Post on 09-Oct-2020
UHPLCUHPLC--MS Basic PrinciplesMS Basic Principlesand Applicationsand Applications
Jitnapa Voranitikul
December, 2017 Product Specialist LC/MS
UHPLCUHPLC--MS Basic PrinciplesMS Basic Principlesand Applicationsand Applications
Jitnapa Voranitikul
December, 2017 Product Specialist LC/MS
• It measures mass betterthan any other technique
• It can give informationabout chemicalstructures.
What does a Mass Spectrometer do?What does a Mass Spectrometer do?
• It measures mass betterthan any other technique
• It can give informationabout chemicalstructures.
What does a Mass Spectrometer do?What does a Mass Spectrometer do?
Applications of Mass SpectrometryApplications of Mass Spectrometry
• Pharmaceutical analysis– Bioavailability studies– Drug metabolism studies,
pharmacokinetics– Characterization of potential drugs– Drug degradation product analysis– Screening of drug candidates– Identifying drug targets
• Biomoleculecharacterization– Proteins and peptides– Oligonucleotides
• Environmental analysis– Pesticides on foods– Soil and groundwater contamination
• Forensic analysis/clinical
• Pharmaceutical analysis– Bioavailability studies– Drug metabolism studies,
pharmacokinetics– Characterization of potential drugs– Drug degradation product analysis– Screening of drug candidates– Identifying drug targets
• Biomoleculecharacterization– Proteins and peptides– Oligonucleotides
• Environmental analysis– Pesticides on foods– Soil and groundwater contamination
• Forensic analysis/clinical
Applications of Mass SpectrometryApplications of Mass Spectrometry
• Biomoleculecharacterization– Proteins and peptides– Oligonucleotides
• Environmental analysis– Pesticides on foods– Soil and groundwater contamination
• Forensic analysis/clinical
• Biomoleculecharacterization– Proteins and peptides– Oligonucleotides
• Environmental analysis– Pesticides on foods– Soil and groundwater contamination
• Forensic analysis/clinical
Fundamental of LiquidFundamental of LiquidChromatographyChromatography
https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ
Fundamental of LiquidFundamental of LiquidChromatographyChromatography
https://www.thermofisher.com/order/catalog/product/IQLAAAGABHFAPUMZZZ?SID=srch-srp-IQLAAAGABHFAPUMZZZ
IntroductionIntroduction –– HPLCHPLC--System: General DesignSystem: General Design
• Pump with Degasser• Autosampler• Column (installed in a Column Compartment)• Detector• Computer with control software
• Pump with Degasser• Autosampler• Column (installed in a Column Compartment)• Detector• Computer with control software
Autosampler
IntroductionIntroduction –– HPLCHPLC--System: General DesignSystem: General Design
• Pump with Degasser• Autosampler• Column (installed in a Column Compartment)• Detector• Computer with control software
• Pump with Degasser• Autosampler• Column (installed in a Column Compartment)• Detector• Computer with control software
Column
ColumnRelated
Terminologies
ColumnRelated
Terminologies
ColumnRelated
Terminologies
ColumnRelated
Terminologies
Sample The Original Representative Material Which Is To Be Analyzed Also
Called The Sample Matrix (Coffee)
Analyte(s)• A Specific Compound(s) Contained In The Sample Which Is(Are) To Be
Separated And Analyzed (Caffeine)
Compound• Pure Chemical Component In A Sample, Also Called An Analyte Or
Solute
Chromatography TerminologyChromatography Terminology
Sample The Original Representative Material Which Is To Be Analyzed Also
Called The Sample Matrix (Coffee)
Analyte(s)• A Specific Compound(s) Contained In The Sample Which Is(Are) To Be
Separated And Analyzed (Caffeine)
Compound• Pure Chemical Component In A Sample, Also Called An Analyte Or
Solute
Sample The Original Representative Material Which Is To Be Analyzed Also
Called The Sample Matrix (Coffee)
Analyte(s)• A Specific Compound(s) Contained In The Sample Which Is(Are) To Be
Separated And Analyzed (Caffeine)
Compound• Pure Chemical Component In A Sample, Also Called An Analyte Or
Solute
Chromatography TerminologyChromatography Terminology
Sample The Original Representative Material Which Is To Be Analyzed Also
Called The Sample Matrix (Coffee)
Analyte(s)• A Specific Compound(s) Contained In The Sample Which Is(Are) To Be
Separated And Analyzed (Caffeine)
Compound• Pure Chemical Component In A Sample, Also Called An Analyte Or
Solute
Stationary Phase The Chromatographic Packing Material Which Is Held In A Fixed
Position Usually Packed Into A Column, Or Coated Onto A Surface. ItPerforms The Chemical Separation (Also Called The Packing Material, TheChromatographic Material, Or The Adsorbent)
Mobile Phase• Carrier Of The Sample, Moving It Through The Stationary
Chromatographic Packing Material. The Mobile Phase Can Be A Liquid(HPLC), Or A Gas (GC)
Chromatography TerminologyChromatography Terminology
Stationary Phase The Chromatographic Packing Material Which Is Held In A Fixed
Position Usually Packed Into A Column, Or Coated Onto A Surface. ItPerforms The Chemical Separation (Also Called The Packing Material, TheChromatographic Material, Or The Adsorbent)
Mobile Phase• Carrier Of The Sample, Moving It Through The Stationary
Chromatographic Packing Material. The Mobile Phase Can Be A Liquid(HPLC), Or A Gas (GC)
Stationary Phase The Chromatographic Packing Material Which Is Held In A Fixed
Position Usually Packed Into A Column, Or Coated Onto A Surface. ItPerforms The Chemical Separation (Also Called The Packing Material, TheChromatographic Material, Or The Adsorbent)
Mobile Phase• Carrier Of The Sample, Moving It Through The Stationary
Chromatographic Packing Material. The Mobile Phase Can Be A Liquid(HPLC), Or A Gas (GC)
Chromatography TerminologyChromatography Terminology
Stationary Phase The Chromatographic Packing Material Which Is Held In A Fixed
Position Usually Packed Into A Column, Or Coated Onto A Surface. ItPerforms The Chemical Separation (Also Called The Packing Material, TheChromatographic Material, Or The Adsorbent)
Mobile Phase• Carrier Of The Sample, Moving It Through The Stationary
Chromatographic Packing Material. The Mobile Phase Can Be A Liquid(HPLC), Or A Gas (GC)
Remember!
