131 Mass Spectroscopy The paper gave a comprehensive overview on Mass Spectroscopy. The paper discussed the principles of mass spectroscopy, techniques and its application in the field of pesticides. Because of high sensitivity of the techniques, mass spectroscopy has become the powerful analytical tool for monitoring environmental pollutants, pesticides residues in food, water and soil upto parts per billion and/or trillion levels. Slide 1 General Applications of Mass Spectrometry •Environmental analysis •Forensic analysis •Clinical research •Proteomics and genomics •Generation of physico-chemical data 1 Slide 2 Identification of unknown compounds • UV Spectroscopy • IR -do- • NMR -do- • Mass Spectrometry Non- spectroscopy Differences: Gas Phase Destructive 2 132 Slide 3 INFORMATION OBTAINED FROM MASS SPECTRA • MOLECULAR WEIGHT • STRUCTURAL CHARACTERISTICS • ELEMENTAL COMPOSITION OF MOLECULAR ION AND FRAGMENT IONS 3 Slide 4 SPECIAL ADVANTAGES • HIGH SENSITIVITY • HIGH ACCURACY • COUPLING OF CHROMATOGRAPHIC TECHNIQUES SUCH AS GC, HPLC, CE ETC., 4 Slide 5 BASIC PRICIPLES GAS PHASE ION CHEMISTRY • PRODUCTION OF IONS • SEPARATION OF IONS • DETECTION OF IONS 5 133 Slide 6 MAJOR ION FOMATION TECHNIQUES • ELECTRON IMPACT IONIZATION (EI) • CHEMICAL IONIZATION (CI) • FAST ATOM BOMBARDMENT (FAB) • ELCTROSPRAY IONIZATION (ESI) • MATRIX ASSISTED LASER DESORPTION IONIZATION (MALDI) 6 Slide 7 M(g) EI M+. a b c d e f g (g) M(s,l,g) CI/ESI MH+ A B C MNa+ (g) MH+ ESI/MALDI MNa+ (s,l) 7 Slide 8 ION ANALYZERS • MAGNETIC (B) • ELCTROSTATIC (E) • QUADRUPOLE (Q) • ION TRAP (Tr) • TIME OF FLIGHT • FOURIER TRANSFORM ION CYCLOTRON RESONANCE (FT-ICR) 8 134 Slide 9 DETECTORS • SECONDARY ELECTRON MULTIPLIER • PHOTOMULTIPLIER • MULTI CHANNEL PLATES 9 Slide 10 SCHEMATIC DIAGRAM OF A MASS SPECTROMETER 10 Slide 11 ELECTRON IMPACT (EI) • METHOD OF IONIZATION BY ELCTRON BOMBARDMENT • SAMPLE NATURE VOLATILE AND THERMALLY STABLE MOLECULAR WEIGHT UPTO 800 Da INFORMATION MOLECULAR WEIGHT STRUCTURAL DETAILS QUANTIFICATION ADDITIONAL FEATURES COUPLING WITH GC 11 135 Slide 12 Schematic representation of an electron ionization ion source. M represents neutral molecules; e-, electrons; M+• , the molecular ion; F+, fragment ions; Vacc, accelerating voltage; and MS, the 12 mass spectrometer analyzer. Slide 13 ELECTRON IMPACT (EI) +. M + e- M + 2e .................. (1) -. M + e- M .................. (2) n+ M + e- M + (n + 1) e ................... (3) 13 Slide 14 ELECTRON IMPACT (EI) +. ABCD + e - ABCD + 2e- 10-14-10-16sec + . + . ABCD ABC + D + . + . ABCD AB + CD + . . ABCD + A + BCD 10-8 sec + . +. ABCD AD + BC + + ABC AB + C . . AD + + A +D 14 136 Slide 15 Ions produced in the Electron impact source CD. D. ABC+ AD+. ABCD+. BCD. A+ A- AB+ 15 Slide 16 Hypothetical electron impact mass spectrum of a compound ABCD 100 + AD + A 75 +. ABCD RA % + AB 50 + + ABC D 25 0 16 m/z Slide 17 NITROGEN RULE -e A-B (A-B)+. (odd electron ion) (even number (odd number of electrons) of electrons) A+ + B. (A-B)+. (odd electron ion) Even electron ion Odd electron ion Even mass number if it contains No nitrogen or Even number of nitrogen atoms Odd mass number if it contains odd number of nitrogen atoms 17 137 Slide 18 Elements C H O S P F Cl Br I N Atomic 12 1 16 32 31 19 35 79 127 14 weight Valency 4 1 2 2 3 1 1 1 1 3 18 Slide 19 Compound Molecular Formula Molecular Weight C6 H6 78 C5 H5 N 79 N N C4 H4 N2 80 N N N C3 H3 N3 81 N 19 Slide 20 Elements Isotopes (abundance %) H 1H (99.99) C 12C (98.9) 13C (1.1) N 14N (99.6) 15N (0.4) O 16O (99.8) 18O (0.2) S 32S (95) 33S (0.7) 34S (4.2) Cl 35Cl (75.5) 37Cl (24.5) 79Br (50.5) 81Br (49.5) Br 19F, 31P, 127I - 100 % each 20 138 Slide 21 (M)+. , (M+1)+. , (M+2)+. (M)+. Each Carbon will contribute~ 1.1 % of (M)+. To (M+1)+. For methane CH4 (16) m/z 17 1.1% of the intensity of m/z 16 (M+1)+. Pm+1 / Pm X 100 = 1.1 (M+2)+. m/z 21 Slide 22 [(PM+1)/( PM)] x 100 = [1.1 x No. of C atoms] + [0.4 x No of N atoms] + [0.7 x No. of S atoms] PM+1 = relative abundance of (M + 1)+· ion PM = relative abundance of M +· ion [(PM+2)/( PM)] x 100 = [(1.1 x No. of C atoms)2/200] + [0.2 x No of O atoms] +[4.2 x No. of S atoms] PM+2 = relative abundance of (M + 2)+· ion 22 Slide 23 Chlorine 35Cl 75.5 37Cl 24.5 ~ 3:1 Chloro benzene 112 M+. 112 (35Cl) Cl (M +2)+. 114 (37Cl) 114 M+. : (M +2)+. = 3:1 m/z 23 139 Slide 24 Chlorine 35Cl 75.5 37Cl 24.5 ~ 3:1 Dichloro benzene 146 M+. 146 (35Cl, 35Cl ) Cl 148 (M +2)+. 148 (35Cl, 37Cl) (37Cl, 35Cl ) 150 Cl (M +4)+. 150 (37Cl, 37Cl) M+. : (M +2)+. : (M+4)+. = 9:6:1 m/z (a+b)2 = a2 + 2ab+ b2 where a =3 and b =1 24 Slide 25 Bromine 79Br 50.5 81Br 49.5 ~ 1:1 156 158 Bromo benzene M+. 156 (79Br) Br (M +2)+. 158 (81Br) M+. : (M +2)+. = 1:1 m/z 25 Slide 26 Bromine 79Br 50.5 81Br 49.5 ~ 1:1 Dibromo benzene 236 M+. 234 (79Br, 79Br ) Br 234 238 (M +2)+. 236 (79Br, 81Br) (81Br, 79Br ) Br (M +4)+. 238 (81Br, 81Br) M+. : (M +2)+. : (M+4)+. = 1:2:1 m/z (a+b)2 = a2 + 2ab+ b2 where a =1 and b =1 26 140 Slide 27 Atomic weight of some elements commonly dealt in Organic Chemistry 12C = 12.0000 1H = 1.00783 31P = 30.97376 10B = 10.01294 32S = 31.97207 14N = 14.00307 35Cl = 34.96885 16O = 15.99491 37Cl = 36.96590 19F = 18.99840 79Br = 78.91839 28Si = 27.97693 81Br = 80.91642 Nominal mass Correct mass BENZENE C6H6 78 78.04698 PYRIDINE C5H5N 79 79.04222 27 Slide 28 Schematic diagram of a GC Injector Detector Column 28 Slide 29 Schematic diagram of a GC-MS Injector Detector MS Column 29 141 Slide 30 Capillary GC-MS interface 30 Slide 31 Abundance TIC: QUE.D 2e+07 Total ion 1.8e+07 chromatogram 1.6e+07 1.4e+07 1.2e+07 1e+07 8000000 6000000 4000000 2000000 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 Time--> 31 Slide 32 Abundance TIC: QUE.D 4.49 4.39 5000000 1.15 Expanded TIC indicating 4500000 solvent peaks 4000000 3500000 3000000 5.49 2500000 2000000 3.89 1500000 1000000 3.52 500000 2.46 2.94 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Time--> 32 142 Slide 33 Library searches from full scan 33 Slide 34 Quantification by mass spectrometry • Total Ion Chromatograms (TIC) • Extracted Ion Chromatograms (EIC) • Selected Ion Recording (SIR) • Multiple Ion Recording (MIR) • Single Reaction Monitoring (SRM) • Multiple Reaction Monitoring (MRM) 34 8.80 NL: 3.96E7 Slide 35 100 5.18 12.09 12.39 21.24 TIC MS data08 50 Full Scan/SIM Analysis 0 8.80 NL: 3.96E7 100 TIC F: + c Full ms 12.09 12.39 5.18 [ 50.00-500.00] 21.24 MS data08 50 0 21.82 NL: 7.57E4 Relative Abundance 100 TIC F: + c SIM ms [ 131.50-132.50] 21.24 MS data08 50 0 14.61 NL: 2.06E3 100 TIC F: + c SIM ms [ 224.50-225.50] MS data08 50 0 11.06 NL: 7.43E3 100 TIC F: + c SIM ms 11.35 [ 178.50-179.50, 263.50-264.50] 50 MS data08 0 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Time (min) Pesticides analysed in strawberry/spinach/pea extract at 35 5 pg/µl 143 Slide 36 Analysis of traces of Phosphorous Pesticides 36 Slide 37 37 Slide 38 38 144 Slide 39 39 Slide 40 Recent references for Pesticide Residue analysis GC-MS or LC-MS ? Mass Spectrometry Reviews, 25, 838-865 (2006) Matrix Effects: Mass Spectrometry Reviews, 25, 881-899 (2006) Pesticides in water: Mass Spectrometry Reviews, 25, 900-916 (2006) Control of Food Safety by LC-MS: Mass Spectrometry Reviews, 25, 917-960 (2006) Micotoxins: Rapid Commun. Mass Spectrom. 20, 2649-2659 (2006) Chloramphenicol: J. Chrom. A 1118, 226-233 (2006) Pesticide Residues in vegetables by low pressure GC J. Chrom. A 1118, 236-243 (2006) 40 145 GAS CHROMATOGRAPHY - Mass Spectroscopy Gas chromatography-mass spectrometry (GC-MS) is a method that combines the features of gas chromatography and mass spectroscopy to identify different substances within a test sample. GC-MS is becoming the tool of choice for tracking organic pollutants in the environment. The paper described the principles, techniques and sample preparation, analysis and application of techniques in pesticide analysis. Slide 1 Chronology of Presentation • Gas Chromatography (GC) – Review of the technique • Mass Spectrometry (MS) – Review of the technique • Coupling of GC and MS • Applications – Sports drug analysis – Pesticides analysis – CWAs analysis • Examples • Conclusion Slide 2 Gas Chromatography • GC is based on distribution of chemicals between two phases: gas and solid or liquid • GC is suitable for chemicals that are volatile and do not degrade below 400oC • Non-volatiles can be derivatized to volatile derivatives • Distribution constant: – The solute partitioning between the two phases in a column can be described as a dynamic equilibrium Distribution constant (Kc) = Cs / Cm Where Cs = concentration of component in stationary phase Where Cm = concentration of component in stationary phase 146 Slide 3 Components of GC Carrier Gas, N2 or He, 1- 2 mL/min Slide 4 Splitless (100:90) vs. Split (100:1) Syringe Syringe Injector Injector He He Purge valve closed Purge valve GC column GC column open Slide 5 Split or splitless • Usually operated in split mode unless sample limited • Chromatographic resolution depends upon the width of the sample plug • In splitless mode the purge valve is close for 30-60 s, which means the sample plug is 30-60 seconds • As we will see, refocusing to a more narrow sample plug is possible with temperature programming 147 Slide 6 Open Tubular Capillary Column 0.32 mm ID Mobile phase (Helium) Liquid flowing at 1 Stationary 0.1-5 µm mL/min phase 15-60 m in length Slide 7 Chromatogram Retention Time Parameter used to identify a sample component Peak Area Parameter used to measure the quantity of the sample component Slide 8 Mass Spectrometry 148 Slide 9 What kind of info can mass spec give you? • Molecular weight • Elemental composition (low MW with high resolution instrument) • Structural info (hard ionization or CID) Slide 10 Parts of a Mass Spec • Sample introduction • Source (ion formation) • Mass analyzer (ion sep.) - high vac • Detector (electron multiplier tube) Slide 11 Sample Introduction/Sources Volatiles • Probe/electron impact (EI),Chemical ionization (CI) • GC/EI,CI Involatiles • Direct infusion/electrospray (ESI) • HPLC/ESI • Matrix Assisted Laser Adsorption (MALDI) Elemental mass spec • Inductively coupled plasma (ICP) • Secondary Ion Mass Spectrometry (SIMS) – surfaces 149 Slide 12 EI process • M + e- M+* f1 f2 f4 f3 This is a remarkably reproducible process M will fragment in the same pattern every time using a 70 eV electron beam Slide 13 Electron Ionization Benefits •Well-understood •Can be applied to virtually all volatile compounds •Reproducible mass spectra •fragmentation provides structural information •Libraries of mass spectra can be searched for EI mass spectral "fingerprint" •Limitations •Sample must be thermally volatile and stable •The molecular ion may be weak or absent for many compounds •Mass range •Low Typically less than 1,000 Da Slide 14 Chemical Ionization •Benefits •Often gives molecular weight information through molecular-like ions such as [M+H]+, even when EI would not produce a molecular ion. •Simple mass spectra, fragmentation reduced compared to EI •Limitations •Sample must be thermally volatile and stable •Less fragmentation than EI, fragment pattern not informative or reproducible enough for library search •Results depend on reagent gas type, reagent gas pressure or reaction time, and nature of sample •Mass range •Low Typically less than 1,000 Da 150 Slide 15 CI Reagent Gases •Methane: •Good for most organic compounds •Usually produces [M+H]+ and [M+29]+ adducts •Adducts are not always abundant •Extensive fragmentation •Isobutane: •Usually produces [M+H]+, [M+C4H9]+ adducts and some fragmentation •Adducts are relatively more abundant than for methane CI •Not as universal as methane •Ammonia: •Fragmentation virtually absent •Polar compounds produce [M+NH4]+ adducts •Basic compounds produce [M+H]+ adducts •Non-polar and non-basic compounds are not ionized Slide 16 Mass Analyzers • Low resolution – Quadrupole – Ion trap • High resolution – TOF time of flight – Sector instruments (magnet) • Ultra high resolution – ICR ion cyclotron resonance Slide 17 151 Slide 18 RESOLUTION • Resolution is the ability of a mass spectrometer to distinguish between ions of different m/z ratios • Greater resolution corresponds directly to the increased ability to differentiate ions • Resolution is inversely proportion to sensitivity Slide 19 The Mass Spectrum Slide 20 Types of Scans • Full Scan – Whole mass range (e.g. 35 – 400) is scanned – If four scans/sec (one scan/0.25 sec) are performed, the time spent in scanning each m/z value is 0.25/400 (0.000625) sec (Dwell time) – Thus total time spent to record each m/z value / sec is 0.25/400 x 4 (0.0025 sec) • Selected ion monitoring (SIM) – Only a few (one to three) characteristic ions are selected – Thus the dwell time (the analyzer remains at given m/z value) is increased – The fraction of these ions reach at the detector is also increased causing the enhanced sensitivity • Multiple Reaction Monitoring (MRM) • Precursor / product ion scan • Neutral ion loss 152 Slide 21 SIM • What it is – Monitoring only m/z ratio containing information • How is it done – Control mass analyzer to only select ions of analytical interest • Why is it done – Greater