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ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
CONCLUSION T h e GRC-MCA developed here can be operated continuously a t 300 "C for periods u p to a year without the need for disassembly, depending on sample composition. Backgrounds are low (-75 cpm) and efficiency is satisfactory. The problem of loss of the proportional counting plateau a t these high temperatures has been solved by carefully honing and electropolishing the flow counter barrel and using a center wire free of defects. Detector contamination by chromatographic column bleed and/or radioactive deposits is removed overnight, in situ, by oxidation of the organic deposits at 325-350 "C with 02. Quantitative analysis is readily achieved via a multichannel analyzer operated in the multiscaling mode and integrating the peaks of interest. When temperature of the proportional tube and the gas flow rates are held constant, the method is capable of a precision of 3% relative in obtaining the dpm of a chromatographic peak. T h e major limitation of the system is that low counting samples of less than 200-300 dpm are not analyzed by this system because of the limited counting (residence) time for a peak, about 0.6 min. For such samples the fraction must be trapped for subsequent liquid scintillation counting. In order to reduce deposition of reactive compounds in the PFC, a gold plated barrel has been prepared for testing.
Table 11. Comparison of I4CAnalyses from Trapping and GRC-MCA Integration %= Fraction Trapping GRC-MCA Benzene Toluene Cycloheptatriene Benzaldehyde Biphenyl plus diphenylmethane Phenyl cycloheptatriene
2.1, 1.8 0.8, 1.1 1.4, 1.2 3.0, 3.1
2.2, 2.1 0.7, 0.7
6.1, 6.3
7.1, 6.9 16.2, 16.3
15.0, 15.7
1.0,l.l 3.4, 3.3
a Percent of carbon-14 in the crude sample found in each fraction (see ref 8).
recovered counts compared to pure t ~ l u e n e - ' ~ CThe . second and more convenient, is to position a 6oCosource next to the proportional counter to yield about 5000-10000 cpm. Then, after the MCA is started, to inject the suspect compound. If quenching occurs, there will be a dip in an otherwise flat display of countsjchannel. Figure 5 shows the effect of acetone, COz, and CHC13 on the counting efficiency. As expected, the halogenated compound is a strong quencher, b u t COz and acetone, both known quenchers in liquid scintillation counting, have no significant effect over a typical range of sample sizes. The COz result is confirmed by Table I, which shows no significant change in the factor from 0.2 t o 2 cm3. Propane as a counting gas seems to be less susceptible to quenching than methane and thus has been used for our counting gas for the past three years. In addition, the critical point is low enough so t h a t at ambient temperature it is a liquid. Thus, a small cylinder will last for several months. Analyses of a typical sample in our work was done by two methods-the conventional trapping method using the collection port to trap each peak for liquid scintillation counting, and the GRC-MCA system described above. Repetitive runs with trapped sample analyses show this technique to have a precision of about 10% for well separated peaks and considerably poorer than this for poorly resolved peaks. Table I1 contains duplicate runs for both types of analyses. There is a general tendency for the GRC-MCA analyses to be higher than trapping for the higher boiling or more reactive samples. This may be due to losses in the collection because of lower trapping efficiency since many high boilers form aerosols which are difficult to trap quantitatively. From our studies of reaction mechanisms, the differences between the two methods are not important, but the far greater convenience of the GRC-MCA method is.
ACKNOWLEDGMENT
L. Biagi of this laboratory prepared the PFC barrels and performed the honing and electropolishing steps. John Griffin constructed the preamplifier and scaler. A. Telfer and M. A. Muhs, Shell Development Co., Houston, Texas, were most generous with their advice on the details of electropolishing.
LITERATURE CITED (1) M . Matucha and E. Smolkova, J . Chromatogr., 127, 163-201 (1976). (2) G. Blyholder, Anal. Chem., 32, 572 (1960). (3) J. K. Lee, E. K. C. Lee, B. Musgrave, Y . N. Tang, J. W. Root, and F. S. Rowland, Anal. Chem., 34, 741 (1962). (4) G. Popjak, A . E. Lowe, and D. Moore, J . Lipid. Res., 3 , 364 (1962). (5) M . A . Muhs, E. L. Bastin, and B. E. Gordon, Int. J . Appl. Rad. Isotopes, 16, 537 (1965). (6) A. T . James and E. A . Piper, Anal. Chem., 35, 515 (1963). (7) A. Karmen, 1. McCaffrey, and R. L. Bowman, J . LipaRes., 3, 372 (1963). (8) L . Swell, Anal. Biochem., 16, 70 (1966). (9) R. M. Lemmon, Acc. Chem. Res., 6, 65 (1973). (10) W. R. Erwin, B. E. Gordon, and R. M. Lemmon. J. f h y s . Chem., 80, 1852 (1976). (11) I. Kiricsi, K . Varga, and P. Fejes, J . Chromatogr., 123, 279 (1976).
