Methods of Analysis of Polychlorinated Biphenyl Congeners Using an Application-Specific Capillary GC Column Frank L. Dorman, Gary B. Stidsen, Chris M. English and Lydia Nolan Restek Corporation, Bellefonte, PA Jack Cochran LECO Corporation, Las Vegas, NV
209 Possible PCB Congeners 7 Indicator Congeners (European): 28,52,101,118,138,153,180 Indicate presence/absence of PCB s 12 Most Toxic Congeners (WHO): 77,81,126,169 105, 114, 118, 123, 156, 157, 167, 189 Have Dioxin-like behavior 136 Congeners in Aroclor Products Frame, et. al. (Fresenius( J. Anal.Chem. 357, 714-22) Add 126, 81, 169, and 209 140 Congeners should satisfy almost everyone
GC-MS Pros: Cons: Analytical Techniques Allows for spectral separation of coeluting congeners of different chlorination level Single column Common instrumentation in many laboratories Moderate sensitivity
GC-ECD Pros: Cons: Analytical Techniques High Sensitivity Simplest instrumentation Common instrumentation in many laboratories Dual column system usually necessary Must have chromatographic separation
Analytical Techniques GCxGC-TOFMS (GCxGC ECD) Pros: Cons: Allows for spectral separation of coeluting congeners of different chlorination level Sensitivity improvement vs- GC-MS Increase in peak capacity for chromatographic separation Instrumentally more complex Commercial instrumentation only recently available
Which GC Column? Identification of all 209 available on many phases Rtx-PCB column designed for this analysis Greatest number of resolved congeners High thermal stability Rtx-XLB column good for GC-MS Rtx-1, 5, CLPesticides,, CLPesticides2, 440,, Dioxin2, 500, 35, 1301, etc
Dioxin-like PCBs PCB# Cl# 77 4 81 4 Cl Pos. 34-34 PCB# 156 Cl# 34 156 6 2345-34 157 167 105 5 234-34 169 6 345-345 114 5 2345-4 118 5 245-34 189 7 2345-345 123 5 345-4 345-24 126 5 345-34 6 6 Cl Pos. 234-345 345 245-345 World Health Organization, non- and mono-ortho ortho substituted
Toxicity of PCBs 2378-TCDD Planar shape Fit in a 3x10Å rectangle Chlorines on the corners Binds with Ah receptor, like dioxins PCB 77 Toxicity Equivalency Factors 2378-TCDD = 1.0 PCB 77 = 0.0001 PCB 126 = 0.1 PCB 169 = 0.01
dlp-cs5 on htc Feb06_02samp1 100 % Toxic PCB Congeners (Carbon Cleanup) first pcb run - identifications not confirmed Cl 7 189 Voltage SIR 25 Channels EI+ 393.8025 3.35e5 21.5 min 0 Feb06_02samp1 100 % Cl 6 167 156 157 157 169 169 Voltage SIR 25 Channels EI+ 357.8444 3.03e5 0 Feb06_02samp1 100 % 118 123 118 114 114 105 126 Cl 5 Voltage SIR 25 Channels EI+ 323.8834 7.03e5 0 Feb06_02samp1 100 81 81 77 Cl 4 % Voltage SIR 25 Channels EI+ 289.9225 1.21e6 0 Time 13.00 13.50 14.00 14.50 15.00 15.50 16.00 16.50 17.00 17.50 18.00 18.50 19.00 19.50 20.00 20.50 21.00 21.50 22.00
GCxGC Schematic From Injector Modulator To Detector 1 st Dimension 2 nd Dimension Two independent separation mechanisms
GCxGC Setup Primary column (1 st dimension) Longer, wider bore, thicker film Non-polar Modulator Thermal in nature Concentrates effluent from primary column Injects this effluent onto secondary column Secondary column (2 nd dimension) Very short, narrow bore, thinner film Polar or selective
GCxGC Column Integral column No press fit 50m x 0.18mm x 0.18µm Rtx-1 5m x 0.18mm x 0.10µm Rtx-PCB Installed so that all 5m of Rtx-PCB was past modulator and in a secondary oven Constant flow He at 1.5 ml/min Splitless injection 1 µl 250 C Purge time 60 sec
GCxGC Conditions Primary oven 120 C (1 min), 5 /min to 330 Modulator (quad jet)* Temperature offset: 40 C Modulation time: 2 sec Secondary oven 140 C (1 min), 5 /min to 350 Run time = 43 min *Built by LECO under license from Zoex Corporation