sensitivity – Better peak shape – Better accuracy and precision • Applications – Trace analysis – Complex matrices – Quantitation Slide 22 Choosing SIM Ions • Use minimum number ions for maximum sensitivity and precision • Choose ions for maximum specificity – High mass – Abundant – Unique to compound • Can choose ions characteristic of compound class for screening purpose Slide 23 Setting Up SIM Acquisition • Choose: – Number of ions/group – Dwell time/ion to obtain requisite number of cycles/peak for good quantitation • Goal: 15 – 25 cycles across a peak • Use equal dwell times for all ions • Use time programming (SIM groups) to minimize number of ions acquired/cycle • Consider mass defect in selecting the ion to get maximum sensitivity 153 Slide 24 Effect of Mass Defect on Maximum Sensitivity in SIM The ion with elemental composition of C23H35O2Si+ has nominal mass of 371 If ∆M ~1 the intensity of detector current as the analyzer scans over this m/z value is a curve having maximum value at 371.25 Setting the instrument at 371 for SIM analysis will result in lower sensitivity than if it set at 371.25 Slide 25 MS/MS OR TANDEM MS ANALYSIS • Widespread use in analytical chemistry for trace analysis in complex matrices • Provides sensitive and selective analysis • Elimination of chromatography – Specificity Slide 26 MS/MS Scan Q1 Q2 Purpose Product / Static (Parent Scanning Detection of all fragment ions Daughter ion ion selected) from a single precursor Precursor / Scanning Static (Daughter Detection of all precursors of a Parent ion ion selected) common fragment ion Neutral loss Scanning Scanning (offset Detection of all precursors by fragment sharing a common mass) neutral fragment SRM / MRM Static (Parent Static (Daughter Detection of a specific fragment mass mass selected ion originating selected) from a specific precursor 154 Slide 27 Triple Quadrupole: MS/MS Product ion Scan Slide 28 Triple Quadrupole: MS/MS Precursor ion Scan Slide 29 Triple Quadrupole: MS/MS SRM / MRM 155 Slide 30 Triple Quadrupole: MS/MS Neutral loss Scan Slide 31 Begin the Mass Spectral Analysis • Switch on the instrument • Check the communication of software with instrument • Wait to reach the required vacuum • Calibrate / Tune the instrument Slide 32 What Does Tuning Do • Set voltages on source elements • Set amu gain and offset for correct peak width • Set EM voltage • Set Mass Axis for proper mass assignment 156 Slide 33 Development of Interfaces between GC and MS GC operates at higher pressure than MS Interface was required to cope up the pressure difference First coupling of GC with MS was done by James and Martin in 1950 First coupling was done using TOF analyzer with GC Initial development were directed to separate the carrier gas molecules from analytes Interfaces working on the principle of differential diffusibility of analytes and carrier gas were developed Slide 34 Applications of GC-MS (Chemical Analysis) • Qualitative analysis – Identification of compounds • Library searches • Interpretation (understanding of fragmentation mechanisms) • Quantitative analysis – Determination of amount of analyte present in a sample • Establish efficient sample preparation protocols Slide 35 Example of Library Search 157 Slide 36 Pesticides Analysis • Pesticides are indispensable chemicals • Poisonous to mankind • Residual analysis in food, water and environment samples is of paramount importance from view point of preventive medicine • Frequently used pesticides are OP and carbamates pesticides • Prerequisite to develop a quantitative determination method using GC-MS is to record the GC-MS and GC-MS/MS spectra of targeted analytes Slide 37 Classes of Pesticides Carbamates Pesticides Slide 38 Comparison of Mass Spectra of Selected Pesticides in Different Ionization modes Iprofenfos 158 Slide 39 Comparison of Mass Spectra of Selected Pesticides in Different Ionization modes MALATHION Slide 40 Comparison of Mass Spectra of Selected Pesticides in Different Ionization modes Ethiofencarb Slide 41 GC-MS in Residual Pesticide Analysis Pesticides Usage • More than 700 pesticides are registered for use WW • About 2.2 billion kg of pesticide used each year WW • 1995 WW pesticide sales = $29 billion • Some very toxic pesticides are banned in many countries but may still be used in others: – Endrin, DDT, lindane, aldrin, chlordane, and many others • No standardization of Maximum Residue Limits (MRLs) in food • Banned or highly restricted pesticides have been “dumped” in developing countries 159 Slide 42 GC-MS in Residual Pesticide Analysis • Analysis of pesticides by GC-MS requires development of extraction protocols • Sample preparation must be applicable to multiple pesticides • The adopted method must eliminate the background without (or at least minimal) loss of analyte Slide 43 Best Approach for Choosing Extraction and Analysis Methods • Choose a method already in use by experienced pesticide analysts –It will already be validated in at least one lab • Make minor adaptations as needed for: –differences in commodities –differences in analytical equipment • Validate the method in your laboratory Slide 44 Where can we find Good Validated Methods? • Florida Department of Agriculture and Consumer services – J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M. Engel (1999) J. AOAC Int. 82, 1419-1435 • California Department of Food and Agriculture (www.cdfa.ca.gov) – Multiresidue Screen for Pesticides in Fruits and Vegetables (1995) California Department of Food and Agriculture, Sacramento, CA, USA summary 1-2 – S.M. Lee, M.L. Papathakis, H.M.C. Feng, G.C. Hunter, J.E. Carr (1991) Fresenius J. Anal. Chem. 339, 376-383 160 Slide 45 Where can we find Good Validated Methods? • Ministry of Public Health, Welfare and Sport, The Netherlands – Analytical Methods for Pesticide Residues in Foodstuffs, 6th ed. (1996) General Inspectorate for Health Protection Ministry of Public Health, Welfare and Sport (The Netherlands) • Pesticide Analytical Manual (PAM) – U. S. Food and Drug Administration Center for Food Safety and Applied Nutrition Office of Plant and Dairy Foods and Beverages 1994; Updated October, 1999 – Can download from the WWW at: • http://vm.cfsan.fda.gov/~frf/pami1.html – Includes a lot of basic information on chromatography Slide 46 General Extraction Protocol for Pesticides Before GC, GC-MS Analysis 5 µL splitless injection inGC, GC-MS Adopted at - Chemical and Veterinary Control Laboratory D – 48147 Muenster, Germany Slide 47 General Extraction Protocol for Pesticides Before GC, GC-MS Analysis 161 Slide 48 New Pesticide Analysis Method Slide 49 Detailed Sample Treatment Before GC- MS Analysis – 10 gram sample – Addition IS – Extraction 10 ml Acetonitril (1% acetic acid) – 4 gram MgSO4 + 1 gram NaAc – Spin 10 min 3000 rpm – 1 ml extract + 25 mg Primary Secondary Amine (PSA) – 150 mg MgSO4 – Spin 5 min 5000 rpm – 700 µl GC vial GC-MS Slide 50 162 Slide 51 Deconvolution software facilitate the analysis *GC/MS in synchronous SIM/Scan mode combined with deconvolution reporting software enables efficient pesticide residue analysis at low µg/Kg in various food commodities in one run *This new method is a powerful tool for multi- residue pesticide analysis Slide 52 Slide 53 Application of GC-MS/MS FOR Pesticide analysis A Comparison of SIM and MRM • Five food matrices cucumber, sweet pepper, grapefruit, wheat flour and curry powder were spiked (0.005 to 0.5 mg/Kg) with 32 pesticides and extracted by QuEChERS method • Extracted samples were analyzed in SIM and MRM modes • Results indicated lesser background and higher sensitivity in case of MRM mode analysis C. Wauschkuhn and P. Hancock; Chemisches und Veterianarsuchungsamt, Stuttgart, Germany; Waters Corporation, Manchester UK, 2006 163 Triple-quad Mass Spectrometry The paper gave a history of the development of Mass spectroscopy, the principles and application of this highly precise techniques in analysis of pesticides at very low level. The paper discussed the principles and advantages of Triple-quad over single quad mass spectroscopy. Slide 1 GC Detectors Thermal Conductivity PMT Flame Photometric Filament pair heats Optical filter selects O when sample dilutes 2 H wavelength specific to P or S REF carrier gas 2 compounds NP Thermionic Flame Ionization N or P compounds Burning produces increase current in plasma Air charged particles which H 2 from vaporized metal salt H 2 collector converts into a current Electron Capture Ion Mass Selective Detector Analyzer Loss of slow electrons Source EM Ionized sample measured by sample absorption by mass analyzer decreases cell current 1 Slide 2 Comparison of GC Detectors TCD FID ECD NPD(N) NPD(P) FPD(S) MSD (SIM) (SCAN) 10-15 10-12 10-9 10-6 10-3 fg pg ng ug mg 1 ng in 1 uL Liquid (sg = 1) is 1 ppm Concentration Mass Selective Detector is both: Specific and Universal 2 164 Slide 3 Functional Components of the MS EXHAUST MECHANICAL PUMP HI VAC PUMP MASS SPECTROMETER ION MASS GC INTERFACE DETECTOR SOURCE FILTER CONTROLLER (ChemStation) 3 Slide 4 GCMS –The components • Inert Ion source BASIC COMPONENTS • Hyperbolic quadrapole • Heated Quadrapole • Triple Axis Detector • vacuum Pump 4 165 Slide 5 Ion Source – Inertness • Improved response for difficult compounds • Improved peak shape for active compounds • Reliable spectral data • Entrance lens design 5 Slide 6 Ion Source – Improved Response Inert Ion source – Improved response 50pg LSD SS Source ance Extracted Ion 253 m/z Abundance Ion 253.00 (252.70 to 253.30): OLDLSD07B.D 105000 100000 30000 Inert Source Ion 253.00 (252.70 to 253.30): INERTLSD13W.D 95 90 85 0 0 0 00 00 00 80000 20000 75000 ~6x 10000 70000 00000 65 60 0 0 00 00 LSD S/N 2.9 90000 improvement! 55000 50000 80000 45000 70000 40000 LSD S/N 16 35000 60000 30000 25000 50000 20000 40000 15000 10000 30000 5000 20000 0 6 .10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 Time--> 10000 0 6.10 6.20 6.30 6.40 6.50 6.60 6.70 6.80 6.90 7.00 7.10 7.20 7.30 7.40 7.50 > 6 166 Slide 7 Ion Source – Improved Peak Shape ｱﾊﾞﾝﾀﾞﾝｽ ｲｵﾝ 277.00 (276.70 ｲｵﾝ 247.00 (246.70 Inert Source ～ 277.70): ～ 247.70): YG0701_1.D YG0701_1.D 15000 14000 277u 13000 12000 Fenitrothion(EIC m/z 277 and 247) 11000 10000 9000 8000 7000 Breakdown ion 247u 6000 5000 4000 3000 2000 1000 0 11.90 12.00 12.10 12.20 12.30 12.40 12.50 12.60 12.70 12.80 Time--> ﾞﾝﾀﾞﾝｽ 7500 SST Source ｲｵﾝ ｲｵﾝ 277.00 247.00 (276.70 (246.70 ～ 277.70): ～ 247.70): YG0703_2.D YG0703_2.D 7000 6500 6000 Breakdown ion Increase in breakdown 5500 SST Source ion reduces the 247u 5000 4500 4000 3500 277u abundance of the ion of interest (277u). 3000 2500 2000 1500 1000 500 me--> 0 11.90 12.00 12.10 12.20 12.30 12.40 12.50 12.60 12.70 12.80 Result: lower sensitivity 7 Slide 8 Monolithic Quartz Quadrupole • Single piece construction • Hyperbolic surface • o Heated upto 200 C –Maintenance free • 8 167 Slide 9 Electron Ionization (EI)-MOST POPULAR IN GC . Ionization: ABC + e - ABC + + 2e - Neutral Excited Molecule Molecular Ion Position of Curve . #ABC+ Depends on IP (ABC) 0 10 70 100 eV Electron Energy Fragmentation: . . ABC + AB + C+ . A+ + BC . AB + +C (loss of neutral) . AC + +B (rearrangement) etc. Resulting Mass Spectrum: + AB+ C Signal + . + + ABC A AC Abundance m/z 9 Slide 10 High Energy Dynode/Electron Multiplier Detector Positive Ions ++ High Energy +- + -- Dynode + --- ++ --- +++ +++ + + ++ + ++ + + + + ++++ + ++ ++ + + ++ -- + + +++++ + + + + + -- Electrons --- -- Electron Multiplier Quadrupole Iris Detector Focus Lens Signal Out 10 168 Slide 11 A Typical Mass Spectrum Dodecane: C12H26 Abundance Average spectrum of dodecane from EVALDEMO.D 57 100 <--[C H ] + 4 9 (Base peak) 90 80 70 71 60 43 + <--[C H ] 5 11 50 85 40 <--[C H ]+ 30 6 13 + . M (Molecular 20 ion) 55 29 98 10 113 170 128 141 159 m/z-> 20 40 60 80 100 120 140 160 180 • Molecular ion (a.k.a. parent ion): loss of one electron • Base peak: most abundant ion in spectrum 11 ADVANTAGES OF SINGLE QUAD • SIMPLE AND EASY TO SETUP • SENSITIVITY AND SELECTIVITY • STRONG SUPPORT OF LIBRARY 12 169 Slide 12 Transmission Quadrupole MS & MS/MS Sensitivity and Selectivity Scale Very Sensitive Most Sensitive Dwell 20-50 ms Fast 12,500 u/s (5975C) Typical < 2,000 u/s Fast 6,250 u/s (7000A) Targets & Non-targets Dwell 1-50 ms Targets & Non-targets Targets only *Selected Reaction Monitoring (similar to SIM – Selected Ion Monitoring Also called MRM for Multiple Reaction Monitoring 13 Slide 13 Transmission Quadrupole MS & MS/MS Sensitivity and Selectivity Scale Very Sensitive Most Sensitive Dwell 20-50 ms Fast 12,500 u/s (5975C) Typical < 2,000 u/s Fast 6,250 u/s (7000A) Targets & Non-targets Dwell 1-50 ms Targets & Non-targets Targets only Very Selective Most Selective Unit mass + AMDIS Q1 1.2 u Unit mass resolution CID product ions Targets only per DBL Q2 1.2 u 14 170 Slide 15 When Is Sensitivity REALLY Important? • When the method requires a lower detection limit • When sample is limited •No option of starting with a larger sample • When there more sample preparations is not a reasonable option • When injecting less sample will extend the life of the inlet liner and column and/or reduce the frequency of source cleaning 15 Slide 16 When Is Selectivity REALLY Important? • When two or more analytes have the same retention time and same ions • When analytes and matrix peaks have the same retention time and same ions Sets of standards will probably not show the benefits of DRS and MS/MS! The chromatographic profile determines the need: DRS for less intense coeluting peaks MS/MS for very intense coeluting peaks Matrix is often the primary source of coelutions. 