Isolation and Purification of Medium Chain Length Poly(3

Sep 15, 2010 - Strategies for recovery and purification of poly[( R )-3-hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass. Martin Koller...
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Biomacromolecules 2010, 11, 2716–2723

Isolation and Purification of Medium Chain Length Poly(3-hydroxyalkanoates) (mcl-PHA) for Medical Applications Using Nonchlorinated Solvents B. Wampfler,*,† T. Ramsauer,† S. Rezzonico,† R. Hischier,† R. Ko¨hling,‡ L. Tho¨ny-Meyer,† and M. Zinn† Empa, Swiss Federal Laboratories for Materials Science and Technology, Lerchenfeldstrasse 5, CH-9014 St. Gallen, Switzerland, and Sigma-Aldrich Production GmbH, Research and Development, Industriestrasse 25, CH-9471 Buchs, Switzerland Received July 9, 2010; Revised Manuscript Received August 31, 2010

A novel process was developed to isolate poly([R]-3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHO) and poly([R]-3-hydroxy-ω-undecenoate-co-3-hydroxy-ω-nonenoate-co-3-hydroxy-ω-heptenoate) (PHUE) from Pseudomonas putida species. Methyl tert-butyl ether (MTBE), ethyl acetate, acetone, and methylene chloride efficiently extracted PHO from freeze-dried biomass. The ratio of solvent to biomass was 15:1 (vol/wt). The nonchlorinated solvents required 18 h of extraction to achieve methylene chloride’s yield of 15 wt % within 60 min. In the case of PHUE, the yield was 15-17 wt % after 60 min of extraction at room temperature, independently of the solvent used. MTBE performed best in life cycle assessment (LCA) if contamination of the environment is avoided. Filtration of the extract containing 8 wt % of raw polyhydroxyalkanoate (PHA) through activated charcoal revealed colorless polymers with less than one endotoxin unit/g. The ratio (v/v) of the solution to activated charcoal was 2:1. The loss (impurities and polymers) amounted up to 50 wt %.

Introduction The discovery of the homopolyester poly(3-hydroxybutyrate) (PHB) by Lemoigne in the 1920s entailed a family of over 100 different aliphatic, biodegradable, and biocompatible polyhydroxyalkanoates (PHAs).1,2 Short chain length PHAs (scl-PHAs) whose 3-hydroxyalkanoate monomers consist of 4-5 carbon atoms are materials of high stiffness and crystallinity.3 They are brittle and break when being strained by more than 10%. Medium chain length PHAs (mcl-PHAs, 6-14 carbon atoms) are semicrystalline thermoplastic elastomers that are suitable for products requiring no mechanical stability. However, when containing at least one double bond in the side chains, they have a significantly larger spectrum of applications. Double bonds can be used for chemical functionalization,4,5 for example, crosslinking to obtain a biodegradable rubber,6-8 covalent binding of natural antifouling agents,9 or the synthesis of organic/ inorganic hybrid polymers.10 Length of side chains and number of double bonds can be controlled to some extent by feeding the cells with appropriate alkanoic and alkenoic acids during biosynthesis in batch cultures11 and more precisely under multiple-nutrient-limited growth conditions in chemostat cultures.12,13 There are currently two protocols used to recover PHA from bacteria. The conventional method involves an organic solvent to extract the polymer from dry biomass and precipitate in a nonsolvent,14 whereas the second one is based on the treatment of the cells with an aqueous mixture of chemical agents and enzymes to lyse bacteria and to gain PHA afterward as latex.15,16 According to the recent review of Jacquel et al.,17 downstream processes have mainly been developed to isolate and purify scl* To whom correspondence should be addressed. E-mail: [email protected]. † Swiss Federal Laboratories for Materials Science and Technology. ‡ Sigma-Aldrich Production GmbH.

