Anal. POPs

Analytical flow chart for POPs Organochlorine Pesticides (OCPs) Samples PCDDs/DFs & co-PCBs Soxhlet extraction Acetone etc Toluene, DCM etc Extracts Solvent exchange to hexane • Florisil cartridge, 25% DCM 100ml • Active Carbon cartridge, hexane 10ml Clean Up Quantification by HR-GC/MS • Multilayered Silica gel column, hexane 220ml • Active Carbon (AC) cartridge, 25% DCM 60ml • Reversed-AC, toluene 100ml General Procedures of POPs analysis Sampling Storage and handling Sample Extraction  Acquisition of suitably representative samples is fundamental to any e nvironmental measurement program and should not be overlooked  “Clean” techniques need to be adopted to avoid contamination  A basic requirement for analytical laboratories involved in the measurement is the availability of freezer and refrigerator capacity for sample storage  should be careful to avoid photo/thermal-degradation, bio-chemical reaction  The main points to consider are to allow adequate exposure time for the solvent system in the sample matrix and to limit sample handing steps  should be considered extraction efficiency Concentration  Solvent evaporation is generally used several times within all analytical methods and successful analyte recoveries critically depend on minimizing losses during this step  Sur standards may be added prior to evaporation to monitor compound losses Column cleanup  Steps for elimination of interferences in sample matrix by adsorption, sizeexclusion column, lipid destruction and sulfur removal Quantification  Separation of POPs will be conducted through gas chromatography  Retention time should match between sample and internal standard;  In general, quantification of the analyte should be done according to the internal standard methodology to correct/compensate the systemic errors General Procedures of POPs analysis Steps Extraction Other Clean up Contents SDE, Soxhlet, ASE, Liquid-liquid, Sonication, etc Acid & Alkali treatment, Copper column Column Clean up Silica gel, Multi-layer silica gel, Florisil, Alumina, Activated-carbon, HPLC, etc Analytical Column DB5, DB5MS, DB17, DB-Dioxin, SP2331, SE54, CP-SilCB, HT8PCB, DB1, etc Instrument Analysis Quantification GC/ECD, GC/MS, HRGC/HRMS EI or NCI mode Peak pattern (t-PCB), Individual Internal standard Method, Isotope dilution Method Pre cleaning Glassware, PUF, Filter all glassware should be washed in detergent solution(i.e.,Decon), thoroughly rinsed with tab water, then with Milli-RO water, then dry in the drying cabinet Before using glassware, Acetone  hexane/DCCM rinse Rinse and Dried separate -funnel Soxhlet washing before extraction Preclean PUF/Thimble filter with SOX Water bottles (Amber) Extraction techniques Liquid-Liquid extraction (LLE) -Liquid samples in separation funnel is extracted with non-polar solvent e.g., DCM, hexane etc Solid-Liquid extraction (Soxhlet Ext) - (semi) soild (ex Soil, sediment, ash, human tissue) + desiccant mixture in glass or paper thimble is leached with warm (condensed) solvent for 4~12 hrs - solvents are, e.g., diethyl ether, DCM, hexane Pressurized Liquid Extraction (PLE) - Sample (usually +desiccant) placed in extraction cartridge and solvent (heated, pressurized) passed through then dispensed in extraction vial (e.g., ASE) - Easy to operate, short extraction time(100~200℃, 1500~2000 psi), low solvent-consuming Column Extraction - Sample placed in large column with filter and stopcock Eluted with large volume of extraction solvent, e.g., hexane:DCM; hexane -In the case of SPE(solid phase extraction) is very useful to save time, less than solvent etc Sonication-assisted Extraction -Sample in open or closed vessel immersed in solvent and heated with ultrasonic radiation using ultrasonic bath or probe Extraction should be standardized with respect to standardization of  type of solvent ( for high selectivity, recovery efficiency)  short extraction time  low solvent-consuming  more convenient to operate  performance of auxiliary equipment  Certified/official methodology Soxhlet (Dean stark) Extraction Soxhlet Dean Stark Soxhlet Liquid – Liquid Extraction Accelerated Solvent Extraction pump oven vent solvent valve nitrogen cylinder extraction cell vent collection vial Extract Column Extraction Solid Phase Microextraction (SPME) Solid Phase Extraction (SPE) Conditioning Loading Rinsing Elution Sampling Injection Desorption Column clean up < Automatic column > < SPE > Column clean up Making the absorbent column Na2SO3 1.0g Copper granule Na2SO3 1.0g 10% AgNO3-silicagel 3.0g Neutral silicagel 0.9g 22% H2SO4-silicagel 6.0g 44% H2SO4-silicagel 4.5g Neutral silicagel 0.9g 2% KOH-silicagel 3.0g Neutral silicagel 0.9g Na2SO3 1.0g After sample loading / elution Evaporation steps Multi Evaporator K-D Apparatus (Kuderna-Danish) Rotary Evaporator Multi-needle concentrator Calibration, Surrogate Standards Calibration Compounds 13C-labeled 0.5~200 ng/ml (4DD/Fs) 2.5~1000 ng/mL(5~7 DD/DFs) 5.0~2000 ng/mL (8DD/DFs) (5 steps) 100 ng/mL Coplanar PCBs (Wellington Lab., WP-CVS) 0.1~800 ng/ml (7 steps) 50 ng/mL marker PCBs (Wellington Lab., EC9605-CVS) 20~2000 ng/ml (5 steps) 400 ng/mL OCPs (HRMS) (CIL ES-5348) 0.4~200 ng/mL (6 steps) 20 ng/mL OCPs (LRMS) (Ultrascientific) (6 steps) 20 ng/mL PCDD/DFs (Wellington Lab., EPA-1613 CVS) Compounds Cleanup spike Syringe spike Wellington Lab., EPA-1613 LCS 200ng/mL, 13C-labeled1,2,3,4/1,2,3,7,8,9 DD (Wellington Lab., EPA-1613 ISS) (1000 ng/ml, 12, 13C-labeled dl-PCBs (Wellington Lab., WP-LCS) (1000 ng/ml, 4, 13C-labeled(#70,111,138,170) dl-PCBs (Wellington Lab., WP-ISS) marker PCBs 2.0 ng//ml, 8, 13C-labeled dl-PCBs (Wellington Lab., EC 9605-SS) 2.0 ng//ml, 2, 13C-labeled(#101,194) dl-PCBs (Wellington Lab., EC 9605-RS) OCPs CIL, ES-5349(HRMS) : 100 ng/mL Pyrene-d10, Ultrascientific CIL, ES-5350 Phenanthrene-d10, Ultrascientific PCDD/DFs Surrogate Unlabeled native Coplanar PCBs General guidance on GC analysis GC detector Analytes Configuration Advantages/ disadvantage Detection limits Capillary GC - with electron capture detection All orthosubsituted PCBs & all OCPs on the POPs list except toxaphene 30 or 60 m×0.25 mm id Col with H2 carrier gas Dual column nonpolar (DB-1) and intermediate polarity columns (DB-5) Relatively inexpensive and easy to operate Similar response factors for most OCs Good sensitivity for all POPs Adequate for routine tasks High potential for misidentification of some POPs due to co-eluting peaks DDT/DDE : pg Quadrupole mass spectrometry in electron ionization (EI) mode All PCBs & all OCPs on the POPs list except toxaphene 30 m×0.25 mm i.d low-bleed columns with He carrier gas Selected ion mode for target POPs Moderately expensive and more complex to operate and maintain Newer instruments (post 1997) have adequate sensitivity for routine POPs monitoring at low pg/μL concentrations Much less potential for misidentification than with ECD DDT/DDE :1~10 pg HCB : ~10 pg Dieldrin : 25 pg Toxaphe : 500 pg (as tech mixture) Quadrupole mass spectrometry in electron capture negative ionization (ECNIMS) mode Toxaphene and other highly chlorinated OCPs and PCB with >4 chlorines 30 m×0.25 mm i.d low-bleed columns with He carrier gas Selected ion mode for target POPs Comparable sensitivity in ECNIMS mode to ECD in SIM mode for some POPs Much less potential for misidentification than with ECD DDT/DDE: 0.1 pg HCB : ∼0.1 pg Dieldrin: ∼1 pg Toxaphene: 10 pg (as tech mixture) Ion trap mass spectrometry using MS/MS mode All PCBs, All OCPs on the POPs list 30 m×0.25 mm i.d low-bleed columns with He carrier gas Same columns as quadrupole MS Comparable sensitivity to ECD in MS/MS mode for some POPs Much less potential for misidentification than with ECD DDT/DDE: pg HCB : pg Dieldrin : pg Toxaphe : 100 pg (as techmixture) High-resolution magnetic sector mass spectrometry in electron ionization (EI) mode All PCBs, all OCPs on the POPs list except toxaphene 30 m×0.25 mm i.d low-bleed columns with He carrier gas Selected ion mode for target POPs at 10,000×resolution Comparable sensitivity to ECD in SIM mode Highly reliable identification at low pg/μL levels DDT/DDE: 0.05 pg HCB : 0.05 pg Dieldrin : 0.1~0.5 pg Toxaphe : 10 pg (as tech mixture) HCB : 0.5 pg Anal Bioanal Chem(2006) 386:769~789 Instrumental Analysis □ Instrument : HP-6890N with CTC PAL □ Column : SP-2331(60m×0.32㎜×0.20㎛) Gas chromatograph (GC) □ Injection Mode : Splitless (inj.vol 1uL) □ Carrier gas : He (99.999%), 1.3 ml/min □ Injection Temp : 260℃ □ Oven Temp : 120℃(1min)→20℃/min→200℃(0min) →5℃/min →260℃(15min) □ Instrument : Autospec Premier (Micromass) □ Principle : Double focusing type □ Resolution : > 10,000 (10% valley) □ Detection method : SIM Mass Spectrometer (MS) □ Ionizing Mode : EI □ Interface Temp : 260℃ □ Detector Temp : 250℃ □ Electron energy : 35eV □ Emission current : 500μA quantification Internal std/Isotope dilution method OCPs Standard (Scan mode, GC/MSD 200 ng) HCB Heptachlor Epoxide B oxychlordane 2,4-DDE, g-chlordane Column DB-5MS (60 m × 0.25 mm ID, 0.25 m) Carrier Helium (99.999%) at 1.0 mL/min Oven 80℃ for 100 - 180℃ at 20℃/min 180 - 280℃ at 2.5℃/min 280℃ for 10 Injector Splitless, 260 ℃, 2.0 uL Detector MSD 230 ℃ ion source, 70 eV Full scan at 50-450 m/z 2,4-DDD, dieldrin 4,4-DDD, cis-nonachlor 4,4-DDE Heptachlor 2,4-DDT 4,4-DDT Mirex a-chlordane Aldrin trans-nonachlor endrin endrin aldehyde endrin ketone Quality Assurance / Quality Control Category Blank Items HRGC/ HRMS Remark Field blank - Assessment of potential contamination by sampling process Glassware blank -Collect the solvent which was rinsed the glassware at last step and then analyze Assessment of potential contamination by glassware Lab blank -Control sample analyze by same procedure with real samples - Need to be done more than sample for sample batch Assessment of potential cross contamination in Laboratory Ambient air - HCB : 40~120%, Aldrin : 30~120% - The others : 50~120% Cleanup std Water, soil, sediment - 50~120% Cleanup std Range - Within quantification range By Sample batch linearity - r2 : > 0.99 RSD of RRF -less than ±15% Ver STD - Less than 20% of RRF Resolution - 10% Valley, > 10,000 PFK mass check - Within 5ppm between theoretical/practical value isotope ratio - Less than ±15% As of quantification lock mass - Within ±20% of initial value If not, re-analysis Recovery Calibration curve Contents Interlaboratory QC check should be done by CRM or RM CS Flow chart for POPs Analysis by K-ESM Korea environmental Standard Method Sampling std spiking Sampling Acidic pretreatment Extraction (HCB & total PCBs) PCDD/DFs Co-PCBs HCB Indicator PCBs Sulfuric treatment Sulfuric treatment Sulfuric treatment Sulfuric treatment Multi-layer silicagel Multi-layer silicagel Multi-layer silicagel Multi-layer silicagel HRGC/HRMS HRGC/HRMS or GC/MS GC/MS or GC/ECD Cleanup std spiking Alumina column Syringe std spiking HRGC/HRMS Flow chart for OCPs Analysis by K-ESM Water Ambient Air  Acetone, 16hrs Soxhlet  Sample 500mL + NaCl 30g  Cleanup std spike  N-hex 50mL, shaking 10min, twice Ultrasonic     Sample 10g + Na2SO3 10g Cleanup std spike Acetone 25mL, shaking 10min, Sonic 10min  concentration  concentration Florisil catridge Florisil column  Rinse : n-hex, 13C POPs spike  Elution : 5% ether/hex, 100mL  Elution : 20% ether/hex, 100mL Elute Liquid/Liquid Ext Soil Florisil catridge  Conditioning : n-hex, 10mL  15% ethyl acetate/n-hex, 20mL  Conditioning : n-hex, 10mL  15% ethyl acetate/n-hex, 20mL Elute  concentration Silicagel column  22% silicagel with impregnated H2SO4  N-hex, 30mL Concentration  Syringe std spike HRGC/HRMS GC/LRMS  Approx 0.5mL Silicagel column Concentration Silicagel column  Approx.50~500 uL  Syringe std spike HRGC/HRMS GC/LRMS Concentration  Approx.50~500 uL  Syringe std spike HRGC/HRMS GC/LRMS POPs monitoring site : ambient air 1day 2day 3day QGF PUF ACF PUF Using High Volume Air Sampler(HVAS),  collection velocity : > 0.7m3/min @ 0℃, 1atm (sample volume : > 1,000 m3  sampling time : days samples collection by 24hrs (sequencing) ) POPs monitoring site : Water/sediment Water  Using 6L amber bottle (considering the depth and width of sampling site), > 20L Sediment  Using Grab sampler, surface layer sediment (upper sediment about 2cm depth) without turbulence ... Similar response factors for most OCs Good sensitivity for all POPs Adequate for routine tasks High potential for misidentification of some POPs due to co-eluting peaks DDT/DDE : pg Quadrupole mass... ionization (EI) mode All PCBs & all OCPs on the POPs list except toxaphene 30 m×0.25 mm i.d low-bleed columns with He carrier gas Selected ion mode for target POPs Moderately expensive and more complex... low-bleed columns with He carrier gas Selected ion mode for target POPs Comparable sensitivity in ECNIMS mode to ECD in SIM mode for some POPs Much less potential for misidentification than with ECD