Safe Use of Hazardous Chemicals

George Lunn1, Gretchen Lawler2

1 null, Baltimore, Maryland, 2 Purdue University West, Lafayette, Indiana
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Appendix 1H
DOI:  10.1002/0471142727.mba01hs58
Online Posting Date:  May, 2002
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Abstract

This appendix presents useful basic information, including common abbreviations, useful measurements and data, characteristics of amino acids and nucleic acids, information on radioactivity and the safe use of radioisotopes and other hazardous chemicals, conversions for centrifuges and rotors, characteristics of common detergents, and common conversion factors.

     
 
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Table of Contents

  • Disposal Methods
  • Basic Protocol 1: Disposal of Benzidine and Diaminobenzidine
  • Alternate Protocol 1: Decontamination of Spills Involving Benzidine and Diaminobenzidine
  • Alternate Protocol 2: Decontamination of Aqueous Solutions of Benzidine and Diaminobenzidine
  • Support Protocol 1: Analytical Procedures to Detect Benzidine and Diaminobenzidine
  • Basic Protocol 2: Disposal of Biological Stains
  • Alternate Protocol 3: Continuous‐Flow Decontamination of Aqueous Solutions of Biological Stains
  • Support Protocol 2: Analytical Procedures to Detect Biological Stain
  • Basic Protocol 3: Disposal of Chlorotrimethylsilane and Dichlorodimethylsilane
  • Basic Protocol 4: Disposal of Cyanides and Cyanogen Bromide
  • Support Protocol 3: Analytical Procedure to Detect Cyanide
  • Basic Protocol 5: Disposal of Dimethyl Sulfate, Diethyl Sulfate, Methyl Methanesulfonate, Ethyl Methanesulfonate, Diepoxybutane, and 1,3‐Propane Sultone
  • Support Protocol 4: Analytical Procedure to Detect the Presence of Dimethyl Sulfate, Diethyl Sulfate, Methyl Methanesulfonate, Ethyl Methanesulfonate, Diepoxybutane, and 1,3‐Propane Sultone
  • Basic Protocol 6: Disposal of Ethidium Bromide and Propidium Iodide
  • Alternate Protocol 4: Decontamination of Equipment Contaminated with Ethidium Bromide
  • Alternate Protocol 5: Decontamination of Ethidium Bromide in Isopropanol Saturated with Cesium Chloride
  • Alternate Protocol 6: Decontamination of Ethidium Bromide in Isoamyl Alcohol and 1‐Butanol
  • Support Protocol 5: Analytical Procedure to Detect Ethidium Bromide or Propidium Iodide
  • Basic Protocol 7: Disposal of Hydrogen Peroxide
  • Basic Protocol 8: Reduction of Iodine
  • Basic Protocol 9: Disposal of Mercury Compounds
  • Alternate Protocol 7: Decontamination of Waste Water Containing Mercury
  • Support Protocol 6: Analytical Procedure to Detect Mercury
  • Basic Protocol 10: Disposal of Sodium Azide
  • Support Protocol 7: Analytical Procedures to Detect Sodium Azide
  • Support Protocol 8: Analytical Procedure to Detect Nitrite
  • Basic Protocol 11: Disposal of Enzyme Inhibitors
  • Support Protocol 9: Analytical Procedures to Detect Enzyme Inhibitors
  • Reagents and Solutions
  • Tables
     
 
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Materials

Basic Protocol 1: Disposal of Benzidine and Diaminobenzidine

  Materials
  • Benzidine or diaminobenzidine tetrahydrochloride dihydrate
  • 0.1 M HCl (for benzidine)
  • 0.2 M potassium permanganate: prepare immediately before use
  • 2 M sulfuric acid
  • Sodium metabisulfite
  • 10 M potassium hydroxide (KOH)
  • Additional reagents and equipment for testing for the presence of aromatic amines (see protocol 4)

Alternate Protocol 1: Decontamination of Spills Involving Benzidine and Diaminobenzidine

  • Glacial acetic acid
  • 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid: prepare immediately before use
  • Absorbent material (e.g., paper towels, Kimwipes)
  • High‐efficiency particulate air (HEPA) vacuum (Fisher)
  • Additional reagents and equipment for testing for the presence of aromatic amines (see protocol 4)
CAUTION: This procedure may damage painted surfaces and Formica.

