J. MATER. CHEM., 1991, 1(4), 643-647 643 Synthesis, Characterization and Biodegradation Test of Nylon 2/6 and Nylon 2/6/6 K.E. Gonsalves,a X. Chen" and T. K.Wongb a Polymer Science Program, Institute of Material Science and Department of Chemistry, U-60, University of Connecticut, Storrs, CT 06269, USA Enzymatics lnc., Horsham, PA 19044, USA Initial results obtained from the characterization and biodegradation tests on poly(6-glycylaminohexanoic acid) (nylon 2/6) and poly[hC(6-glycylaminohexyl)adipamicacid] (nylon 2/6/6) are presented. Nylon 2/6 was synthesized via a condensation reaction and nylon 2/6/6 by interfacial polymerization. Their intrinsic viscosities were 0.03 and 0.09 dm3 g-', respectively. Thermal analysis and X-ray diffraction methods were applied to characterize these two nylons.The biodegradation of nylon 2/6 and nylon 2/6/6 films was observed by using a qualitatively fast screening test, which showed that nylon 2/6/6 is more readily degradable than nylon 2/6 under attack by fungi. Keywords: Nylon; Biodegradation; Intrinsic viscosity 1. Introduction A series of glycine-containing nylons, such as poly(6-glycylaminohexanoic acid) (nylon 2/6), poly[N-(6- glycylaminohexy1)adipamic acid] (nylon 2/6/6) and poly [N-(6-glycylaminohexylaminocarbonylmethyl)adipamicacid] (nylon 2/6/2/6), were reported to be biodegradable based on the measurement of the release of carbon dioxide from slurry samples under attack by fungi and bacteria.'v2 It therefore appears that these biodegradable nylons have the potential to be used in replacing commercial nylons in packaging and marine applications.Hence, it was considered necessary to conduct investigations on the thermal and physical character- istics of these polymers to assess their processability, as well as to determine their degradability in end products such as films and fibres. We reported previously an alternative syn- thesis of nylon 2/6.3 In this paper, we report a modified synthesis of nylon 2/6/6, the thermal and X-ray diffraction characterizations of nylon 2/6 and nylon 2/6/6, as well as a fast screening test to ascertain the biodegradability of films of these gl ycine-containing polymers. 2. Results and Discussion 2.1 Synthesis 2.1.1 Nylon 2/6 (fNHCH2CONH(CH2),C~,) As described previ~usly,~ nylon 2/6 was made uia conden-sation of a pentachlorophenol (PCP) active ester.Its molecular weight can be inferred to be low because of the low intrinsic viscosity [q] of 0.029 dm3 g- in 90% formic acid. Note that in the earlier synthesis of nylon 2/6,4i5 low [q] values were also reported. In order to increase the molecular weight of nylon 2/6, different substituted phenols were used in the process of active-ester formation. As shown in Table 1, all of the three active esters gave polymers with similar values of [q1.It can be qualitatively concluded that only relatively low-molecular- weight nylon 2/6 can be obtained uia these condensation polymerizations. 2.1.2 Nylon 2/6/6(fHNCH2CONH(CH2)6NHCO(CH2)4 CWn) This polymer was first synthesized by Wu6 with an intrinsic viscosity of 0.086 dm3 g-' in dichloroacetic acid at 298.2 K by the following method (Scheme 1, note tert-BOC is the tert-butyloxycarbonyl group).By applying a dicyclohex ylcarbodiimide (DCC) peptide-linking method instead of the slow (1 week) aminolysis reaction, we synthesized nylon 2/6/6 with [q] of 0.093 dm3 g-' in 90% formic acid at 298.2 K as shown in Scheme 2. 2.2 Characterization 2.2.1 Diflerential Scanning Calorimetry, Thermogravimetric Analysis and Thermal Chromatography-Mass Spectrometry For comparison, poly[N-(6-aminohexyl)adipamic acid] (nylon 6/6) was prepared by the same method used for making nylon 2/6/6 described in section 3.3. The <(glass transition) and T, Table 1 Intrinsic viscosities [q] of nylon 2/6 condensed from different active esters phenol [ttiw3g -0.029 ~ ~ ~ ~ ~ ~0.020 $ ~ ~ pen tafluorophenol 0.034 a In 90% formic acid.rerr-BOC azide ClCH COCI H2N(CH2),NH2 -tert-BOC-NH(CH2),NH2 NHJH20 conc. HC1 tert-BOC-NH(CH,),NHOCCH2cl-tert-B0C-NH(CH2),NHCOCH2NH2- J. MATER. CHEM., 1991, VOL. 1 di-fert-butyl pyrocarbonate H2N(CH 2) 6NH 2 + tert-BOC-NH(CH2),NH2HC1 tert-BOC glycine, DCC ' tert-BOC-NHCH2CONH(CH2)6NH-tert-BOC HCl/acetic acid ClH,NCH2CONH(CH2)6NH3CI (melting point) of the former were shown to be 338 and 538 K, respectively. As seen in Table2, the Tg and T, of nylon 2/6 are close to those of nylon 6/6, whereas those of nylon 2/6/6 are apparently lower.It is shown in Table2 that the onset decomposition temperatures of both nylon 2/6 and nylon 2/6/6, measured by thermogravimetric analysis (TG), are 50 K higher than their melting points. To elucidate further the thermal stabilities of these two nylons, the thermal chromatog- raphy-mass spectrometry technique (TC-MS) was applied. Details of the TC-MS instrument have been described pre- vio~sly.~Here, only the results from level I-FID analysis of 3000 2 2000 i:0 LL 1000 -/,L0-'-1 ', TC-MS (i.e. total gas production us. temperature) are dis- cussed. The experimental runs were conducted in helium (20 cm3 min-') at a heating rate of 30 K min-' from 303 to 873 K. As shown in Fig. 1, the onset temperatures for gas product formation from nylon 2/6/6 and nylon 2/6 are ca.620 and 570 K, respectively. Nylon 2/6 shows a small decompo- sition peak also at ca. 420 K. Both TG and TC-MS results, based on the measurements of weight loss and gas product formation, respectively, indicate -303 423 573 723 873 that these nylons decompose only at temperatures higher than 570 K. As mentioned above, the melting points of nylon 2/6 and nylon 2/6/6 are 549 and 501 K, respectively. Therefore, it should be possible to process these two nylons by regular approaches such as melt-moulding or melt-spinning. However, nylon 2/6 was discoloured after melt-moulding at 550 K in N2. The film and fibre samples of nylon 2/6/6 were also slightly discoloured upon moulding and melt-spinning at 500 K.Further studies on the thermal decomposition modes of these glycine-containing nylons are therefore necessary and will be reported separately. 2.2.2 X-Ray Difruction Preliminary powder X-ray diffraction patterns of nylon 2/6 and nylon 2/6/6 samples, crystallized from formic acid-n- butanol at 368 K, are shown in Fig. 2. A strong reflection at spacing 4.15A was observed, which is the same as that reported by Puigalli et al.' for nylon 2/6. This result probably indicates that both of these nylons have hexagonal crystal structures as proposed by Puigalli et aL5 Table 2 Physical properties of nylon 216 and nylon 21616 property nylon 216" nylon 21616 method IIr11/dm3g-0.029 0.093 in 90% formic acid, 298.2 K TB/K 333 327 DSC, 15 K min-' TmIK 549 501 DSC, 10 K min-' Gb/K in N, 599 611 TGA, 20 K min-in air 600 601 a Nylon 216 was synthesized from PCP ester; Td is the onset decomposition temperature measured by TGA.L g 2000 -1000 . i Oh ' ~303 423 573 723 873 l------cell temperature/K cool pyrocell Fig. 1 FID response of nylons by TC-MS analysis: (a)nylon 216, (b) nylon 21616 2.3 Biodegradation Test Poly[oxy( 1-oxohexamethylene)] film was tested as a positive control in this experiment. After 35 days of incubation, it became so fragile that it could hardly be picked up from the Petri dish for cleaning and SEM observation. The degradation on nylon films was not detectable visually. Biodegradation invovles the loss of structural characteristics and mass of a material as C02 and water-soluble components.There are many methods to test the biodegradation of syn- thetic Most measurements are based on C02 production, O2 consumption, weight loss and changes in structural characteristics. As pointed by Wool and Cole" most of these methods are technically difficult and may yield inconclusive or misleading results. In this experiment, we estimated the surface erosion of nylon films by using a qualitative scanning electron microscopy method. As seen in Fig. 3, nylon 6 maintained a smooth surface after 14 days of incubation, while obvious degradation occurred on the surface of nylon 2/6/6 after 31 days of incubation. These results confirmed the fact that the commercial nylons like nylon 6 are non-biodegradable, and also indicated that nylons con- J.MATER. CHEM., 1991, VOL. 1 (a) Fig. 2 Powder X-ray diffraction patterns of nylon (the innermost ring corresponds to the spacing 4.15 A): (a)nylon 2/6; (b)nylon 2/6/6 (a1 (b) Fig. 3 SEM pictures of nylon film surface, x 10 000: (a)nylon 6 after 14 days of incubation; (b)nylon 2/6/6 after 31 days of incubation taining glycine units in the backbone could be potentially biodegradable. Fig. 4 and Fig. 5 show the time progression of surface erosion on nylon 2/6 and nylon 2/6/6 films caused by fungal attack. From these SEM micrographs it was observed that nylon 2/6/6 degrades more readily than nylon 2/6 under the conditions mentioned above. 