METHOD 3640A GEL-PERMEATION CLEANUP 1.0 SCOPE AND APPLICATION 1.1 Gel-permeation chromatography (GPC) is a size exclusion cleanup procedure using organic solvents and hydrophobic gels in the separation of synthetic macromolecules (1). The packing gel is porous and is characterized by the range or uniformity (exclusion range) of that pore size. In the choice of gels, the exclusion range must be larger than the molecular size of the molecules to be separated (2). A cross-linked divinylbenzene-styrene copolymer (SX-3 Bio Beads or equivalent) is specified for this method. 1.2 General cleanup application - GPC is recommended for the elimination from the sample of lipids, polymers, copolymers, proteins, natural resins and polymers, cellular components, viruses, steroids, and dispersed high-molecular- weight compounds (2). GPC is appropriate for both polar and non-polar analytes, therefore, it can be effectively used to cleanup extracts containing a broad range of analytes. 1.3 Specific application - This method includes guidance for cleanup of sample extracts containing the following analytes from the RCRA Appendix VIII and Appendix IX lists: ______________________________________________________________________________ Compound Name CAS No.a ______________________________________________________________________________ Acenaphthene 83-32-9 Acenaphthylene 208-96-8 Acetophenone 98-86-2 2-Acetylaminofluorene 53-96-3 Aldrin 309-00-2 4-Aminobiphenyl 92-67-1 Aniline 62-53-3 Anthracene 120-12-7 Benomyl 17804-35-2 Benzenethiol 108-98-5 Benzidine 92-87-5 Benz(a)anthracene 56-55-3 Benzo(b)fluoranthene 205-99-2 Benzo(a)pyrene 50-32-8 Benzo(ghi)perylene 191-24-2 Benzo(k)fluoranthene 207-08-9 Benzoic acid 65-85-0 Benzotrichloride 98-07-7 Benzyl alcohol 100-51-6 Benzyl chloride 100-44-7 alpha-BHC 319-84-6 beta-BHC 319-85-7 CD-ROM 3640A - 1 Revision 1 September 1994 ______________________________________________________________________________ Compound Name CAS No.a ______________________________________________________________________________ gamma-BHC 58-89-9 delta-BHC 319-86-8 4-Bromophenyl phenyl ether 101-55-3 Butyl benzyl phthalate 85-68-7 2-sec-butyl-4,6-dinitrophenol (Dinoseb) 88-85-7 Carbazole 86-74-8 Carbendazim 10605-21-7 alpha-Chlordane 5103-71-9 gamma-Chlordane 5566-34-7 4-Chloro-3-methylphenol 59-50-7 4-Chloroaniline 106-47-8 Chlorobenzilate 510-15-6 Bis(2-chloroethoxy)methane 111-91-1 Bis(2-chloroethyl) ether 111-44-4 Bis(2-chloroisopropyl) ether 108-60-1 2-Chloronaphthalene 91-58-7 2-Chlorophenol 95-57-8 4-Chlorophenol 106-48-9 3-Chlorophenol 108-43-0 4-Chlorophenyl phenyl ether 7005-72-3 3-Chloropropionitrile 542-76-7 Chrysene 218-01-9 2-Cresol 95-48-7 3-Cresol 108-39-4 4-Cresol 106-44-5 Cyclophosphamide 50-18-0 DDD 72-54-8 DDE 72-55-9 DDT 50-29-3 Di-n-butyl phthalate 84-74-2 Diallate 2303-16-4 Dibenzo(a,e)pyrene 192-65-4 Dibenzo(a,i)pyrene 189-55-9 Dibenz(a,j)acridine 224-42-0 Dibenz(a,h)anthracene 53-70-3 Dibenzofuran 132-64-9 Dibenzothiophene 132-65-0 1,2-Dibromo-3-chloropropane 96-12-8 1,2-Dibromoethane 106-93-4 trans-1,4-Dichloro-2-butene 110-57-6 cis-1,4-Dichloro-2-butene 1476-11-5 1,2-Dichlorobenzene 95-50-1 1,3-Dichlorobenzene 