Alternate Protocol 2: Decontamination of Aqueous Solutions of Benzidine and Diaminobenzidine

  • Aqueous solution of benzidine or diaminobenzidine
  • 1 N HCl or NaOH
  • 3% (v/v) hydrogen peroxide
  • recipeHorseradish peroxidase (see recipe)
  • 1:1 (v/v) 0.2 M potassium permanganate/2 M sulfuric acid
  • 5% (w/v) ascorbic acid
  • Porous glass filter or Sorvall GLC‐1 centrifuge or equivalent
  • Additional reagents and equipment for testing for the presence of aromatic amines (see protocol 4)

Support Protocol 1: Analytical Procedures to Detect Benzidine and Diaminobenzidine

  Materials
  • Decontaminated aromatic amine solution
  • 10:30:20 (v/v/v) acetonitrile/methanol/1.5 mM potassium phosphate buffer (1.5 mM K 2HPO 4/1.5 mM KH 2PO 4) (benzidine) or 75:25 (v/v) methanol/1.5 mM potassium phosphate buffer (diaminobenzidine)
  • 250‐mm × 4.6‐mm‐i.d. Microsorb C‐8 reversed‐phase HPLC column (Varian) or equivalent
  • Additional reagents and equipment for reversed‐phase liquid chromatography (Snyder et al., )

Basic Protocol 2: Disposal of Biological Stains

  Materials
  • Amberlite XAD‐16 resin (Supelco)
  • 100 µg/ml biological stain in water
  • Additional reagents and equipment for testing for the presence of biological stain (see protocol 7)
For batch decontamination of 20 ml stain

Alternate Protocol 3: Continuous‐Flow Decontamination of Aqueous Solutions of Biological Stains

  • 25 µg/ml biological stain in water
  • Methanol (optional)
  • 300‐mm × 11‐mm‐i.d. glass chromatography column fitted with threaded adapters and flow‐regulating valves at top and bottom nut and insert connectors, and insertion tool (Ace Glass) or 300‐mm × 15‐mm‐i.d. glass chromatography column (Spectrum 124010, Fisher)
  • Glass wool
  • 1.5‐mm‐i.d. × 0.3‐mm‐wall Teflon tubing
  • Waring blender (optional)
  • Rubber stopper fitted over a pencil
  • QG 20 lab pump (Fluid Metering)
  • Additional reagents and equipment for testing for the presence of biological stain (see protocol 7)

Support Protocol 2: Analytical Procedures to Detect Biological Stain

  Materials
  • Filtrate or eluate from biological stain decontamination (see protocol 5 or protocol 6)
  • recipepH 5 buffer (see recipe)
  • 1 M KOH solution
  • 20 µg/ml calf thymus DNA in TBE electrophoresis buffer, pH 8.1 ( appendix 22)

Basic Protocol 3: Disposal of Chlorotrimethylsilane and Dichlorodimethylsilane

  Materials
  • Cyanide (e.g., NaCN) or cyanogen bromide (CNBr)
  • 1 M NaOH
  • 5.25% (v/v) sodium hypochlorite (NaOCl; i.e., standard household bleach)
  • Additional reagents and equipment for testing for the presence of cyanide (see protocol 10)

Basic Protocol 4: Disposal of Cyanides and Cyanogen Bromide

  Materials
  • Cyanide or cyanogen bromide decontamination reaction mixture (see protocol 9)
  • recipePhosphate buffer (see recipe)
  • 10 mg/ml sodium ascorbate in water: prepare fresh daily
  • 100 mg/liter NaCN in water: prepare fresh weekly
  • 10 mg/ml chloramine‐T in water: prepare fresh daily
  • recipeCyanide detection reagent (see recipe)
  • Sorvall GLC‐1 centrifuge or equivalent