PHAs. There is little work that deals with methods for separating mcl-PHA from lipophilic biomass components. In a recent study, six organic solvents were compared with methylene chloride in terms of mcl-PHA’s endotoxicity and yield of extraction from freeze-dried biomass.18 Ethyl acetate and acetone showed the same power of extraction as methylene chloride but led to a polymer containing significantly less endotoxin. Low endotoxin poly(3-hydroxyoctanoate) (PHO) was obtained when freezedried biomass was extracted with n-hexane at 50 °C and cooled down to 0-5 °C after filtration.19 Temperature-controlled redissolution and precipitation in 2-propanol resulted in a purity over 99 wt %. Jiang et al.20 optimized the solvent/nonsolvent system for PHA extraction from Pseudomonas putida and recommended a pretreatment of the dry biomass with methanol for 5 min followed by Soxhlet extraction for 5 h, followed by precipitation of PHA in cold methanol. Purity determinations revealed averages of about 90 wt %, accompanied by the typical uncertainty of gas chromatographic analysis after derivatization. For the large-scale production of PHO, extraction or treatment of the biomass, respectively, using acetone, chloroform, and sodium dodecyl sulfate was investigated.21 Acetone was found to give the highest yield. Purity of 99 ( 0.2% was determined when the PHO concentrate had been precipitated in a volumetrically prepared mixture of 35% methanol, 35% ethanol, and 30% water. Some other studies were designed to avoid the use of organic solvents. Hereby, bacterial cells were treated with a mixture of enzymes and detergents to destroy and remove cell walls, nucleic acids, and peptides.16,22-24 Due to insufficient purity of the obtained mcl-PHA, such water-based extraction could not prevail. The present work was focused on the evaluation of the most environmentally friendly solvent to extract poly([R]-3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHO) and poly([R]-3-hydroxy-ω-undecenoate-co-3-hydroxy-ω-nonenoate-co-3-hydroxyω-heptenoate) (PHUE; Figure 1) from freeze-dried biomass and

10.1021/bm1007663  2010 American Chemical Society Published on Web 09/15/2010

mcl-PHA for Medical Applications

Figure 1. Chemical structure of poly([R]-3-hydroxyoctanoate-co-3hydroxyhexanoate) (PHO) produced by Pseudomonas putida KT2440 when cultured on octanoic acid (top) and of poly([R]-3-hydroxy-ωundecenoate-co-3-hydroxy-ω-nonenoate-co-3-hydroxy-ω-heptenoate) (PHUE) produced by Pseudomonas putida GPo1 when cultured on ω-undecenoic acid (bottom).

to purify the crude extract for use in medicine. Solvent evaluation was approached by a comparison of three nonchlorinated solvents, namely, methyl tert-butyl ether (MTBE), ethyl acetate, and acetone with methylene chloride. Critical parameters were duration of extraction, yield of raw and pure polymers, and impact on the environment described by a life cycle analysis. When PHA is isolated for medical applications, special attention must be paid to the contamination by pyrogenic compounds, that is, endotoxins (lipopolysaccharides) from the outer cell membrane of Gram-negative production strains.25 The United States Pharmacopeia approves maximum 20 endotoxin units (EU) per medical device.26 According to Pegues et al.,27 endotoxin can be removed from aqueous solutions by filtration through activated charcoal. In the field of PHA, activated charcoal has also been used for the purpose of purification, for example, to decolorize and detoxify growth media prior to fermentation,28 to decolorize solutions of hydroxy acid methyl esters obtained by transesterification of PHA and intended for GC analysis,29 or to purify PHB dissolved in chloroform.30 Here, we investigated the potential of activated charcoal to remove endotoxins and other impurities from PHO and PHUE. The overall goal was to develop basics for an efficient industrial process.

Experimental Section Materials. PHO was produced from octanoic acid in a chemostat culture (D ) 0.15 h-1) of Pseudomonas putida KT2440 under dual (carbon, nitrogen) limited growth conditions (C/N ) 15 g g-1). The