16 171 Slide 17 How Different Modes Complement More Sensitivity Scan SIM (SIM/Scan) More Selectivity Scan DRS More Selectivity SIM MS/MS Larger sample SIM More Sensitivity Larger injection MS/MS Sharper peaks 17 Slide 18 Synchronous SIM/Scan Comparison of PAHs SIM Scan 45-450u Benz[a]anthracene Triphenyl phosphate 5.55 cycles/s 5.55 cycles/s Scan: Chrysene Poorer S/N but targets and non- targets detected SIM: Better S/N but only target analytes 0.2 ppm ? Application 5989-4184EN 18 172 Slide 19 The matrix Raw ions results spectrum in false negative Deconvoluted DRS enables a spectrum positive identification p,p’-DDE Library spectrum 19 Slide 20 Complex Matrices Show the Benefit of MS/MS 100 fg HCB in Clean Matrix 300 fg HCB in Diesel Single MS: SIM 283.8 S/N=26:1 RMS SIM about equal to MS/MS 15x better than SIM in MS/MS in clean matrix complex matrix – and better baseline MS/MS: 283.8:213.9 S/N=37:1 RMS 20 173 Slide 21 Assessment of Needs and Limitations what is the budget? higher cost OK need lower cost Scan MS/MS MS-DRS SIM This may not be your first question, but it must be asked what detection limit is required? ng & mid-pg OK need low pg & fg Scan MS/MS MS-DRS SIM MS/MS gives consistently lower detection limits for simple and very complex samples SIM/Scan how important are non-target peaks? only analyze targets need lbr searches SIM Scan MS/MS MS-DRS If non-targets (unknowns) are important Scan is essential; SIM/Scan may be the best choice for an analysis with both targets and non-targets 21 Slide 22 Assessment of Needs and Limitations how complex are the samples? how much coelution is expected? less complex matrix very complex matrix Scan MS-DRS MS/MS SIM Least selective Most selective how critical is sample preparation? MS-DRS sample preparation = time, money, errors more smp prep OK need less smp prep Scan MS-DRS MS/MS SIM Do you like spending time on sample prep? how critical is a unique quant ion? important very, very important Scan MS/MS SIM can you afford an error? MS-DRS Ionization mode Ionization mode + CID determines uniqueness determines uniqueness 22 Slide 23 Cost of Operation Benefits of GC/MS/MS • GC/MS/MS can be much, much cheaper to operate: • Smaller sample size and/or less sample preparation • Faster analyses (more samples/hour) • Simple and faster data review (less confusion) • Methods are more complex to build, but just as easy to run • The extra purchase price of MS/MS may be recovered in a few years . . . of the guaranteed 10-year life • During which time, more accurate results have been also generated! • What is the “cost” of false negatives and positives (errors)? 23 174 Slide 24 Summary of Relative Performance Factors GC/MSD GC/MSD GC/MSD GC/QQQ Scan SIM Scan-DRS MS/MS S from analyte + +++ + +++ S from matrix -- -- - 0 N from neutrals = = = Ultra low N from matrix --- --- - 0 N from "bleed" Low Low Lower 0 MDL (clean) Very Low Lowest Lower Lowest MDL (very dirty) Low Low Lower Lowest Quant Error Low Low Lower Lowest Sanalyte MDL = But Scan is Nneutral + Nmatrix + NGC “bleed” essential for non-targets Smatrix (unknowns)! Quant Error = Sanalyte + Smatrix S= Signal N=Noise 24 Slide 25 Application Alignment with MS Modes Non target compounds Drinking water Synthesis confirmation Scan Industrial samples “Street” drug samples Flavor and Fragrances it y sel itiv ec t ns iv it se y High purity water Waste water Air Analysis Food matrices Toxicology screens SIM DRS Tox of complex samples Pharma residual solvents y s el vit e ct siti iv it sen y Highly contaminated water MS/MS Very complex food matrices Trace tox of very complex samples 25 Slide 26 Basic Questions – Which GC/MS Solution? • Target analysis only? • Scan with libraries • SIM with ion ratios • MS/MS with ion ratios • Analysis of non-targets • Scan MS with SQ, TOF, or (unknowns)? tandem MS in SQ mode with • Orders of magnitude higher libraries detection limits than SIM or MS/MS • How much chemical noise • DRS or MS/MS from the matrix? • Backflush or 2D GC • (More sample prep) 26 175 Slide 27 General Information about GC/MS/MS Hyperlinked menu • Why Quadrupole GC/MS/MS? • Description of MS/MS process • Analytical benefits of MS/MS • General Examples of MS/MS Data High Sensitivity Fast SRM Speed MassHunter Software Agilent Reliability 27 Slide 28 Why a Quadrupole GC/MS/MS System? • MS/MS provides lower S/N in complex matrices than single quadrupole scan or SIM • MS/MS allows for the accurate quantitation of target compounds even in high chemical background samples • MS/MS selectivity means less sample prep • Sample prep must meet requirements of the GC inlet and column • Quadrupole MS/MS has better precision and linearity than ion trap MS/MS • Newer regulations in some markets specify the analytical power of GC/MS/MS 28 Slide 29 GC/MS Triple Quad (QQQ) for GC/MS/MS Collision Gas (Ar, N2, He) Carrier Gas (He, H2 ) Ion Source Detector Quad 1 Quad 2 Quad 3 •Ionize Mass Collision Mass Analysis Cell Analysis Mean Free Long Short Long Path Collisions No Yes No MS MS 29 176 Slide 30 Selected Reaction Monitoring (SRM) Quad Mass Filter (Q1) Collision Cell Quad Mass Filter (Q2) Spectrum with Q1 lets only Collision cell Q2 monitors only background target ion 210 breaks ion 210 characteristic ions (from EI) pass through apart fragments 158 and 191 from ion 210 210 210 for quant and qual. 222 158 158 268 280 165 191 210 191 160 190 170 210 250 290 190 210 150 170 190 210 no chemical background 30 Slide 31 MS/MS Eliminates Scan and SIM Interferences Triple Quad MS Single Quad MS Precursor selectivity same as MS but selectivity proportional to high probability that one or more of the spectral resolution product ions will be a unique dissociation no selectivity against ions interference product of the precursor only with same m/z AND NOT the interference analyte Product 2 Product 1 interference Product 3 unit mass resolution analyte 31 Slide 32 MS/MS Ensures Lowest Detection Limits EI: spectrum of analyte can also include ions from matrix, column bleed, gases, etc. Product 2 Product 1 isolate precursor Product 3 Q1 SIM before CID chemical noise from these other CID + Lower m/z Product Q2 SIM Ions measured ions is eliminated against zero chemical noise 32 177 Slide 33 Eliminates the “Invisible” Interferences of SIM 103 EI-SIM Scale Unit mass resolution change Removed by SIM EI-SIM 20 x 103 Unit mass resolution filters the intense ion that is 1 m/z lower, BUT NOT the isotope peak from that intensity ion—this can be a common Isotope ion interference in very ‘dirty’ not removed samples by SIM But what happens when a much more intense ion MS/MS eliminates of a multi-carbon compound has this interference analyte ion an ion 1 m/z lower? Note: in complex matrices, this isotope interference creates incorrect SIM ratios and SIM reports with false negatives 33 Slide 34 MS/MS Succeeds Where MS Fails GC/MS Single Quad SIM Interfering matrix peaks = chemical noise GC/MS Triple Quad SRM A chromatographer’s dream: single peak on flat baseline 34 Slide 35 As Matrix Increases - MS/MS is More Valuable 100 fg HCB in Clean Matrix 300 fg HCB in Diesel Single MS: SIM 283.8 S/N=26:1 RMS SIM about equal to MS/MS 15x better than MS/MS in clean matrix SIM in complex matrix MS/MS: 283.8:213.9 S/N=37:1 RMS 35 178 Slide 36 SIM (5973 Single Quad) vs SRM (7000A Triple Quad) a-HCH 3.2 pg injected SIM target m/z 219 -> 147 m/z 181 RMS S/N 222 : 1 RMS S/N 30 : 1 * 36 Slide 37 Lindanes: SRM Quant Transition 219 147 3.2 pg injected 1.6 pg injected 0.4 pg injected 0.2 pg injected 37 Slide 38 Agilent 7000A (QHQ) Design Collision Gas (N2 ) Ion Source Quad 1 Hexapole Quad 2 Detector Collision Cell The hexapole field has excellent transmission efficiency for precursor and product ions 38 179 Slide 39 Why a Hexapole: Comparison of Transmission Characteristics Mass Range Transmission Quadrupole Hexapole Octopole 1.2 1 0.8 0.6 0.4 o i m n a r T s 0.2 GC/MS m/z=1050 0 0 500 1000 1500 m/z Quadrupoles are the best mass filters (analyzers) Hexapoles and octapole are the best transmission devices 39 Slide 40 500 Transitions/sec: Why Is This Important? • Narrow chromatographic peaks (GC <1-2 s) • Sufficient data points (15) to perform acceptable/accurate quantification • Many compounds to monitor in single run (multiresidue) when coeluting peaks are quite frequent • Regulated environment where QC checks are required to ensure data validity • Confirmatory transitions according to the 96/23/CE directive 40 Slide 41 Why Helium Quenching? Collision Cell Process: Typical Description Collision Cell 1 ml/min N2 Collision Gas Quad Analyzer Quad Analyzer Precursor Product Source Detector Ions In collision induced dissociation Ions Out This is a good description for LC/MS/MS, but it is not complete for GC/MS/MS 41 180 Slide 42 Why Helium Quenching? Collision Cell Process: Full Description for GC/MS/MS Collision Cell 1 ml/min N2 At the detector, Collision Gas metastable helium generates neutral noise. Quad Analyzer Quad Analyzer Precursor Product Source He* + + He* Detector Ions In collision induced dissociation Ions Out A high population of In GC/MS, neutral noise highly energetic helium is buried in much higher metastables are produced chemical noise. in an EI source; since metastable helium is not In GC/MS/MS, chemical charged, it can pass noise is greatly reduced through mass analyzer so neutral noise is a field into the collision cell critical source of noise. and through to the HED- EM 42 Slide 43 Agilent Collision Cell Process with Quench Gas Collision Cell 1 ml/min N2 Collision Gas Quad Analyzer Quad Analyzer Precursor Product Source He* + + He* Detector Ions In collision induced dissociation Ions Out Transmission of metastable helium to the detector is He Buffer Gas greatly reduced; the Triple-Axis detector further He* + He → 2 He + heat reduces neutral noise for ultra- low neutral noise. 43 Slide 44 7000A GC/MS/MS Specifications Installation Checkout Specs EI SRM 100:1 for 100 fg OFN PCI SRM (CH4) 20:1 for 100 fg BZP Typical Sensitivity Spec EI Scan 300:1 for 1 pg OFN EI SIM 10:1 for 25 fg OFN PCI Scan (CH4) 100:1 for 100 pg BZP PCI SIM (CH4) 10:1 for 1 pg BZP NCI Scan (CH4) 500:1 for 200 fg OFN NCI SIM (CH4) 10:1 for 1 fg OFN OFN = octafluoronaphthalene BZP = benzophenone 44 181 Slide 45 Pesticide Analysis Most Popular Application using GCQQQ • Pesticides in Traditional Chinese Medicine (TCM) – Exceptional quantitative performance across a wide concentration range – Exceptional precision for qualitative ion ratios • Pesticides in carrot – MS/MS succeeds where SIM has failed 45 Page 45 Slide 46 Chlorpyrifos (28 ppb) Easily Detected and Quantitated by GC/MS/MS – Incurred Carrot 197.0 -> 169.0 , 197.0 -> 98.0 Chlorpyrifos - 11 Levels, 11 Levels Used, 11 Points, 11 Points Used, 0 QCs x10 7 y = -0.0083 * x ^ 2 + 2515.4505 * x - 2432.4293 Counts Responses x10 4 Ratio=14.7 2.4 1.6 R^2 = 0.99980926 2.2 1.4 2 1.8 1.2 1.6 1 1.4 1.2 0.8 1 0.6 0.8 0.6 0.4 0.4 0.2 0.2 0 0 -0.2 9.3 9.4 9.5 9.6 9.7 9.8 0 1000 2000 3000 4000 5000 6000 7000 Acquisition Time (min) Concentration (ng/ ml) 46 182 Slide 47 7000A-Designed for Performance and Reliability Making femtogram level sensitivity and high speed SRM accessible to a wide range of users – Leading sensitivity: 100fg of OFN at 100:1 RMS S/N – High performance SRM (MRM) with 500 transitions /sec speed – New proprietary hexapole collision cell technology – Reliable, heated gold plated hyperbolic quartz quadrupoles – Agilent 7890 GC with Capillary Flow technology – MassHunter Software 47 Page 47 183 HPLC & LC/MS Analysis of Pesticide/Residues The paper discussed the liquid chromatography and compared the High Performance Chromatography with Gas Chromatography. The presentation explained fundamental principles of separation and its uses and application with respect to pesticides analysis. Slide 1 CHROMATOGRAPHY (Chromatography was discovered by MS Tswett in 1903) Separation technique Involves two phases Stationary phase and Mobile phase Separation occurs due to differential affinity of components of mixture towards stationary phase Slide 2 Classification of Chromatographic Techniques Mobile phase Stationary phase Chromatographic technique Based on type of stationary and mobile phase Gas Liquid GC or GLC Gas Solid GC or GSC Liquid Liquid LC or LLC Liquid Solid LC or LSC Based on nature (affinity) of stationary phase Polar Normal phase Non-polar Reverse phase 184 Slide 3 Liquid-liquid chromatography by Martin and Synge in 1940 Chromatographic technique when coupled with detection system can be used for quantitative analysis HIGH PRESSURE LIQUID CHROMATOGRAPHY (HPLC or LC) Moving phase is Liquid Stationary phase could be Liquid or Solid Coupled with detection system which can detect at very low concentration Can be