The Stationary Phase and The Mobile Phasewill have OPPOSITE PROPERTIES to Set-Up Competition
For Sample Matrix and Analytes
Remember!
The Stationary Phase and The Mobile Phasewill have OPPOSITE PROPERTIES to Set-Up Competition
For Sample Matrix and Analytes
Remember!
The Stationary Phase and The Mobile Phasewill have OPPOSITE PROPERTIES to Set-Up Competition
For Sample Matrix and Analytes
Remember!
The Stationary Phase and The Mobile Phasewill have OPPOSITE PROPERTIES to Set-Up Competition
For Sample Matrix and Analytes
IntroductionIntroduction –– The ChromatographicThe Chromatographic ProcessProcess
Continous stream of mobile phase
Column Stationary Phase
• The stationary phase retains analytes due to various interactions.• When different chemical components pass through the column at
different rates they become separated in single zones.
Column Stationary Phase
IntroductionIntroduction –– The ChromatographicThe Chromatographic ProcessProcess
Continous stream of mobile phase
Stationary Phase Mobile Phase
• The stationary phase retains analytes due to various interactions.• When different chemical components pass through the column at
different rates they become separated in single zones.
Stationary Phase Mobile Phase
Characteristics of a ChromatogramCharacteristics of a Chromatogram
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50
100
200
300
400
500
600
90,0
100,0
110,0
120,0
130,0
140,0
1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_12 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_PressuremAU bar
min
1
1 -
5,3
63
2 -
6,5
73
3 -
8,8
63
4 -
12
,93
0
2
Flow: 0,50 ml/minMethanol: 70,0 %%C: 0,0 %%D: 0,0 %
Detectorsignal
Peak Height
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50
100
200
300
400
500
600
90,0
100,0
110,0
120,0
130,0
140,0
1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_12 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_PressuremAU bar
min
1
1 -
5,3
63
2 -
6,5
73
3 -
8,8
63
4 -
12
,93
0
2
Flow: 0,50 ml/minMethanol: 70,0 %%C: 0,0 %%D: 0,0 %
Peak area
Characteristics of a ChromatogramCharacteristics of a Chromatogram
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50
100
200
300
400
500
600
90,0
100,0
110,0
120,0
130,0
140,0
1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_12 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_PressuremAU bar
min
1
1 -
5,3
63
2 -
6,5
73
3 -
8,8
63
4 -
12
,93
0
2
Flow: 0,50 ml/minMethanol: 70,0 %%C: 0,0 %%D: 0,0 %
Pressuresignal and
ripple
Retention time
Peak Height
0,0 1,0 2,0 3,0 4,0 5,0 6,0 7,0 8,0 9,0 10,0 11,0 12,0 13,0 14,0 15,0-50
100
200
300
400
500
600
90,0
100,0
110,0
120,0
130,0
140,0
1 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 UV_VIS_12 - Parabene_Summit_071002 #5 Parabene isocratic 70B Summit 5 Pump_PressuremAU bar
min
1
1 -
5,3
63
2 -
6,5
73
3 -
8,8
63
4 -
12
,93
0
2
Flow: 0,50 ml/minMethanol: 70,0 %%C: 0,0 %%D: 0,0 %
Baseline
Pressuresignal and
ripple
Theory of ChromatographyTheory of Chromatography
tR2
Resolution R of two peaks: Goal of every chromatographic method!
Time, s
tR1
Sig
nal
Peak 1
Peak 2
w1 w2
Theory of ChromatographyTheory of Chromatography
• Distance between the peakcenters of two peaks divided bythe average base width of thepeaks.
• From theory R > 1.50 indicatesbaseline separation.
• In real life R ≥ 2 is usually thegoal (requested in regulatedenvironment).
• Much more resolution than 2does not improve separationquality but increases analysistime.
Resolution R of two peaks: Goal of every chromatographic method!• Distance between the peak
centers of two peaks divided bythe average base width of thepeaks.
• From theory R > 1.50 indicatesbaseline separation.
• In real life R ≥ 2 is usually thegoal (requested in regulatedenvironment).
• Much more resolution than 2does not improve separationquality but increases analysistime.
uCu
BAH
Pla
te h
eig
ht
(HE
TP)
VanVan DeemterDeemter PlotPlot
Eddy dispersion A
Longitudinal diffusion B/u
Resistance to mass
transfer C u
Pla
te h
eig
ht
(HE
TP)
Mobile phase velocity (u)
AA
u
BB
VanVan DeemterDeemter PlotPlot
Eddy dispersion A
Longitudinal diffusion B/u
Resistance to mass
transfer C u
Mobile phase velocity (u)
u
BB
CC
A TermA Term –– Eddy DiffusionEddy Diffusion
http://www.chromacademy.com/lms/sco3/Theory_Of_HPLC_Band_Broadening.pdf
Band broadening due to EddyDiffusion (A Term) in columns withlarge and small particleslarge and small particles – effects onchromatographic peak shape(Efficiency (N))
A TermA Term –– Eddy DiffusionEddy Diffusion
Band broadening due to EddyDiffusion (A Term) in columns withlarge and small particleslarge and small particles – effects onchromatographic peak shape(Efficiency (N))
B TermB Term –– Longitudinal DiffusionLongitudinal Diffusion
http://www.chromacademy.com/lms/sco3/Theory_Of_HPLC_Band_Broadening.pdf
Band broadening due to LongitudinalDiffusion (B Term) in columns withlow and high mobile phase linearlow and high mobile phase linearvelocityvelocity – effects on chromatographicpeak shape (Efficiency (N))
B TermB Term –– Longitudinal DiffusionLongitudinal Diffusion
Band broadening due to LongitudinalDiffusion (B Term) in columns withlow and high mobile phase linearlow and high mobile phase linearvelocityvelocity – effects on chromatographicpeak shape (Efficiency (N))
C TermC Term –– Mass TransferMass Transfer
Cs – term, describing thecontributions to peakbroadening in stationary phase
https://chem.libretexts.org/Core/Analytical_Chemistry/Chromedia/01Gas_Chromotography_(GC)/Gas_Chromotography%3A_Basic_Theory/13The_Van_Deemter_equation
C TermC Term –– Mass TransferMass Transfer
Cm – term, describing thecontributions to peakbroadening in mobile phase
Cs – term, describing thecontributions to peakbroadening in stationary phase
https://chem.libretexts.org/Core/Analytical_Chemistry/Chromedia/01Gas_Chromotography_(GC)/Gas_Chromotography%3A_Basic_Theory/13The_Van_Deemter_equation
C TermC Term –– Mass TransferMass Transfer
http://www.chromacademy.com/lms/sco3/Theory_Of_HPLC_Band_Broadening.pdf
C TermC Term –– Mass TransferMass Transfer
Band broadening due to MassTransfer (C Term) in columns withmobile phase linear velocity andmobile phase linear velocity andstationary phase particle sizestationary phase particle size –effects on chromatographic peakshape (Efficiency (N))
Band broadening due to MassTransfer (C Term) in columns withmobile phase linear velocity andmobile phase linear velocity andstationary phase particle sizestationary phase particle size –effects on chromatographic peakshape (Efficiency (N))
Particle Size EvolutionParticle Size Evolution
Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter
10 min.