Modulator and Secondary Oven Secondary Oven Modulator
TOFMS Conditions LECO Pegasus 4D Source temperature: 225 C Electron ionization: 70 ev Stored mass range: 120 to 520 u Acquisition rate: 50 spectra/sec
Dioxin-like PCBs Rtx-PCB > m/z 292 326 360 394 126 169 189 81 77 114 105 167 156 157 123 118 Contour Plot Rtx-1 1 >
Dioxin-like PCBs vs. Other PCBs Rtx-PCB > m/z 292 326 360 394 126 169 189 77 105 156 157 81 114 167 118 123 Rtx-1 1 >
Dioxin-like PCBs in an Aroclor Mix Rtx-PCB > m/z 292 326 360 394 126 169 189 77 105 156 157 81 114 167 118 123 Rtx-1 1 >
Dioxin-like PCBs in an Aroclor m/z 292 326 360 394 Mix 156 128 105 180 177 77 138 163 132 118 Surface Plot
Non-ortho ortho PCBs in Siscowet Rtx-PCB > m/z 292 326 360 394 126 BDE 47 169 77 105 128 Rtx-1 1 > C81900-0019 0019
Mono-ortho ortho PCBs in Siscowet Rtx-PCB > m/z 292 326 360 394 189 105 156 157 118 114 167 123 Rtx-1 1 > C81900-0019 0019
Non-ortho ortho PCBs in Siscowet PCB HR 77 170 81 22 126 610 TOF 170 169 250 270 HR TOF 150 31 1100 490 HR TOF 170 24 570 270 140 30 1200 550 640 83 1700 470 630 84 1400 440 Results in pg/g for three different Siscowet from Lake Superior Based on extracted amount and final extract volume: 20 pg/g = ~2.5 pg/µl 200 pg/g = ~25 pg/µl C81900-0018,19,20 0018,19,20
Conclusions GCxGC-TOFMS is a viable way to determine dioxin-like PCBs in fish Sensitive: modulation enhances detection Selective: 2 nd dimension separations Full mass spectra provide powerful confirmations Peak find and spectral deconvolution enable location, identification, and quantification (?) of other environmentally significant compounds
GC-TOFMS Analysis of Indicator Polychlorinated Biphenyls
Experiment Determine retention times for all 209 PCBs on a new low-bleed capillary column Analyze individual congener standards Hexachlorobenzene for relative retention times Analyze mix of Aroclors 1221:1242:1254:1262 1:2:2:2 ratio Show chromatogram with congeners labeled GC-MS results obtained using a time-of of-flight flight mass spectrometer to allow for possible spectral deconvolution
GC-TOFMS Conditions Gas Chromatography One microliter splitless 250 C, 60 sec valve 60 m x 0.18 mm x 0.18 µm Restek Rtx-PCB column Constant flow helium, 1.5 ml/minute 70 C (1 min), 50 /min to 120, 3 /min to 360 (1 min) Mass Spectrometry LECO Pegasus III Source temperature: 225 C Electron ionization: 70 ev Stored mass range: 120 to 520 u Acquisition rate: 5 spectra/sec
Aroclor Mix Chromatogram Overlaid ion chromatograms are plotted For example, hexachlorobiphenyl is 360 Peaks are labeled with congener numbers Text size and format is only for space and not meant to indicate anything about PCBs, except: European indicator congeners are in red Chromatograms are not to one scale Each panel is adjusted so most of the PCB peaks can be seen
1 3 2
8 4+10 6 9 7 5
18 15 17 16 32 19 12 27 13 24
31 28 52 33 22 49 26 34 23 54 29 25 53 20 51 45 46 43 48
70 95 44 64 74 37 47 75 59 42 71 41 72 35 96 103 40 94 67 102 57 98 63 93 76
110 101 66 56 99 87 91 92 89+84 60 97 136 117 85 55 119 83 125 86 154 115
132 179 141 118 153 147+149 105 151 135 82 144 77 124 139 123+109 134 133 146 114 131 122 176 137
138 187 174 163 183 128 177 164 178 130 158 129 175 166 185 202 167 201 171 172 173 197
180 199 156 157 170 196 203 193 200 190 191 198 208
194 195 206 207 189 205
Important Congener Separations European indicator PCBs May coelute on 5% phenyl methylsilicone and other column phases
31 28 Trichlorobiphenyls 33 20
153 Hexachlorobiphenyls 132
138 Hexachlorobiphenyls 163