used for all types of compound Most suitable for compounds which are polar in nature and heat labile Slide 4 ADVANTAGES OF HPLC No limitation of polarity and Heat Stability Can be used for both qualitative and quantitative analysis High sensitivity Fast Reproducible Quantitative sample recovery Pesticide Referral Laboratory Slide 5 Preparative HPLC – Separation, Isolation, Cleanup Analytical HPLC - Qualitative and Quantitative analysis Modes of HPLC depending on stationary phase Normal phase Reverse phase Ion exchange Size exclusion or Gel permeation 185 Slide 6 SCHEMATIC DIAGRAM OF HPLC SYSTEM Slide 7 COMPONENTS OF HPLC INSTRUMENT o Solvent reservoir and Delivery system (pumps) o Sample introduction/ loading : injector, auto sampler o Separation system : column, oven with temperature control o Detection system – Detectors o System control, data recording, data analysis system o Accessories: fraction collector, guard column, online derivatization, etc Slide 8 Solvent Delivery System A) Solvent Reservoirs B) Online Degasser C) Pumps D) Mixer Isocratic system – Single pump Gradient system – binary or quaternary Pressure Low/medium pressure – MPLC (<3000 psi) High pressure – HPLC (3000-5000 psi) Ultra high pressure – UPLC (upto 50,000 psi) 186 Slide 9 SAMPLE INTRODUCTION/ LOADING (Injector & Autosamplers) Sample can be introduced in liquid (solution) form Rheodyne injector – Manual – different size loops (20 – 1000 µl) -- graduated microsyringe (10 – 1000 µl) Slide 10 AUTOSAMPLERS Automation, Unattended operation Automatic Liquid Sampler (ALS) Vials containing sample solution placed in sampler tray Sampler identifies position as per command Autoinjector fills syringe up to required volumn or pump required volume Advantage : Improved precision and reproducibility Slide 11 Separation System (Column and Oven) Separation occurs on stationay phase packed in column Normally operated at ambient temperature Fluctuation in temperature during day time Separation and retention time may vary due to temperature variation Column is housed in thermostated oven where temperature can be accurately controlled and maintained Better repeatability Better reproducibility Some applications may require cryogenic conditions 187 Slide 12 COLUMNS Main component of HPLC where separation occurs Stationary phase packed in suitable tubing High pressure – stainless steel tubing Sizes Length : 50 mm – 250 mm Diameter : 2-5 mm Slide 13 SUPPORT MATERIAL (To hold stationary liquid phase and provide large surface area) Material : high purity and porous synthesized silica Columns have a distribution of particle sizes Reported “particle diameter” is an average Broader distribution ---> broader peaks Particle Size : 3 -10 µm (UPLC- < 2 µm) Surface area : 150 and 250 sq.m per gram Shape : Sphere; Irregular Porosity : mean pore diameter of 150Å Slide 14 Liquid Chromatography Stationary Phases Silica Gel : Silica gel is manufactured by releasing silicic acid from a strong solution of sodium silicate by hydrochloric acid. Na2SiO3 +H2O + 2HCl = Si(OH)4 + 2NaCl Bonded Phases : C8,C18, cyano, amino, phenyl, etc Chiral Stationary Phases Macroporous Polymers (Cyclodextrin) Polystyrene/Divinylbenzene – Based Resins 188 Slide 15 CHEMISTRY: Bonded Phases BONDED HYDROCARBON:C-18, C-8, C-4, C-1 Slide 16 Slide 17 End Capping Free OH groups on silica Large hydrocarbons (C8, C18) not able to reach and react with OH groups which are inside smaller cavities Such groups are deactivated by methylation Improves reverse phase properties of the stationary phase 189 Slide 18 RP Mechanisms Hydrophobic Theory Partition Theory Adsorption Theory Slide 19 Hydrophobic Theory Nonpolar (nonspecific) interactions of analyte with hydrophobic adsorbent surface (-C18, C8, Phenyl, C4) Difference in analyte sorption affinities results in their separation More polar analytes retained less Analytes with larger hydrophobic part are retained longer Almost no separation of structural isomers Slide 20 Adsorption Theory Analytes “land” on surface - do not penetrate Non-polar interactions between analyte hydrophobic portion and bonded phase Weak interactions dipole-dipole dipole-induced dipole induced dipole-induced dipole 190 Slide 21 Partition Theory Analyte distributes between aqueous mobile phase and organic stationary phase Correlation between log P and retention Does not explain shape selectivity effects Slide 22 Important Reversed Phase Parameters Solvent (mobile phase) Strength: gradient (proportion) Choice of Solvent : polar like acetonitrile, methanol Mobile Phase pH : suppress ionization (neutral, buffered) Silanol Activity : minimum Slide 23 Solvent Strength 191 Slide 24 Solvent Strength • Water is “weak” solvent for organic compounds • Increased organic portion ---> decreased retention • Organic must be miscible with water Slide 25 Effect of Solvent Slide 26 pH (2-8) • Affects ionizable compounds – organic acids – organic bases • In reversed phase we need to suppress ionization as much as possible • May need very precise pH control 192 Slide 27 Factors Influencing HPLC Separation Parameters affecting efficiency: • Flow rate • Column length • Particle diameter • Particle size distribution Parameters affecting retention factor: • Eluent type • Eluent composition • Stationary phase type • Analyte nature Parameters affecting selectivity: • Stationary phase type • Analyte nature • Eluent additives • Temperature • Eluent composition (ionizable analytes) Slide 28 Efficiency Slide 29 Column Efficiency Column length is a compromise between the efficiency and backpressure Column efficiency is proportional to the column length Specific efficiency (# of particles per one plate) decreases with an increase of column length Length Particle Efficiency, Specific [cm] Dia. [um] N Efficiency, h 10 3 11111 3 10 5 10526 1.9 15 5 13636 2.2 25 5 15625 3.2 25 10 10000 2.5 193 Slide 30 DETECTING SYSTEM DETECTORS Sensors which detect presence of compounds in column effluent Selective Response proportional to concentration/ amount Detection limit Linearity range Slide 31 DETECTORS FOR PESTICIDE ANALYSIS UV/VIS Absorption Refractive Index Fluorescence Mass Detector (MS) Slide 32 Refractive Index Detector The refractive index of a medium is the ratio of the speed of light in a vacuum to the speed in the medium. The detector measure the change in refractive index in the eluent as the solute passes through the sample cell. less sensitive than UV detection 194 Slide 33 Fluorometric Detector Solute is excited by UV radiation at a particular wavelength The emission wavelength is detected. Can be used with naturally fluorescent compounds Compounds can be reacted to produce fluorescent derivatives. Slide 34 MASS DETECTOR (LC-MS) o Non-specific (Can be used for all types of compounds) o Mainly used for confirmation and structure elucidation PRINCIPLE: Positive ions are deflected when passed through magnetic or electric field. The magnitude of deflection is related to mass/ charge ratio Slide 35 COMPONENTS OF MASS SPECTROMETER i. Vacuum generating system ii. Sample inlet system (interface) iii. Ionization system iv. Ion analyzer or separating system v. Ion collector or detecting system vi. Recording system 195 Slide 36 Vacuum Generating System Mass operated under high vacuum (10-4 to 10-7 torr) Initial vacuum by rotary pump Final vacuum by oil diffusion pump or turbomolecular pump Slide 37 IONIZATION SYSTEM (To convert compounds into charged ions) Chemical Ionization (CI Mode) – APCI ESI EI Slide 38 ION ANALYZER OR SEPARATING SYSTEM (ION FILTERS) (Separation of ions according to their mass/ charge ratio) 1) Quadrupole mass analyzer 2) Ion trap 3) Time of flight 196 Slide 39 Modes Of Operation Quantitative analysis : Single ion monitoring (SIM) mode Confirmation : Scan mode Multi-ion monitoring Slide 40 Applications HPLC/ UPLC as Analytical tool – Formulation analysis - Residue analysis Semi-prep HPLC/MPLC/GPC - Cleanup technique LC-MS - Confirmation technique – LC-MS-MS- Residues at ppt level 197 Eco-Analytix Solution towards the environmental Health & Safety The paper discussed the Slide 1 PerkinElmer EcoAnalytix Solution towards the Environmental Health and Safety” Slide 2 Our mission is to expand… Personal Health Environmental Health Technology and solutions to address the challenges of dangerous contaminants that threaten the environment Initial focus on water purity, food and consumer products safety, and biofuels and alternative energy development …our leadership in personal and environmental health 198 Slide 3 Protecting our global ecosystem is key… Food Food 1000’s of global food products 1000’s of global food products rejected contaminated by rejected contaminated by Consumer Products Consumer Products Water aflatoxins, mercury, melamine, aflatoxins, mercury, melamine, >25 million items Water >25 million items 55 million people die million people die cadmium, etc. recalled globally 2007 cadmium, etc. recalled globally 2007 from water borne from water borne due to safety concerns due to safety concerns diseases each year. diseases each year. including lead including lead contamination. contamination. Air Air Food 4.6 million people die Food 4.6 million people die >2 million people die each year from causes >2 million people die each year from causes annually due to food directly attributable to annually due to food directly attributable to safety & quality issues. air pollution. safety & quality issues. air pollution. Sustainable Energy Sustainable Energy Global warming and greenhouse Global warming and greenhouse gas emissions deplete gas emissions deplete protective ozone and pollute air protective ozone and pollute air we breathe we breathe Source: WHO, CDC, CPSC …to ensuring health & well being of future generations Page 3 Slide 4 Analytical Sciences… Inorganic: Chromatography: The Leader in AA, High throughput GC ICP & ICP-MS & GC/MS Systems Lab Data Systems: Tailored to Specific Market Segments Mol. Spectroscopy: Thermal/Mechanical: A Leader in IR & High Diversified Materials Performance UV Analysis Products …a leading measurement solutions provider, worldwide Slide 5 Analytical Sciences Mission … Food/Consumer Renewable Products Energy Environmental Forensics Be the global leader in application-focused measurement solutions to improve the quality of our environment, food, energy sources and new materials, for global and human health and well being Pharmaceutical Semiconductor Development & Manufacturing Chemicals & Material Hydrocarbon Characterization Processing … to measure & protect the Ecosystem 199 Slide 6 Solving the critical challenges of the world … Awareness and Understanding Strong Infrastructures Monitoring and Analytical Measurements … requires focus, commitment and partnerships Page 6 Slide 7 Global EcoAnalytix Application Development Centers Seer Green, UK Shelton, CT, USA Application Development Center Application Development Center Shanghai, China Application Development Center Mumbai, India Application Development Center Application development, method development, SOPs Platform capabilities Page 7 Slide 8 Solving the critical challenges of the world … Thought Leadership Partnerships Platform & Service Application Thought Platforms Centers Partnerships Leadership EcoAnalytix EcoAnalytix Know More, Do More … requires focus, commitment and partnerships Page 8 200 Gas Chromatographic Analysis of Pesticides/Residues The paper discussed the principles, instrumentation, separation techniques and different parameters of the compounds that are involved in the separation process by gas chromatography. The paper explained and discussed results of some of the pesticides analysis carried out by gas chromatography. Slide 1 PESTICIDES SCENARIO IN INDIA REGISTERED PESTICIDES = 219 BANNED = 31 PESTICIDE WITHDRAWN = 7 LIST OF PESTICIDES REFUSED REGISTRATION = 18 PESTICIDES RESTRICTED FOR USE IN INDIA = 10 Slide 2 MAJOR CONSUMER CROPS OF PESTICIDES IN INDIA AND GLOBALLY ____________________________________________________________ Share (%) Crop India Global ____________________________________________________________ Cotton 37 9 Paddy/Rice 20 12 Chilli 6 Wheat 6 Vegetable/fruits 5 25 Tea 5 Pulses/ other cereals 3 15 Potato, Grapes and oilseeds 2+2+2 Maize 11 Soya 9 Sugar beet 3 Others 12 16 _________________________________________________________ Ministry of Petrochemicals (2005) 201 Slide 3 CONSUMPTION OF AGROCHEMICALS IN INDIA Year Pesticides (T) Year Pesticides 1955 - 56 2353 2002 - 03 48350 1961 - 62 10300 2003 - 04 41020 1971 - 72 29535 2004 - 05 40672 1981 - 82 60878 2005 - 06 44177 1991 - 92 72133 2006 - 07 42017 2001 - 02 47022 2007 - 08 35809 Slide 4 AGROCHEMICAL USE IN KEY COUNTRIES/AREAS OF THE WORLD Country or Area Pesticide (Kgha-1) Republic of Korea 16.56 Italy 13.35 Hungary 12.57 Japan 10.8 China 2.0-2.5 Europe 1.9 USA 1.5* Mexico 1.38 Thailand 1.37 Indonesia 0.58 India 0.38 Turkey 0.3 Argentina 0.29 Latin America 0.22 Oceania 0.20 Africa 0.13 *Low consumption in USA could be due to use of the newer low volume and high value pesticides. Slide 5 PESTICIDE RESIDUE Any substance in food for human or animals resulting from the use of pesticides. It also