Late 1960’s40µm pellicular non-porous coated100-500 psi (7-40 bar)5,000 plates/meter
10 min.
Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter
10 min.
Particle Size EvolutionParticle Size Evolution
Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter
Late 1960’s40µm pellicular non-porous coated100-500 psi (7-40 bar)5,000 plates/meter
10 min.
Early 1970’s10µm Irregular micro-porous1000-2500 psi (70-180 bar)40,000 plates/meter
1980’s to present day3.5 - 5µm spherical micro-porous1500-4000 psi (110-280 bar)80,000 - 115,000 plates/meter
Thermo Analytical LC SystemsThermo Analytical LC Systems
UltimateTH 3000Max Pressure 1000 bar
Thermo Analytical LC SystemsThermo Analytical LC Systems
VanquishTH
Max Pressure 1517 bar
System ContributionSystem Contribution –– What Do We Want?What Do We Want?
• Qualitative analysis– Resolved analytes
• Quantitative Analyis– Reproducible peak areas
We want a reliable method working on a reliable system!
• Qualitative analysis– Resolved analytes
• Quantitative Analyis– Reproducible peak areas
We want a reliable method working on a reliable system!
System ContributionSystem Contribution –– What Do We Want?What Do We Want?
• Qualitative analysis– Resolved analytes
• Quantitative Analyis– Reproducible peak areas
We want a reliable method working on a reliable system!
• Qualitative analysis– Resolved analytes
• Quantitative Analyis– Reproducible peak areas
We want a reliable method working on a reliable system!
• Accuracy is the degree of closeness of a measured quantity to its true value relevance for method transfer
• Precision is the degree of further measurements show the same results(reproducibility) deviation of repeated measurements
• The target analogy:
• A valid method or system is accurate and precise!
System ContributionSystem Contribution –– Accuracy and PrecisionAccuracy and Precision
• Accuracy is the degree of closeness of a measured quantity to its true value relevance for method transfer
• Precision is the degree of further measurements show the same results(reproducibility) deviation of repeated measurements
• The target analogy:
• A valid method or system is accurate and precise!
accurate and precise(ideal result)
accurate, but not precise(random errors)
• Accuracy is the degree of closeness of a measured quantity to its true value relevance for method transfer
• Precision is the degree of further measurements show the same results(reproducibility) deviation of repeated measurements
• The target analogy:
• A valid method or system is accurate and precise!
System ContributionSystem Contribution –– Accuracy and PrecisionAccuracy and Precision
• Accuracy is the degree of closeness of a measured quantity to its true value relevance for method transfer
• Precision is the degree of further measurements show the same results(reproducibility) deviation of repeated measurements
• The target analogy:
• A valid method or system is accurate and precise!
accurate, but not precise(random errors)
precise, but not accurate(systematic error)
Neither accurate nor precise(useless)
TheThe UltiMateUltiMate™™ 30003000 LC SystemsLC Systems
Pumps
Autosampler
Binary
StandardBasic Automated
Isocratic
Column Compartments
Detectors
Standard
VWD MWD/DAD
TheThe UltiMateUltiMate™™ 30003000 LC SystemsLC Systems
Quaternary Dual-Gradient
Thermostatted + Fractionation
With ValvesStandard
Fluorescence Corona Coulochem
https://www.thermofisher.com/order/catalog/product/TSQ02-10001?SID=srch-srp-TSQ02-10001
Fundamental of Mass SpectrometryFundamental of Mass Spectrometryhttps://www.thermofisher.com/order/catalog/product/TSQ02-10001?SID=srch-srp-TSQ02-10001
Fundamental of Mass SpectrometryFundamental of Mass Spectrometry
“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”
What is Mass Spectrometry?What is Mass Spectrometry?
“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”
Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry:Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.
“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”
What is Mass Spectrometry?What is Mass Spectrometry?
“The basis in mass spectrometry (MS) is the productionof ions, that are subsequently separated or filteredaccording to their mass-to-charge (m/z) ratio, anddetected. The resulting mass spectrum is a plot of the(relative) abundance of the produced ions as a functionof the m/z ratio.”
Niessen, W. M. A.; Van der Greef, J., Liquid Chromatography–Mass Spectrometry:Principles and Applications, 1992, Marcel Dekker, Inc., New York, p. 29.
