The importance of polymeric materials possessing biodegradability increasingly has been recognized; in particular, biodegradable water-soluble polymers are desired in terms of the earth's environment because water-soluble polymers, which are used in scale inhibitors and dispersing agents, rarely are recovered or collected after use. Therefore, the macromolecular design and synthesis of such polymers actively have been studied in recent years.1–3 Poly(amino acid), which has a protein-like amide linkage, is known to be biodegradable and thus is used in medical, cosmetic, fabric, and metal absorbent materials.4–5 For example, poly(aspartic acid) (PASP) is expected to be one of the promising water-soluble and biodegradable polymer, which commonly is obtained from the hydrolysis of poly(succinimide) (PSI) prepared by the thermal bulk polycondensation of aspartic acid6–7 or ammonium salts of maleic acid and malic acid.8 Although a few preparative methods for PASP have been proposed,9–10 they have several disadvantages, including a long reaction time, high reaction temperature, and coloring of the obtained polymers. Neri et al.11 reported a high molecular weight PSI was prepared by the polycondensation of L-aspartic acid (ASP) in a large amount of o-phosphoric acid as the catalyst and solvent under reduced pressure. However, this synthetic method had the disadvantage of isolating PSI from the reaction mixture because the remaining phosphoric acid was difficult to remove.
Recently, we reported the acid-catalyzed polycondensation of ASP in a mixed solvent leading to a high molecular weight linear PSI and the metal-cation-chelating ability of sodium polyaspartate (PASP-Na).12 Thus, we were interested in investigating a commercially available method for producing PSI based on the acid-catalyzed polycondensation of ASP; for example, a solvent-free system and a continuous process are desirable conditions. In this study, we report the bulk polycondensation of ASP with an acid catalyst for the practical production of PSI; that is, batch and continuous methods were utilized for the bulk polycondensation (Scheme 1). In addition, we discuss the relationship between the structures of PASP-Nas obtained by different methods and their properties, such as biodegradability and calcium-ion-chelating ability.
Materials and Measurements
ASP was obtained from Mitsubishi Chemical Corporation (Japan). N,N-Dimethylformamide (DMF), 85% o-phosphoric acid, and sodium hydroxide were commercially available and used without further purification.
The molecular weight of PSI was estimated in DMF containing LiBr (20 mmol · L−1) by gel permeation chromatography [GPC; column, PL gel 5 μm MIXED-C × 2 (Polymer Laboratories Ltd., U.K.); detector, refractive index; standard, polystyrene]. The residual amount of ASP after the polycondensation was measured in water containing 2.5 g · L−1 of H3PO4 and 31.2 g · L−1 of NaH2PO4 · 2H2O by liquid chromatography [column, Shim-pack ISC-07/S1504 (Shimadzu Corporation, Japan); detector, UV 210 nm]. Proton nuclear magnetic resonance (1H NMR) spectra were measured using a JNM-GSX400 MHz spectrometer (JEOL, Japan). Sample solutions were prepared by dissolving 100 to 200 mg of the polymer in 0.6 mL of DMSO-d6 in 5-mm NMR tubes. All spectra were recorded at 60°C, and tetramethylsilane was used as the internal standard. Thermal gravity analysis (TGA) was measured with TG/DTA 220 (Seiko Instrument Inc., Japan). Ten mg of ASP with or without o-phosphoric acid was heated from 50 to 300°C under flowing N2 at a heating rate of 10°C/min.
Acid-Catalyzed Bulk Polycondensation of ASP
The general procedure is as follows: ASP (100 g, 0.752 mol) and 85% o-phosphoric acid (10 g, 86.7 mmol) were mixed for 15 min at room temperature in a blender (Oster Osterizer, U.S.A.). The mixture was transferred to a 200-mL four-necked round-bottomed flask equipped with a thermometer, cooler, mechanical stirrer, and N2 inlet and was heated at 200°C under a N2 atmosphere. After 30 min, the reaction mixture changed to a mixture of light-yellow powder and clumps and was ground to a fine powder with a blender. The reaction mixture was heated at 200°C for 6.5 h. The conversion of ASP was 99 wt %. The product was washed several times with water (200 mL) until it was neutral and with methanol (200 mL) and then was dried at 85°C under reduced pressure to yield PSI. Residual ASP was not detected in the PSI.