includes any specified derivatives such as degradation and conversion products and metabolites which are considered to be of toxicological significance 202 Slide 6 PESTICIDE RESIDUE ANALYSIS 1.Extraction 2. Clean-up of interfering materials 3. Concentration of the sample 4. Analysis (Identification and Quantification) Slide 7 CHROMATOGRAPHY Physical separation method based on the differential migration of analytes in a mobile phase as they move along a stationary phase. Mechanisms of Separation Partitioning Adsorption Exclusion Ion Exchange (Affinity) Chromatographic Separations Based on the distribution (partitioning) of the solutes between the mobile and stationary phases, described by partition coefficient, K: K = Cs/Cm where Cs is the solute concentration in the stationary phase and Cm is its concentration in the mobile phase. Slide 8 A QUICK HISTORICAL PERSPECTIVE • 1938 Paper and TLC • 1952 Gas-liquid chromatography (GLC) • 1968 High performance liquid chromatography (HPLC) • 1980s Super critical fluid chromatography (SFC) 203 Slide 9 GAS CHROMATOGRAPHY Gas Chromatography (GC) A chromatographic technique where mobile phase is gas. most popular methods for separating and analyzing pesticides. Because of high resolution, low limits of detection, speed, accuracy and reproducibility. Slide 10 GAS CHROMATOGRAPHY Two molecules can be separated from one another based on: 1. Volatility 2. Polarity 3. Molecular size 4. Charge Slide 11 SCHEMATIC DIAGRAM OF GC 204 Slide 12 Slide 13 COMPONENTS OF GC INSTRUMENT 1) Gases – carrier gas (Nitrogen, Helium) & flame gases (hydrogen and air)– pressure regulation and flow control 2) Sample introduction/ loading – auto sampler 3) Separation system – oven (temperature control), column 4) Detection system – Detectors 5) Recording system – Integrator or computer Slide 14 GC CARRIER GASES (THE MOBILE PHASE) • Usually “inert” gases (don’t react with analytes ) • Purpose – sweep sample through the column – protect column from oxygen exposure at temperature – assist with function of the detector • Most common – Helium (available relatively pure without extensive purification after it leaves a compressed gas cylinder) – Nitrogen (usually requires an oxygen and water trap) – Hydrogen • normally used only with flame ionization detectors (FID) since the FID needs it as fuel for the flame • still rarely used due to safety concerns (and chromatographic ones) 205 Slide 15 GC INJECTION…. • Samples are injected through a septum – keeps oxygen out of the column – provides a seal to keep the carrier gas pressure up at the head of the column • carrier gas flow rate is determined by the pressure or the gas at the opening of the column – Many different (mostly proprietary) materials • red rubber (bleeds at about 250 C) • Thermogreen (good up to about 300 C) • High-temperature blue (good a little over 300 C) • The injector is usually lined with a de-activated glass liner – prevents metal injector-sample reactions that would alter analytes or damage the metal of the injector – Can be cleaned/replaced regularly Slide 16 INJECTOR PORT Split Injector the injection is split, with only a portion of the sample (usually 1% - 20%) actually making it to the column the most common method of injecting samples onto small diameter, open-tubular columns Not good for analytes with a wide range of boiling points Splitless injector Sample is vaporized in the injector and all of the sample is swept onto the column by the carrier gas Relatively small samples injected (10 µ L or less in capillary GC) Sample spends a large amount of time in the injector which helps volatilize the analytes Best for trace (1 -100 ppm range) concentrations of high boiling point On-column inlet used widely in packed-column GC, less in capillary GC sample is deposited directly on the column Good for thermally unstable compound and for quantitative analysis at low concentrations BUT, can inject only a relatively small amount of sample in capillary GC Modern capillary GCs come with a Split/Splitless injectors standard one can switch between modes by changing the split vent gas flow and using a different injection liner. PTV injector; Temperature-programmed sample introduction. It is a method for the introduction of large sample volumes (up to 250 µL) in capillary GC. Slide 17 INJECTION TYPES 206 Slide 18 HEADSPACE GAS CHROMATOGRAPHY ANALYSIS • Headspace GC (HSGC) analysis employs a specialized sampling and sample introduction technique, making use of the equilibrium established between the volatile components of a liquid or solid phase and the gaseous / vapor phase in a sealed sample container. Aliquots of the gaseous phase are sampled for analysis • Examples of HSGC are the forensic analysis of blood and urine alcohol levels, quality and production control of diesel fuel and beer constituents. Aromatic flavors and trace volatiles in foods and soft-drinks are also readily analyzed. and HSGC analysis of volatile free fatty acids produced by bacteria, particularly anaerobic bacteria, enables a fingerprint of the particular microorganisms to be obtained, which assists in the identification of the bacteria. Slide 19 COLUMNS Main component of GC where separation occurs Three types: 1) Packed 2-4 mm diameter 2) Megabore 0.53 mm diameter 3) Capillary 0.1-0.25 mm diameter Slide 20 207 Slide 21 Slide 22 1. Column “frame” constructed of fused silica tubing 2. Polyamide coating on the outside gives it strength 3. Liquid stationary phases coated or bonded to the inside of the tubing 4. 0.1 - 0.53 mm + ID, 5-100 meters in length, stationary phases usually 0.10 to 1.5 µm in thickness 5. Mounted on a wire cage to make them easier to handle 6. 5-150 meters long. Slide 23 CAPILLARY VS. PACKED COLUMNS • Capillary Columns: • Packed Columns – Higher resolution (R) – Greater sample capacity – Greater HETP and N – Lower cost (can make your own) – Shorter analysis time – More rugged – Greater sensitivity – Most common in process labs or – Most common in analytical separating/determining major laboratory GC instruments components in a sample (prep – Smaller sample capacity GC) – Higher cost/column – Limited lengths reduces R and N – Columns more susceptible – Not compatible with some GC to damage detectors 208 Slide 24 OVEN • Programmable • Isothermal- run at one constant temperature • Temperature programming - Start at low temperature and gradually ramp to higher temperature – More constant peak width – Better sensitivity for components that are retained longer – Much better chromatographic resolution – Peak refocusing at head of column Slide 25 GC INSTRUMENTS - DETECTORS A chromatography detector is a device that locates in the dimensions of space and time, the positions of the components of a mixture that has been subjected to a chromatographic process and thus permits the senses to appreciate the nature of the separation. • Characteristics of a “good” detector – Sensitivity appropriate to sample – Large linear dynamic range – Useful at a range of temperatures – Rapid response time – Easy to use (idiot proof?) – Stable, Predictable response – Nondestructive (probably least important) Slide 26 DETECTORS USED FOR PESTICIDE ANALYSIS Flame Ionization Detector (FID) Alkali Flame Ionization Detector (AFID) Nitrogen Phosphorus Detector (NPD) Electron Capture Detector (ECD) Flame Photometric Detector (FPD) Mass Detector (MS) 209 Slide 27 Slide 28 FLAME IONIZATION DETECTOR Teflon insulating ring Coaxial cable to Analog to Digital Gas outlet converter Collector Ions Flame Sintered disk Platinum jet Air Hydrogen FID (Nanogram - ng) Capillary tube (column) High temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame. The ions collected on collector or electrode and were recorded on recorder due to electric current. Slide 29 ALKALI FLAME IONIZATION DETECTOR (AFID) - Specific -Burnt in presence of alkali salt to enhance ionization of pesticides containing P, S & N Alkali salts used Alkali salt Potassium chloride Sodium sulfate Cesium bromide Rubidium chloride 210 Slide 30 NITROGEN PHOSPHORUS DETECTOR (NPD) (Also called TSD, TID, etc.) Specific for Nitrogen and Phosphorus compounds • Instead of complete flame, plasma flame • Sensitive to P&N containing compounds Flame gases H2 : 3-5 ml/min Air : 100-150 ml/min Slide 31 FLAME PHOTOMETRIC DETECTOR (FPD) Specific for P&S containing Compounds Atoms in compound excited when burnt in flame Return to ground state releasing energy in the form of light Emitted light detected by photo-multiplier tube Filters allow only specific light to pass, others get absorbed Flame gases H2 : 50-75 ml/min Air : 100-150 ml/min P : 526 & S : 394 nm Slide 32 ELECTRON CAPTURE DETECTOR (ECD) Electrons from radioactive source ECD detects ions in the exiting from the gas chromatographic column by the anode electrode. 3H or 63Ni which emits β particles. Ionization : N2 (Nitrogen carrier gas) + β (e) = N2+ + 2e These N2+ establish a “base line” X (F, Cl and Br) containing sample + β (e) X- Ion recombination : X- + N2+ = X + N2 Decrease in current The “base line” will decrease and this decrease constitutes the signal. Electron-capture detectors are highly sensitive and have the advantage of not altering the sample significantly. 211 Slide 33 THERMAL CONDUCTIVITY DETECTOR The carrier gas has a known thermal • conductivity. As the thermal conductivity of the column eluent (gas flow in) changes, the resistance of the filament changes. The presence of analyte molecules in the carrier gas alter the thermal conductivity of the gas (usually He) There is normally a second filament to act as a reference (the carrier gas is split) Increased sensitivity with decreasing temperature (detector), flow rate and applied current. Filaments will burn out (oxidized) in the Non-destructive presence of oxygen if hot! Slide 34 MASS DETECTOR (GC-MS) o Non-specific (Can be used for all types of compounds) o Mainly used for confirmation and structure determination PRINCIPLE: Positive ions are deflected when passed through magnetic or electric field. The magnitude of deflection is related to mass/ charge ratio Slide 35 DETECTION LIMIT AND MARKING LIMIT QUALITYANALYSIS Is a signal equal to the lowest analyte LIMIT OF DETECTION concentration which can be detected with the (LOD) probabiloity of 99% In practice such a limit allows the analyst to decide if a signal of a very low intnsivness is a signal of the analyte or of the interferent, that is PROTOCOL to define the presence or the absence of the analyte QUANTITY ANALYSIS LIMIT OF QUANTITATION (LOQ) After the confirmation of analyte presence, it is a minimum analyte concentration which can by measured by the analyst with acceptable accuracy and precision(within the limits of acceptable deviation) LOQ = 10*LOD Practical a Qantification Limit (PQL) is a limit obtained by different NOTES O: LOD & Laboratories with acceptable accuracy and precision, in routine LOQ conditions PQL = 5*LOD 212 Slide 36 APPLICATION OF GLC IN PESTICIDE ANALYSIS RESIDUES AND THEIR DEGRADATION PRODUCTS FROM FOOD VEGETABLES FROM SERUM FROM WATER FROM SOIL FROM AIR DAIRY PRODUCTS HUMAN TISSUES PESTICIDE FORMULATION SUCH AS AEROSOL WETTABLE POWDER EMULSION LIQUID CONCENTRATE PESTICIDES AND IMPURITIES Slide 37 PESTICIDES • Analysis of pesticide residues in soil, water, and food is crucial for maintaining safe levels in the environment. • ECD mode is highly selective for monitoring electron capturing compounds such as chlorinated pesticides and other halogens. • Sample: Pesticide calibration mix • Detector mode: Electron capture • Detector temp: 330°C • Column: 25 m x 0.32 mm x 25 µm, HP-5 • Column temp: 150°C to 300°C at 10°C/min • Sample volume: 1 µL, 10:1 split Discharge gas: Helium, 30 mL/min Dopant gas: 5% methane in helium, 2.4 mL/min Attenuation: 1 Pesticide separations Slide 38 CHLORINATED PESTICIDES DDT, HCH and ITS ISOMERS, CYCLODIENES (ENDOSULFAN, ALDRIN) FROM FOOD AND VEGETABLES EXTRACTION FROM MATRICES WITH SOLVENTS SUCH AS ACETONITRILE, ACETONE, ISOPROPANOL CLEANUP BY A FLORISIL COLUMN (10% ACETONE:HEXANE) ANALYSIS BY GLC 213 GLC CONDITIONS Slide 39 COLUMN: OV – 101/ 210/ 17 (Cappillary PESTICIDE SEPARATIONS column 0.22mm id, 30 m) DETECTOR: ECD INJECTOR TEMPERATURE : 300 DETECTOR TEMPERATURE : 300 COLUMN TEMPERATURE : 100 for 5 min to 270 @ 5 0C/min Carrier gas: Nitrogen Retention time (sec) Slide 40 HALOGENATED PESTICIDES/ HERBICIDES IN DRINKING WATER BY LIQUID- LIQUID EXTRACTION AND GAS CHROMATOGRAPHY WITH ELECTRON- CAPTURE DETECTION A 50 mL sample aliquot is extracted with 3 mL of methyl tertiary butyl ether or 5 mL of pentane. Injector temperature: 200°C Detector temperature: 290°C. Column A - 0.25 mm ID x 30 m fused silica capillary with chemically bonded methyl polysiloxane phase (J&W, DB-1, 1.0 m film thickness or equivalent). The column oven is temperature programmed as follows:  HOLD at 35°C for 22 minutes  INCREASE to 145°C at 10°C/min and hold at 145°C for two minutes  INCREASE to 225°C at 20°C/min and hold at 225°C for 15 minutes  INCREASE to 260°C at 10°C/min and hold at 260°C for 30 minutes or until all expected compounds have eluted. Slide 41 Separation by NPD detector 214 Slide 42 CHLORINATED PESTICIDES ANALYSIS BY CAPILLARY GC 16 Chlorinated Pesticides Separated in 20 Minutes Slide 43 CHLORINATED PESTICIDES BY SPME/ CAPILLARY GC Slide 44 EXTRACTION OF PESTICIDES FROM FRUITS AND VEGETABLES Homogenize 50g chopped sample with 100mL acetonitrile Add 10g sodium chloride (Homogenize 5 min) Transfer ~acetonitrile (top) layer to centrifuge tube. Add ~3g sodium sulfate to remove water. Centrifuge at high speed for 5 min. Transfer 10mL aliquot (= 5g of sample) to a clean 15mL tube. Evaporate to 0.5mL under clean nitrogen (water bath, 35°C). Transfer to ENVI-Carb SPE tube (6mL tube, 500mg packing). Elute pesticides with 20mL acetonitrile/ toluene (3:1). concentrate sample to ~2mL. Add 2 x 10mL acetone, concentrating the material to ~2mL after each addition, to make a solvent exchange to acetone. Transfer quantitatively to a clean 15mL tube. Add 50µL internal standard (50ng/µL cis-chlordane in acetone), then bring volume to 2.5mL with acetone (final concentrations = 2g/mL extract, 1.0ng/µL cis-chlordane). 