Mass spectrometry CharacteristicsMass spectrometry Characteristics
•Operate at very low pressure (10-5 to 10-7 torr)
(Atmosphere = 760 torr)
•Mass spectrometer work with IONSIONS
•Measure gas-phase ions
•Determine the mass are separated according to their mass-to-charge
(m/z) ratio
•Operate at very low pressure (10-5 to 10-7 torr)
(Atmosphere = 760 torr)
•Mass spectrometer work with IONSIONS
•Measure gas-phase ions
•Determine the mass are separated according to their mass-to-charge
(m/z) ratio
Mass spectrometry CharacteristicsMass spectrometry Characteristics
•Operate at very low pressure (10-5 to 10-7 torr)
(Atmosphere = 760 torr)
•Mass spectrometer work with IONSIONS
•Measure gas-phase ions
•Determine the mass are separated according to their mass-to-charge
(m/z) ratio
•Operate at very low pressure (10-5 to 10-7 torr)
(Atmosphere = 760 torr)
•Mass spectrometer work with IONSIONS
•Measure gas-phase ions
•Determine the mass are separated according to their mass-to-charge
(m/z) ratio
Mass Spectrometry “Simplified”Mass Spectrometry “Simplified”
GenerateMoveSelectMoveSelectDetect
The lifetime of an ion from the point of formationto detection is approximately 50 to 100 microseconds
Mass Spectrometry “Simplified”Mass Spectrometry “Simplified”
Ion Production
Ion OpticsIon Optics
Analyser (Quadrupole, Orbitrap)
Electron Multiplier
The lifetime of an ion from the point of formationto detection is approximately 50 to 100 microseconds
Mass SpectrometryMass Spectrometry –– Block DiagramBlock Diagram
LiquidChromatography
Ionization
• ESI• APCI• APPI
Very important!- Many columns- Many solvent systems
Detector/Data
Collection
Mass SpectrometryMass Spectrometry –– Block DiagramBlock Diagram
Ionization Mass AnalyserDetector/
DataCollection
•Triple Quadrapoles•Ion-Traps•Orbitrap
Q1Hyperquad
Ion SourceInterface
TSQ Quantum ComponentsTSQ Quantum Components
Q00 Q0Q1
Hyperquad
Ion SourceInterface
Ion Transfer Tube
Q1Hyperquad
Q3Hyperquad
DetectionSystem
TSQ Quantum ComponentsTSQ Quantum Components
Q1Hyperquad
Q3Hyperquad
Q2Collision Cell
Mass Analyzer
ION SOURCEION SOURCE
IONIZATION TECHNIQUESIONIZATION TECHNIQUES
ION SOURCEION SOURCE
IONIZATION TECHNIQUESIONIZATION TECHNIQUES
Type of Ionization techniquesType of Ionization techniques
•Electron impact (EI)•Chemical Ionization (CI)
•Atmospheric Pressure Ionization (API)•Electrospray Ionization (ESI)•Atmospheric Pressure Chemical Ionization (APCI)•Atmospheric Pressure Photo-Ionization (APPI)
•Matrix Assisted Laser Desorption/Ionization (MALDI)
•Electron impact (EI)•Chemical Ionization (CI)
•Atmospheric Pressure Ionization (API)•Electrospray Ionization (ESI)•Atmospheric Pressure Chemical Ionization (APCI)•Atmospheric Pressure Photo-Ionization (APPI)
•Matrix Assisted Laser Desorption/Ionization (MALDI)
Type of Ionization techniquesType of Ionization techniques
•Electron impact (EI)•Chemical Ionization (CI)
•Atmospheric Pressure Ionization (API)•Electrospray Ionization (ESI)•Atmospheric Pressure Chemical Ionization (APCI)•Atmospheric Pressure Photo-Ionization (APPI)
•Matrix Assisted Laser Desorption/Ionization (MALDI)
•Electron impact (EI)•Chemical Ionization (CI)
•Atmospheric Pressure Ionization (API)•Electrospray Ionization (ESI)•Atmospheric Pressure Chemical Ionization (APCI)•Atmospheric Pressure Photo-Ionization (APPI)
•Matrix Assisted Laser Desorption/Ionization (MALDI)
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
Three Fundamental Processes:
1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation.
Three Fundamental Processes:
1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation.
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
Three Fundamental Processes:
1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation.
Three Fundamental Processes:
1. Production of charged droplets.2. Droplet size reduction, and fission.3. Gas phase ion formation.
ESI Needle
+/- 3-5 kV
Solvent evaporation andIon desolvation
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
Taylor Cone
Ion Transfer TubeSolvent evaporation andIon desolvation
Electrospray Ionization (ESI)Electrospray Ionization (ESI)
ESIESI -- Ion Max SourceIon Max SourceESIESI -- Ion Max SourceIon Max Source
• Gas phase ionization via corona discharge• APCI is a three-step process:
1. High voltage (via corona needle) interacts with both the nitrogen carrier gas and the vaporizedHPLC solvent to produce primary ions:
O2 + e- → O2+. + 2e-
N2 + e- → N2+. + 2e-
2. Through a complex series of reactions primary ions react with solvent molecules formingreagent ions, H3O+ and CH3OH2
+
3. Reagent ions react with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- innegative ion mode:
H3O+ + Analyte → (Analyte + H)+ + H2OOH- + Analyte → (Analyte – H)- + H2O
Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)
• Gas phase ionization via corona discharge• APCI is a three-step process:
1. High voltage (via corona needle) interacts with both the nitrogen carrier gas and the vaporizedHPLC solvent to produce primary ions:
O2 + e- → O2+. + 2e-
N2 + e- → N2+. + 2e-
2. Through a complex series of reactions primary ions react with solvent molecules formingreagent ions, H3O+ and CH3OH2
+
3. Reagent ions react with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- innegative ion mode:
H3O+ + Analyte → (Analyte + H)+ + H2OOH- + Analyte → (Analyte – H)- + H2O
• Gas phase ionization via corona discharge• APCI is a three-step process:
1. High voltage (via corona needle) interacts with both the nitrogen carrier gas and the vaporizedHPLC solvent to produce primary ions:
O2 + e- → O2+. + 2e-
N2 + e- → N2+. + 2e-
2. Through a complex series of reactions primary ions react with solvent molecules formingreagent ions, H3O+ and CH3OH2
+
3. Reagent ions react with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- innegative ion mode:
H3O+ + Analyte → (Analyte + H)+ + H2OOH- + Analyte → (Analyte – H)- + H2O
Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)
• Gas phase ionization via corona discharge• APCI is a three-step process:
1. High voltage (via corona needle) interacts with both the nitrogen carrier gas and the vaporizedHPLC solvent to produce primary ions:
O2 + e- → O2+. + 2e-
N2 + e- → N2+. + 2e-
2. Through a complex series of reactions primary ions react with solvent molecules formingreagent ions, H3O+ and CH3OH2
+
3. Reagent ions react with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- innegative ion mode:
H3O+ + Analyte → (Analyte + H)+ + H2OOH- + Analyte → (Analyte – H)- + H2O
Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)
© 2004 Dr. Paul Gates
University of Bristol
Atmospheric Pressure Chemical Ionization (APCI)Atmospheric Pressure Chemical Ionization (APCI)
Ion Max Source DesignIon Max Source Design -- APCI ProbeAPCI ProbeIon Max Source DesignIon Max Source Design -- APCI ProbeAPCI Probe
• It depends on the exact application.
• Increasing polarity and molecular weight and thermal instabilityfavors electrospray.
