131
Mass Spectroscopy
The paper gave a comprehensive overview on Mass Spectroscopy. The paper discussed the
principles of mass spectroscopy, techniques and its application in the field of pesticides. Because
of high sensitivity of the techniques, mass spectroscopy has become the powerful analytical tool
for monitoring environmental pollutants, pesticides residues in food, water and soil upto parts
per billion and/or trillion levels.
Slide 1
General Applications of
Mass Spectrometry
•Environmental analysis
•Forensic analysis
•Clinical research
•Proteomics and genomics
•Generation of physico-chemical data
1
Slide 2
Identification of unknown compounds
•
•
•
•
UV
IR
NMR
Mass Spectrometry
Spectroscopy
-do-doNon- 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+
MNa+
A
B
C
(g)
MH+
MNa+
ESI/MALDI
(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
•
SAMPLE NATURE
BY ELCTRON BOMBARDMENT
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 + eM + e-
M + 2e
-.
M
.................. (1)
M + e-
M + (n + 1) e ................... (3)
.................. (2)
n+
13
Slide 14
ELECTRON IMPACT (EI)
ABCD + e -
+.
ABCD + 2e-
+ .
ABCD
+ .
ABCD
ABC
AD +
.
+
AB + CD
.
+
A
+ BCD
+ .
ABCD
.
+
ABC + D
+ .
ABCD
10-14-10-16sec
10-8 sec
+.
AD + BC
+
+
AB + C
.
+
.
A +D
14
136
Slide 15
Ions produced in the Electron impact source
CD.
ABC+
A-
D.
ABCD+.
AD+.
A+
BCD.
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
A-B
-e
(A-B)+. (odd electron ion)
(odd number of electrons)
(even number
of electrons)
(A-B)+. (odd electron ion)
Odd electron ion
A+
B.
+
Even 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
weight
12
1
16
32
31
19
35
79
127
14
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
C4 H4 N2
80
C3 H3 N3
81
N
N
N
N
N
N
19
Slide 20
Elements
Isotopes (abundance %)
H
1H
C
12C
(98.9)
13C
(1.1)
N
14N
(99.6)
15N
(0.4)
O
16O
(99.8)
18O
(0.2)
(95)
33S
(0.7)
(99.99)
32S
S
34S
(4.2)
Cl
35Cl
(75.5)
Br
79Br
(50.5)
37Cl
(24.5)
19F, 31P, 127I
- 100 % each
81Br
(49.5)
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)+.
(M+2)+.
Pm+1 / Pm X 100 = 1.1
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
~ 3:1
112
Chloro benzene
Cl
24.5
M+. 112 (35Cl)
(M +2)+. 114 (37Cl)
114
M+. : (M +2)+. = 3:1
m/z
23
139
Slide 24
Chlorine
35Cl
37Cl
75.5
24.5
~ 3:1
Dichloro benzene
146
M+.
Cl
146 (35Cl, 35Cl )
148
(M +2)+. 148 (35Cl, 37Cl)
(37Cl, 35Cl )
Cl
150
(M +4)+. 150 (37Cl, 37Cl)
m/z
M+. : (M +2)+. : (M+4)+. = 9:6:1
(a+b)2 = a2 + 2ab+ b2 where a =3 and b =1
24
Slide 25
Bromine
79Br
81Br
50.5
49.5
~ 1:1
156 158
Bromo benzene
Br
M+. 156 (79Br)
(M +2)+. 158 (81Br)
M+. : (M +2)+. = 1:1
m/z
25
Slide 26
Bromine
79Br
81Br
50.5
49.5
~ 1:1
Dibromo benzene
236
M+.