RECEIVED for review September 1, 1977. Accepted October 25, 1977. This research was supported by the Division of Biomedical and Environmental Research of the U.S. Energy Research and Development Administration.
AIDS FOR ANALYTICAL CHEMISTS Isolation of Xenobiotic Chemicals from Tissue Samples by Gel Permeation Chromatography Douglas W. Kuehl" and Edward
N. Leonard
U.S. Environmental Protection Agency, Environmental Research Laboratory-Duiuth, 620 7 Condgon Boulevard, Duluth, Minnesota 55804
Buytenhuys (6) had previously reviewed applications of GPC for the separation of a variety of organics on Bio-Beads or Sephadex LH-20 with various organic solvents. An excellent review on the fundamental gel network structure, its ability
Since automatic gel permeation chromatographic (GPC) systems were first described for the cleanup of samples with high fat content ( I , 2 ) ,efforts have been made to characterize ( 3 ) and utilize ( 4 , 5 ) these systems more fully. Mulder and 0003-2700/78/0350-0182$01 0010
C
1977 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Table I. Gel Permeation Chromatography Retention Volume and Recovery Data CH2C1, Cyclohexane/CH,Cl,
I
Compound
Retn vol, mL
Recovery, 7%
Retn vol, mL
Aroclor 1254 Aroclor 1016 Hexachlorobenzene Naphthalene Hexachlorobutadiene p,p-DDT o-Chlorophenol Pen tachloroanisole 2,4,6-Tribromoanisole 2,4-Dibromoanisole p-Bromoanisole o-Bromophenol 2,4-Dibromophenol 2,4-Dichlorophenol 2,4,6-TrichIorophenol Pentachlorophenol 3,4-Dichloroaniline Diphenylamine 1-Naphthylamine m-Chloroaniline 2,4,6-Tribromophenol 5-Bromoindole 1,2,4-Trichlorobenzene p-Chlorophenol p-Bromophenol Pyrene Phenanthrene
160-190 150-1 86 168-199 168-195 144-168 144-171 162-178 156-174 158-176 152-169 156-174 156-181 162-183 170-1 95 172-197 166-196 170-195 154-180 166-192 172-200 147-161 167-181 174-200 180-210 154-176 176-200 168-195
100 95
160-190 150-188 168-198 170-195 148-169 152-178 170-198 172-196 172-198 168-202 174-200 174-208 182-211 190-218 192-244 186-216 200-255 184-214 2 0 6 - 2 37 216-246 194-214 224-270 234-266 240-284 220-244 252-294 250-284
LOO 100
90 100
100 100
89 84 95 98 94 100
90 95 94 89 100 100
100 100
75 93 100 100 99
100 72 84 86 87 5'7 100 75 98 6 '7 60 84 100 87 8 :l 90 9 11 83 60 84 80 61 64 100 9b 87 87
n
3
111
I?,
,I, ELUTION
*bo VOLUME
,lo
216
3Io
(ml1
GPC chromatogram showlng separation capabilities of methylene chloride-cyclohexane mixed solvent system on a Bio-Rad SX-2 column to fractionate on the basis of steric exclusion, and the chemical contributions of chromatographic affinity has been presented by Freeman ( 7 ) . Gel permeation, gel filtration, or molecular sieve chromatography are synonymous terms for separation by steric exclusion, Le., differences in solute molecular size. In theory, inert gels have the ability to function as a sort of mass spectrometer where the degree of permeation varies inversely with solute molecular size. However, chromatographic affinity due principally to hydrogen bonding between the solute and the gel network has been shown to have a definite effect upon column performance (8). The finding of an orderly relationship between measured affinity and solute proton-donor strength suggests a new framework for studying hydrogen bonding, for measuring the proton-donor strengths of chemicals and for performing chemical separations (9). The objective of this work was to develop an efficient, rapid method for the isolation of low molecular weight polar organics in fatty tissue for subsequent gas-liquid chromatographic (GLC) - mass spectrometric (MS) analysis. We describe a 2-step GPC cleanup procedure for samples with high fat content that uses first the steric exclusion principle and then takes advantage of the combination of the steric exclusion and chromatographic affinity phenomenon. EXPERIMENTAL Procedure. Blend ground fathead minnow (Pimephales
Figure 1.