– Most drugs of abuse are highly polar and are easilyanalyzed using electrospray.
– High molecular weight proteins also require electrospray
• Lower polarity and molecular weight favors APCI or APPI.• Lower background, but compounds must be more
thermally stable.
Which is Best?Which is Best?
• It depends on the exact application.
• Increasing polarity and molecular weight and thermal instabilityfavors electrospray.
– Most drugs of abuse are highly polar and are easilyanalyzed using electrospray.
– High molecular weight proteins also require electrospray
• Lower polarity and molecular weight favors APCI or APPI.• Lower background, but compounds must be more
thermally stable.
• It depends on the exact application.
• Increasing polarity and molecular weight and thermal instabilityfavors electrospray.
– Most drugs of abuse are highly polar and are easilyanalyzed using electrospray.
– High molecular weight proteins also require electrospray
• Lower polarity and molecular weight favors APCI or APPI.• Lower background, but compounds must be more
thermally stable.
Which is Best?Which is Best?
• It depends on the exact application.
• Increasing polarity and molecular weight and thermal instabilityfavors electrospray.
– Most drugs of abuse are highly polar and are easilyanalyzed using electrospray.
– High molecular weight proteins also require electrospray
• Lower polarity and molecular weight favors APCI or APPI.• Lower background, but compounds must be more
thermally stable.
Mass SpectrometryMass Spectrometry –– Block DiagramBlock Diagram
LiquidChromatography
IonizationDetector/
DataCollection
Mass SpectrometryMass Spectrometry –– Block DiagramBlock Diagram
Ionization Mass AnalyserDetector/
DataCollection
•Triple Quadrapoles•Ion-Traps•Orbitrap
MASSMASS ANALYSERANALYSER
QUADRUPLEQUADRUPLE
MASSMASS ANALYSERANALYSER
QUADRUPLEQUADRUPLE
MASSMASS ANALYSERANALYSER
QUADRUPLEQUADRUPLE
MASSMASS ANALYSERANALYSER
QUADRUPLEQUADRUPLE
• Operate under high vacuum (keeps ions from bumping into gasmolecules)
• Actually measure mass-to-charge ratio of ions (m/z)
• Key specifications are resolution, mass measurementaccuracy, and sensitivity.
• Several kinds exist: for ion traps, quadrupole, time-of-flight andorbitrap are most used.
Mass analyzers separateMass analyzers separate ionsions
• Operate under high vacuum (keeps ions from bumping into gasmolecules)
• Actually measure mass-to-charge ratio of ions (m/z)
• Key specifications are resolution, mass measurementaccuracy, and sensitivity.
• Several kinds exist: for ion traps, quadrupole, time-of-flight andorbitrap are most used.
• Operate under high vacuum (keeps ions from bumping into gasmolecules)
• Actually measure mass-to-charge ratio of ions (m/z)
• Key specifications are resolution, mass measurementaccuracy, and sensitivity.
• Several kinds exist: for ion traps, quadrupole, time-of-flight andorbitrap are most used.
Mass analyzers separateMass analyzers separate ionsions
• Operate under high vacuum (keeps ions from bumping into gasmolecules)
• Actually measure mass-to-charge ratio of ions (m/z)
• Key specifications are resolution, mass measurementaccuracy, and sensitivity.
• Several kinds exist: for ion traps, quadrupole, time-of-flight andorbitrap are most used.
TSQTSQ Quantiva MSQuantiva MS——Powered by AIM TechnologyPowered by AIM Technology
Systematic optimization of all electric fields,in concert, to produce breakthroughperformance.
Active Ion Management (AIM)
TSQTSQ Quantiva MSQuantiva MS——Powered by AIM TechnologyPowered by AIM Technology
Systematic optimization of all electric fields,in concert, to produce breakthroughperformance.
Active Ion Management (AIM)
TSQTSQ QuantivaQuantiva Animation (available on YouTube)Animation (available on YouTube)
http://www.youtube.com/watch?v=LFB14D8pkoc
TSQTSQ QuantivaQuantiva Animation (available on YouTube)Animation (available on YouTube)
http://www.youtube.com/watch?v=LFB14D8pkoc
Scan Modes
Scan Mode Q1 Q2
Full Scan Scanning Pass All
SIM Fixed m/z Pass All
Product Fixed m/z Pass All (+ CE)
SRM Fixed m/z Pass All (+ CE)
Neutral Loss Scanning Pass All (+ CE)
Precursor Scanning Pass All (+ CE)
Scan Modes
Q2 Q3 Purpose
Pass All Pass All MW Info.
Pass All Pass All Quantitation
Pass All (+ CE) Scanning Structural Info.