106-46-7 1,4-Dichlorobenzene 541-73-1 3,3'-Dichlorobenzidine 91-94-1 2,6-Dichlorophenol 87-65-0 2,4-Dichlorophenoxyacetic acid (2,4-D) 94-75-7 2,4-Dichlorophenol 120-83-2 CD-ROM 3640A - 2 Revision 1 September 1994 ______________________________________________________________________________ Compound Name CAS No.a ______________________________________________________________________________ 2,4-Dichlorotoluene 95-73-8 1,3-Dichloro-2-propanol 96-23-1 Dieldrin 60-57-1 Diethyl phthalate 84-66-2 Dimethoate 60-51-5 Dimethyl phthalate 131-11-3 p-Dimethylaminoazobenzene 60-11-7 7,12-Dimethyl-benz(a)anthracene 57-97-6 2,4-Dimethylphenol 105-67-9 3,3-Dimethylbenzidine 119-93-7 4,6-Dinitro-o-cresol 534-52-1 1,3-Dinitrobenzene 99-65-0 2,4-Dinitrophenol 51-28-5 2,4-Dinitrotoluene 121-14-2 2,6-Dinitrotoluene 606-20-2 Diphenylamine 122-39-4 Diphenyl ether 101-84-8 1,2-Diphenylhydrazine 122-66-7 Disulfoton 298-04-4 Endosulfan sulfate 1031-07-8 Endosulfan I 959-98-8 Endosulfan II 33213-65-9 Endrin 72-20-8 Endrin aldehyde 7421-93-4 Endrin ketone 53494-70-5 Ethyl methane sulfonate 62-50-0 Ethyl methacrylate 97-63-2 Bis(2-ethylhexyl) phthalate 117-81-7 Famphur 52-85-7 Fluorene 86-73-7 Fluoranthene 206-44-0 Heptachlor 76-44-8 Heptachlor epoxide 1024-57-3 Hexachlorobenzene 118-74-1 Hexachlorobutadiene 87-68-3 Hexachlorocyclopentadiene 77-47-4 Hexachloroethane 67-72-1 Hexachloropropene 1888-71-7 Indeno(1,2,3-cd)pyrene 193-39-5 Isodrin 465-73-6 Isophorone 78-59-1 cis-Isosafrole 17627-76-8 trans-Isosafrole 4043-71-4 Kepone 143-50-0 Malononitrile 109-77-3 Merphos 150-50-5 Methoxychlor 72-43-5 3-Methylcholanthrene 56-49-5 CD-ROM 3640A - 3 Revision 1 September 1994 ______________________________________________________________________________ Compound Name CAS No.a ______________________________________________________________________________ 2-Methylnaphthalene 91-57-6 Methyl parathion 298-00-0 4,4'-Methylene-bis(2-chloroaniline) 101-14-4 Naphthalene 91-20-3 1,4-Naphthoquinone 130-15-4 2-Naphthylamine 91-59-8 1-Naphthylamine 134-32-7 5-Nitro-o-toluidine 99-55-8 2-Nitroaniline 88-74-4 3-Nitroaniline 99-09-2 4-Nitroaniline 100-01-6 Nitrobenzene 98-95-3 2-Nitrophenol 79-46-9 4-Nitrophenol 100-02-7 N-Nitrosodi-n-butylamine 924-16-3 N-Nitrosodiethanolamine 1116-54-7 N-Nitrosodiethylamine 55-18-5 N-Nitrosodimethylamine 62-75-9 N-Nitrosodiphenylamine 86-30-6 N-Nitrosodi-n-propylamine 621-64-7 N-Nitrosomethylethylamine 10595-95-6 N-Nitrosomorpholine 59-89-2 N-Nitrosopiperidine 100-75-4 N-Nitrosopyrolidine 930-55-2 Di-n-octyl phthalate 117-84-0 Parathion 56-38-2 Pentachlorobenzene 608-93-5 Pentachloroethane 76-01-7 Pentachloronitrobenzene (PCNB) 82-68-8 Pentachlorophenol 87-86-5 Phenacetin 62-44-2 Phenanthrene 85-01-8 Phenol 108-95-2 1,2-Phenylenediamine 95-54-5 Phorate 298-02-2 2-Picoline 109-06-8 Pronamide 23950-58-5 Pyrene 129-00-0 Resorcinol 108-46-3 Safrole 94-59-7 1,2,4,5-Tetrachlorobenzene 95-94-3 2,3,5,6-Tetrachloronitrobenzene 117-18-0 2,3,5,6-Tetrachlorophenol 935-95-5 2,3,4,6-Tetrachlorophenol 58-90-2 Tetraethyl dithiopyrophosphate (Sulfotep) 3689-24-5 Thiosemicarbazide 79-19-6 2-Toluidine 106-49-0 4-Toluidine 95-53-4 CD-ROM 3640A - 4 