Recovery, 7%
F R I C T I O N S C O L l ECTED
Retn vol shift 0 0 0
2 4 8 8
16 14 16 18 18
20 20 20 20 30 30 40 44 50 58
60 60 66 76 82
183
184
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Table 11. Compounds Identified in Fish Tissue by GC/MS after Sample Cleanup by Two-step GPC Method Fractions 3 and 4 Methylnaphthylene C-alkylnaphthylene C-alkylnaphthylene Biphenyl Dichlorobiphenyl Trichlorobiphenyl Tetrachlorobiphenyl Trichlorobenzene Tetrachloroanisole Tribromoanisole cis-Chlordane trans-Chlordane cis-Nonachlor trans-Nonachlor p,p’-DDE
Fractions 5 , 6, and 7 Chlorophenol Indole Bromoindole Dibromoindole Tribromoindole Dibromomethylindole Trichloromethylindole Pentachlorophenol Trichlorophenylphenol Nicotinamide 1,3-Diphenylpyrazoline Dichlorodibenzofuran
Fractions 8, 9, and 1 0 Anisole Dibromoanisole Tribromoanisole Chlorobromoanisole Tetrachloroanisole Phenol Chlorophenol Dibromophenol Bromodichlorophenol Chlorodibromophenol Ethylphenol Dibromocresol Dichlorobenzene Trichlorobenzene Chloronaphthalene Dichloronaph thalene Dibenzofuran Benzthiazole Methylbenzothiaphene Dibenzothiaphene Naphthylamine Me th ylcarbazole Indole Dibromoindole Tribromoindole Dibromomethylindole Tribromomethylindole Tetrabromomethylindole Pentachloroaniline Dibromomethylbenzothiazole
Varian MAT Spectrosystem-100 MS data system was used for data acquisition and processing. Standards. All standard compounds used for retention volume and recovery studies (Table I) were from the Lab Assist kit (Chemservices, Inc., West Chester, Pa.). Solutions of 1 mg/mL CHzClzwere prepared.
RESULTS AND DISCUSSION T h e isolation of nonpolar xenobiotic organics from fatty tissue on Bio-Rad SX-2 (copoly(styrene-270divinylbenzene)) with cyclohexane as the solvent is a very efficient sample preparation technique for subsequent GLC-MS analysis ( 4 ) . Recoveries of PCBs, for instance, are generally better than 95%. A disadvantage of this system, however, is the low recovery of polar organics such as pentachlorophenol, which was only 10%. On this system, however, a mixture of equal quantities of corn oil, Aroclor 1254, and pentachlorophenol can be completely separated. Excellent chromatographic resolution can therefore be obtained if low recoveries of the polar chemicals are considered acceptable. Polar solvent systems will reduce retention volumes, reduce band broadening, and increase recoveries of polar compounds to acceptable levels. Johnson e t al. (5) observed that polm solvent systems such as mixtures of toluene and ethyl acetate will result in high percentage recoveries of both polar and nonpolar chemicals. This system, however, did not provide any chromatographic resolution of polar and nonpolar compounds. Nonpolar solvents readily elute nonpolar solutes from the gel network, whereas larger quantities of solvent are required to eventually elute polar solutes at low yields and both polar and nonpolar solutes co-elute at high yields with polar solvents. It was necessary then to develop a GPC system that would yield high recoveries of both polar and nonpolar chemicals and would give good chromatographic resolution of polar and nonpolar chemicals. I n addition, the solvent system had to be highly volatile so that the more volatile chemicals isolated from samples would not be lost during solvent removal. Retention volume and recovery studies for various polar and nonpolar organics ranging from p-chlorophenol to PCBs were conducted for the following solvent systems: (a) 100%