234 (79Br, 79Br )
Br
(M +2)+. 236 (79Br, 81Br)
234 238
(81Br, 79Br )
Br
(M +4)+. 238 (81Br, 81Br)
M+. : (M +2)+. : (M+4)+. = 1:2:1
(a+b)2 = a2 + 2ab+ b2 where a =1 and b =1
m/z
26
140
Slide 27
Atomic weight of some elements commonly dealt in
Organic Chemistry
12C
= 12.0000
1H
31P
= 1.00783
= 10.01294
14N = 14.00307
16O = 15.99491
19F = 18.99840
28Si = 27.97693
= 30.97376
= 31.97207
35Cl = 34.96885
37Cl = 36.96590
79Br = 78.91839
81Br = 80.91642
10B
BENZENE
C6H6
PYRIDINE
C5H5N
32S
Nominal mass
78
79
Correct mass
78.04698
79.04222
27
Slide 28
Schematic diagram of a GC
Injector
Detector
Column
28
Slide 29
Schematic diagram of a GC-MS
Detector
Injector
Column
MS
29
141
Slide 30
Capillary GC-MS interface
30
Slide 31
Abundance
TIC: QUE.D
2e+07
Total ion
chromatogram
1.8e+07
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
4.39
4.49
5000000
1.15
4500000
Expanded TIC indicating
solvent peaks
4000000
3500000
5.49
3000000
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
Slide 35
NL: 3.96E7
8.80
100
TIC MS data08
12.09 12.39
5.18
21.24
Full Scan/SIM Analysis
50
0
NL: 3.96E7
TIC F: + c Full ms
[ 50.00-500.00]
MS data08
8.80
100
12.09 12.39
5.18
21.24
50
Relative Abundance
0
NL: 7.57E4
TIC F: + c SIM ms
[ 131.50-132.50]
MS data08
21.82
100
21.24
50
0
NL: 2.06E3
TIC F: + c SIM ms
[ 224.50-225.50]
MS data08
14.61
100
50
0
NL: 7.43E3
11.06
100
TIC F: + c SIM ms
[ 178.50-179.50,
263.50-264.50]
MS data08
11.35
50
0
6
8
10
12
14
16
18
20
Time (min)
22
24
26
28
30
32
Pesticides analysed in strawberry/spinach/pea extract at35
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, 12 mL/min
Slide 4
Splitless (100:90) vs. Split (100:1)
Syringe
Syringe
Injector
Injector
He
He
Purge valve
closed
GC column
Slide 5
GC column
Purge valve
open
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
Mobile
phase
(Helium)
flowing at 1
mL/min
0.32 mm ID
Liquid
Stationary
phase
0.1-5 µm
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+*
• M + e-
f1
f2
f3
f4
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
analysis
sensitive
and
selective
• Elimination of chromatography
– Specificity
Slide 26
MS/MS
Scan
Q1
Q2
Purpose
Product /
Daughter ion
Static (Parent
ion selected)
Scanning
Detection of all
fragment ions
from a single
precursor
Precursor /
Parent ion
Scanning
Static (Daughter
ion selected)
Detection of all
precursors of a
common fragment
ion
Neutral loss
Scanning
Detection of all
Scanning (offset
precursors
by fragment
sharing a common
mass)
neutral fragment
SRM / MRM
Static (Parent
mass
selected)
Static (Daughter
mass selected
Detection of a
specific fragment
ion originating
from a specific
precursor
154
Slide 27
Slide 28
Slide 29
Triple Quadrupole: MS/MS Product
ion Scan
Triple Quadrupole: MS/MS
Precursor ion Scan
Triple Quadrupole: MS/MS SRM /
MRM
155
Slide 30
Slide 31
Triple Quadrupole: MS/MS
Neutral loss Scan
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
Consumer services
of
Agriculture
and
– J. Cook, M.P. Beckett, B. Reliford, W. Hammock, M.