Revision 1 September 1994 ______________________________________________________________________________ Compound Name CAS No.a ______________________________________________________________________________ Thiourea, 1-(o-chlorophenyl) 5344-82-1 Toluene-2,4-diamine 95-80-7 1,2,3-Trichlorobenzene 87-61-6 1,2,4-Trichlorobenzene 120-82-1 2,4,6-Trichlorophenol 88-06-2 2,4,5-Trichlorophenol 95-95-4 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T) 93-76-5 2,4,5-Trichlorophenoxypropionic acid (2,4,5-TP) 93-72-1 Warfarin 81-81-2 ______________________________________________________________________________ a Chemical Abstract Services Registry Number. Table 1 presents average percent recovery and percent RSD data for these analytes, as well as the retention volumes of each analyte on a single GPC system. Retention volumes vary from column to column. Figure 1 provides additional information on retention volumes for certain classes of compounds. The data for the semivolatiles were determined by GC/MS, whereas, the pesticide data were determined by GC/ECD or GC/FPD. Compounds not amenable to GC were determined by HPLC. Other analytes may also be appropriate for this cleanup technique, however, recovery through the GPC should be >70%. 1.4 Normally, this method is most efficient for removing high boiling materials that condense in the injection port area of a gas chromatograph (GC) or the front of the GC column. This residue will ultimately reduce the chromatographic separation efficiency or column capacity because of adsorption of the target analytes on the active sites. Pentachlorophenol is especially susceptible to this problem. GPC, operating on the principal of size exclusion, will not usually remove interference peaks that appear in the chromatogram since the molecular size of these compounds is relative similar to the target analytes. Separation cleanup techniques, based on other molecular characteristics (i.e., polarity), must be used to eliminate this type of interference. 2.0 SUMMARY OF METHOD 2.1 The column is packed with the required amount of preswelled absorbent, and is flushed with solvent for an extended period. The column is calibrated and then loaded with the sample extract to be cleaned up. Elution is effected with a suitable solvent(s) and the product is then concentrated. 3.0 INTERFERENCES 3.1 A reagent blank should be analyzed for the compound of interest prior to the use of this method. The level of interferences must be below the estimated quantitation limits (EQLs) of the analytes of interest before this method is performed on actual samples. CD-ROM 3640A - 5 Revision 1 September 1994 3.2 More extensive procedures than those outlined in this method may be necessary for reagent purification. 4.0 APPARATUS 4.1 Gel-permeation chromatography system - GPC Autoprep Model 1002 A or B, or equivalent, Analytical Biochemical Laboratories, Inc. Systems that perform very satisfactorily have also been assembled from the following components - an HPLC pump, an auto sampler or a valving system with sample loops, and a fraction collector. All systems, whether automated or manual, must meet the calibration requirements of Sec.