
Synthesis of Active Poly(methyl methacrylate) (Active PMMA)
In a pyrex tube, a given amount of methyl methacrylate and the oligoperoxide were charged separately. Argon was intro-duced through a needle into the tube for about 3 min to expel the air. The tightly capped tube was then put in an oil bath at 80 8C. After the required time, the contents of the tube were coagulated in methanol. The active PMMA sample was dried over night under vacuum at 30 8C. Table 1 shows the characteristic data of the methyl methacrylate polymeriza-tion.
Grafting Reactions
For grafting reaction, a solution was prepared from a mixture of 0.5 g of PHA-soybean, 0.25 g of active PS or active PMMA and 10 ml of chloroform (as a solvent). The solution was stirred, spread into petri dishes and air dried. Gel forma-tion was carried out on this glass plate by introducforma-tion to an oven preheated to 80 8C for 2 h. The grafted polymer mixture was extracted with chloroform for 24 h for sol gel analysis. The gels were removed, washed with methanol and dried under vacuum at 30 8C for 2 d. The results of the grafting procedure are listed in Table 2 and Table 3.
Fractional Precipitation of PHA-g-PMMA and PHA-g-PS The graft copolymer was isolated from the homopolymer of styrene or methyl methacrylate and unreacted PHA-soybean by fractional precipitation method. c Values were calculated as the ratio of the total volume of MeOH used for each
frac-tion of CHCl3in which graft copolymer was dissolved. The
polymer fractionated was dried under vacuum. Table 2 and Table 3 include results of fractional precipitation.
Polymer Characterization
FT-IR spectra (such as that shown in Figure 1) were obtained using Perkin-Elmer 177 IR spectrometers.
1H NMR were recorded in CDCl
3 at 17 8C with a
tetra-methylsilane internal standard using a Bruker 200 MHz
NMR AC 200 L.1H NMR spectra of graft copolymer
sam-ples are shown in Figure 2 and 3.
The molecular weight of the polymeric samples was deter-mined by gel permeation chromatography (GPC) with a Waters model 6000A solvent delivery system with a model 401 refractive index detector and a Mode 730 data module and with two Ultrastyragel linear columns in series. Tetrahy-drofuran was used as the elution at a flow rate of
Table 2. Grafting reactions of active PS on PHA-soybean.
Run No. Feed Polymer yield Swelling ratio
BC PHA-Soybean g Active PS g Yield g Crosslinked polymer wt:-% Soluble pure graft polymer wt:-% a,b) in CHCl3 qv 31-61 1 0.2009 0.1007 0.2879 64 15 14.7 31-61 2 0.1006 0.1003 0.1919 53 34 16.9 31-62 1 0.2008 0.1005 0.2992 37 60 20.8 31-62 4 0.2012 0.2005 0.3872 38 55 19.9 31-62 2 0.2002 0.2002 0.3797 34 45 12.0 31-64 1 0.2048 0.0540 0.2460 70 20 20.7 31-64 2 0.1090 0.0516 0.1162 39 20 15.2 31-64 3 0.2000 0.2000 0.3091 38 36 30.3 31-66 1 0.2020 0.0532 0.2414 72 15 17.3 31-66 2 0.2001 0.1011 0.2837 61 20 19.1 31-66 3 0.2013 0.2016 0.3727 45 40 17.3
a) Pure graft copolymer precipitated in c: 0.8–1.4 after fractional precipitation (solvent: CHCl
3, nonsolvent: MeOH). chomo PS: 0.4–
0.8; chomo PHA: 1.4–2.2.
b) The rest of the percentage is made up of the related homopolymers (e. g., for Run no. 31-61-1: 64 + 15% = 79 wt.-%. 21% is the
related homopolymers.).
Table 3. Grafting reactions with active PMMA on PHA-soybean. Run No.
