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MASS SPECTROMETRY for
pesticides residue analysis
Lecture(3)
“Electrospray Ionization and Atmospheric pressure chemical ionization”
Contents Lecture 1: Pesticides classification with an introduction to mass spectrometry& vacuum system for GC MS/MS and LC MS/MS
Lecture 2 : Electron ionization and Chemical ionization
Lecture 3 : Electrospray Ionization and Atmospheric pressure chemical ionization
Lecture 4 : The commonly used mass to charge analyzer for pesticides residue analysis
Types of IonizationThere are Different ionization techniques that can be used for mass spectrometry. In
these series of lectures , the most commonly used ionization techniques for mass
spectrometry analysis of pesticides residue will be discussed, especially:
• Electron Ionization (EI) as an example for Hard ionization technique.
• Chemical ionization (CI), Atmospheric pressure chemical ionization (APCI) and Electron spray ionization (ESI) as examples for Soft ionization techniques.
• Used for analysis of thermo labile and nonvolatile analytes (reverse to EI)
• produce ions by passing a solution into narrow charged electrode (protonated /deprotonated)
• Produce multi charged ions (with lower m/z ratio) for analyte molecules of high molecular weight (above 1000 u) with multi active atoms
• ESI is applicable for a wide range of analyte
molecules varying in mwt and polarity
Electrospray ionization (ESI)
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011
The first electrospray–mass spectrometry interface (Fenn group,1980s)
• Fenn and his group have showed the formation of multiply charged ions from proteins allowing their molecular weight to be determined with instruments of low mass range
• ESI was first used for protein analysis after that for polymers, co-polymers and for smaller molecules
E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)https://en.wikipedia.org/wiki/Electrospray_ionization
Mechanism of Electrospray Ionization• ESI works by online HPLC MS/MS, Where a liquid is converted to an charged aerosol
droplets by passing through a metal capillary which also maintained at high voltage (around 4 kV) in a chamber held near atmospheric pressure
• ESI can work in positive and negative modes• Positive ESI produce both positive and negative charges only positive charged
particles will be attracted to mass vacuum (the reverse in –ve ESI)
P. Kebrale, Electrospray: From ions in solution to ions in the gas phase, what we know now, Mass Spectrometry Reviews, 2009https://www.youtube.com/watch?v=r6TGvG7RUyo
Shape of the charged droplets
• The shape of liquid passed through 0.1-mm-ID horizontally positioned capillary at :
No voltage ------------------------drops fall off under (gravity)
Moderate voltage --------------drops with reduced size are formed
Higher potentials (Von)----------------reduced size droplets are formed withsharp point at its tip, the droplet divided with spray formation (Taylor cone, the formed droplets attracted to the counter electrode)
At further higher potentials------- liquid cone vanishes and a fine mist of droplets is produced (avoid discharge)
W. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)
Shape of the charged droplets
• Calculation of the onset voltages (Von)
σ is the surface tension of the liquidrc is the inner diameter of needled is the distance between capillary and counter electrode
For rc = 0.1 mm and d = 40 mm, Von = 2.2 kV (MeOH), 2.5 kV (Acetonitrile) , 4.0 kV (Water)
• For stable ESI performance, the voltage should be set a few hundred volts higher than the onset voltage. But, too high potential cause discharge especially when applying Negative ESI mode
W. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)
Formation of charged ions
J. Throck, Introduction to Mass Spectrometry Instrumentation, John Wiley & Sons Ltd, England (2007)E.Hoffmann, Mass Spectrometry Principles and Applications, John Wiley & Sons Ltd, England (2007)El-Aneed A., Mass spectrometry review of the basics : Electrospray, maldi and commonly used mass analyser, APPLIED SPECTROSCOPY REVIEWS (2009)
• As the droplet size decrease the repulsive forces between the excess charged ions increase. Which result in “coulomb explosion” of the droplet before reaching the Rayleigh limit (the point at which repulsive forces between like charges in an electrolytic solution overcome the cohesive forces of the solvent).
• Finally, when the solvent molecules have evaporated, quasi molecular ions of the analyte will be formed; protonated for positive ESI, protons attach at sites of high Lewis basicity. While, deprotonated for -ESI
Desolvation of the charged droplets
J. Throck, Introduction to Mass Spectrometry Instrumentation, John Wiley & Sons Ltd, England (2007)
1.0073
Positive ESI Mass spectrum of lysozyme
ESI For high flow rate• ESI introduce aerosol from liquid of low flow rates of 1–20 μl/ min, Therefore
further modification have been established
• Applying a constant flow inert gas (N2) around the electrospray needle (Nebulizer Gas) to reduce the influence of the surface tension of the used solvent.
• Although, ultrasonic nebulizer handles liquid flow rates of 50–1000 μl/min but, the ultrasonically created droplets are comparatively large and this hinders ion formation
Pneumatically assisted ESI
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011
Improved geometries for ESI• In order to decrease the system contamination (orifice,…) different geometries for
ESI are produced
j.gross, Mass Spectrometry, a text book, Springer-Verlag Berlin Heidelberg 2004, 2011
Increase the desolvation of the charged droplets
• A fast desolvation for the charged droplets can be obtained by introducing a heated inert gas. Which,
ionization of the sample.