215 Slide 45 ANALYSIS OF SYNTHETIC PYRETHROIDS BY GLC SYNTHETIC PYRETHROIDS (Permethrin, cypermethrin, decamethrin, fenvalerate) Acetone extract (GLC) Column Stationary pace Column tem. Detector Packed OV-101/17/210 270 ECD Capillary HP-17/1/101 250 ECD Limit of detection 0.01µg Slide 46 ANALYSIS OF HALOGENATED PESTICIDES BY GLC HALOGENATED PESTICIDES (BHC to methoxychlor) Acetone extract(GLC) Column Stationary pace Column tem. Detector Packed OV-101/17/210 200 ECD Capillary HP-17/1/101 200 ECD More than 140 pesticides and metabolites with halogen chromatograph from 0.3 to 7 relative to chlorpyriphos Limit of detection 0.01µg Slide 47 ANALYSIS OF ORGANOPHOSPHORUS PESTICIDES BY GLC ORGANOPHOSPHORUS PESTICIDES Acetone extract(GLC) Column Stationary pace Column tem. Detector Packed OV-101/17/210 200-220 FPD PACKED DEGS 200-200 FPD Capillary HP-17/1/101 150-200 FPD More than 120 pesticides and their metabolites DEGS : polar such as Acephate, dimethoate and monocrotophos Limit of detection 0.01µg 216 Slide 48 Reversed Liquid—Liquid Partition in Determination of Poly chlorinated Biphenyl and Chlorinated Pesticides in Water A new method based on an application of the common reversed Iiquid—Iiquid partition for the extraction of chlorinated pesticides from water. water is passed through a filter (3 grams) containing a mixture of n-undecane and Carbowax 4000 monostearate on Chromosorb W, and the absorbed pesticides are eluted with petroleum ether (10 ml). detected by GC-ECD sensitivity is 10 ng/m1 of lindane with a sample size of 200 liters. Recovery of added pesticides was 50—100% (DDT, 80%) , PCB, 93—100%. Slide 49 GC MULTI-RESIDUE METHOD FOR ANALYSING PESTICIDE RESIDUES IN SAMPLES OF PLANT ORIGIN • extraction of pesticide residues from homogenized sample (with ethyl acetate i, acetone/ethyl acetate/cyclohexane and acetone/dichloromethane), • GPC (SX-3) clean-up using cyclohexane/ethyl acetate 1:1 v/v as eluent, • additional clean-up with SPE minicolumns in case of difficult matrices, • GC determination, • With this method up to 145 pesticide residues can be determined. Slide 50 Luke Multiresidue Method Sample Blend with acetone Filter Residues Filterate Extract with Pet.ether/DCM Pet.Ether/DCM Aq. filterate [discard] Concentrate with pet.ether Concentrated extract [dilute and shoot] GC with element selective detectors •NPD – nitrogen and phosphorus containing compounds •Halls electrolytic conductivity detector [halogen] •FPD – phosphorus and sulphur containing compound •Column cleanup optional-must for [ECD] 217 Slide 51 Mills Multiresidue Method Sample Extract with acetonitrile [low fat] Or petroleum ether [high fat] Residues Extract Pet-ether/acetonitrile partitioning Acetonitrile Pet.ether [discard] Add salt water Extract with petroleum ether Aqueous acetonitrile Petroleum ether [discard] Florisil column Elution with pet. ether-diethyl ether Slide 52 Electron capture gas chromatographic determination of Kepone® residues in environmental samples River sediment, soil, water, shellfish, and finfish Rigorous extraction techniques by Soxhlet apparatus and Polytron® tissue homogenizer Cleanup by gel permeation chromatography to remove most of the lipid material followed by a micro Florisil® column elution Cleanup of shellfish and other environmental samples was accomplished with a micro Florisil column only. GC-ECD was used to analyze the sample extracts. Recoveries of Kepone from fortified samples averaged 84% or greater Slide 53 Multi-residue Methods used for Pesticides Residues Analysis of Fruits and Vegetables Extraction with ethyl acetate NaHC0 3 and Na2S04 Filtration Determination GC (ITD, ECD) LC-MS/MS about 180 analytes about 120 analytes 218 Slide 54 Capillary column gas chromatographic determination of trace residues of the herbicide chlorsulfuron in agricultural runoff water. water sample is acidified with acetic acid extracted with methylene chloride derivatized to its monomethyl derivative Florisil column cleanup methylated chlorsulfuron is determined by GC-ECD Detection limit of the method is 25 ng chlorsulfuron/L water (25 ppt Slide 55 Calculations The concentration of the pesticide in the sample (on a dry weight basis) is— Level (mg/kg) = C x A 1 x (W 1 + 125) x 50 WxA2xV1xV2 where C = concentration of the standard (µg/mL) A 1 = peak area of the sample A 2 = peak area of the standard W 1 = weight (in g) of water contained by the test portion of soil V 1 = recorded volume of the methanol/water filtrate V 2 = recorded volume of n-hexane recovered W = weight (in g) of the test portion of air dried soil assuming 100 mL of methanol and 25 mL of water are used to extract the soil sample, and 50 mL of n-hexane is used in the partitioning step. 219 Bioefficacy and Field Trials The paper describes in detail the methods involved in bio-evaluation of pesticides formulations. The presentation discussed bioassay, field trials and data collection process and reporting of the data for presentation of bio-evaluation. 220 Acceptability of GC & HPLC data by NABL and the concept of uncertainty The presentation discussed the minimum standard requirements by the NABL for the acceptance of data generated through Gas chromatographic and High Performance Liquid Chromatographic methods. The paper explained how various equipments in the laboratory need to be calibrated and how often this exercise need to repeated. Slide 1 Accreditation • Accreditation has grown rapidly in the last 25 to 30 years and now is the norm amongst quality conscious laboratories. • The accreditation is essentially a “designation” of competence after some type of evaluation or audit of the laboratory has been performed by a third party. • NABL is an accreditation body that accredits testing and calibration laboratories in India. • Accreditation depends heavily on formal proof of how the measurements was carried out. Slide 2 Technical requirements for NABL accreditation • Knowledge of the labor atory personnel • Tec hnical validity of the methods • Adequacy and calibr ation of equipments • Sampling and handling of test items • Envir onmental factor s • Tr aceability of measur ements to the inter national system of units (SI) • Unc er tainty in the measurements • Assur ing the quality of tests 221 Slide 3 Equipment calibration • A set of oper ation that established under spec ified condition, the r elationship between the values indicated by an item of test equipment and the cor r esponding tr ue values realized by r efer enc e standar ds. • Deter mining how wr ong the equipment is, and then assigning cor r ec tions; or assigning values to the scale of an instr ument. • Calibr ation of equipment is per for med – After ever y assigned dur ation of calibration – After any br eakdown in the instr ument except software pr oblem Slide 4 Equipment calibration (continue) • GC and HPLC is the most widely used analytical tec hnique for estimation of pesticides in macr o as well as micr o quantities. It offer both qualitative and quantitative analysis with speed, accur acy, r epr oducibility and sensitivity. • Pur pose of this talk is to discuss methods and par ameter s need to be calibr ated in GC and HPLC for NABL ac cr editation. Slide 5 GC Calibration • Data of the following four par ameter s ar e r equir ed in GC calibr ation – Flow ac curacy – Temper ature – Repeatability – Linearity 222 Slide 6 Flow accuracy Column Flow Detector Flow Connect a calibrated flow meter Connect a calibrated flow meter to the Inlet to the detector outlet Set the carrier pneumatic flow Set the gas flow rate from Instrument Software individually from Instrument Software Record the values & Check for accuracy Record the values & Check for accuracy Slide 7 Temperature Slide 8 Repeatability • Choose a validated method for any reference standard (pesticide standard) • Prepare a reference standard solution of concentration x ppm depending upon the sensitivity of the detector • Inject the same solution 5 times • Record the area and RT • Calculate the coefficient of variation CV(%) = (SD/ x)*100 Note: Peak area CV(%) ≤ 2%, Retention time CV(%) ≤ 0.5% 223 Slide 9 Linearity Linearity is defined as the range of mass flow rates over which the response remains constant. Linear range of a detector is the ratio of the largest to the smallest concentrations within which the detector response is linear. For a quantitative application it is important that a detector operates within its linear range. Therefore, the linear range of the detectors should be checked in case of starting up detector as well as after a long break in operation. Slide 10 Linearity (continue) Table 1 : Areas of Standards injected in GC-FID Fig 1 : Linearity Curve Concentration S.No. (ppm) Area Avg. area V.F. 1237013 1 500 1238536 1237774.89 0.122905 2488213 2 1000 2481722 2484967.5 0.26087 3657366 3 1500 3663132 3660249 0.157406 5160948 4 2000 5158690 5159819 0.043752 7396915 R2 value = 0.9981 shows good performance 5 3000 7384668 7390791.5 0.165569 of instrument An example of linearity calculation for a GC-FID with packed glass column Slide 11 HPLC Calibration • Data of the following parameters are required in HPLC calibration – Flow accuracy test – Composition accuracy test or Gradient concentration test – Wavelength accuracy test – Noise/Drift test – Repeatability – Linearity 224 Slide 12 Flow accuracy test • To prove that flow rate selected on the pump is delivered in the correct time. • Set the pump flow for 1 ml/min. • Let the pump run for 5 minutes for stabilization • Then collect the water for 5 minutes in a tare beaker • Weigh the beaker with the water The weight should be 5 g ± 0.005 g 1 ml/min. * 5 min. = 5 g (water weighs 1g/ml) • Calculate the volume of water delivered in a one minute interval • Likewise set the pump at 2ml, & 5ml, repeat the process and record the values Note: Flow accuracy limit ≤ 1.0 % of setting Slide 13 Composition accuracy test or Gradient concentration test • To prove that the proportioning valves are operating correctly. This is done by varying the amounts of solvent entering the pump through proportioning valves • Make 0.2% solution of acetone in water (note: 0.1% solution for PDA detector) • Place 500ml of water as liquid A (channel A) and the acetone solution prepared as liquid B,C & D as the mobile phases • Degas all the mobile phases • Bypass the column • Set the condition as follows and continue till baseline stabilizes: T. flow = 5ml/min., B. Conc. = 0%, C. Conc. = 0%, D. Conc. = 0%, wavelength = 265nm Slide 14 Composition accuracy test or Gradient concentration test (continue) • After zeroing the baseline, read the signal levels at the concentrations 0% (B. Conc. = 0), 10% (B. Conc. = 10), 50% (B. Conc. = 50), 90% (B. Conc. = 90),and 100% (B. Conc. = 100) (note: measure the signal level at the point where concentration stabilizes) • Calculate the actual concentration as follows: 10% actual concentration = (B. Conc. 10 level) - (B. Conc. 0 level) X 100 (B. Conc. 100 level) - (B. Conc. 0 level) Similarly, calculate for the 50% and 90% actual concentrations • Repeat the gradient performance test for liquids A & C and liquids A & D Note: Composition accuracy limit ± 1.0 % @ 5 ml/min. 225 Slide 15 Wavelength accuracy test • Make 1% of caffeine solution in water • Place HPLC water in one reservoir and caffeine solution in other reservoir • Purge the pump lines • Bypass the column • Set the detector wavelength to 300nm • Start the pump flow rate at 1 ml/min. with water and pump for 5 minutes • Set the wavelength to 266nm • Start pump at 1 ml/min. with dilute caffeine solution check the absorbance and record the displayed value • Similarly change the wavelength from 267nm to 277nm in 1nm step, and record the corresponding displayed values • Determine the wavelength where the maximum displayed value occurs Note: Maximum absorbance wavelength is 272 ± 2 nm Slide 16 Noise test • The noise associated with a detector is defined as the maximum amplitude of the combined short and long term noise measured over a period of about 10 minutes. • Connect the detector to the column • Let the mobile phase passed through the column over the period of measurement. • Set the