BC
Feed Polymer yield Fractional
precipitation PHA-soybean g Active PMMA g Polymer yield g Crosslinked polymer Soluble polymeraÞ % c : 2:2ÿ 3:2 wt:-% 31-712 2 0.4983 0.2515 0.745 – 100 46.7 31-712 1 0.4990 0.4993 0.7534 – 100 57.6 31-712 3 0.4985 0.9977 1.4755 – 100 76.1 a) c
1.0 mL N min– 1. A calibration curve was generated with six polystyrene standards.
Differential scanning calorimetry (DSC) and Thermogra-vimetric analysis (TGA) of the polymers obtained were per-formed on a DuPont 2910 instrument to determine the glass
transition temperatures (Tg), melting transition temperatures
(Tm) and thermal degradation. For DSC analysis, samples
were heated from –100 to +200 8C in a nitrogen atmosphere
at a rate of 10 8C N min– 1. Thermal analysis results of the
graft copolymers were shown in Figure 4 and 5.
Swelling degrees of polymers at equilibrium were
deter-mined by gravimetry at room temperature in CHCl3.
Swel-ling ratios (qv) were calculated using the volume ratio of
swollen polymer (vswollen polymer) to dry polymer (vdry polymer).[33]
Results and Discussion
A new polymeric initiator, OTP, was synthesized by interfacial condensation reaction of terephthaloyl chloride with 2,5-dimethyl-2,5-dihydroperoxy hexane. It was solu-ble in common solvents when compared to the terephtha-loyl peroxide obtained from sodium peroxide reported by Figure 1. FT-IR spectrum of the copolymer samples. (BC: 31-62-1)
Figure 2. 1H NMR spectrum of graft copolymer samples. (BC:
31-62-1) Figure 3. 1H NMR spectrum of graft copolymer samples. (BC:
Pechmann and Vanino.[28] The OTP chain was estimated to have three repeating units from the peroxygen analysis results (18 wt.-%). OTP was used as initiator in the poly-merization of styrene and methyl methacrylate to obtain active PS and active PMMA which contain undecom-posed peroxide groups in the main chain. Oligoperoxide synthesis and polymerization steps are shown in Scheme 1. Active polymers were obtained in low yield
and with short polymerization times but quite high per-oxygen content (Table 1). They can thermally initiate the grafting reactions on the unsaturated bacterial polyester as designed by the route shown in Scheme 2. PS-P and Figure 4. Thermogravimetric trace of graft copolymers. (BC:
31-62-4)
Figure 5. Thermogravimetric trace of graft copolymers. (BC: 31-712-1).
Scheme 1.
PMMA-P thermally give peroxy radicals and the most likely route is hydrogen abstraction with the formation of an allylic radical. Allylic radicals couple with each other and the peroxy-derived radicals. Grafting reaction condi-tions and results are collected in Table 2 and Table 3. In case of grafting reactions with active PS (Table 2), cross-linked polymer was formed for 38 to 72 wt.-% of the total polymer yield. Swelling degrees of the polymers at equi-librium were determined by gravimetry at room tempera-ture in CHCl3. Swelling ratios of highly elastomeric crosslinked copolymers varied from 12.0 to 30.3, which can be attributed to large network structure. Soluble frac-tions were fractionally precipitated to isolate pure graft copolymers from their related homopolymers. Unreacted homo-PS and homo-PHA were precipitated in the c range 0.4–0.8 and 1.4–2.2, respectively, while copolymer frac-tions were precipitated in that of 0.8–1.4. These pure graft copolymer fractions were also highly elastomeric samples.
Table 3 includes the results and conditions of the graft-ing procedure at 80 8C. The PMMA-g-PHA films were highly transparent, strong and brittle and also wholly soluble. The mode of termination in PS polymerization is combination, whereas the mode of termination for PMMA is disproportionation (at T A 330 K). This gives the active polymers proposed in Scheme 1. PMMA only has one end carrying the peroxides. This may also account for the interesting differences in results to have in gel formation for PS and soluble graft for PMMA. c ranges of PHA-soybean and active PMMA were 1.4–2.2 and 3.2–3.7, respectively. c values of copolymer samples were between those of the related homopolymers, e. g., 2.6–3.2, which confirms the graft copolymer structure. In this manner, GPC traces of the pure graft copolymers were all unimodal.