CHZCL,; (b) 10% CH&l,/SO% CsH12; (c) 3370 CHzC12/67% C6Hle;(d) 5070 CHZClz/50%C6HI2;and (e) 75% CH2Cl2/25% C6HI2. The 50:50 mixture was the best compromise of high percentage recoveries and compound separations. Figure 1 shows an example of the resolution obtained with the 50:50 mixture. The retention volume and percentage recovery data with the 50350 mixture and 100% CHzClz are presented in Table I. All compounds tested eluted a t low retention volumes in narrow bands and at approximately the same retention volume when 100% CH2Cl2was the solvent. This system, therefore, can be used as a rapid and efficient technique for the bulk separation of lipids from low molecular weight organic chemicals (LMWO). T h e unique elution of compounds from the mixed solvent GPC system can then be used to fractionate the LMU’Os into polar and nonpolar organics. This 2-step procedure has been demonstrated for an extract of fish that had previously been exposed to a bromine chloride-disinfected wastewater effluent (10). Figure 2 shows a GPC trace for the lipid/LMWO separation (step l), and a GPC trace for fractionation of the LMWO by polarity (step 2). After each sample was screened on the GC, fractions 3 and 4 were combined; 5, 6, and 7 were combined; and 8, 9, and 1c) were combined for GLC-MS analysis. T h e qualitative results are presented in Table 11. These fractions contained compound types such as phenols, anisoles, and heterocyclic aromatics (dibenzofurans, dibenzothiaphenes, indoles, etc.). Fractions 1 and 2 were analyzed separately and were basically PCBs and chlorobenzenes. Commercially available GPC units can be used for both steps of this technique. Typically a timing unit is used to provide “waste”, “collect”, and “wash” cycles for repetitive processing of a single sample. Step 1 can therefore be completed, after sample loading, without operator assistance. Step 2 can also be automated by routing the effluent to a fraction collector during the “collect” cycle. However, since step 2 is done only once for each sample, and each sample may be slightly different, it may be advantageous to allow the operator to decide when each fraction should be collected.
ANALYTICAL CHEMISTRY, VOL. 50, NO. 1, JANUARY 1978
Future developments involving automatic GPC units for the cleanup of fatty tissue are the addition of an expanded fraction-collection system, the addition of gradient solventelution capabilities, and the use of a variety of detectors for a more complete characterization of the GPC elution pattern of a sample.
LITERATURE CITED (1) R. C. Tindle and D. L. Stalling, Anal. Chem., 44, 1768-1773 (1972) (2) D. L. Stalling, R. C. Tindle, and J. L. Johnson, J , Assoc. Off. Anal. Chem.. 55, 32-38 (1972). (3) K. R. Griffin and J. C . Craun, J . Assoc. Off. Anal. Chem.. 57, 168-172 (1974).
(4)
G.D. Veith, D. W. Kuehi, and J. Rosenthail, J. Assoc. 58, 1-5 (1975).
185
Off. Anal. Chem.,
(5) L. D. Johnson, R. H. Waltz, J. P. Ussary, and F. E. Kaiser, J . Assoc. O f f . Anal. Chem., 59, 174-179 (1976). ( 6 ) J. L. Mulder and F. A. Buytenhuys, J . Chfomatogr.,51, 459-477 (1970). (7) D. H. Freeman, J . Chromatogr., S c i , 11. 175-180 (1973). (8) D. H. Freeman and R. M. Angeles, J . Chromatogr., Sci., 12, 730-735 (1974). (9) D. A. Freeman, R. M. Angeles, D. P. Enagonio, and W. My, Anal. Chem., 45, 768-474 (1973). (10) H. L. Kopperman, D. W. Kuehi, and Ei. E. Glass, Proceedings of the Conference on Environmental Impact of Water Chlorination., Oak Ridge National Laboratory. Oak Ridge, Tenn., 0c:tober 22-24, 1975, pp 327-345.
RECEIVED for review August 26, 1977. Accepted October 17, 1977.
High Performance Liquid Chromatographic Determination of Unreacted Acrylamide in Emulsion or Aqueous Homopolymers or Emulsion Copolymers F. J. Ludwig, Sr.”