Pass All (+ CE) Fixed m/z TargetedQuantitation
Pass All (+ CE) Scanning Analyte Screening
Pass All (+ CE) Fixed m/z Analyte Screening
R T : 0 . 0 0 - 2 0 . 0 2
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0T i m e ( m i n )
0
51 0
1 5
2 0
2 5
3 0
3 5
4 04 5
5 0
5 5
6 0
6 5
7 0
7 58 0
8 5
9 0
9 5
1 0 0
Relative
Abundan
ce
1 . 2 4
4 . 6 0
2 . 6 2
3 . 7 9
3 . 1 05 . 3 8
7 . 7 87 . 5 8
1 8 . 3 41 6 . 9 3 1 8 . 9 41 5 . 9 58 . 2 3 1 5 . 0 91 4 . 0 58 . 9 0 1 2 . 7 39 . 8 1
N L :7 . 3 5 E 7T I C M SH S - h e l i n -1 0 2 4 - 1
One Click
Chromatogram
Full Scan ModeFull Scan Mode
One Click
Spectrum
H O
H O
O H
6060 8080 100100 120120 140140 16016000
100100
%%
R T : 0 . 0 0 - 2 0 . 0 2
0 2 4 6 8 1 0 1 2 1 4 1 6 1 8 2 0T i m e ( m i n )
0
51 0
1 5
2 0
2 5
3 0
3 5
4 04 5
5 0
5 5
6 0
6 5
7 0
7 58 0
8 5
9 0
9 5
1 0 0
Relative
Abundan
ce
1 . 2 4
4 . 6 0
2 . 6 2
3 . 7 9
3 . 1 05 . 3 8
7 . 7 87 . 5 8
1 8 . 3 41 6 . 9 3 1 8 . 9 41 5 . 9 58 . 2 3 1 5 . 0 91 4 . 0 58 . 9 0 1 2 . 7 39 . 8 1
N L :7 . 3 5 E 7T I C M SH S - h e l i n -1 0 2 4 - 1
Chromatogram
Full Scan ModeFull Scan Mode
H +Base peak
at m/z 240 (MH+)N H tB uH
180180 200200 220220 240240 260260 280280 300300
240
241
Full Scan ModePurpose: Survey scan of a chromatographic peak
Q1 Scanning RF Only Q3 RF Only
Full scan Q1:
Full Scan (QFull Scan (Q11 or Qor Q33))
Q1 RF Only RF Only Q3 Scanning
Full scan Q1:
Full Scan Q3:
Full Scan ModePurpose: Survey scan of a chromatographic peak
Q1 Scanning RF Only Q3 RF Only
Full Scan (QFull Scan (Q11 or Qor Q33))
Q1 RF Only RF Only Q3 Scanning
SIM ModePurpose: Quantitation on a specific m/z range of ions
Selected Ion MonitoringSelected Ion Monitoring –– SIMSIM
Q1 Set RF Only + CE Q3 RF Only
SIM Q1:
Q1 RF Only RF Only + CE Q3 Set
SIM Q1:
SIM Q3:
SIM ModePurpose: Quantitation on a specific m/z range of ions
Selected Ion MonitoringSelected Ion Monitoring –– SIMSIM
Q1 Set RF Only + CE Q3 RF Only
Q1 RF Only RF Only + CE Q3 Set
SIM is in essence a full scan acquisition on a relatively narrow masswindow (defined as center mass / scan width)
Fixed m/z Pass All
Selected Ion MonitoringSelected Ion Monitoring –– SIMSIM
Advantages Targeted analyte monitoring High duty cycle
Disadvantages Can suffer from interferences Not as sensitive or selective as SRM
SIM is in essence a full scan acquisition on a relatively narrow masswindow (defined as center mass / scan width)
Pass All Pass All
Selected Ion MonitoringSelected Ion Monitoring –– SIMSIM
Disadvantages Can suffer from interferences Not as sensitive or selective as SRM
RT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
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0
10
20
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40
50
60
70
80
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Rel
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52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + cNSI Full ms [400.00-1800.00]MS data14
NL: 7.97E7Base Peak m/z=1030.90-1031.90 F:+ c NSI Full ms [400.00-1800.00]MS data14
Full Scan
Full Scan versus SIMFull Scan versus SIMRT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
0
10
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40
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100
Rel
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0
10
20
30
40
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Rel
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e A
bund
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52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + cNSI Full ms [400.00-1800.00]MS data14
NL: 7.97E7Base Peak m/z=1030.90-1031.90 F:+ c NSI Full ms [400.00-1800.00]MS data14
SIM
RT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
0
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50
60
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100
Rel
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0
10
20
30
40
50
60
70
80
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100
Rel
ativ
e A
bund
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52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + cNSI Full ms [400.00-1800.00]MS data14
NL: 7.97E7Base Peak m/z=1030.90-1031.90 F:+ c NSI Full ms [400.00-1800.00]MS data14
Full Scan versus SIMFull Scan versus SIMRT: 0.00 - 75.04 SM: 7G
0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75Time (min)
0
10
20
30
40
50
60
70
80
90
100
Rel
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0
10
20
30
40
50
60
70
80
90
100
Rel
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e A
bund
ance
52.33
47.88
31.3055.14
34.47 50.24
39.421.00 18.87 23.568.09 24.156.50 17.2211.51 65.28 70.26 72.6363.6542.17 44.24 56.03
31.30
39.8538.39 47.883.23 30.99 40.53 59.413.45 64.64 67.2452.44 73.5755.5327.2610.36 21.9019.6614.03
NL: 2.91E8Base Peak F: + cNSI Full ms [400.00-1800.00]MS data14
NL: 7.97E7Base Peak m/z=1030.90-1031.90 F:+ c NSI Full ms [400.00-1800.00]MS data14
Pass AllFixed m/z
Q1 Q2
Selected Reaction Monitoring (SRM)Selected Reaction Monitoring (SRM)
Advantages Targeted analyte monitoring High duty cycle “Simultaneous” monitoring of
multiple transitions
Fixed m/zPass All
Q2 Q3
Selected Reaction Monitoring (SRM)Selected Reaction Monitoring (SRM)
Advantages Targeted analyte monitoring High duty cycle “Simultaneous” monitoring of
multiple transitions
Disadvantages No structural information
The Need for True MS/MSThe Need for True MS/MSThe Need for True MS/MSThe Need for True MS/MS
RT: 2.28 - 5 .89 S M: 15G
2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)
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RT: 5 .37S N: 1093
RT: 5 .37S N: 27528
RT: 5 .37S N: 201353020
NL: 8 .14E7m/z= 191 .50-192 .50MS G enesis F u ll-MS2
NL: 1 .38E8m/z= 191 .50-192 .50 F :+ c EI Q 1MS[191 .945-191 .995 ] MSG enesisSIM-1_111128173605
NL: 3 .27E7T IC F : + c EI SRM ms2191 .950[126 .935-126 .985 ] MSG enesisF u ll-SRM-Survey-1
x300
Full MS Scan
Noise Level
SRM Selectivity in Complex MatricesSRM Selectivity in Complex Matrices
RT: 2.28 - 5 .89 S M: 15G
2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)
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RT: 5 .37S N: 1093
RT: 5 .37S N: 27528
RT: 5 .37S N: 201353020
NL: 8 .14E7m/z= 191 .50-192 .50MS G enesis F u ll-MS2
NL: 1 .38E8m/z= 191 .50-192 .50 F :+ c EI Q 1MS[191 .945-191 .995 ] MSG enesisSIM-1_111128173605
NL: 3 .27E7T IC F : + c EI SRM ms2191 .950[126 .935-126 .985 ] MSG enesisF u ll-SRM-Survey-1
x300
SIM Scan
SRM Scan
Noise Level
RT: 2.28 - 5 .89 S M: 15G
2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)
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RT: 5 .37S N: 1093
RT: 5 .37S N: 27528