• The higher the liquid flow or the higher of its aqueous composition, the higher heater temperature and gas flow required.
• Temperature lead to a premature vaporization of the solvent, chemical background noise.
• Heater Gas flow can produce a noisy, or unstable signal.
API LC/MS/MS TurboIonSpray Ion Source Manual
Declustering using curtain gas
API LC/MS/MS Turbo IonSpray Ion Source ManualW. Niessen, Liquid Chromatography– Mass Spectrometry, Taylor and Francis Group, LLC (2006)
• Curtain gas (inert gas (N2) is used as an insulator between the ion source (prevent entering of air, solvent, non polar matrices) and vacuum chamber. So, it should be optimized at highest possible setting.
• This gas is introduced between curtain plate and orifice plate, Part of this gas passes through the orifice nozzle into the differentially pumped interface, while the remaining gas flows back through the aperture in the curtain plate to make a declusting collisions.
• The resulted ionized gas molecules are in a cluster form (with some residue from solvent molecules). A cluster may be resulted from the cooling that happened after solvent evaporation or by incomplete evaporation.
• Introducing curtain gas in the way of these ion clusters leads to a complete separation for the analyte ions from solvent molecules.
Declustering using potential and ion focusing
API 4000™ LC/MS/MS System Hardware Manualhttp://sciex.com/products/mass-spectrometers/triple-quad-systems/triple-quad-6500-system-x36184
• A cluster may also be resulted from the cooling that happened under the first vacuum stage (after passing orifice plate) that induce adiabatic expansion that favor cluster formation.
• Applying a potential (declustring potential, DP ) on the orifice is needed to decluster the aggregated ions between orifice and skimmer or between orifice and Qjet .
• DP should be high enough to reduce chemical noisy with avoiding fragmentation especially, for MRM analysis.
• The entrance potential (EP) guides and focuses the ions through Q0 region.
Turbo V™ ESI
API LC/MS/MS TurboIonSpray Ion Source ManualTurbo V™ Ion Source Operator Guide
Turbo heater
Electrode adjustment cap
Retaining ring
X-axisY-axis
Turbo heater
Exhaust
Spray is directed away from orifice
Probe
Electrode
Turbo IonSpray ESI (operating conditions for the at three different flow rates)
Turbo V™ Ion Source Operator GuideAPI LC/MS/MS TurboIonSpray Ion Source Manual
Spray is directed away from orifice
• The solvent composition usedfor optimization was 1:1 water: acetonitrile
• ISV : Positive mode 4000 to 5500 V. Negative mode usually lower
-3000 to - 4500 V. (P ISV of 4500 for 6500 system)
• For too high ISV, a blue glow can be seen at the tip of the TurboIonSpray (a corona discharge) decreasing the sensitivity.
• Curtain Gas flow should be as high as possible. Start with 20 for 4000 and 4500 & 25 for 5000 and 5500 & 30 for 6500
Turbo V™ APCI (APCI Heated nebulizer)
API 4000™ LC/MS/MS System Hardware ManualTurbo V™ Ion Source operator guideT. Portol, Potential of atmosphericpressure chemical ionization source in GCQTOF MS for pesticide residue analysis, journal of mass spectrometry (2010).
• The liquid sample is nebulized in the heated tube into finely dispersed droplets. Where, a corona discharge needle ionize first the solvent molecules (most abundant) which subsequently collision ionize the analyte molecules with minimum thermal decomposition (least fragmentation) at API. (like Chemical ionization)
• This interface can be used for GC or LC
• This ion source combines the two most common ionization techniques : ESI probe and APCI probe in a single source housing
• Increase the applicability of Triple TOF™ 5600 system for a wider range of analytes.
Z. Rhoades, Broader Coverage and Automatic Mass Calibration Using the TripleTOF™ 5600 System with DuoSpray™ Ion Source, AB SCIEX, Foster City, CA, USA and Concord, Ontario, Canada
The DuoSpray™ Ion Source
Comparing the Extracted ion chromatogram and mass spectra of dieldrin in TOF MS EI source Vs TOF MS APCI source.
T. Portol, Potential of atmosphericpressure chemical ionization source in GCQTOF MS for pesticide residue analysis, journal of mass spectrometry (2010).
The influence of electrospray ion source design on matrix effects
H. Stahnke, The influence of electrospray ion source design on matrix effects, J. Mass. Spectrom. 2012, 47, 875–884
• The matrix effect by these ESI designs for 45 pesticides (in different matrices include: grapefruit, orange, pear and sweet pepper) doesn’t largely differ
• The Jet Stream ESI (orthogonal geometry) With heated sheath gas Give a higher sensitivity With a stronger signal suppression than Without using sheath gas
Positive ESI Mass spectrum of Atrazine using ABSCIEX 6500TIC of +Q3: from Sample 92 (Boscalid_0.10_SCan_Q3) of 131114_Boscalid.wiff (Turbo Spray IonDrive) Max. 1.1e10 cps.
