General Applications of Mass Spectrometry by qvg16642

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                                        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

                                                                                  アバンダンス



                                                                                                                   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-->




          ゙ンダンス




             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




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                                                 Environmental Health and Safety”




Slide 2       Our mission is to expand…




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                         …our leadership in personal and environmental health
                                                                                                                                  198


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                                                                        Food
                                                                         Food
                                                           1000’s of global food products
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                                                              rejected contaminated by
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                                                                                                        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.
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                                                                                                           including lead
                                                                                                         contamination.
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                                Air                                                                            Food
                    4.6 million people die                                                                      Food
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                   directly attributable to                                                             annually due to food
                     directly attributable to                                                         safety & quality issues.
                         air pollution.                                                                safety & quality issues.
                          air pollution.                        Sustainable Energy
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                                                           Global warming and greenhouse
                                                              gas emissions deplete
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                                                          protective ozone and pollute air
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                                                                     we breathe
                                                                      we breathe
                       Source: WHO, CDC, CPSC




                                                 …to ensuring health & well being of future generations
          Page 3




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                                                                                                                  199


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                                                                             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:
           [1] HOLD at 35°C for 22 minutes
           [2] INCREASE to 145°C at 10°C/min and hold at 145°C for two minutes
           [3] INCREASE to 225°C at 20°C/min and hold at 225°C for 15 minutes
           [4] 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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