3. Experimental 3.1 Synthesis of Nylon 2/6/6 3.1.1 Protection of Hexanediamine with tert-BOC Group A solution of di-tert-butyl pyrocarbonate (43.6 g, 0.2 mol) in dioxane (70 cm3) was added slowly to a solution of hexanedia- mine (75 g, 0.65 mol) and NaOH (8 g, 0.2 mol) in 1 : 1 H20-dioxane (220 cm3) held in an ice-water bath while stirring.After ca. 3 h addition, the solution was allowed to warm to room temperature and was left overnight. Dioxane was removed by vacuum evaporation, and the residue solution was saturated with NaCl and then extracted with EtAc. The oily material, obtained by removing EtAc from the organic phase, was dissolved in H20 (100 cm3) and acidified to pH 3-4 with 1 mol dmP3 HCl. A white precipitate was formed on the top of the solution after it was saturated with NaCl.The solid was dissolved in hot ethanol. Three times its volume of ether was added to this solution and a white solid was formed. 21 g (83.1 mmol, 41.3%) of solid tert-BOC-HN(CH2),NH3C1 (N-tert-BOC-hexanediamine hydrochloride, I) was obtained upon vacuum drying, m.p. 433-435 K. 3.1.2 N-Glycylhexanediamine Dihydrochloride Solid I (21 g), tert-BOC-glycine (14.6 g, 83.5 mmol), freshly distilled dicyclohexylcarbodiimide (DCC) (1 7.4g, 83.5 mmol) and 1,4-diazobicyclo[2.2.2]octane (7.9 g, 70 mmol) were dis- solved in 400cm3 of dry EtAc in an ice-water bath while stirring. The mixture was stirred at room temperature over- night followed by addition of 5 cm3 glacial acetic acid to decompose the excess of DCC.After cooling in ice water for 30 min, the mixture was filtered. The filtrate was washed thoroughly with 40 cm3 cool 1 mol dmP3 HCl, water, satu- rated NaHCO, and water. The solution was dried with Na,SO, and the solvent removed by rotory evaporation under reduced pressure. To the oily residue of tert-BOC-HN(CH2),NHOCCH2NH-tert-BOC (11), 120 cm3 7% HC1-acetic acid was added to remove the blocking tert- BOC groups. A white precipitate was formed after adding 400 cm3 acetone. The solid was recrystallized from 1 : 1 meth-anol-acetone giving 15.7g (63.6 mmol, 76.6%) C1H3NCH2CONH(CH2),NH3C1 (N-glycylhexanediamine dihydrochloride, 111), with m.p. 433-434 K. 3.1.3 Nylon 21616 via Interfacial Polymerization In a 1 dm3 capacity blender, 15.7 g of solid I11 and 10.4 g (0.26 mol) of NaOH were dissolved in 400 cm3 H20.To this solution, 150 cm3 of a solution of 9.45 cm3 (65 mmol) adipoyl chloride in CCl, were added slowly with vigorous stirring. The nylon 2/6/6 solid was washed successively with many portions of H20, 1 :1 EtOH-H20 and EtOH, and then dried at 363 K in a vacuum oven overnight. 8.15 g nylon 21616 (yield 45.2%) were obtained. Its intrinsic viscosity in 90% formic acid at 298.2 K was 0.093 dm3 g-'. Elemental analysis results for the polymer are as follows. Found: C, 57.79; H, 9.22; N,14.36%. Calc. for [C14H25N303]n: C, 59.34; H, 8.89; N, 14.83 and for [C14H25N303-H20]n: C, 56.47; H, 9.01; N,14.11%. 3.2 Characterizations The intrinsic viscosities [q] of nylon 2/6 and 2/6/6 were measured in 5 g dm-, solution of 90% formic acid at 298.2 K.No change in [q] was observed after 1 week for both nylons. Samples for powder X-ray diffraction were prepared as fol- Fig. 4 SEM pictures of biodegraded nylon 2/6 film, x 5000: (a) before test; (b)7 day test; (c) 31 day test n-butanol was added to an equal volume of a 0.2% polymer solution of 90% formic acid at 368 K followed by cooling slowly to room temperature overnight. The crystals were separated by centrifugation, washed twice with n-butanol, and finally dried in a vacuum for 2 days. Powder X-ray diffraction diagrams were recorded using Cu-Ka radiation with an Ni filter at 30 kV and 20 mA. Perkin-Elmer DSC-2 and Perkin-Elmer TGS-2 instruments were used for DSC and TG measurements, respectively.TC-MS runs were made in helium using the Pyran System (Ruska Labs).’ 