Engel (1999) J. AOAC Int. 82, 1419-1435
• California
Department
of
Agriculture (www.cdfa.ca.gov)
Food
and
– 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
Slide 49
New Pesticide Analysis Method
Detailed Sample Treatment Before GCMS 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 Î 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 multiresidue 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
REF
Thermal Conductivity
Filament pair heats
when sample dilutes
carrier gas
Air
H
2
PMT
O
2
H
2
Flame Photometric
Optical filter selects
wavelength specific to P or S
compounds
Flame Ionization
Burning produces
charged particles which
collector converts into a
current
H
Electron Capture
Loss of slow electrons
by sample absorption
decreases cell current
NP Thermionic
N or P compounds
increase current in plasma
from vaporized metal salt
2
Ion
Source
Analyzer
EM
Mass Selective Detector
Ionized sample measured
by mass analyzer
1
Slide 2
Comparison of GC Detectors
TCD
FID
ECD
NPD(N)
NPD(P)
FPD(S)
MSD
(SIM)
10-15
fg
10-12
pg
(SCAN)
10-9
ng
10-6
ug
10-3
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
GC
INTERFACE
ION
SOURCE
MASS
FILTER
DETECTOR
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
Extracted Ion 253 m/z
Abundance
ance
Ion 253.00 (252.70 to 253.30): OLDLSD07B.D
105000
Inert Source
Ion 253.00 (252.70 to 253.30): INERTLSD13W.D
30000
100000
95000
90000
85000
80000
20000
75000
10000
00000
90000
80000
~6x
improvement!
70000
60000
50000
70000
65000
60000
LSD S/N 2.9
55000
50000
45000
40000
LSD S/N 16
35000
30000
25000
20000
40000
30000
15000
10000
5000
20000
10000
0
6.10
6.20
6.30
6.40
6.50
6.60
6.70
6.80
6.90
7.00
Time-->
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
7.10
166
Slide 7
Ion Source – Improved Peak Shape
ｱﾊﾞﾝﾀﾞﾝｽ
ｲｵﾝ 277.00 (276.70
ｲｵﾝ 247.00 (246.70
Inert Source
～ 277.70):
～ 247.70):
YG0701_1.D
YG0701_1.D
15000
14000
277u
13000
12000
Fenitrothion(EIC m/z 277 and 247)
11000
10000
9000
Breakdown ion
8000
7000
247u
6000
5000
4000
3000
2000
1000
0
11.90
12.00
12.10
12.20
12.30
12.40
12.50
12.60
12.70
12.80
Time-->
ﾞﾝﾀﾞﾝｽ
SST Source
ｲｵﾝ
ｲｵﾝ
7500
277.00
247.00
(276.70
(246.70
～ 277.70):
～ 247.70):
YG0703_2.D
YG0703_2.D
7000
Increase in breakdown
ion reduces the
abundance of the ion of
interest (277u).
Breakdown ion
6500
6000
SST Source
5500
5000
4500
277u
4000
3500
247u
3000
2500
2000
1500
1000
500
0
11.90
12.00
12.10
12.20
12.30
12.40
12.50
12.60
me-->
12.70
12.80
Result: lower sensitivity
7
Slide 8
Monolithic Quartz Quadrupole
•
•
Single piece construction
Hyperbolic surface
•
Heated upto 200 C –Maintenance free
o
•
8
167
Slide 9
Electron Ionization (EI)-MOST POPULAR IN GC
.
ABC + + 2e -
ABC + e Neutral
Molecule
Ionization:
Excited
Molecular Ion
Position of Curve
.
#ABC+
Depends on IP (ABC)
0
10
70
100
eV
Electron Energy
Fragmentation:
ABC +
.
.
AB
A+
.
AB +
.
AC +
+ C+
.
+ BC
+C
(loss of neutral)
(rearrangement)
+B
etc.
Resulting Mass Spectrum:
AB+
+
C
Signal
A+
Abundance
+
AC
+.
ABC
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 ] +
(Base peak)
4 9
90
80
70
71
43
60
+
<--[C H ]
5 11
50
85
40
<--[C H ]+
6 13
30
M
20
10
m/z->
20
+
.