Support Protocol 3: Analytical Procedure to Detect Cyanide

  Materials
  • Dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3‐propane sultone
  • 5 M NaOH
  • Additional reagents and equipment for testing for the presence of dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3‐propane sultone (see protocol 12)

Basic Protocol 5: Disposal of Dimethyl Sulfate, Diethyl Sulfate, Methyl Methanesulfonate, Ethyl Methanesulfonate, Diepoxybutane, and 1,3‐Propane Sultone

  Materials
  • Reaction mixture containing dimethyl sulfate, diethyl sulfate, methyl methanesulfonate, ethyl methanesulfonate, diepoxybutane, or 1,3‐propane sultone
  • 98:2 (v/v) 2‐methoxyethanol/acetic acid
  • 5% (w/v) 4‐(4‐nitrobenzyl)pyridine in 2‐methoxyethanol
  • Piperidine
  • 2‐Methoxyethanol

Support Protocol 4: Analytical Procedure to Detect the Presence of Dimethyl Sulfate, Diethyl Sulfate, Methyl Methanesulfonate, Ethyl Methanesulfonate, Diepoxybutane, and 1,3‐Propane Sultone

  Materials
  • Ethidium bromide– or propidium iodide–containing solution in water, buffer, or 1 g/ml cesium chloride
  • 5% (v/v) hypophosphorous acid: prepare fresh daily by diluting commercial 50% reagent 1/10
  • 0.5 M sodium nitrite: prepare fresh daily
  • Sodium bicarbonate
  • Additional reagents and equipment for testing for the presence of ethidium bromide or propidium iodide (see protocol 17)

Basic Protocol 6: Disposal of Ethidium Bromide and Propidium Iodide

  Materials
  • Equipment contaminated with ethidium bromide
  • recipeDecontamination solution (see recipe)
  • Sodium bicarbonate
  • Additional reagents and equipment for testing for the presence of ethidium bromide (see protocol 17)

Alternate Protocol 4: Decontamination of Equipment Contaminated with Ethidium Bromide

  Materials
  • Ethidium bromide in isopropanol saturated with cesium chloride
  • recipeDecontamination solution (see recipe)
  • Sodium bicarbonate
  • Additional reagents and equipment for testing for the presence of ethidium bromide (see protocol 17)

Alternate Protocol 5: Decontamination of Ethidium Bromide in Isopropanol Saturated with Cesium Chloride

  Materials
  • Ethidium bromide in isoamyl alcohol or 1‐butanol
  • recipeDecontamination solution (see recipe)
  • Activated charcoal
  • Sodium bicarbonate
  • Separatory funnel
  • Additional reagents and equipment for testing for the presence of ethidium bromide

Alternate Protocol 6: Decontamination of Ethidium Bromide in Isoamyl Alcohol and 1‐Butanol

  Materials
  • Reaction mixture containing ethidium bromide or propidium iodide
  • TBE buffer, pH 8.1 ( appendix 22)
  • 20 µg/ml calf thymus DNA in TBE buffer, pH 8.1

Support Protocol 5: Analytical Procedure to Detect Ethidium Bromide or Propidium Iodide

  Materials
  • 30% hydrogen peroxide
  • 10% (w/v) sodium metabisulfite
  • 10% (w/v) potassium iodide
  • 1 M HCl
  • 1% (w/v) starch indicator solution

Basic Protocol 7: Disposal of Hydrogen Peroxide

  Materials
  • Iodine
  • 10% (w/v) sodium metabisulfite
  • 1 M HCl
  • 1% (w/v) starch indicator solution

Basic Protocol 8: Reduction of Iodine

  Materials
  • Solution containing ≤1600 ppm mercuric acetate or ≤1350 ppm mercuric chloride
  • Dowex 50X8‐100 ion‐exchange resin or Amberlite IRA‐400(Cl) ion‐exchange resin
  • Additional reagents and equipment to test for the presence of mercury (see protocol 22)