RT: 5 .37S N: 201353020
NL: 8 .14E7m/z= 191 .50-192 .50MS G enesis F u ll-MS2
NL: 1 .38E8m/z= 191 .50-192 .50 F :+ c EI Q 1MS[191 .945-191 .995 ] MSG enesisSIM-1_111128173605
NL: 3 .27E7T IC F : + c EI SRM ms2191 .950[126 .935-126 .985 ] MSG enesisF u ll-SRM-Survey-1
x300
Noise Level
SRM Selectivity in Complex MatricesSRM Selectivity in Complex Matrices
RT: 2.28 - 5 .89 S M: 15G
2.5 3.0 3.5 4.0 4.5 5.0 5.5Time (m in)
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RT: 5 .37S N: 1093
RT: 5 .37S N: 27528
RT: 5 .37S N: 201353020
NL: 8 .14E7m/z= 191 .50-192 .50MS G enesis F u ll-MS2
NL: 1 .38E8m/z= 191 .50-192 .50 F :+ c EI Q 1MS[191 .945-191 .995 ] MSG enesisSIM-1_111128173605
NL: 3 .27E7T IC F : + c EI SRM ms2191 .950[126 .935-126 .985 ] MSG enesisF u ll-SRM-Survey-1
x300
Noise Level
RT: 17.13 - 27.39
18 19 20 21 22 23 24 25 26 27Time (min)
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RT: 23.76
24.01
25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 6.37E4m/z= 236.50-237.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 2.67E4m/z= 271.50-272.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EISRM ms2 237.000[207.999-208.001] MSGenesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EISRM ms2 272.000[236.999-237.001] MSGenesis Probe20F
SIM
Comparison of SIM and SRMComparison of SIM and SRMRT: 17.13 - 27.39
18 19 20 21 22 23 24 25 26 27Time (min)
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RT: 23.76
24.01
25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 6.37E4m/z= 236.50-237.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 2.67E4m/z= 271.50-272.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EISRM ms2 237.000[207.999-208.001] MSGenesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EISRM ms2 272.000[236.999-237.001] MSGenesis Probe20F
SRM
RT: 17.13 - 27.39
18 19 20 21 22 23 24 25 26 27Time (min)
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RT: 23.76
24.01
25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 6.37E4m/z= 236.50-237.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 2.67E4m/z= 271.50-272.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EISRM ms2 237.000[207.999-208.001] MSGenesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EISRM ms2 272.000[236.999-237.001] MSGenesis Probe20F
Comparison of SIM and SRMComparison of SIM and SRMRT: 17.13 - 27.39
18 19 20 21 22 23 24 25 26 27Time (min)
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RT: 23.76
24.01
25.5223.55 24.11 24.55 25.3523.2822.9722.52 25.6522.17 25.87 26.45 26.7821.6721.3021.0920.4020.27
RT: 23.76
25.37
23.5327.3522.93
26.6220.19 23.43 24.55 25.4824.6721.25 26.2622.41 24.0220.57 22.1719.61
RT: 23.76
24.5923.13 24.17 25.18 25.3422.93 27.0926.31 26.4623.37
21.89 22.5221.6721.1520.5719.65 20.15
RT: 23.77
RT: 23.77
NL: 6.37E4m/z= 236.50-237.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 2.67E4m/z= 271.50-272.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 9.94E3m/z= 306.50-307.50 F: + c SIMms [236.50-237.50,271.50-272.50, 306.50-307.50] MS probe20f_sim
NL: 6.10E5m/z= 207.50-208.50 F: + c EISRM ms2 237.000[207.999-208.001] MSGenesis Probe20F
NL: 1.06E6m/z= 236.50-237.50 F: + c EISRM ms2 272.000[236.999-237.001] MSGenesis Probe20F
Superior Selectivity
Free from sample matrix
Applications of TripleApplications of TripleQuadrupole LCQuadrupole LC--MS/MSMS/MS
Applications of TripleApplications of TripleQuadrupole LCQuadrupole LC--MS/MSMS/MS
Applications of TripleApplications of TripleQuadrupole LCQuadrupole LC--MS/MSMS/MS
Applications of TripleApplications of TripleQuadrupole LCQuadrupole LC--MS/MSMS/MS
Why HighWhy High--throughput LCthroughput LC--MS/MS for Drugs of Abuse Analyses?MS/MS for Drugs of Abuse Analyses?Why HighWhy High--throughput LCthroughput LC--MS/MS for Drugs of Abuse Analyses?MS/MS for Drugs of Abuse Analyses?
Thermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLCThermo Scientific Vanquish Horizon UHPLC
Experimental DesignExperimental Design –– Liquid ChromatographyLiquid ChromatographyExperimental DesignExperimental Design –– Liquid ChromatographyLiquid Chromatography
Experimental DesignExperimental Design –– Mass SpectrometryMass SpectrometryExperimental DesignExperimental Design –– Mass SpectrometryMass Spectrometry
ApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMSApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMS
ApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMSApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMS
ApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMSApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMS
ApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMSApproximateApproximate 100100 Drugs of Abuse on Thermo Scientific TSQDrugs of Abuse on Thermo Scientific TSQ QuantisQuantis MSMS
HIGH RESOLUTION MASSHIGH RESOLUTION MASS ANALYSERANALYSER
ORBITRAPORBITRAP
HIGH RESOLUTION MASSHIGH RESOLUTION MASS ANALYSERANALYSER
ORBITRAPORBITRAP
HIGH RESOLUTION MASSHIGH RESOLUTION MASS ANALYSERANALYSER
ORBITRAPORBITRAP
HIGH RESOLUTION MASSHIGH RESOLUTION MASS ANALYSERANALYSER
ORBITRAPORBITRAP
Mass Analyzers ParametersMass Analyzers Parameters
•Nominal Mass
The mass of an ion with a given empirical formula calculated using theinteger mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2
+
•Exact Mass
The mass of an ion with a given empirical formula calculated using theexact mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479
•Nominal Mass
The mass of an ion with a given empirical formula calculated using theinteger mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2
+
•Exact Mass
The mass of an ion with a given empirical formula calculated using theexact mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479
Mass Analyzers ParametersMass Analyzers Parameters
•Nominal Mass
The mass of an ion with a given empirical formula calculated using theinteger mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2
+
•Exact Mass
The mass of an ion with a given empirical formula calculated using theexact mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479
•Nominal Mass
The mass of an ion with a given empirical formula calculated using theinteger mass numbers of the most abundant isotope of each element
Ex : M=249 C20H9+ or C19H7N+ or C13H19N3O2
+
•Exact Mass
The mass of an ion with a given empirical formula calculated using theexact mass of the most abundant isotope of each element
Ex : M=249 C20H9+ 249.0070C19H7N+ 249.0580C13H19N3O2+ 249.1479
MASS RESOLUTIONMASS RESOLUTIONMASS RESOLUTIONMASS RESOLUTION
• Ability of a mass spectrometer to distinguish between ions ofnearly equal m/z ratios (isobars).