Bulk Polycondensation of ASP with an Acid Catalyst Using a Twin-Screw Extruder
The general procedure is as follows: ASP (37.6 mol) and 85% o-phosphoric acid (3.76 mol) were mixed for 5 min at room temperature under a N2 atmosphere in a blender (Kawata Super Mixer). A twin-screw extruder [KRC Kneader (Kurimoto, Ltd., Japan); diameter = 50 mm, length = 600 mm) was used for polycondensation. The temperature of the barrel was 260°C, the rotation speed of the screw was 30 rpm, and the amount of the ASP feed was 1.0 kg · h−1 (the average retention time was 16 min). The conversion of ASP was 99 wt %. The product (100 g) was washed several times with water (200 mL) until it was neutral and with methanol (200 mL) and then was dried at 85°C under reduced pressure to yield PSI. Residual ASP was not detected in PSI.
Preparation of PASP-Na
The hydrolysis of PSI was carried out as follows: PSI (3 g), a solution of sodium hydroxide (1.4 g, equivalent per succinimide residue), and deionized water (20 mL) under cooling were added to a 100-mL beaker with a stirring bar. After the mixture was stirred for 1 h, the solution was poured into methanol (300 mL), and the precipitate then was filtered and dried at 40°C under reduced pressure to yield PASP-Na.
Biodegradability of PASP-Na
The biodegradability of PASP-Na was estimated with the OECD 301C method (a modified MITI test). A sample was treated with the standard activated sludge, which was obtained from the Chemicals Inspection & Testing Institute, Japan, at 25 ± 1°C for 28 days. Aniline was used as the standard to check the activity of the standard activated sludge. Biological oxygen demand (BOD) and the amount of total organic carbon (TOC) indicated the consumption of oxygen and the amount of TOC during the evaluation, respectively (both generally are used for evaluating biodegradability). BOD and TOC were measured using an OM3001 coulometer (Ohkura Electric Co., Ltd., Japan) and a TOC-5000A total carbon analyzer (Shimadzu Corporation, Japan), respectively. The amount of removed TOC was calculated from the difference between the amount of TOC before and after the evaluation of biodegradability.
Calcium-Ion-Chelating Ability of PASP-Na
The calcium-ion-chelating ability of the various polymers was determined using a calcium-ion electrode and an ion meter in accordance with the description in a previous article.13 A sample (10 mg) was dissolved in 50 mL of an aqueous solution that had been adjusted to give a calcium chloride concentration of 1.0 × 10−3 mol · L−1 and a potassium chloride concentration of 0.08 mol · L−1. The resulting mixture was stirred at 30°C for 10 min, and the calcium ions in the solution were determined with a calcium-ion electrode (Orion Model 93-20, U.S.A.) and an ion meter (Orion Model 720A, U.S.A.).
RESULTS AND DISCUSSION
Synthesis of PSI
In order to characterize the bulk polycondensation of ASP, the weight loss of ASP (i.e., the process for the dehydration of ASP) with or without an acid catalyst was observed using TGA. As shown in Figure 1, without o-phosphoric acid, a weight loss occurred at about 220°C and then rapidly progressed, whereas the addition of o-phosphoric acid obviously caused the starting temperature of the weight loss to be lower. Although the temperature decreased from about 200 to about 170°C with an increase in the amount of o-phosphoric acid, there was no obvious difference in the dehydration rate among the reactions with 10 ∼ 20 wt %.
The synthesis of PSI by the bulk polycondensation of ASP was examined with or without an acid catalyst under batch reaction conditions. Table I lists typical polycondensation results. The polycondensation in the absence of an acid catalyst needed a higher reaction temperature to achieve a high conversion of ASP; that is, the conversion increased from 41% for 200°C to 96% for 260°C, although the weight-average molecular weight (Mw) of the PSI obtained did not vary significantly (e.g., 7400 and 9000). For the polycondensation with an acid catalyst, the reaction mixture immediately began to form hard clumps, so it was ground to a fine powder with a blender for further polycondensation. Sulfuric acid (H2SO4), p-toluenesulfonic acid (p-CH3C6H4SO3H), methanesulfonic acid (CH3SO3H), and o-phosphoric acid (H3PO4) were effective for producing PSI in a high conversion of ASP, as listed in Table I. On the other hand, the type of acid catalyst apparently affected the Mw of the resulting polymers; that is, the Mw value was 7,200 for H2SO4, 7,800 for p- CH3C6H4SO3H, and 8,800 for CH3SO3H but 19,000 for H3PO4. In addition, for 10 wt % H3PO4, the highest Mw obtained was 24,000 with quantitative conversion.
Table I. Bulk Polycondensation of L-Aspartic Acida
Determined by high-performance liquid chromatography analysis.
Determined by GPC in DMF containing LiBr with polystyrene as the standard.
The continuous process for the bulk polycondensation of ASP was examined using a twin-screw extruder. Table II lists representative results. In the absence of an acid catalyst, the polycondensation slightly proceeded at 260°C; that is, the conversion of ASP was 16%. On the other hand, the conversion was 79% for the polycondensation with 10 wt % o-phosphoric acid even at 200°C and greater than 99% at 260°C. In addition, when the feed rate of ASP was 3.0 kg · h−1, the polycondensation proceeded quantitatively. The Mw values were 22,000 ∼ 24,000, similar to the results for the polycondensation under the batch condition listed in Table I. These results indicate that the continuous method, using a twin-screw extruder, is suitable for the large-scale synthesis of PSI.