wavelength to 240 nm • Set the response to 1000 mS • Leave the detector for at least 20 minutes for stabilization. • Record noise for 10minutes Slide 17 Noise test Continue Calculate the noise Measurement of detector noise level Note : Cf = Conversion factor = 1 AU/ 1V Noise ≤ 2.0 x 10-5 AU 226 Slide 18 Drift test • The drift does not obscure the eluted peak. Drift can result from slowly changing output from the power supply to the detector, lamp aging, changes in ambient temperatures; often due to changes in the composition of the column eluent - incomplete equilibrium. • Connect the detector to the column • Let the mobile phase passed through the column over the period of measurement. • Set the wavelength to 240 nm • Set the response to 1000 mS • Leave the detector for at least 20 minutes for stabilization. • Record drift for 15 minutes Slide 19 Drift test • Calculate the drift Drift (AU/hr) = (Highest mean value – Lowest mean value) mV x 60 min. x 1V x Cf 1hr x 1000 mV Note : Cf = Conversion factor = 1 AU/ 1V Drift ≤ 6.0 x 10-4 AU/h Slide 20 Repeatability • Choose a validated method for any reference standard (pesticide standard) • Prepare a reference standard solution of concentration 10 ppm or less depending upon the sensitivity of the detector and dimension of the column • Inject the same solution 5 times • Record the area and RT • Calculate the coefficient of variation CV(%) = (SD/ x)*100 Note: Peak area CV(%) ≤ 1%, Retention time CV(%) ≤ 0.5% 227 Slide 21 Linearity • Take standards of a range of concentrations • Inject in HPLC, record the areas • Draw a linearity curve • Get the R2 value Slide 22 Estimation of uncertainty of measurements why? how? Slide 23 Content What is uncertainty? What is the relationship of error and uncertainty? How is the estimation process done? Bottom-up and top-down approach The law of error propagation work Example: Calculation of the uncertainty in the preparation of a working standard solution 228 Slide 24 What is uncertainty of measurement a parameter, generally a standard deviation associated with the result of a measurement characterizes the dispersion of values attributed to the measurand built-up of many components, type A and B other simple definitions: the doubt about the exactness of a measurement result the +- after the result Slide 25 Errors • ISO definition: the result of a measurement minus the true value of the measurand. • distinction between – gross error (typically arises through human failure or instrument malfunction) – random error (typically arises from unpredictable variations of influence quantities) – systematic error (component of error which, in the course of a number of analysis of the same measurand, remains constant or varies in a predictable way) Slide 26 Difference between error and uncertainty • Error is a single value (it is an idealised concept, cannot be known exactly, in principle the systematic part can be corrected) • They are not synonyms, but represent completely different concepts • Uncertainty takes the form of a range (no part of it can be corrected for) • The uncertainty of the results of a measurement should never be interpreted as representing the error itself, nor the error remaining after correction. 229 Slide 27 BASIC TERMS • Uncertainty component (sources) • Standard uncertainty: u(y) • Relative standard uncertainty • Combined standard uncertainty: uc(y) • Expanded uncertainty: U • Coverage factor: k Slide 28 Estimation Specification Process Identify Uncertainty Sources Specification: Quantify Uncertainty Components Defining the measurand: write down a clear statement of what is being measured and the Convert to Standard Deviation relationship between it and the parameters on which it depends Calculate the Combined as far as possible express the Uncertainty relationship in an equation as a function of the imput parameters YES Do the Significant y=f(p,q, T, A..) Re-evaluation of the Components Need Significant Components Re-evaluation? split the measurement process in a series of steps and assess them NO separately END Slide 29 Identifying uncertainty sources • list sources of uncertainty for each part of the process or each parameter • examples of sources of uncertainty – sampling – storage conditions – instrument effects – reagent purity – measurement conditions – computational effects ( i.e. calibration) – operator effects – random effects the sources are not necessarily independent 230 Slide 30 Quantification of uncertainty components preliminary simplification: if possible use method validation data to neglect the smaller components estimate the size of each component by: doing experimental work: repeatability experiments, (reference materials) Or utilisation of data and results from certificates, suppliers,QA/QC data Or using the judgment of the analyst based on experience Slide 31 Conversion to standard deviations • Experimental evaluation of the uncertainty can be easily expressed in terms of standard deviation (repeatability experiments; for the contribution to uncertainty in averaged measurements, the standard deviation to the mean is used) • If a confidence interval is given with a level of confidence • balance reading is within + 0.2 mg at 95%. This interval is calculated using 1.96 s (value from the standard tables of percentage points on the normal distribution). u(y)=0.2/1.96=0.1 mg • balance reading at 99% u(y)=0.2/2.58=0. 08 mg Slide 32 Conversion to standard deviations • If a confidence interval (± y) is given without a confidence level – extreme values are not likely assume a rectangular distribution with a standard deviation of y / √3 – extreme values are unlikely (or small errors are more likely than large errors) assume a triangular distribution with a standard deviation of y / √6 231 Slide 33 Distribution functions Slide 34 Calculating the combined standard uncertainty • Identify significant components • Combine the uncertainty components algebraically • Use the law of error propagation • KISS concept: break down in several Blocks • possibly obtain information on combined effect of several components (i.e. sample processing and extraction) RETAIN SUFFICIENT NUMBER OF DECIMALS Slide 35 Reporting results Calculate the expanded uncertainty: It provides a confidence interval within which the value of the measurand is expected to lie. • reporting uncertainty: it is better to provide too much information on how the uncertainty was calculated rather than too little • this allows to re-evaluate the results if new data becomes available 232 Slide 36 Bottom-up approach helps reducing the total lab uncertainty by optimizing those steps that contribute significantly helps improving the knowledge of analytical techniques and skills however large number of sources of error in chemical measurement (random and systematic) and difficult estimations not always the sources are independent time consuming despite this RECOMMENDED by many organizations Slide 37 Top-down approach seeks to use the results of proficiency testing schemes in a range of laboratories to give estimates of the overall uncertainties of the measurements – without necessarily trying to identify every individual source of error can only be applied if data from properly run proficiency schemes are available is rapidly expanding in number and may thus provide a real alternative to "bottom-up" methods Slide 38 Identify the Uncertainty sources Standard purity Pipette Temperature Calibration Repeatability Concentration Temperature Temperature Calibration Calibration Calibration Repeatability Reproducibility Repeatability Volumetric Mass (balance) Syringe flask Cause and effect diagram (Fish Bone Diagram) showing uncertainty components in preparation of standard 233 Slide 39 QUANTIFYING THE UNCERTAINTY COMPONENTS PURITY of standard, 0.1 g The purity of the analytical standard is quoted in the supplier’s certificate as 99.9 +- 0.1% (= 0.9990.001). The supplier gives no further information concerning the uncertainty in the catalogue. Therefore the quoted uncertainty is taken as having a rectangular distribution, so that the standard uncertainty u(P) is 0.001/√3= 0.00057735. RSD= 0.000577928 Slide 40 Evaluation of Uncertainty in Chlorpyrifos Active Ingredients (A.I.) measurement A GC Assay Slide 41 Anaysis steps • Preparation of stock solution of standard and internal standard • Preparation of stock solution of sample • Analysis by injecting standard and sample into GC-FID system • Calculate the A.I. Content 234 Slide 42 Identify the Uncertainty sources Standard purity Volume Temperature Calibration Repeatability Standard Calibration Mass (balance) A.I. Content Temperature Chlorpyrifos Calibration Calibration Repeatability Volume Mass (balance) Repeatability (Instrument GC) Cause and effect diagram (Fish Bone Diagram) showing uncertainty components in A.I. analysis Slide 43 QUANTIFYING THE UNCERTAINTY COMPONENTS • Uncertainty in measurement has to be evaluated in two parts – Uncertainty in preparation of calibration/standard solution and – Uncertainty in measurement Assumption:- Uncertainty due to internal standard can be nullify as it is added to the sample and standard in the same way Slide 44 QUANTIFYING THE UNCERTAINTY COMPONENTS • Standard – Std. purity = 98.5 ± 1.5% – Uncertainty component = 1 ± 0.015% – U(P) = 0.015/√3 = 0.009 (Type B, Rectangular distribution) • Mass (Balance) – Uncertainty of measurement = ± 0.001g (mention in certificate) – U(M) = 0.001/2 = 0.0005 (Type B, Normal distribution) Note: Type A – Uncertainty measured in laboratory by own experimental data Type B – Uncertainty from literature, record and past experience 235 Slide 45 QUANTIFYING THE UNCERTAINTY COMPONENTS Volume (50 ml volumetric flask) (a) Uncertainty in the stated internal volume of the flask (Type B, Triangular distribution) – Uncertainty = ± 0.05g (mention in certificate) – U(M) = 0.05/ √6 = 0.02 (b) Variation in filling the flask to the mark (Type A) – Ten fill and weigh experiment give the std. deviation of 0.0059. This can be directly used as a std. uncertainty – U(R) = 0.0059 (c) Temperature (Type B, Rectangular distribution) – Flask calibrated at = 27 °C (taken from records) – Laboratory temperature varies b/w = 27 ± 4 °C – Coefficient of volume expansion for water = 2.1 X 10-4 / °C – Variation in volume = 50 X 4 X 2.1 X 10-4 = 0.042 – U(T) = 0.042/√3 = 0.024 Total uncertainty for the volume [U(V)] is = √[U(M)]2 + [U(R)]2 + [U(T)]2 = √(0.02)2 + (0.0059)2 + (0.024)2 U(V) = 0.001 236 SAFE PESTICIDE APPLICATION TECHNOLOGY A detailed presentation was made to describe the importance of safe pesticide application. The paper describes the various factors responsible for efficient pesticide application. The importance of droplets in relation to drift was also discussed. The latest technology available/being developed were also discussed. Slide 1 P O P U L A T I EP O N D E N S I T Time Y Slide 42 P O P EIL U L A T ET I O N D EP E N S I T Y Time 237 Slide 3 Pests 14 Diseases 12 10 Weeds 14 Pests in store Estimated losses in crops (percentage of potential yield) Slide 4 CONTROL METHODS • CULTURAL • BIOLOGICAL • INTERFERENCE • CHEMICAL • INTEGRATED CONTROL • PEST MANAGEMENT • INTEGRATED CROP MANAGEMENT Slide 5 SUCCESS OF PLANT PROTECTION MEASURE • QUALITY OF THE PRODUCT • CORRECT TIMING • ACCURACY & SKILL IN APPLICATION TECHNIQUES 238 Slide 6 INSECTICIDE Non target deposition Target deposition 99% 1% Slide 7 HERBICIDE Target deposition 30% Non target deposition 70% Slide 8 UTILISATION OF CROP PROTECTION CHEMICALS ____________________________________________________________________ Pesticide Methods Pest/Crop Efficiency of utilization ---------------------------------------------------------------------------------------------------------------- DIMETHOATE Foliar spray Aphid on field beans 0.03% ETHIRIMOL Seed treatment Barley (for mildew control) 2.20% DISULFOTAN Soil incorporation Wheat (aphid control) 2.90% LINDANE / Arial spraying of Locusts 6.00% DIELDRIN swarms PARAQUAT Spray Grass up to 30% __________________________________________________________________ 239 Slide 9 Biology of the pest Bionomics of the target pest Slide 10 LEAF AREA INDEX Slide 11 A spraying system is said to be efficient if 240 Slide 12 * DEPOSITING A REASONABLE PROPORTION OF THE APPLIED CHEMICAL ON THE TARGET * KEEPING NON-TARGET CONTAMINATION TO A MINIMUM * ENSURING THAT THE TREATMENT IS COST EFFECTIVE Slide 13 EFFECTIVE PESTICIDE APPLICATION Define and know the target Slide 14 EFFECTIVE PESTICIDE APPLICATION * Coverage * Dosage * Timing 241 Slide 15 COVERAGE Efficacy of a treatment depends on * Droplet size * Droplet density * Spray retention * Concentration * Organisms susceptibility Slide 16 OPTIMUM DROPLET SIZE RANGE FOR SELECTED TARGETS ______________________________________________ Target droplet size (um) ______________________________________________ Flying insects 10 - 50 Insects on foliage 30 - 50 Foliage 40 - 100 Soil and where drift is to 250 -500 be avoided ______________________________________________ Slide 17 EFFECTIVE DROPLETS PER CM.2 Insecticides minimum 20 -30 Fungicides minimum 50 -70 Pre-emergence herbicides minimum 20 - 30 Other herbicides minimum 