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content of PHO (crude extract obtained with methylene chloride) was found to be 15-17% in relation to the freeze-dried biomass. Gas chromatographic analysis revealed the following composition: 9.2 mol % of 3-hydroxyoctanoate and 90.8 mol % of 3-hydroxyhexanoate. PHUE was produced from ω-undecenoate in a chemostat culture (D ) 0.1 h-1) of Pseudomonas putida GPo1 (ATCC 29347) under dual (carbon, nitrogen) limited growth conditions (C/N ) 12.5 g g-1).13 The content of PHUE (crude extract obtained with methylene chloride) was found to be 16-17% in relation to the freeze-dried biomass. Gas chromatographic analysis revealed the following composition: 14 mol % of 3-hydroxy-ω-undecenoate, 63 mol % of 3-hydroxy-ω-nonenoate, and 23 mol % of 3-hydroxy-ω-heptenoate. Methyl tert-butyl ether (MTBE) and methylene chloride, both of technical quality, were purchased from VWR International, Dietikon, Switzerland. Other solvents were of Ph. Eur. quality and were obtained from Ha¨nseler AG, Herisau, Switzerland. Activated charcoal (granular, about 1.5 mm, extra pure, food grade) was purchased from Merck, Darmstadt, Germany. Isolation of the Raw Polymers. PHO and PHUE were each extracted from freeze-dried biomass with the following parameters being varied: temperature of suspension (ambient temperature and 35 °C), solvent (MTBE, methylene chloride, acetone, and ethyl acetate), and period of extraction while stirring. Each experiment was performed three times. A portion of 10-20 g of freeze-dried biomass was manually crushed to small pieces and mixed with the 15-fold amount (vol/wt) of solvent (i.e., 15 mL of solvent per g biomass). Preliminary tests with acetone revealed that extraction could be performed with much lower ratios of solvent to biomass, for example, with 1 mL solvent per g biomass. However, lower ratios than 15 mL g-1 involved timeconsuming filtration steps and plugging of the filter occurred when ratios below 5 mL g-1 were chosen. The solids in the 15-fold amount of solvent were further crushed using a SilentCrusher M (Heidolph, Kelheim, Germany) equipped with a disperser 18 G/M. Afterward, the suspension was stirred for 1, 3, and 18 h, respectively. Extractions at 35 °C were performed in a temperature-controlled water bath. After stirring, the phases were separated by pressure filtration (air, 1 bar) through a metallic lace tissue Duplex 15 (diameter of 47 mm, G. Bopp, Zurich, Switzerland) using a 200 mL pressure vessel (SM 16249, Sartorius, Go¨ttingen, Germany). The filtrate was concentrated in a rotary evaporator. Residual solvent was removed by drying the gel-like solid in a vacuum dryer (VTR 5036, Heraeus, Hanau, Germany) for 24 h at 40 °C and 200 mbar. The raw polymer was weighted and afterward stored at -20 °C to prevent autoxidation.31 The solvent from the concentration step was reused for further extractions. The yield of the raw polymers was calculated in wt% with respect to the dry biomass. Purification of the Raw Polymers. A defined portion of the raw polymer was dissolved in the 15-fold quantity of acetone (15 mL of acetone per g of raw polymer) by stirring for at least 2 h at ambient temperature. The solution was slowly filtered twice through a column filled with activated charcoal (0.5 mL of charcoal per mL of solution to be filtered). When containing small amounts of activated charcoal, the filtrate was subjected to a pressure filtration, as described above; however, a membrane filter 0.45 µm (RC 55, Whatman, Go¨ttingen, Germany) was used and an overpressure of 1-4 bar was set. The filtrate was concentrated in a rotary evaporator at 40 °C and 300 to 400 mbar until the solution became viscous. To precipitate the PHA, the viscous solution was added dropwise under stirring to a 6-fold quantity of ethanol previously cooled down to 4 °C. After standing for 30 min, the mixture was decanted and the solid phase was dried in a vacuum dryer for 24 h at 40 °C and 200 mbar. The pure PHA was stored at -20 °C. Acetone from the concentration step was reused for further processing. Methods of Characterization. Weight average molecular weight and molecular weight distribution of the samples were determined by size exclusion chromatography (SEC) using a differential refractive index detector (SEC apparatus: Viscotek, Houston, TX). About 30 mg of each sample was dissolved in 10 mL of tetrahydrofuran (THF).