Basic Protocol 9: Disposal of Mercury Compounds

  Materials
  • Iron powder, 60 mesh
  • Waste water containing ≤2.5 ppm mercury
  • 6‐mm‐i.d. column

Alternate Protocol 7: Decontamination of Waste Water Containing Mercury

  Materials
  • Sodium azide or solution containing sodium azide
  • Ceric ammonium nitrate
  • 10% (w/v) potassium iodide
  • 1 M HCl
  • 1% (w/v) starch indicator solution
  • Sodium nitrite
  • 4 M sulfuric acid
  • Additional reagents and equipment to test for the presence of sodium azide (see protocol 24) or nitrite (see protocol 25)

Support Protocol 6: Analytical Procedure to Detect Mercury

  Materials
  • Reaction mixture from sodium azide treated with ceric ammonium nitrate or sodium nitrite
  • 1 M KOH
  • Acetonitrile
  • recipeSodium azide indicator solution (see recipe)
  • 0.2 M HCl
  • 20% (w/v) sulfamic acid
  • 3,5‐dinitrobenzoyl chloride
  • 50:50 (v/v) acetonitrile/water
  • Sorvall GLC‐1 centrifuge or equivalent
  • 25‐cm × 4.6‐mm‐i.d. Microsorb C‐8 reversed‐phase HPLC column (Varian) or equivalent
  • Additional reagents and equipment for reversed‐phase liquid chromatography (Snyder et al., )

Basic Protocol 10: Disposal of Sodium Azide

  Materials
  • α‐Naphthylamine
  • 15% (v/v) aqueous acetic acid
  • recipeSulfanilic acid solution (see recipe)
  • Reaction mixture treated to remove excess nitrite (see protocol 24, step )

Support Protocol 7: Analytical Procedures to Detect Sodium Azide

  Materials
  • Solutions of APMSF, AEBSF, PMSF, DFP, TLCK, or TPCK in buffer, DMSO, isopropanol, or water
  • 1 M NaOH
  • Glacial acetic acid
  • Additional reagents and equipment for testing for the presence of the enzyme inhibitors (see protocol 27)

Support Protocol 8: Analytical Procedure to Detect Nitrite

  Materials
  • Decontaminated enzyme inhibitor solutions
  • Acetonitrile (HPLC grade)
  • Water (HPLC grade)
  • 0.1% (v/v) trifluoroacetic acid in water
  • 10 mM phosphate buffer, pH 7
  • 250‐mm × 4.6 mm‐i.d. Microsorb C‐8 reversed‐phase HPLC column (Varian) or equivalent
  • Additional reagents and equipment for reversed‐phase liquid chromatography (Snyder et al., )
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Figures

