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Chapter 22 Peptide Synthesis Waste Reduction and Reclamation of Dichloromethane Richard V. Joao, Ilona Linins, and Edward L. Gershey Rockefeller University, 1230 York Avenue, New York, NY 10021 Waste management has two important benefits. Obviously, reducing volume decreases costs and liabilities and it may also effect generator status. Reclaiming chemicals is a cost effective means of minimizing waste. Peptide synthesis, an important process in academic research and industrial production laboratories, produces large amounts of chemical waste, the primary component of which is methylene chloride (dichloromethane, DCM). Other significant components are ethanol, methanol, dimethylformamide, and trifluoroacetic acid. Due to the high halogen content of the mixture, disposing of this is costly. At this university reclaiming DCM reduces the volume of chemicals shipped as waste by 25%. Separating and purifying DCM is a four part process involving distillation, aqueous extraction, sieving, and analysis. Recovery of the distillate between 37 and 40.5°C results in an azeotropic mixture which is approximately 95% DCM, 5% methanol, and trace amounts of other components. Most of the methanol is removed by two 1:1 water washes. The remaining trace amounts of methanol and water are removed by 4 angstrom molecular sieves. High sensitivity capillary gas chromatography andflameionization detection indicate that the product is greater than 99.9% pure. Reclamation and recycling of solid phase peptide synthesis effluents are important means of waste reduction. In recent years automated peptide synthesis has become an important and widely practiced biochemical technique. However, a single investigator working with an automated peptide synthesizer can generate 20 to 40 liters of waste per week. The waste produced is approximately 60% dichloromethane (DCM). Assuming that 75% of the DCM can be recovered, a yearly waste reduction of 936L of halogenated solvent and a sixteen hundred dollar decrease in waste disposal costs per investigator can be realized. Also, at a cost of approximately eight dollars per liter for HPLC grade DCM, a recycling program could save approximately seven thousand dollars per investigator, annually. Besides decreasing costs, reducing wastes also lowers liabilities. RCRA places a "cradle to grave" responsibility and provides for penalties of up to $50,000 per day and/or by imprisonment for up to two years (1). The liabilities associated with waste disposal include not only the potential criminal penalties but also the cost of negative 0097-6156/90A)422-0378$06.00/0 © 1990 American Chemical Society In Emerging Technologies in Hazardous Waste Management; Tedder, D. William, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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22. JOAOETAL.
Waste Reduction atid Reclamation of Dichloromethane
public perception of waste generators. DCM has been found in 11% of the waste disposal sites on the National Priorities List (2). Media attention to improper waste disposal practices has resulted in a public hesitant to accept waste handling facilities. This has led to costlier disposal solutions, higher transportation costs and restricted access to these facilities. Peptide synthesis (3, 4) produces a complex waste stream that is primarily dichloromethane (DCM). It also contains Ν,Ν-dimethylformamide (DMF), methanol (MeOH), ethanolamine trifluoroacetate (EATFA), and diïsopropylethylamine (DiEA). DCM and DMF are used throughout the peptide synthesis process. DiEA is used to accelerate the esterification step. MeOH is used to wash away excess reactants between steps. TFA is used to remove protecting groups from the amino acids which are being added to the peptide chain. Ethanolamine is used to neutralize the TFA to produce a free base which can be coupled with the next amino acid. This neutralization results in the production of EATFA. For recycling to a peptide synthesizer, it is crucial that the purified DCM be anhydrous and uncontaminated with these reagents. EXPERIMENTAL DISTILLATION. Material was distilled with a spinning band type still (B/R model 8400, Pasadena, MD). This distillation apparatus has a separation capability of 30 theoretical plates. The mantle rate was set at 75% of the maximum. An equilibrium time of thirty minutes was selected. Test samples were taken over a temperature range from 35°C to 45°C at 10 minute intervals. The head and pot temperatures were also recorded. The still was then programmed to begin collecting material at 37°C and finish at 41°C (the boiling point of DCM is 40.1°C). The reflux ratio was set at 10. A maximum of 10L of peptide synthesis waste may be distilled at a time and requires approximately 12 hours. AQUEOUS EXTRACTION. Much of the MeOH and other water-soluble contaminants were removed by aqueous extraction. The distillate was extracted twice at a ratio of 1:1 (distillate:water) by shaking in 4L separatory funnels. Aqueous extraction was tried before and after distillation, the number of extractions was varied from one to three, and the ratios of 1:2, 1:1, and 2:1 (distillate:water) were investigated. DESICCATION. Trace water and MeOH were removed by batch adsorption with 4Â molecular sieves (Aldrich, Milwaukee, Wi). Desiccation using 3À molecular sieves and anhydrous calcium chloride was also examined. GAS CHROMATOGRAPHIC ANALYSIS. All samples were run on a Gas Chromatograph (Varian Model 3700, Sugar Land, TX) modified for use with capillary columns; data were analyzed with an automated integrator (Shimadzu Chromatopac C-R3A, Kyoto, Japan). Two protocols were used. A screening protocol was used to analyze peptide synthesis waste and monitor the product throughout the recycling process, 0.2 microliters of sample were introduced into a direct injector at 250°C connected to a capillary column (Supelco SPB-35 Glass Capillary, 60m χ 0.75mm χ 1.0 micron film). Helium was used as a carrier gas (Matheson Ultra High Purity - 99.999%). A Thermal Conductivity Detector (TCD) was used, with its temperature set at 270°C, the TCD range was set at 0.5mV. The filament temperature was set at 290°C. The oven temperature was held at ambient for 5 minutes and then increased to 250°C at a rate of 15°C/min. In the second protocol, for quality control of the final product,
In Emerging Technologies in Hazardous Waste Management; Tedder, D. William, et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1990.
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EMERGING TECHNOLOGIES IN HAZARDOUS WASTE MANAGEMENT
0.5 microliters (unsplit) of sample were introduced into an injector set at 150°C and onto a column (Hewlett-Packard Ultra 2 Capillary column, 25m χ 0.32mm χ 0.52 micron film). Helium was used as a carrier gas. A Flame Ionization Detector (FID) was used, its temperature set at 150°C and its range set at 10" amp/mV. 12
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GAS CHROMATOGRAPHY-MASS SPECTROMETRY fGC-MSY Peptide synthesis waste samples were analyzed and components identified on a HewlettPackard 5890 Gas Chromatograph, outfitted with a column described in GC protocol 2 above, coupled with a VG Analytical Quadrupole Mass Spectrometer. This work was performed by the Rockefeller University Mass Spectrometry Service Laboratory. RESULTS AND DISCUSSION ANALYSIS OF PEPTIDE SYNTHESIS WASTE. Figure la illustrates the composition of a typical sample of peptide synthesis waste. DCM accounts for 56.7%, DMF constitutes 35.9%, MeOH 6.7%, DiEA 0.5%, EATFA 0.2%. Due to the nature of the peptide synthesis process, the quantity of a given solvent can vary significantly from one waste sample to the next. The specific solvents used also vary depending on the synthesis chemistry used. Analysis of many samples from different sources of peptide synthesis waste show that certain generalizations can be made. DCM is consistently the largest component of all peptide synthesis wastes, varying from 50% to 80%. The second largest component, DMF makes up between 20% and 35% of the waste volume. The amount of methanol varies from 3% to 15% and the next significant component, EATFA, comprises from