30 - 40 242 Slide 18 DOSAGE * Temperature * Humidity * Air velocity Slide 19 TIMING Slide 20 COMPOSITION OF SPARY 243 Slide 21 DROPLET SIZE IS HIGHLY IMPORTANT IF PESTICDES ARE TO BE APPLIED EFFICIENTLY WITH MINIMUM CONTAMINATION OF THE ENVIRONMENT Slide 22 Wide range of droplets 20-400 um through hydraulic system Slide 23 20 um - 1 unit of pesticide 400 um - 8000 units of pesticide 244 Slide 24 Classification of spray according to droplet size _______________________________________________ vmd Droplet size (um) classification ---------------------------------------------------------------------- < 25 Fine aerosol 26-50 Coarse aerosol 51 -100 Mist 101 -200 Fine spray 201 - 400 Medium spray > 400 Coarse spray _______________________________________________ Slide 25 PARAMETERS OF DROPLET SIZE vmd : divide a sample of droplets of spray into equal parts by volume; one half of the volume contains droplet smaller than the a droplet whose diameter is vmd and the other half of the volume contain larger droplets nmd : divides droplets into two equal parts by number without reference to the volume thus emphasising the small droplets Slide 26 PRODUCTION OF DROPLET SIZE BY VARIOUS NOZZLES Output Pressure Vmd Type l/min Vmd : nmd kPa um Hydraulic Fan 1.1 300 250 22.7 Low pressure fan 0.6 100 350 5.2 Centrifugal Spinning disc 1.0 2000 262 1.5 0.1 5000 94 1.4 Electrodynamic kV Electrodyn 0.01 25 63 1.02 245 Slide 26 TRANSPORT TO THE TARGET * Path * Velocity * Size at the target T A R Sprayer Droplet G E T * Atomisation * Drop size formulation * Distance to the target * Evaporation & Vaporisation Slide 28 Droplets are influenced by • Gravity • Meteorological factors : wind, temperature and humidity. Slide 29 MOVEMENT OF DROPLET Effect of evaporation • Surface area of spray liquid increases enormously when broken into smaller droplets especially when the diameter is less than 50 um. • Vaporization of volatile part i.e. water • Evaporation increases at higher temperature & lower humidity. • Smaller droplets become aerosol particles which are more prone to drift. Initial droplet Lifetime to extinction size (um) 20o C 80%RH 30o C 50%RH 50 14 s 4s 100 57 s 16 s 200 227 s 65 s 246 Slide 30 MOVEMENT OF DROPLET Effect of meteorological factors • Temperature • Wind velocity • Wind direction • Relative humidity Slide 31 COARSE DROPLETS FINE DROPLETS * Narrow swath * Wider swath * Less under leaf coverage * More under leaf coverage * More spray volume required * Less spray volume required * Particle coalesce and run off * Particle do not coalesce and run off * Less loss due to wind, thermal current * More loss due to wind, thermal current * Poor biological efficacy * Good biological efficacy * Spray pattern like rain * Spray pattern like mist Slide 32 FACTOR AFFECTING THE SPRAY DISTRIBUTION ON THE TARGET • Method of carrying the sprayer • Pressure in the knapsack sprayer • Presence of dirt particles in the tank • Walking speed of the operator • Distance between nozzle & top of the plant • Swath • Air current • Nozzle 247 Slide 33 Improvement in application efficiency 1. Maintaining pressure – Spray management Valve 2. Selection of right nozzle 3. Calibration of the application equipment Slide 34 CONTROLLED DROPLET APPLICATION SYSTEM Slide 35 ADVANTAGES OF CDA 1. Sprayer inexpensive and simple to maintain 2. Labour costs reduced 3. Water carrier not necessary 4. Large area coverage in short time 5. Physical damage to crop is negligible 6. Wider swath 7. Smaller droplets - better distribution due to better penetration 248 Slide 36 Electrodyn System Slide 37 T A R Spraye Droplet G r route E T CONVENTIONAL 1. Initial velocity of each droplet 2. Size of droplet 3. Air conditions ELECTROSTATICS 4. A charged drop will induce an opposite charge on earthed object that it approaches & will therefore be attracted to that object 5. Because of the same polarity droplets would repel each other results - uniform spray cloud - wrap-around effect Slide 38 What is pesticide drift?? • the movement of the pesticide away from the target area. • physical drift • vapor drift 249 Slide 39 Spray drift is undesirable!! • inefficient use of equipment and time • under-application/ineffective control • unintentional contamination of foodstuffs • air/water pollution • livestock and human health/safety Slide 40 Physical Drift • movement of pesticide away from target during application • droplet size • boom height • weather Slide 41 Relationship of Particle Size to Drift Drop Diameter Particle Drift (microns) Type Distance 400 Course 8.5 150 Medium 22.0 100 Fine 48.0 10” Based upon 10” fall in 3 MPH winds 250 Slide 42 Physical Drift Droplet Size • nozzle selection • Drift Guard Nozzles * reduces volume of droplets below 200 microns up to 65% • Raindrop Nozzles * 0.7% - 200 microns or less Slide 43 Physical Drift Weather • wind speed/direction * most important • temperature • humidity • inversions Slide 44 Physical Drift Other Factors to Consider • nozzle orientation • spray pressure • spray volume 251 Slide 45 Physical Drift Spray Volume • most effective means to increase spray volume is to increase nozzle orifice size Slide 46 Pesticide Drift Management Equipment Modifications • shielded spray boom • covered boom • hooded-sprayers • air-assist systems • controlled solution application (CSA) • controlled oil droplet application (CODA) • electrostatic spraying Slide 47 Tips for influencing droplet size nozzle selection lookout for inversions reduce pressure use additives lower boom height calibrate sprayer increase nozzle size use common sense avoid spraying when winds exceed 10 MPH 252 IX. RECOMMENDATIONS The workshop adopted the following recommendations: 1. Programme on the whole was satisfactory and informative. However, more practical session on formulation development and quality assurance need to be included in the future programmes. 2. Participants from the member countries on return may organize national workshops/training programmes on the same lines to train concerned technical personnel. 3. There is need for large scale promotion of eco and user friendly formulations and for this the pesticide industry would be benefitted if similar programmes are organized at selected centers where there are large concentrations of pesticide industry. 4. Dissemination of wealth of information on pesticides, its formulations and alternatives at the Conference of Parties of the Stockholm Convention on POPs may be organized by UNIDO/RENPAP. 5. National coordinators of RENPAP may consider inviting representative from local pesticide industry for future workshops/training programme of UNIDO/RENPAP in order to promote user and environment friendly pesticide formulations at their cost. 253 X. EVALUATION OF THE WORKSHOP Evaluation of the workshop was carried out according to UNIDO Standard Questionnaire. Fifteen participants filled in the questionnaire and the salient features of their answers are summarized below: 1 Duration of Workshop Too long Just right Too short 1 12 2 2 Did training To a small To a large Very large To sufficient correspond to your extent extent extent Extent present need 1 2 8 4 3 General Technical Too low Adequate Too high Much too high Level of Workshop - 13 2 - 4 Most valuable topic WDG Application Formulation Country papers covered Technology – overview 9 6 8 8 5 Least valuable Micro Analytical Practical Nil emulsion - - - 12 6 Any topic not Yes No adequately covered 3 12 7 Did you have sufficient Workshop Faculty Fellow Participants time for professional exchange of views Yes No Yes No with 11 4 12 3 8 Changes in method of Lecture Practical Instruction More 2 13 Less 4 - No change 9 1 9 Participating in Workshop benefited To sufficient To great Very great extent professionally extent extent 3 10 2 254 ANNEXURE - I Workshop on Production of User and Environment Friendly Pesticide Formulations, Quality Assurance and Instrumental Methods of Analysis List of Participants India (Host Country) Mr. Bijoy Chatterjee, Secretary, Department of Chemicals & Petrochemicals, Ministry of Chemicals & Fertilisers, Shastri Bhawan, New Delhi Mr. Surjit Bhujabal, Director, Department of Chemicals & Petrochemicals, Ministry of Chemicals & Fertilisers, Shastri Bhawan, New Delhi Mr. K. Harikumar, Chairman & Managing Director, Hindustan Insecticides Limited Scope Complex, Lodi Road, New Delhi Mr. Rajju Sharof, Chairman & Managing Director, United Phosphorus Limited Mumbai Dr. M. Vairamani, Director, Institute of Pesticide Formulation Technology, Udyog Vihar, Sector 20, Gurgaon 255 Delegates S.No. Participant’s Name Country Mr. Chen Yinghui 1. P.R. China Ms. Eva Dasmita 2. Indonesia Ms. Thipphavanh SILIPANYO 3. Lao PDR Mr. Sahadev Prasad Humagain, 4. Nepal Ms. Bella Fe Carmona 5. Philippines Ms. Duangrat Wilasinee 6. Thailand 7. Mrs. Albertina Benza Canda Angola Mrs. Elizeth Luzola Costa Godinho 8. Angola Goncalves Ms. Bhekiwe Hlope 9. Swaziland Mr. D. Khumalo 10. Swaziland Mr. Sushil Kumar 11. India Mr. G. Kaliyamoorthy 12. India Mr. Nitin Patel 13. India Mr. Anil Kumar Saini 14. India Dr. A. Jha 15. India 256 UNIDO Mr. Philippe R. Scholtès Representative & Head, Regional Office for South Asia, United Nations Industrial Development Organization, New Delhi, India Dr. S.P. Dhua, Regional Coordinator, RENPAP UNIDO, New Delhi, India Dr. Y.P. Ramdev Assistant Regional Coordinator, RENPAP UNIDO, New Delhi, India 257 Workshop on Production of User and Environment Friendly Pesticide Formulations, Quality Assurance and Instrumental Methods of Analysis March 2-9, 2009 SCHEDULE MONDAY. MARCH 2. 2009 09.30 - 10.00 Registration 10.00- 11.00 Inaugural Session 11.00 - 11.30 Tea / Coffee Country Paper Session 11.30- 12.00 Election of Office Bearers & Adoption of Agenda 12.30 - 13.00 Country Papers 13.00 - 14.00 Lunch 14.00 - 15.00 Country Papers (contd.) 15.00 -15.30 Tea/Coffee 15.30 -17.30 Country Papers (contd.) TUESDAY. MARCH 3. 2009 09.30 - 10.30 Pesticide Formulations: An Overview- Dr. P. K. Patanjati 10.30 - 10.45 Tea / Coffee 10.45- 11.30 Role of Surfactants in Pesticide Formulation - Dr. A. Sarangi 11.30- 12.15 WP & Granular Formulations - Dr. L. C. Rohilla 12.15- 13.00 Emulsifiable Concentrates Dr. Anil Gupta 13.00- 14.00 Lunch 258 Laboratory Exercises 14.00- 15.00 Wettable Powder Formulations - Dr. Amrish Agrawal 15.00-15.15 Tea/Coffee 15.15-16.15 Emulsifiable Concentrates - Ms. SIDotiKala 16.15- 16.45 Granular Formulations - Mr. Dipak Kr. Hazra WEDNESDA Y. MARCH 4.2009 09.30 - 10.30 . Concentrated Emulsions & Microemulatios - Dr. P. K. Patanjali 10.30 -10.45 Tea/Coffee 10.45 - 11.45 Suspension Concentrates - Dr. Amrish Agrawal 11.45 - 12.45 Water Dispersible Granules - Dr. Jitender Kumar 12.45 - 13.00 Discussion 13.00 - 14.00 Lunch Laboratory Exercises 14.00-15.00 Concentrated Emulsions& Microemulatios- Ms. SmritiKala 15.00-15.15 Tea / Coffee 15.15-16.15 S~spensionConcentrates - Dr.AmrishAgrawal 16.15 -16.45 Water Dispersible Granules - Mr. Dipak Kr. Hazra THURSDA Y. MARCH 5. 2009 09.30 - 10.30 Concentrated Emulsions & Microemulatios - Dr. P. K. Patanjali 10.30 -10.45 Tea/Coffee 10.45 -11.45 Recent Advances in Vector Control & Household Formulations - Dr.Partibhan 11.45 - 12.45 Registration Aspects Pesticide Formulations- Dr. P. S. Chandurkar 12.45 - 13.00 Discussion 259 13.00 - 14.00 Lunch Laboratory Exercises 14.00 -15.00 Controlled Release Formulations - Dr. Amrish Agrawal 15.00 - 15.30 Tea / Coffee 15.30 - 16.30 Formulations ofBio - botanical Pesticides - Dr. SurabhDubey FRIDA Y. MARCH 6. 2009 09.30 - 10.30 Mass Spectroscopy - An Overview Dr. M. Vairamani, IPFT 10.30 - 10.45 Tea/Coffee 10.45 - 11.45 Gas Chromatography- Mass spectrometric Analysis Dr. D. K. Dubey, DRDE 11.45- 12.45 Triple-quad Mass Spectrometry: Principles and advantages over Single Quad mass Spectrometry. Ms Agilent 12.45 - 13.00 Discussion 13.00-14.00. Lunch Laboratory Exercises 14.00-15.00 Solutions for Pesticide ~esidue Analysis - The Latest in ChromatographyTechniques By Hui-Loo Lai Chin, Shimadzu 15.00-15.30 Tea/Coffee 15.30- 16.30 Demo and Uses ofRTL and DRS in GC/MS Sh. Anil KumarlSh. Samsul Alam SATURDAY. MARCH 7. 2009 09.30 -10.30 HPLC& LC/MS Analysis of Pesticides/residues. Dr. V. K. Gajbhiye, IAR! 10.30 -10.45 Tea/Coffee 10.45- 11.45 Eco-Analytix Solution towards the EnvironmentalHealth & Safety By Dr. Asit Dutta, Perkin Elmer 260 11.45- 12.45 Gas Chromatographic Analysis of Pesticides/residues By Prof. P. Dureja, IARI 12.45 - 13.00 Discussion 13.00-14.00 Lunch Laboratory Exercises 14.00 - 15.00 Lab exercises with HPLC/Demo Exp with LC-MS Sh. Ann Kumar & Ms Anita Rani, IPFT 15.00 - 15.30 Tea/Coffee 15.30 - 16.30 Lab exercises with HPLC/Demo Exp with LC-MS SUNDAY. MARCH 8. 2009 Visit to United Phosphorus Limited, Vapi MONDAY. MARCH 9. 2009 09.30 - 10.30 Bio-evaluation and residue studies of pesticides Dr. A. S. Tomar, IPFT 10.30- 10.45 Tea/Coffee 10.45 - 11.45 Acceptability of GC & HPLC data by NABL and the concept uncertainty Ms Anita Rani, IPFT 11.45 -12.45 Safe Pesticide Application Technology Dr. Y.P. Ramdev, RENPAP 12.45 - 13.00 Discussion 13.00 - 14.00 Lunch 14.00 - 16.30 Conduding Session/Remarks etc.
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