Conclusions Greater than 140 Aroclor PCBs can be determined with the new column and mass spectrometry The seven European indicator PCBs can be determined individually Spectral deconvolution available with TOFMS allows qualitative identification for coeluting PCBs
GC-ECD Analysis Columns should be complementary As many congeners resolved as possible Different coelutions (nothing will resolve all 140 environmentally-significant congeners) Similar thermal properties Operation under same temperature and pressure programs
Rtx-PCB (40m x 0.18mm, 0.18um film) 10.958 12.521 12.373 13.239 15.245 14.990 15.152 15.954 16.785 16.504 18.804 17.667 20.516 20.340 20.986 20.700 21.542 22.368 24.263 24.513 25.467 25.643 27.809 30.334 31.146 33.037 35.632 35.851 23.385 42.431 46.983 24.115 25.308 28.630 28.961 29.372 29.634 30.900 31.956 34.270 39.530 44.388 27.399 38.828 21.972 24.852 26.086 30.773 31.464 33.344 28.366 36.296 PCB 209 RT = 48.95 10 15 20 25 30 35 40 45 50min 300ppb Aroclor 1242, 1254, 1262 100 C(1 min) 20 C/min 200 C(0 min) 2 C/min 320 C(1 min)
Rtx-440 (40m x 0.18mm, 0.18um film) 10 15 20 25 30 35 40 45 min 29.128 10.987 12.482 15.158 16.933 10.295 12.217 14.441 17.831 18.343 19.279 21.033 36.368 40.992 13.488 16.748 19.741 20.870 24.290 24.042 24.515 26.908 28.717 16.586 22.442 33.119 33.274 33.800 33.944 38.465 18.053 28.122 20.330 21.610 25.649 26.466 23.660 30.879 PCB 209 RT = 42.82 300ppb Aroclor 1242, 1254, 1262 100 C(1 min) 20 C/min 200 C(0 min) 2 C/min 320 C(1 min)
Quantitation of Congeners Rule#1: If compound elutes as single on both columns report lower value Rtx-PCB Rtx-440 Peak PCB 28 PCB 28
Quantitation of Congeners Rule#2: If compound elutes as single on one column and multiple congeners on the second report the single column result if confirms on second column Rtx-PCB Rtx-440 Peak PCB 53 + 31 PCB 31
Quantitation of Congeners Rule #3: If there are complicating coelutions on both columns calculate the results using normalized responses (ex. PCB 136) Rtx-PCB Rtx-440 Peak PCB 136 + 117 PCB 115 + 136 PCB 115
Quantitation of Congeners Rule #3 continued: PCB 136 Concentration = ( [PCB 115+136] X (RF115 + RF136) 2 x RF115 - [PCB 115] ) RF115 RF136 RF = response factor of congener on ECD
Rtx-PCB (40m x 0.18mm, 0.18um film) 10 12 14 16 18 min 19 29 9 25 46 6 7 17 27+12 13+24 8 15 32 45 75 41 26 43 47 42 22 16 71+35 18 20+51+33 49 53+31 44 52 28 102 300ppb Aroclor 1242, 1254, 1262 64 37 40 5 34 67 48 59
Rtx-440 (40m x 0.18mm, 0.18um film) 300ppb Aroclor 1242, 1254, 1262 9 10 11 12 13 14 15 16 17 min 9+7 6 19 15 27 34 29 51 46+73 5+8 16+32 25 26 35+42 71 40 67 63+102 53 33+20 45+22 47+75 48 49 37+64 18 17+12 31 44 66+91 28 70 43+52 74+95 13 24 59 41
Rtx-440 Rtx-PCB PCB# s or m PCB# 1 s 1 s 1 3 s 3 s 1 PCB Congener Separation 50 72 19 141 Case s or m 1 2 3 4 m 4 m 1 4+10 reported together 5 m 5 s 1 6 s 6 s 1 7 m 7 s 1 8 m 8 s 1 9 m 9 s 1 10 m 10 m 1 4+10 reported together 12 m 12 m 1 ok 13 s 13 m 1 15 s 15 s 1 16 m 16 s 1 17 m 17 s 1 18 s 18 s 1 19 s 19 s 1 20 m 20 m 1 33+20 reported together 22 m 22 s 1
GC-ECD Summary Rtx-PCB and Rtx-440 Combination Quantification of the 141 PCB congeners possible using the 3-case 3 quantification system High thermal stability Good choice for dual column operation
Overall Conclusions GC-MS analysis with Rtx-PCB column European indicator unbiased WHO-12 with carbon cleanup GCxGC-TOFMS TOFMS has acquisition speed necessary to characterize the narrow peaks WHO-12 resolved from non-toxic without carbon cleanup Dual-Column GC-ECD Also successful, with high sensitivity All congeners of significance can be quantified