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Aliquots of 100 µL of the polymer solution were injected and separated on three sequentially coupled SEC columns (300 × 8 mm, pore sizes 103, 105, and 106 Å, PSS, Mainz, Germany) at 35 °C and applying a flow rate of 1 mL/min with THF. Calibration was performed with 10 narrow standard polystyrene samples from PSS (2 × 103 g/mol to 2.13 × 106 g/mol). The partitions of 3-hydroxyalkanoates in PHO and PHUE were determined by gas chromatography after conversion of the polymer into the methyl ester of the monomers using boron trifluoride in methanol.32 The analyses of the resulting hydroxyalkenoic and hydroxyalkanoic acid methyl esters were performed with a gas chromatograph (GC Trace 2000 Series, CE Instruments, Rodano, Italy) equipped with a flame ionization detector and a capillary column DB-Wax (30 m × 0.32 mm, film thickness: 0.3 µm, Agilent, Santa Clara, CA). UV/vis absorption spectra were taken from solutions of PHO and PHUE in methylene chloride at ambient temperature using a Cary 50 Bio UV-visible spectrophotometer from Varian, Palo Alto, CA. NMR spectra were measured with a Bruker BioSpin 600 MHz NMR spectrometer (Bruker Biospin AG, Fa¨llanden, Switzerland) at 300 K using a 5 mm broad-band probe. A total of 10 mg of polymer was dissolved in 0.7 mL of CDCl3. Chemical shifts are given in ppm relative to the remaining signals of chloroform as internal reference (1H NMR: 7.26 ppm; 13C NMR: 77.0 ppm). 1H NMR spectrum was recorded at 600.2 MHz with the following parameters: 6.5 µs 90° pulse length, 10823 Hz spectral width, 64k data points, 24 scans, and relaxation delay 20 s. 13C NMR spectra were recorded at 150.92 MHz with 1H WALTZ decoupling. Other parameters were chosen as follows: 3.2 µs 45° pulse length, 37594 Hz spectral width, 64k data points, 500 scans, relaxation delay 10 s, and decoupling field 2.5 kHz. Nitrogen was determined according to Kjeldahl (Apparatus: Bu¨chi 321, Bu¨chi, Flawil, Switzerland). About 300 mg of the sample was digested by means of concentrated sulphuric acid (Merck, for the determination of nitrogen) in the presence of a copper catalyst (Merck, Kjeldahl tablet). After addition of 10 M sodium hydroxide to the solution, ammonia liberated from the reaction was steam-distilled and absorbed in a solution of 4% (w/v) of boric acid (puriss, Sigma-Aldrich, Buchs, Switzerland) in water. The resulting ammonium borate was visually titrated with 0.01 M hydrochloric acid (FIXANAL, SigmaAldrich) using a Tashiro indicator (solvent: ethanol). Endotoxicity was determined by means of the Limulus Amebocyte Lysate (LAL) assay ENDOSAFE-PTS (Charles River, L’Arbresle Cedex, France). Samples were prepared by dissolving 200 mg of polymer in 13 mL of chloroform and casting into depyrogenized glass Petri dishes (d ) 4 cm), followed by drying under nearly saturated atmosphere at 40 °C and 200 mbar for 24 h. To extract endotoxins, samples were incubated with 3 mL of endotoxin-free water (LAL Reagent Water ENDOSAFE) at 37 °C for 24 h. Aliquots of 200 µL were withdrawn and each mixed with 100 µL of dispersing agent BD100 (ENDOSAFE) and 100 µL of endotoxin-free water. The resulting solutions were measured according to the manufacturer’s instructions with the spectrophotometer ENDOSAFE-PTS using cartridges PTS2001. PTS cartridges allow testing of sample and positive sample control in duplicates. When endotoxin of the raw polymers was determined, water extracts were diluted 10 times with endotoxin-free water and no dispersing agent was added. In this case, measurements were taken with the cartridge ENDOSAFE PTS2005. Evaluation of Results. Tests were evaluated using robust statistics. Results are given as median, and repeatability is specified as median of the absolute deviation (MAD). To determine MAD,33 the single values were first subtracted from the median. These deviations were then converted into absolute values from which the median was determined () MAD). Combined standard uncertainty was evaluated according to the GUM (Guide to the expression of uncertainty in measurement).34

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Figure 2. Yields of extraction and purification. Crude extracts obtained from freeze-dried biomass at ambient temperature with MTBE, methylene chloride, ethyl acetate, and acetone (1, 3, and 18 h) and mass fractions of PHA recovered thereof by purification over activated charcoal (purified). Values of crude extract are represented as median ( MAD (n ) 3). Values of pure PHA are single values with combined standard uncertainties.