Table II. Bulk Polycondensation of L-Aspartic Acid (ASP) Using a Twin-Screw Extrudera
Figure 2 shows the 1H NMR spectra of PSIs prepared through different synthetic methods. For PSI prepared through polycondensation in mesitylene/sulfolane, signals at 2.7 and 3.2 ppm and 5.3 ppm, which were assigned to the methylene and methine protons of the main chain of PSI, respectively, were observed. On the other hand, the 1H NMR spectrum of the PSI prepared by acid-catalyzed bulk polycondensation was similar to that of the PSI prepared by the solvent method, except that there was absorption at 6 ∼ 7 ppm and 8 ∼ 10 ppm, which were assigned to the olefin protons of the fumaramic acid end-group and fumaramide units and the amide protons of the branched and/or opened amide groups, respectively.14 In addition, for the PSI prepared by bulk polycondensation without the acid catalyst, a signal at 11.5 ppm, which was assigned to the imide proton of the succinimide end group,14 strongly was observed. These results indicated that the scission of the polymer chain scarcely occurred throughout the acid-catalyzed bulk polycondensation as well as throughout the acid-catalyzed polycondensation with the mixed solvent because there was no signal because of the succinimide end-group, which was formed through the polycondensation without the acid catalyst.
Biodegradability and Calcium-Ion-Chelating Ability of PASP-Na
PSI easily was hydrolyzed in an alkali medium for conversion into PASP-Na. PSI (Mw = 64,300), which previously was prepared by the polycondensation of ASP in a mixed solvent was used for PASP-Na-1. The other PASP-Nas were prepared from PSIs synthesized by bulk polycondensation with and without an acid catalyst: PASP-Na-2 from PSI with an Mw value of 9000 (Table I), PASP-Na-3 from PSI with an Mw value of 24,000 (Table I), and PASP-Na-4 from PSI with an Mw value of 24,000 (Table II). For all PASP-Nas, the α/β amide ratio and D/L ratio of the aspartic units were about 30/7012 and about 50/50, respectively. PSIs did not exhibit optical activity, indicating that the racemization essentially occurred during the acid-catalyzed polycondensation.
The biodegradability was estimated by the OECD 301C method (a modified MITI test), and the calcium-ion-chelating ability of PASP-Na was measured. The results are summarized in Table III. PASP-Na-1 showed a higher biodegradability (TOC = 89%), which was caused by the highly regulated structure of PSI, as shown in Figure 2(a). On the other hand, for PASP-Na-2 prepared from PSI with the succinimide end-group [Fig. 2(c)], the TOC value was 46%; its biodegradability was half that of PASP-Na-1. Although PSI from the acid-catalyzed bulk polycondensation contained branched and/or opened amide groups [Fig. 2(b)], the TOC value of PASP-Na-3 was 86%, corresponding to that for PASP-Na-1. In addition, the biodegradability of PASP-Na-4 was similar to that for PASP-Na-1 and PASP-Na-3. These results indicated that the difference in PSI synthesized by the acid-catalyzed polycondensation of ASP under different conditions (i.e., the bulk and solution methods and the batch and continuous methods) had little effect on the biodegradability of PASP-Na.
Table III. Biodegradability and Calcium-Ion Chelating-Ability of PASP-Na
Although the calcium-ion-chelating abilities of PASP-Na-3 and PASP-Na-4 were slightly lower than that for PASP-Na-1, they were higher than that for PASP-Na-2. This means that the use of the acid catalyst was important for producing linear PSI as the precursor of PASP-Na, resulting in a higher ability for calcium-ion chelation as well as biodegradability.
The bulk polycondensation of ASP with an acid catalyst was demonstrated to be a practical production method for PSI. o-Phosphoric acid was the most suitable acid catalyst for preparing PSI, in which the scission of the polymer chain scarcely occurred throughout the acid-catalyzed bulk polycondensation as well as throughout the acid-catalyzed polycondensation using the mixed solvent. Additionally, the acid-catalyzed bulk polycondensation using a twin-screw extruder was developed as a continuous method for the large-scale synthesis of PSI. PASP-Na exhibited higher biodegradability and calcium-ion-chelating properties, and the difference in PSI synthesized by the acid-catalyzed polycondensation of ASP under different conditions (i.e., the bulk and solution methods and the batch and continuous methods) had little effect on the biodegradability and calcium-ion-chelating ability of PASP-Na.