55
29
98
113
128
40
60
80
100
120
(Molecular
ion)
170
141
140
159
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)
Fast 6,250 u/s (7000A)
Targets & Non-targets
Typical < 2,000 u/s
Targets & Non-targets
Dwell 1-50 ms
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)
Fast 6,250 u/s (7000A)
Targets & Non-targets
Typical < 2,000 u/s
Targets & Non-targets
Very Selective
Most Selective
Unit mass + AMDIS
Unit mass resolution
Dwell 1-50 ms
Targets only
Targets only per DBL
Q1 1.2 u
CID product ions
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
Scan
More Sensitivity
SIM (SIM/Scan)
Scan
More Selectivity
DRS
SIM
More Selectivity
MS/MS
SIM
MS/MS
More Sensitivity
Larger sample
Larger injection
Sharper peaks
17
Chrysene
Benz[a]anthracene
Synchronous SIM/Scan Comparison of PAHs
Triphenyl phosphate
Slide 18
SIM
5.55 cycles/s
Scan 45-450u
5.55 cycles/s
Scan:
Poorer S/N but
targets and nontargets detected
SIM:
Better S/N but
only target
analytes
0.2 ppm
?
Application 5989-4184EN
18
172
Slide 19
The matrix
ions results
in false
negative
Raw
spectrum
Deconvoluted
spectrum
DRS enables a
positive
identification
p,p’-DDE
Library
spectrum
19
Complex Matrices Show the Benefit of MS/MS
Single MS: SIM 283.8
100 fg HCB in Clean Matrix
MS/MS: 283.8:213.9
Slide 20
300 fg HCB in Diesel
S/N=26:1
RMS
SIM about equal to
MS/MS in clean matrix
MS/MS 15x better than SIM in
complex matrix – and better baseline
S/N=37:1
RMS
20
173
Slide 21
Assessment of Needs and Limitations
what is the budget?
higher cost OK
need lower cost
MS/MS
Scan
SIM
MS-DRS
This may not be your first question, but it must be asked
what detection limit is required?
need low pg & fg
ng & mid-pg OK
Scan
MS-DRS
MS/MS
SIM
MS/MS gives consistently lower detection limits for
simple and very complex samples
how important are non-target peaks?
only analyze targets
SIM
MS/MS
MS-DRS
SIM/Scan
need lbr searches
Scan
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
Scan
SIM
very complex matrix
MS/MS
MS-DRS
Least selective
MS-DRS
Scan
SIM
Most selective
how critical is sample preparation?
sample preparation = time, money, errors
more smp prep OK
need less smp prep
MS-DRS
MS/MS
Do you like spending
time on sample prep?
how critical is a unique quant ion?
important
Scan
SIM
MS-DRS
Ionization mode
determines uniqueness
very, very important
MS/MS
can you afford an error?
Ionization mode + CID
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
S from analyte
S from matrix
N from neutrals
N from matrix
N from "bleed"
GC/MSD
Scan
+
-=
--Low
GC/MSD
SIM
+++
-=
--Low
GC/MSD
Scan-DRS
+
=
Lower
GC/QQQ
MS/MS
+++
0
Ultra low
0
0
MDL (clean)
Very Low
Lowest
Lower
Lowest
Low
Low
Low
Low
Lower
Lower
Lowest
Lowest
MDL (very dirty)
Quant Error
MDL =
Sanalyte
Nneutral + Nmatrix + NGC “bleed”
Smatrix
Quant Error =
But Scan is
essential for
non-targets
(unknowns)!
Sanalyte + Smatrix
S= Signal
N=Noise
24
Application Alignment with MS Modes
Drinking water
Industrial samples
Flavor and Fragrances
Scan
iv it
ec t
y
se
sel
ns
High purity water
Air Analysis
Toxicology screens SIM
Pharma residual solvents
Waste water
Food matrices
Tox of complex samples
iv it
e ct
siti
s el
vit
y
DRS
y
sen
Non target compounds
Synthesis confirmation
“Street” drug samples
itiv
it y
Slide 25
MS/MS
Highly contaminated water
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
(unknowns)?