Experimental Results Influence of the Solvent and of Activated Charcoal on the Amount of PHO and PHUE Extracted from FreezeDried Biomass. Four different PHA solvents were compared in a first step to optimize the downstream processing. The mass fractions extractable from freeze-dried biomass at ambient temperature are depicted in Figure 2, together with the fractions of the polymers subsequently purified with activated charcoal. The solvents were arrayed according to their positions in the eluotropic row:35 MTBE (polarity 2.5) < methylene chloride (3.1) < ethyl acetate (4.4) < acetone (5.1). As can be seen in Figure 2, the mass fractions of raw PHO depended on the solvent used. Methylene chloride yielded the highest amounts and the mass fraction after 1 h (15 wt %) was close to the one after 18 h (17 wt %). The mass fractions obtained with ethyl acetate and acetone increased with increasing periods of extraction and reached maximum values of 12-13 wt %, which was in the case of acetone still significantly below the one of methylene chloride. An increase in yield with increasing extraction time could also be observed with MTBE whose maximum value after 18 h was comparable to the values of methylene chloride. Increase in the temperature up to 35 °C did not cause any effect when the extraction was performed with MTBE and acetone for 1 h (results not shown). In contrast to PHO, the mass fraction of raw PHUE depended neither on the solvent nor on the duration of extraction; medians randomly varied between 15 and 17.5 wt %. Similar values were found for extractions at 35 °C (Figure S1, Supporting Information). For both polymers PHO and PHUE, the loss of material by purification over activated charcoal was mostly between 47 and 53 wt %. A loss of more than 60% was observed for PHO obtained with MTBE, probably indicating coextraction of high amounts of lipophilic cell components that were adsorbed by activated charcoal. In short-term extractions, highest amounts of pure PHO were obtained with methylene chloride followed by the nonchlorinated solvents. In case of PHUE, the amount of the pure polymer neither depended on the solvent used for extraction nor on the period of extraction. SEC Analysis. Samples of raw and purified PHO and PHUE were analyzed regarding weight average molecular weight (Mw)

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Table 1. Weight Average Molecular Weight (Mw) and Polydispersity Index (PDI) of PHO and PHUE Extracted from Freeze-Dried Biomass at Room Temperature with Four Different Solventsa PHO solventb MTBE methylene chloride ethyl acetate acetone

quality of polymer Mwd (kDa)

PHUE PDIf

Mwd (kDa)

PDIf

raw purec raw

119 ( 3 155 ( 3 128 ( 5

3.9 ( 0.1 1.8 ( 0.05 3.8 ( 0.2

252 ( 10 288 243 ( 3

3.2 ( 0.3 2.1 2.4 ( 0.3

purec raw purec raw purec

156 ( 1 116 ( 3 132 ( 3 117 ( 4 138 ( 3

1.9 ( 0.1 3.7 ( 0.1 1.7 ( 0.05 3.9 ( 0.1 1.7 ( 0.05

248 234 ( 3 247 252 ( 4 274

2.6 2.6 ( 0.1 2.0 2.9 ( 0.3 2.1

a Data are medians of 4-10 individual experiments. Errors are indicated as medians of the absolute deviations (MAD). Results without indication of MAD are single values. b Solvent used to extract the polyesters from freeze-dried biomass. For the treatment with activated charcoal, all samples were dissolved in acetone. c Purification with activated charcoal. d Mw ) weight average molecular weight (1 Da ) 1 g mol-1). f PDI ) polydispersity.

Figure 4. SEC chromatograms of PHUE obtained by extraction from freeze-dried biomass at ambient temperature and at 35 °C. SEC profiles were normalized to identical peak areas after baseline correction: (a) raw PHUE extracted with MTBE at ambient temperature for 1 h, (b) raw PHUE extracted with methylene chloride and acetone, respectively, at 35 °C for 3 h, (c) raw PHUE extracted with ethyl acetate at ambient temperature for 2 h, and (d) pure PHUE extracted with ethyl acetate at 35 °C for 18 h after additional purification with activated charcoal.