3.3 Biodegradation 3.3.1 Samples and Cultures The cultures used were Aspergillus niger, Aspergillus Jauus, Penicillum notatum, Saccharomyces cereuisiae, Sordaria Jimic- ola and Schizosaccharomyces octospores. J. MATER. CHEM., 1991, VOL. 1 Fig. 5 SEM pictures of biodegraded nylon 2/6/6 film: (a) before test, x 10 000; (b)after 7 day test, x 5000; (c) 14 day test, x 5000 Films of nylon 2/6/6 and nylon 6, with a thickness of ca. 0.1 mm, were moulded at 500 K by fast heating and cooling within 1 min. Nylon 2/6 was moulded at 550 K and turned yellow. Poly[oxy( 1-oxohexamethylene)], which is a well known biodegradable polymer, was moulded at 340 K to be used as a positive control, and nylon 6 film as a negative control.3.3.2 Test Procedures After each fungal culture was grown in its own medium in agar for 1 week, one loopful was transferred to a tube containing 1 cm3 of liquid nutrient medium. This mixed fungal culture was incubated at 298 K for 1 day. Liquid nutrient agar was poured into a Petri dish and cooled to a semi-solid state, and a piece of sample film (ca. 0.5 cm2) was placed onto the agar <0.5 mm below the surface. After the agar was solidified, the mixed cultures were laid directly on top of the film area. Incubation was performed at J. MATER. CHEM., 1991, VOL. I 298 K in a humidified environment for 7, 14 and 31 days. During the incubation, the growth of the cultures was observed by optical microscopy.After incubation, each film was subjected to soaking in 5moldm-3 NaOH and clorox solution, and then washed thoroughly with distilled water. Scanning electron microscopy (SEM) was used for testing the surface erosion by the cultures in order to assess qualitatively the degree of biodegradation. 4. Conclusions Only a low molecular weight of nylon 2/6 can be obtained from polycondensation reactions of azide or active ester, whereas, by interfacial polymerization, nylon 2/6/6 with a moderately high molecular weight can be made. Here, a fast method for making the diamine monomer of nylon 2/6/6 was developed. Although the thermal analysis (TG and TC-MS) showed that nylon 2/6 decomposes only at temperatures 50 K beyond its melting point, the nylon 2/6 sample was noticeably degraded after melt-moulding.Nylon 2/6/6 also showed slight discolouration after melt-moulding or melt-spinning at 500 K. The difference of thermal stability between nylon 2/6 and nylon 2/6/6 upon melt processing can be explained from the fact that both contain a glycine group which is assumed to be relatively thermally unstable and the melting temperature of nylon 2/6/6 is lower than that of nylon 2/6 (see Table 2). Results from the fast screening test of biodegradation described here show that nylon 2/6 and nylon 2/6/6 are biodegradable. This method can be applied as a general approach to measure the biodegradability of other polymer fiims and fibres qualitatively.Refinement of the tests are underway and will be reported subsequently. References I D. Ennis and A. Krammer, J. Food Sci., 1975,40, 181 2 W. J. Bailey and B. Gapud, Ann. N.Y. Acad. Sci., 1985, 446,42. 3 K. E. Gonsalves and X. Chen, Polym. Commun., 1990, 31, 3 12. 4 W. J. Bailey, Y. Okamoto, W. Kuo and T. Narita, Proc. 3rd Znt. Biodegradation Symp., Applied Science Publishers, Barking, 1976, p. 765. 5 J. Puiggali, S. Munoz-Guerra and B. Lotz, Polymer, 1987, 28, 209. 6 Y. S. Wu,Ph.D. Thesis, University of Maryland, 1977. 7 K. E. Gonsalves, S. S. Stivala, L. Reich, S. H. Patel and D. H. Trivedi, Polym. Muter. Sci. Eng., 1990, 63, 962. 8 Standard Practice for Determining Resistance of Synthetic Poly- meric Materials to Fungi, G21-70, Annual Book of ASTM Standards, American Society of Testing and Materials, 1985. 9 S. H. Huang, in Comprehensive Polymer Science: Synthesis, Characterization, Reactions and Applications of Polymers, Perga-mon Press, Oxford, 1989, vol. 6, ch. 21, p. 597. 10 R. P. Wool and M. A. Cool, ASM Engineering Materials Hand- book, Vol. 2: Engineering Plastics, Plenum Press, New York, 1988, p. 783. Paper 1/00964H; Received 1st March, 1991