and M. F. Besand
Research Laboratory, Petrolite Corporation, Tretolite Division, St. Louis, Missouri 63 1 19
Aqueous solutions of polyacrylamides or emulsion homopolymers or copolymers of acrylamide with comonomers such as ethylenic-unsaturated carboxylic acid salts or ethylenicunsaturated quaternary ammonium salts are used as flocculants, filter-aids, mobility-control agents, etc. in waste water treatment plants and in oilfield waterfloods. The emulsion copolymers, such as described by J. W. Vanderhoff and R. M. Wiley ( I ) or L. P. Koskan (21, consist of a petroleum hydrocarbon solvent as external phase and water-swollen polymer as internal phase. These polymeric products may contain some residual acrylamide monomer which is toxic. Therefore, determination of the amounts of unreacted acrylamide in such water-treating products is necessary to protect public health. In addition, for product development and process improvement studies, to measure the rates of solution polymerization or emulsion copolymerization of acrylamide, a procedure is needed to determine unreacted acrylamide contents which may vary from 20 to 0.01YO.These polymerizations may require more than 6 h for completion. Hence, analyses should be completed in less than an hour after the reaction mixture is sampled. Methods of determination of trace quantities of acrylamide in aqueous or dispersed phase polymeric systems have been described by A. Hashimoto ( 3 ) ,E. R. Husser et al. ( 4 ) , and B. T. Croll (5,6). None of these procedures was suitable for the determination of residual acrylamide in our water-in-oil emulsion copolymers of acrylamide and certain cationic comonomers. We have been able to achieve quantitative precipitation of these emulsion copolymers or of polyacrylamide from aqueous solution by dropwise addition to acetone. Since no centrifugation was necessary, this step required only about 20 min. T h e GLC separation of acrylamide from the other components of the emulsion on our column was too lengthy for rate measurements and became less accurate a t concentrations less than about 0.01 ’70because of adsorption effects. When we used the Partisil 10 PAC liquid chromatography column which was recommended by E. R. Husser et al. ( 4 ) with a 10% methano1-90% methylene chloride mobile phase, we obtained satisfactory separation of acrylamide, benzamide, the internal standard, and the other components of the acetone solution within 9 min after injection. Hence, an analysis of a water-in-oil emulsion copolymer of acrylamide with either a cationic or anionic comonomer can be completed in about 45 min with an accuracy 0003-2700/78/0350-0187$01.OO/O
and precision suitable for kinetic studies and quality control of the finished product.
EXPERIMENTAL Instrumentation. The Waters Associates Model 202 Liquid Chromatograph with the U6K Injector and M-6000 Pump, and the Schoeffel Instrument Corp. Model SF770 UV Spectrophometer were used. The chromatograms were recorded on a Texas Instrument Inc. Servo/Riter I1 Portable Recorder. Reagents. The distilled-in-glasschromatography solvents were obtained from Burdick & Jackson, Labs., Inc. Other compounds were of reagent grade and were used without further purification. The polymers were either purchased from commercial sources or synthesized in this laboratory. Column. The 250 X 4.6 mm Partisill0 PAC prepacked column was purchased from Whatman, Inc. Liquid Chromatography Conditions. The mobile phase was a 90% methylene chloride-1070 methanol (v/v) filtered solution. The mobile phase flow rate was 1.0 mLimin at 600 psi pressure. The spectrophotometer was set at 240 nm and an absorbance range of 0 to 0.04 for the lowest acrylamide concentrations. The recorder chart speed was 0.75 inch/min. Procedure. A solution of 1.000 x J.0-3 g/mL of benzamide in acetone is prepared. A 1.00- to 3.00-mL aliquot of this solution and 99 mL of acetone are added t o a 150-mL beaker. A 4.000to 6.000-g aliquot of the polymer emulsion or solution is added dropwise over a 3- to 4-min interval to the rapidly stirred acetone solution. The suspension is then stirred for an additional 15 min. About 1-2 mL of the supernate is filtered through a 0 . 5Millipore ~ filter. A 10- to 1 5 - ~ aliquot L of the filtrate is injected into the UfiK Injector. For those emulsions which gave a more readily filterable solution after precipitation of the polymer in methanol, the standard benzamide solution is prepared in methanol, an aliquot diluted with 99 mL of methanol, and the polymeric product added dropwise to this solution. Calculations. The heights of the benzamide and acrylamide peaks are measured from a baseline which is drawn between minimum peak amplitudes. To determine a response factor, standard methanolic solutions of acrylamide and benzamide were prepared, and measured volumes were added to an emulsion which contained hydrocarbon oil, water, emulsifier, and initiator in the same proportions as in the polymerization. The resulting mixture was added to an 80% methylene chloride-20% methanol solution to produce one phase. An aliquot of this was filtered through a 0 . 5Millipore ~ filter and injected into the chromatograph. The response factor was calculated from the equation C 1977 American Chemlcal Soclety