• Scan MS with SQ, TOF, or
tandem MS in SQ mode with
libraries
• How much chemical noise
from the matrix?
• DRS or MS/MS
• Backflush or 2D GC
• (More sample prep)
• Orders of magnitude higher
detection limits than SIM or
MS/MS
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
9
High Sensitivity
9
Fast SRM Speed
9
MassHunter Software
9
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
•Ionize
Mean Free
Path
Collisions
Quad 1
Mass
Analysis
Quad 2
Collision
Cell
Quad 3
Mass
Analysis
Long
Short
Long
No
Yes
No
MS
Detector
MS
29
176
Slide 30
Selected Reaction Monitoring (SRM)
Quad Mass Filter (Q1)
Collision Cell
Q1 lets only
target ion 210
pass through
Spectrum with
background
ions (from EI)
210
Quad Mass Filter (Q2)
Collision cell
breaks ion 210
apart
210
222
268
165
170
210
250
158
280
290
Q2 monitors only
characteristic
fragments 158
and 191 from ion
210 for quant and
qual.
158
191 210
150
190 210
170
190
191
160
210
190
no chemical
background
30
Slide 31
MS/MS Eliminates Scan and SIM Interferences
Triple Quad MS
Single Quad MS
selectivity proportional to
spectral resolution
no selectivity against ions
with same m/z
interference
Precursor selectivity same as MS but
high probability that one or more of the
product ions will be a unique dissociation
product of the precursor only
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
Q1 SIM
isolate precursor
before CID
chemical noise
from these other
ions is eliminated
Product 3
CID +
Q2 SIM
Lower m/z Product
Ions measured
against zero chemical
noise
32
177
Slide 33
Eliminates the “Invisible” Interferences of SIM
103
EI-SIM
Unit mass resolution
Scale
change
Removed by SIM
EI-SIM
20 x 103
But what happens when a
much more intense ion
of a multi-carbon compound has
an ion 1 m/z lower?
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
interference in very ‘dirty’
samples
MS/MS eliminates
this interference
Isotope ion
not removed
by SIM
analyte ion
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
As Matrix Increases - MS/MS is More Valuable
Single MS: SIM 283.8
100 fg HCB in Clean Matrix
MS/MS: 283.8:213.9
Slide 35
300 fg HCB in Diesel
S/N=26:1
RMS
SIM about equal to
MS/MS in clean matrix
MS/MS 15x better than
SIM in complex matrix
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
Collision Cell
Quad 2
Detector
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
sn
o
m
ra
iT
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
Source
Precursor
Ions In
Quad Analyzer
collision induced dissociation
Product
Ions Out
Detector
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
Collision Gas
At the detector,
metastable
helium generates
neutral noise.
Quad Analyzer
Source He* +
Quad Analyzer
Precursor
Ions In
collision induced dissociation
Product
Ions Out
A high population of
highly energetic helium
metastables are produced
in an EI source; since
metastable helium is not
charged, it can pass
through mass analyzer
field into the collision cell
and through to the HEDEM
+
He* Detector
In GC/MS, neutral noise
is buried in much higher
chemical noise.
In GC/MS/MS, chemical
noise is greatly reduced
so neutral noise is a
critical source of noise.