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Literature Cited

Literature Cited
   Bretherick, L. (ed.) 1986. Hazards in the Chemical Laboratory, 4th ed. Royal Society of Chemistry, London.
   Bretherick, L., Urben, P.G., and Pitt, M.J. 1999. Bretherick's Handbook of Reactive Chemical Hazards, 6th ed. Butterworth‐Heinemann, London.
   Castegnaro, M., Barek, J., Dennis, J., Ellen, G., Klibanov, M., Lafontaine, M., Mitchum, R., van Roosmalen, P., Sansone, E.B., Sternson, L.A., and Vahl, M. (eds.) 1985. Laboratory Decontamination and Destruction of Carcinogens in Laboratory Wastes: Some Aromatic Amines and 4‐Nitrobiphenyl. IARC Scientific Publications No. 64. International Agency for Research on Cancer, Lyon, France.
   Cunniff, P. (ed.) 1995. Official Methods of Analysis of the Association of Official Analytical Chemists, 16th ed., Ch. 4 p. 14. Association of Official Analytical Chemists, Arlington, Va.
   Degenhardt‐Langelaan, C.E.A.M. and Kientz, C.E. 1996. Capillary gas chromatographic analysis of nerve agents using large volume injections. J. Chromatogr. A. 723:210‐214.
   Forsberg, K. and Keith, L.H. 1999. Chemical Protective Clothing Performance Index Book, 2nd ed. John Wiley & Sons, New York.
   Furr, A.K. (ed.) 2000. CRC Handbook of Laboratory Safety, 5th ed. CRC Press, Boca Raton, Fla.
   Lewis, R.J. Sr. 1999. Sax's Dangerous Properties of Industrial Materials, 10th ed. John Wiley & Sons, New York.
   Lunn, G. and Sansone, E.B. 1985a. Destruction of cyanogen bromide and inorganic cyanides. Anal. Biochem. 147:245‐250.
   Lunn, G. and Sansone, E.B. 1985b. Validation of techniques for the destruction of dimethyl sulfate. Am. Ind. Hyg. Assoc. J. 46:111‐114.
   Lunn, G. and Sansone, E.B. 1987. Ethidium bromide: Destruction and decontamination of solutions. Anal. Biochem. 162:453‐458.
   Lunn, G. and Sansone, E.B. 1989. Decontamination of ethidium bromide spills. Appl. Ind. Hyg. 4:234‐237.
   Lunn, G. and Sansone, E.B. 1990a. Validated methods for degrading hazardous chemicals: Some alkylating agents and other compounds. J. Chem. Educ. 67:A249‐A251.
   Lunn, G. and Sansone, E.B. 1990b. Degradation of ethidium bromide in alcohols. BioTechniques 8:372‐373.
   Lunn, G. and Sansone, E.B. 1991a. The safe disposal of diaminobenzidine. Appl. Occup. Environ. Hyg. 6:49‐53.
   Lunn, G. and Sansone, E.B. 1991b. Decontamination of aqueous solutions of biological stains. Biotech. Histochem. 66:307‐315.
   Lunn, G. and Sansone, E.B. 1991c. Decontamination of ethidium bromide spills‐author's response. Appl. Occup. Environ. Hyg. 6:644‐645.
   Lunn, G. and Sansone, E.B. 1994a. Destruction of Hazardous Chemicals in the Laboratory, 2nd ed. John Wiley & Sons, New York.
   Lunn, G. and Sansone, E.B. 1994b. Safe disposal of highly reactive chemicals. J. Chem. Educ. 71:972‐976.
   Lunn, G. and Sansone, E.B. 1994c. Degradation and disposal of some enzyme inhibitors. Scientific note. Appl. Biochem. Biotechnol. 48:57‐59.
   Lunn, G. and Sansone, E.B. 1994d. Safe disposal of diisopropyl fluorophosphate (DFP). Appl. Biochem. Biotechnol. 49:165‐171.
   Lunn, G., Klausmeyer, P.K., and Sansone, E.B. 1994. Removal of biological stains from aqueous solution using a flow‐through decontamination procedure. Biotech. Histochem. 69:45‐54.
   Manufacturing Chemists Association. 1973. Laboratory Waste Disposal Manual. p. 136. Manufacturing Chemists Association, Washington, D.C.
   Mason, K.G. 1967. Hydrogen azide. In Mellor's Comprehensive Treatise on Inorganic and Theoretical Chemistry, Vol.VIII (Suppl. II) pp. l‐15. John Wiley & Sons, New York.
   National Research Council. 1983. Prudent Practices for Disposal of Chemicals from Laboratories, p. 88. National Academy Press, Washington, D.C.