0 2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.00
1.00e9
2.00e9
3.00e9
4.00e9
5.00e9
6.00e9
7.00e9
8.00e9
9.00e9
1.00e10
1.10e10
In
te
ns
it
y,
c
ps
20.3722.96
21.37
23.30 24.34 27.8626.30
20.03
19.6218.16
17.4216.3815.510.040.73 14.4313.451.49 2.26 12.392.99 11.47
+Q3: 20.074 to 20.651 min from Sample 92 (Boscalid_0.10_SCan_Q3) of 131114_Boscalid.wiff (Turbo Spray IonDrive), Centroided Max. 7.0e8 cps.
335.0 336.0 337.0 338.0 339.0 340.0 341.0 342.0 343.0 344.0 345.0 346.0 347.0 348.0 349.0 350.0m/z, Da
0.0
5.0e7
1.0e8
1.5e8
2.0e8
2.5e8
3.0e8
3.5e8
4.0e8
4.5e8
5.0e8
In
te
ns
it
y,
c
ps
343.0
345.0
346.9346.1344.0
341.2 348.0339.1335.1 337.1 349.1338.2336.2 340.2
N
O
NH
Cl
Cl Flow rate 30μ l/minCUR: 20.00IS: 5500.00TEM: 400.00GS1: 35.00GS2: 35.00DP 102.00EP 10.00CXP 26.00
342
342+ 1
QCAP Egypt
TIC of -Q3: from Sample 5 (Boscalid_Only_Coverd15M_) of 131121_Boscalid.wiff (Turbo Spray IonDrive) Max. 4.9e7 cps.
0 2 4 6 8 10 12 14 16 18 20 22 24 26Time, min
0.0
5.0e6
1.0e7
1.5e7
2.0e7
2.5e7
3.0e7
3.5e7
4.0e7
4.5e7
4.9e7
In
te
ns
it
y,
c
ps
20.21
1.23 21.16
1.87 21.6523.15
22.242.65 3.87 24.564.39 5.19 6.02 27.4026.906.7512.80 14.46 14.8311.59 17.4911.17 15.768.26 10.04 18.18
-Q3: 20.224 min from Sample 5 (Boscalid_Only_Coverd15M_) of 131121_Boscalid.wiff (Turbo Spray IonDrive), Centroided Max. 1.8e7 cps.
333.0 334.0 335.0 336.0 337.0 338.0 339.0 340.0 341.0 342.0 343.0 344.0 345.0 346.0 347.0 348.0 349.0 350.0m/z, Da
0.0
2.0e6
4.0e6
6.0e6
8.0e6
1.0e7
1.2e7
1.4e7
1.6e7
1.8e7
In
te
ns
it
y,
c
ps
340.9
342.9
341.8 343.8 344.9
346.0
N
O
NH
Cl
Cl Flow rate 30μ l/minCUR: 20.00TEM: 400.00GS1: 35.00GS2: 35.00IS: -4500.00DP -102.00EP -10.00CXP -26.00342
342- 1
Negative ESI Mass spectrum of Atrazine using ABSCIEX 6500
QCAP Egypt
Negative ESI Mass spectrum of Atrazine
https://www.researchgate.net/figure/258856265_fig10_Figure-1-Mass-spectrum-of-atrazine-with-a-chlorine-adduct-(reprinted-with-permission [accessed Oct 15, 2015]
215+35 Cl adduct
215
215-1
SummaryIn an ESI; a liquid is converted to an charged aerosol (containing both positive and negative ions) by passing through a metal capillary which at high voltage (around 4-5.5 kV P ESI, 3- 4.5 kV N ESI)
A Nebulizer Gas reduce the influence of the surface tension of the used solvent (allow high flow rate with fast desolvation)
ESI needle should be at suitable geometry with the orifice plate( for example : orthogonal, a way from the orifice aperture) In order to minimize the system contamination
A Higher heater temperature and gas flow values are required for higher liquid flow of high aqueous composition (Avoid fragmentation)
The huge presence of vapors (evaporated solvent, volatile matrices) and gases (nebulizer, heat gas,..) are separated from the vacuum system by applying a curtain gas (between curtain plate and orifice) should be optimized to a high value
Applying a declustering potential is needed to decluster the aggregation of the charged ions after its passing through the first vacuum region (between orifice and skimmer)
Videos :ESI : https://www.youtube.com/watch?v=r6TGvG7RUyoGC APCI MSMS : https://www.youtube.com/watch?v=lnAwbfiMfUEESI with APCI : https://www.youtube.com/watch?v=9QraBDViT2YLC MSMS 6500 : https://www.youtube.com/watch?v=um_ELG02wpY&list=PLRe-FFrwYkWD216bgzEUE1amMrMEem-mD
Note : The optimizing information that found in this lecture is used only for education and may simplify your understanding to the corresponding parameters, Not to be used for optimizing your system.
This lecture and all lectures in these series are introduced for QCAP Egypt colleagues, we have 5 LC MS MS (Ab Sciex) and 5 GC MS MS (Agilent). So, I have mad a more detailed information about optimizing parameter on AB SCIEX LC MS MS and that for GC MS MS from the available information online .
Thanks for AB SCIEX and Agilent for presenting many manuals, Guides,.. available online