42
Slide 43
Agilent Collision Cell Process with Quench Gas
Collision Cell
1 ml/min N2
Collision Gas
Quad Analyzer
Source He* +
Quad Analyzer
Precursor
Ions In
collision induced dissociation
Product
Ions Out
He Buffer Gas
He* + He
→
2 He + heat
+
He*
Detector
Transmission of
metastable
helium to the
detector is
greatly reduced;
the Triple-Axis
detector further
reduces neutral
noise for ultralow 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
Chlorpyrifos (28 ppb) Easily Detected and
Quantitated by GC/MS/MS – Incurred Carrot
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
1.6 R^2 = 0.99980926
Responses
197.0 -> 169.0 , 197.0 -> 98.0
x10 4 Ratio=14.7
2.4
2.2
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
-0.2
9.3
9.4
9.5
9.6
9.7
9.8
Acquisition Time (min)
Counts
Slide 46
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
1000
2000
3000
4000
5000
6000
7000
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)
9
9
9
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
9
9
Slide 4
ADVANTAGES OF HPLC
9
No limitation of polarity and Heat Stability
9
Can be used for both qualitative and quantitative
analysis
9
High sensitivity
9
Fast
9
Reproducible
9
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
¾ Normal phase
¾ Reverse phase
¾ Ion exchange
¾ 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
¾
Normally operated at ambient temperature
¾
Fluctuation in temperature during day time
¾
Separation and retention time may vary
due to temperature variation
Column is housed in thermostated oven where temperature
can be accurately controlled and maintained
¾
¾
¾
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
¾
Free OH groups on silica
¾
Large hydrocarbons (C8, C18) not able to
reach and react with OH groups which are
inside smaller cavities
¾
Such groups are deactivated by methylation
¾
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
z
More polar analytes retained less
z
Analytes with larger hydrophobic part
are retained longer
z
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
z
z
z
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
10
15
25
25
3
5
5
5
10
11111
10526
13636
15625
10000
3
1.9
2.2
3.2
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
¾
The refractive index of a medium is the ratio of
the speed of light in a vacuum to the speed in
the medium.
¾
The detector measure the change in refractive
index in the eluent as the solute passes through
the sample cell.
¾
less sensitive than UV detection
194
Slide 33
Fluorometric Detector
Solute is excited by UV radiation at a particular
¾
wavelength
¾
The emission wavelength is detected.
¾
Can be used with naturally fluorescent
compounds
Compounds can be reacted to produce
¾
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)
¾
Chemical Ionization (CI Mode) – APCI
¾
ESI
¾
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
¾
HPLC/ UPLC as Analytical tool
– Formulation analysis
- Residue analysis
¾
Semi-prep HPLC/MPLC/GPC - Cleanup technique
¾
LC-MS
¾
LC-MS-MS- Residues at ppt level
- Confirmation technique –
197
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Slide 1
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Slide 2
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Protecting our global ecosystem is key…
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199
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Page 8
200
Gas Chromatographic Analysis of Pesticides/Residues
The paper discussed the principles, instrumentation, separation techniques and
different parameters of the compounds that are involved in the separation
process by gas chromatography. The paper explained and discussed results of
some of the pesticides analysis carried out by gas chromatography.
Slide 1
PESTICIDES
SCENARIO IN
INDIA
REGISTERED PESTICIDES
=
219
BANNED
=
31
PESTICIDE WITHDRAWN
=
7
LIST OF PESTICIDES REFUSED REGISTRATION
=
18
PESTICIDES RESTRICTED FOR USE IN INDIA
=
10
Slide 2
MAJOR CONSUMER CROPS OF PESTICIDES
IN INDIA AND GLOBALLY
____________________________________________________________
Share (%)
Crop
India
Global
____________________________________________________________
Cotton
37
9
Paddy/Rice
20
12
Chilli
6
Wheat
6
Vegetable/fruits
5
25
Tea
5
Pulses/ other cereals
3
15
Potato, Grapes and oilseeds
2+2+2
Maize
11
Soya
9
Sugar beet
3
Others
12
16
_________________________________________________________
Ministry of Petrochemicals (2005)
201
Slide 3
CONSUMPTION OF AGROCHEMICALS IN INDIA
Year
Slide 4
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
AGROCHEMICAL USE IN KEY COUNTRIES/AREAS OF THE WORLD
Country or Area
Pesticide
(Kgha-1)
Republic of Korea
Italy
Hungary
Japan
China
Europe
USA
Mexico
Thailand
Indonesia
India
Turkey
Argentina
Latin America
Oceania
Africa
16.56
13.35
12.57
10.8
2.0-2.5
1.9
1.5*
1.38
1.37
0.58
0.38
0.3
0.29
0.22
0.20
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)
chromatographic technique where mobile phase is
gas.