   O'Neil, M.J. (ed.) 2001. The Merck Index, 13th ed. Merck & Co., Whitehouse Station, N.J.
   Patnode, W. and Wilcock, D.F. 1946. Methylpolysiloxanes. J. Am. Chem. Soc. 68:358‐363.
   Shirakashi, T., Nakayama, K., Kakii, K., and Kuriyama, M. 1986. Removal of mercury from laboratory waste water with iron powder. Chem. Abstr. 105:213690y.
   Snyder, L.R., Kirkland, J.J., and Glajch, J.L. 1997. Practical HPLC Method Development, 2nd ed. John Wiley & Sons, New York.
Key References
   American Chemical Society, Committee on Chemical Safety. 1995. Safety in Academic Chemistry Laboratories, 6th ed. American Chemical Society, Washington, D.C.
  The following are good general references for laboratory safety.
   Castegnaro, M. and Sansone, E.B. 1986. Chemical Carcinogens. Springer‐Verlag, New York.
   DiBerardinis, L.J., First, M.W., Gatwood, G.T., and Seth, A.K. 2001. Guidelines for Laboratory Design, Health and Safety Considerations, 3rd ed. John Wiley & Sons, New York.
   Fleming, D.D., Richardson, J.H., Tulis, J.J., and Vesley, D. 1995. Laboratory Safety, Principles and Practices, 2nd ed. American Society for Microbiology, Washington, D.C.
   Freeman, N.T. and Whitehead, J. 1982. Introduction to Safety in the Chemical Laboratory. Academic Press, San Diego.
   Fuscaldo, A.A., Erlick, B.J., and Hindman, B. (eds.) 1980. Laboratory Safety, Theory and Practice. Academic Press, San Diego.
   Lees, R. and Smith, A.F. (eds.) 1984. Design, Construction, and Refurbishment of Laboratories. Ellis Horwood, Chichester, United Kingdom.
   Montesano, R., Bartsch, H., Boyland, E., Della Porta, G., Fishbein, L., Griesemer, R.A., Swan, A.B., and Tomatis, L. (eds.) 1979. Handling Chemical Carcinogens in the Laboratory, Problems of Safety. IARC Scientific Publications No. 33. International Agency for Research on Cancer, Lyon, France.
   National Research Council. 1995. Prudent Practices in the Laboratory: Handling and Disposal of Chemicals. National Academy Press, Washington, D.C.
   Occupational Health and Safety. 1993. National Safety Council, Chicago.
   Pal, S.B. (ed.) 1991. Handbook of Laboratory Health and Safety Measures, 2nd ed. Kluwer Academic Publishers, Hingham, Mass.
   Rosenlund, S.J. 1987. The Chemical Laboratory: Its Design and Operation: A Practical Guide for Planners of Industrial, Medical, or Educational Facilities. Noyes Publishers, Park Ridge, N.J.
   Young, J.A. (ed.) 1991. Improving Safety in the Chemical Laboratory: A Practical Guide, 2nd ed. John Wiley & Sons, New York.
Internet Resources
   http://www.ilpi.com/msds/index.html
  Where to find MSDSs on the internet. Contains links to general sites, government and nonprofit sites, chemical manufacturers and suppliers, pesticides, and miscellaneous sites.
   http://www.OSHA.gov
  OSHA web site.
   http://www.osha‐slc.gov/OshStd_data/1910_1450.html
  Text of OSHA Standard 29 CFR 1910.1450: Occupational Exposure to Hazardous Chemicals in Laboratories.
   http://www.osha‐slc.gov/OshStd_data/1910_1000_TABLE_Z‐1.html
  Tables of permissible exposure limits (PELs) for air contaminants.
   http://www.osha‐slc.gov/OshStd_data/1910_1000_TABLE_Z‐2.html
  Tables of PELs for toxic and hazardous substances.
   http://hazard.com/msds/index.php
  Main site for Vermont SIRI. One of the best general sites to start a search. Browse manufacturers alphabetically (for sheets not in the SIRI collection) or do a keyword search in the SIRI MSDS database. Lots of additional safety links and information.
   http://siri.uvm.edu/msds
  Alternate site for Vermont SIRI.
   http://tis.eh.doe.gov/docs/osh_tr/ch5.html
  DOE OSH technical reference chapter on personal protective equipment.
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