Figure 5. UV/vis spectra of raw and pure PHO and PHUE. Figure 3. SEC chromatograms of PHO obtained by extraction from freeze-dried biomass at ambient temperature. SEC profiles were normalized to identical peak areas after baseline correction: (a) raw PHO extracted with acetone for 1 h, (b) raw PHO extracted with acetone for 18 h, (c) raw PHO extracted with methylene chloride for 1 and 18 h, respectively, and (d) pure PHO extracted with MTBE for 18 h.

and polydispersity index (PDI). It was found that the dependence of Mw and PDI values on the duration of extraction was low. Therefore, all single values from the different extraction periods were pooled and the medians were determined for each solvent. This was performed for the results obtained at both temperatures of extraction. Both PHO and PHUE extracted with an individual solvent at ambient temperature had about the same average Mw value as the corresponding one extracted with the same solvent at 35 °C. Typically, at ambient temperature, the overall values varied from 116 (ethyl acetate) to 128 kDa (methylene chloride) for PHO and from 234 (ethyl acetate) to 252 kDa (MTBE) for PHUE, respectively (Table 1). Samples of raw PHO contained more polymeric impurities (Figure 3) than the ones of raw PHUE (Figure 4). The peaks at 30.2 (PHO) and 31.4 mL (PHUE) were based on polymeric impurities having molecular weights Mw below 10 kDa. When the period of the extraction of PHO with acetone was increased from 1 to 18 h, the area of the peak at 30.2 mL decreased in favor of the PHO peak, which on his part shifted slightly toward higher molecular weights (Figure 3, chromatograms a and b). Similar but less pronounced behavior was observed with ethyl acetate and MTBE as well. Impurities were obviously dissolved faster than PHO of high molecular weight. Methylene chloride revealed the best dissolving properties for PHO; this solvent excelled due to nearly identical yields (Figure 2) and chromatograms (c in Figure 3) when periods of extraction were varied, thus, indicating fast

kinetics of dissolution. The comparison of chromatograms of raw PHUE showed a nearly perfect overlap of the peaks independent of the temperature (Figure 4). The slight variation of the peak areas was due to the second peak at 31.4 mL, which varied independently of the duration of extraction and of the solvent chosen. The variation of the impurity caused a variation of PDI between 2.4 and 3.2 (Table 1). An opposite behavior was found when PHUE was extracted from dry biomass with acetone using the Soxhlet method: an Mw of only 150 kDa was determined after an extraction period of 16 h. A slight decrease of Mw was observed with ethyl acetate as well: Mw of PHUE was found to be 200 kDa when the biomass had been extracted for 24 h (data not shown). Independently of the solvent, filtration through activated charcoal enhanced the molecular weight Mw of raw PHO and PHUE that were obtained by classical extraction (Table 1). In the case of PHO, the deviation of the Mw values between pure and raw PHO was based on the disappearance of the high content of impurities (Figure 3). In the case of PHUE, the main reason was rather a loss of molecules of lower molecular weight; the PHUE peak shifted toward lower retention volumes (Figure 4). The measured PDI values ranging from 1.7 to 1.9 (PHO) and from 2.0 to 2.6 (PHUE) indicate normal distributions because polyesters undergo transesterification that leads to a broadening of the partition.36 UV/Vis Absorption of Raw and Pure PHA. Some UV/vis spectra are depicted in Figure 5. The spectra of the raw polymers show a relative maximum between 275 and 280 nm, which is characteristic for proteins, that is, for a combination of the amino acids tyrosine, tryptophan, and arginine. There is no indication for the presence of nucleic acids with their strong absorption at 260 nm. The absorption of the raw polymers slightly decreased from 300 nm into the energy-rich part of the visible spectrum,

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Table 2. Content of Nitrogen in Raw and Purified PHO and PHUE Extracted from Freeze-Dried Biomass at Ambient Temperature Using Four Different Solventsa

solvent

PHO (µg g-1)

PHUE (µg g-1)

rawb

pureb

rawc

pured

36 ( 18 35 ( 11 40 ( 23 67 ( 43

730 ( 20 840 ( 5 900 ( 25 800 ( 100

330 ( 30 240 ( 5 270 ( 5 360 ( 5

MTBE 740 ( 440 methylene chloride 1880 ( 150 ethyl acetate 1130 ( 590 acetone 1260 ( 450

a Errors are given as MAD. b Median of two single values. c Median of five averages of duplicates. d Median of four single values.

Table 3. Content of Endotoxin in Raw and Purified PHO and PHUE Extracted from Freeze-Dried Biomass at Ambient Temperature Using Four Different Solvents endotoxin in PHO (EU g-1)

endotoxin in PHUE (EU g-1)

solvent

raw

pure

rawa

pure

MTBE methylene chloride ethyl acetate acetone

600 >750 >750 >750