most popular methods for separating and analyzing
pesticides. Because of
A
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:
Slide 20
1) Packed
2-4 mm diameter
2) Megabore
0.53 mm diameter
3) Capillary
0.1-0.25 mm diameter
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
laboratory GC instruments
– Smaller sample capacity
separating/determining major
components in a sample (prep
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
–
–
–
–
Slide 25
More constant peak width
Better sensitivity for components that are retained longer
Much better chromatographic resolution
Peak refocusing at head of column
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
Gas outlet
Collector
Sintered disk
Coaxial cable to
Analog to Digital
converter
Ions
Flame
Platinum jet
Air
Hydrogen
Capillary tube (column)
FID (Nanogram - ng)
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 salt
Alkali salts used
ƒ Potassium chloride
ƒ Sodium sulfate
ƒ Cesium bromide
ƒ 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
excited
flame
in
compound
when burnt in
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
Slide 32
P : 526 & S : 394 nm
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) Æ XIon 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
presence of oxygen if hot!
Non-destructive
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
concentration which can be detected with the
probabiloity of 99%
LIMIT OF DETECTION
(LOD)
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
to define the presence or the absence of the
analyte
PROTOCOL
QUANTITY ANALYSIS
LIMIT OF QUANTITATION (LOQ)
NOTES O: LOD &
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
Laboratories with acceptable accuracy and
precision, in routine
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
PESTICIDES
Slide 37
•
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
Slide 39
PESTICIDE SEPARATIONS
GLC CONDITIONS
COLUMN: OV – 101/ 210/ 17 (Cappillary
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 LIQUIDLIQUID EXTRACTION AND GAS CHROMATOGRAPHY WITH ELECTRONCAPTURE 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
Slide 44
CHLORINATED PESTICIDES BY SPME/ CAPILLARY GC
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
Slide 46
0.01µg
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
Slide 47
0.01µg
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
[discard]
Slide 52
Petroleum ether
Florisil column
Elution with pet. ether-diethyl ether
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)
about 180 analytes
LC-MS/MS
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
•
Calibr ation of equipment is per for med
cor r ec tions; or assigning values to the scale of an instr ument.
– 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
from Instrument Software
individually
the
gas
from
flow
rate
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
S.No.
Concentration
(ppm)
1
500
Area
Avg. area
Fig 1 : Linearity Curve
V.F.
1237013
1238536
1237774.89
0.122905
2484967.5
0.26087
3660249
0.157406
5159819
0.043752
7390791.5
0.165569
2488213
2
1000
2481722
3657366
3
1500
4
2000
3663132
5160948
5158690
7396915
5
3000
7384668
R2 value = 0.9981 shows good performance
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
ƒ
ƒ
ƒ
ƒ
ƒ
ƒ
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
ƒ
ƒ
ƒ
a parameter, generally a standard deviation
associated with the result of a measurement
characterizes the dispersion of values attributed to the
measurand
ƒ
ƒ
built-up of many components, type A and B
other simple definitions:
ƒ the
doubt
about
the
exactness
of
a
measurement result
ƒ 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
Process
Specification
Identify Uncertainty
Sources
Specification:
ƒ
ƒ
Slide 29
Convert to Standard
Deviation
Calculate the Combined
Uncertainty
as far as possible express the
relationship in an equation as a
function of the imput parameters
y=f(p,q, T, A..)
ƒ
Quantify Uncertainty
Components
Defining the measurand: write
down a clear statement of what is
being
measured
and
the
relationship between it and the
parameters on which it depends
split the measurement process in a
series of steps and assess them
separately
YES
Re-evaluation of the
Significant Components
Do the Significant
Components Need
Re-evaluation?