Characteristic FT-IR spectra of the BC: 31-62-1 sam-ples show absorption peaks of phenyl group at 1 600 cm– 1 and signals of double bonds in PHA-soybean at 800, 2 900 cm– 1. Carbonyl stretching appears between 1 735 and 1 750 cm– 1. Figure 1 shows the FT-IR spectrum of PHA-g-PS.
The1H NMR spectrum of the soluble sample of PHA-g-PS (BC: 31-62-1; c: 0.8–1.4) show characteristic peaks (d ppm): 0.84 (1CH3), 2.6 (1CH21COO1), 5.2–5.4 (1CH1O1), 6.4–6.6 and 7.0–7.2 (phenyl group of OTP) (Figure 2). For this sample, PHA inclusion was found to be 39.4 mol-%. The 1
H NMR spectra of the copolymer samples contained characteristic peaks (d ppm): (1COOCH3) of PMMA at 3.5 and (1O1CH1) of polyester at 5.1. A typical 1H NMR spectrum of a graft copolymer, BC: 31-712-1, can be seen in Figure 3. When the NMR spectrum of polyester was compared with BC: 31-712-1 and BC: 31-712-2, for PHA-g-PMMA samples, PHA inclusions were found to be in the range 7 and 21 mol-% of biopolyester, respectively.
GPC was used to determine the molecular weights and polydispersity of the graft copolymers; PMMA-g-PHA samples gave unimodal GPC chromatograms. This can be attributed to the proper separation of graft copolymer by fractional precipitation in this work.
Thermal analysis of graft copolymers was performed by DSC and TGA. The results of the thermal analysis were also collected in Table n4. DSC results revealed that the immiscibility of the segments. The degradation of PHA-g-PS appeared to be 175 8C, 270 8C and 400 8C (Fig-ure 4). Maximum weight loss occurred at 270 8C. The degradation of PHA-g-PMMA graft copolymer started above 180 8C (Figure 5). Degradation appeared to be a three-stage process occurring from 180 8C, 305 8C and 375 8C. Maximum weight loss occured at 375 8C.
Conclusion
Biopolyesters obtained (PHA-soybean) were soft and sticky materials so they are difficult to handle. Grafting vinyl monomers on PHAs was carried out via active vinyl polymers. The morphology of the polymers change and gain biodegradability. The viscose, sticky bacterial poly-ester can be handle in form of copolymeric material. As a conclusion, graft copolymers obtained can be combine advantages of natural and synthetic polymers.
Table 4. PHA content and GPC measurement of the graft copolymers.
Run No. Molecular weight 6 105 PHA content Thermal analysis weight loss
M—w M — n MWD in copolymer mol-% Td1 % a) Td 2 % a) Td 3 % a) BC: 31-712-1 4.01 1.26 3.19 7b) 180 (15) 305 (25) 375 (60) BC: 31-712-2 5.43 2.23 2.44 21b) BC: 31-62-1 Crosslinked 75c) 175 (2.0) 270 (75) 400 (23) BC: 31-62-4 Crosslinked 70c) 175 (2.5) 270 (70) 400 (27.5) BC: 31-66-1 Crosslinked 89.5c) 175 (1.5) 270 (89.5) 400 (10) a) Td
1, Td2and Td3are the temperatures of the first, second and third decomposition, respectively. b) From1H NMR spectrum.
Acknowledgement: This work was financially supported by Zonguldak Karaelmas University Research Fund. The Authors thank the TUBITAK-Marmara Research Center for their instru-mentation facilities.
Received: March 30, 2001 Revised: August 28, 2001
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