NO
END
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
suppliers,QA/QC data
data
and
results
from
certificates,
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
Repeatability
Reproducibility
Volumetric
flask
Mass (balance)
Calibration
Repeatability
Syringe
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
Chlorpyrifos
Temperature
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
– U(P)
= 1 ± 0.015%
= 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)
(c) Temperature (Type B, Rectangular distribution)
= 0.0059
–
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
EP
I
O
N
D
E
N
S
I
T
Time
Y
Slide 42
P
O
EIL
P
U
L
A
ET
T
I
O
N
EP
D
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
ETHIRIMOL
Seed treatment
Barley (for mildew control)
DISULFOTAN
Soil incorporation Wheat (aphid control)
LINDANE /
DIELDRIN
Arial spraying of
swarms
PARAQUAT
Spray
Locusts
Grass
0.03%
2.20%
2.90%
6.00%
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
be avoided
250 -500
______________________________________________
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
Type
Output
l/min
Pressure
kPa
Vmd
um
Vmd : nmd
Hydraulic
Fan
1.1
300
250
22.7
Low pressure fan
0.6
100
350
5.2
1.0
2000
262
1.5
0.1
5000
94
1.4
63
1.02
Centrifugal
Spinning disc
kV
Electrodynamic
Electrodyn
0.01
25
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
less than 50 um.
is
•
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
size (um)
20o C 80%RH
50
14 s
4s
100
57 s
16 s
200
227 s
65 s
Lifetime to extinction
30o C 50%RH
246
Slide 30
MOVEMENT OF DROPLET
Effect of meteorological factors
•
Temperature
•
Wind velocity
•
Wind direction
•
Relative humidity
Slide 31
COARSE DROPLETS
Slide 32
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
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
Spraye
r
T
A
R
G
E
T
Droplet
route
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
(microns)
400
Particle
Type
Course
Drift
Distance
8.5
150
Medium
22.0
100
Fine
48.0
Based upon 10”
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
Ò nozzle selection
µ lookout for inversions
Ó
Ô
Õ
Ö
¶ use additives
· calibrate sprayer
¸ use common sense
reduce pressure
lower boom height
increase nozzle size
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
2
3
4
Duration of Workshop
Did training
correspond to your
present need
General Technical
Level of Workshop
Most valuable topic
covered
Too long
Just right
Too short
1
12
2
To a small
extent
To a large
extent
Very large
extent
To sufficient
Extent
1
2
8
4
Too low
Adequate
Too high
Much too high
-
13
2
-
WDG
Application
Technology
6
Formulation
– overview
8
Country papers
Analytical
Practical
Nil
-
-
12
9
5
Least valuable
6
Any topic not
adequately covered
7
Did you have sufficient
time for professional
exchange of views
with
8
9
Micro
emulsion
-
8
Yes
3
No
12
Workshop Faculty
Fellow Participants
Yes
No
Yes
No
11
4
12
3
Lecture
Practical
More
2
13
Less
4
-
No change
9
1
To sufficient
extent
3
To great
extent
10
Changes in method of
Instruction
Participating in Workshop benefited
professionally
Very great extent
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
1.
Mr. Chen Yinghui
P.R. China
2.
Ms. Eva Dasmita
Indonesia
3.
Ms. Thipphavanh SILIPANYO
Lao PDR
4.
Mr. Sahadev Prasad Humagain,
Nepal
5.
Ms. Bella Fe Carmona
Philippines
6.
Ms. Duangrat Wilasinee
Thailand
7.
Mrs. Albertina Benza Canda
Angola
8.
Mrs. Elizeth Luzola Costa Godinho
Goncalves
Angola
9.
Ms. Bhekiwe Hlope
Swaziland
10.
Mr. D. Khumalo
Swaziland
11.
Mr. Sushil Kumar
India
12.
Mr. G. Kaliyamoorthy
India
13.
Mr. Nitin Patel
India
14.
Mr. Anil Kumar Saini
India
15.
Dr. A. Jha
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
Concentrated Emulsions & Microemulatios - Dr. P. K. Patanjali
09.30 - 10.30 .
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.