Mass Resolution: What is it?Mass Resolution: What is it?
• m - measured mass• Δm - peak width measured at
50% peak intensity (Full WidthHalf Maximum)
- or the mass differencebetween two adjacent peaksof equal intensity, in this casepw @ 10% valley definition isused.
• m - measured mass• Δm - peak width measured at
50% peak intensity (Full WidthHalf Maximum)
- or the mass differencebetween two adjacent peaksof equal intensity, in this casepw @ 10% valley definition isused.
• Ability of a mass spectrometer to distinguish between ions ofnearly equal m/z ratios (isobars).
Mass Resolution: What is it?Mass Resolution: What is it?
• m - measured mass• Δm - peak width measured at
50% peak intensity (Full WidthHalf Maximum)
- or the mass differencebetween two adjacent peaksof equal intensity, in this casepw @ 10% valley definition isused.
• m - measured mass• Δm - peak width measured at
50% peak intensity (Full WidthHalf Maximum)
- or the mass differencebetween two adjacent peaksof equal intensity, in this casepw @ 10% valley definition isused.
Resolution & Peak WidthResolution & Peak WidthResolution & Peak WidthResolution & Peak Width
• At minimum the resolution of the mass analyzer should besufficient to separate two ions differing by one mass unitanywhere in the mass range scanned (unit mass resolution).
• Typical values of resolution for low resolution mass analyzers(e.g. quadrupoles and ion traps) are below 5000.
• High resolution instruments have a resolution exceeding 15000.
Mass ResolutionMass Resolution: What is it?: What is it?
• At minimum the resolution of the mass analyzer should besufficient to separate two ions differing by one mass unitanywhere in the mass range scanned (unit mass resolution).
• Typical values of resolution for low resolution mass analyzers(e.g. quadrupoles and ion traps) are below 5000.
• High resolution instruments have a resolution exceeding 15000.
• At minimum the resolution of the mass analyzer should besufficient to separate two ions differing by one mass unitanywhere in the mass range scanned (unit mass resolution).
• Typical values of resolution for low resolution mass analyzers(e.g. quadrupoles and ion traps) are below 5000.
• High resolution instruments have a resolution exceeding 15000.
Mass ResolutionMass Resolution: What is it?: What is it?
• At minimum the resolution of the mass analyzer should besufficient to separate two ions differing by one mass unitanywhere in the mass range scanned (unit mass resolution).
• Typical values of resolution for low resolution mass analyzers(e.g. quadrupoles and ion traps) are below 5000.
• High resolution instruments have a resolution exceeding 15000.
Mass ResolutionMass Resolution: What is it?: What is it?
Mass spectrometerFT-ICR-MS
OrbitrapHR-ToFHR-ToF
Magnetic SectorsQuadrupole / IonTrap in UltraZoom mode
Quadrupole / IonTrap
Mass ResolutionMass Resolution: What is it?: What is it?
Resolving Power (FWHM)1,000,000500,00060,00060,00010,000
Quadrupole / IonTrap in UltraZoom mode 5,0001,000
Commercial High Resolution MS Technology RaceCommercial High Resolution MS Technology RaceM
ass re
solu
tion (
FW
HM
)
300000
350000
400000
450000
500000
Orbitrap Tof / QTof
ORBITRAP’s spectacular climbin performance in a decade!
Time progression (year)
Mass re
solu
tion (
FW
HM
)
Bendix Tof0
50000
100000
150000
200000
250000
1955 1965 1975 1985
Commercial High Resolution MS Technology RaceCommercial High Resolution MS Technology Race
Tribrid Orbitrap
ORBITRAP’s spectacular climbin performance in a decade!
Q-Orbitrap*
New Tribrid Orbitrap
LIT-Orbitrap ETD
Time progression (year)
1995 2005 2015
Ion Trap-Orbitrap
Quad Orbitrap
First Q-Tof
New Q-Orbitrap
Entry Q-Orbitrap
MASS ACCURACYMASS ACCURACYMASS ACCURACYMASS ACCURACY
• Mass accuracy is the precision of which the mass is measured bythe mass spectrometer.
• Typical way of reporting mass error in ppm (relative mass error):
• Absolute mass error can be used (mDa).
• Main advantage: the possibility to determine the elementalcomposition of individual molecular or fragment ions, a powerfultool for the structural elucidation or confirmation.
Mass Accuracy: What is it?Mass Accuracy: What is it?
• Mass accuracy is the precision of which the mass is measured bythe mass spectrometer.
• Typical way of reporting mass error in ppm (relative mass error):
• Absolute mass error can be used (mDa).
• Main advantage: the possibility to determine the elementalcomposition of individual molecular or fragment ions, a powerfultool for the structural elucidation or confirmation.
• Mass accuracy is the precision of which the mass is measured bythe mass spectrometer.
• Typical way of reporting mass error in ppm (relative mass error):
• Absolute mass error can be used (mDa).
• Main advantage: the possibility to determine the elementalcomposition of individual molecular or fragment ions, a powerfultool for the structural elucidation or confirmation.
Mass Accuracy: What is it?Mass Accuracy: What is it?
• Mass accuracy is the precision of which the mass is measured bythe mass spectrometer.
• Typical way of reporting mass error in ppm (relative mass error):
• Absolute mass error can be used (mDa).
• Main advantage: the possibility to determine the elementalcomposition of individual molecular or fragment ions, a powerfultool for the structural elucidation or confirmation.
• Accurate mass measurements take advantage of the fact that the combination ofelements contained in a molecule have a very specific, non-nominal molecularweight:
– Carbon has a mass of 12.0000– Hydrogen has a mass of 1.0078– Oxygen has a mass of 15.9949– Nitrogen has a mass of 14.0031
Mass accuracy depends on resolutionHigher resolution allows for better mass accuracy
Mass AccuracyMass Accuracy• Accurate mass measurements take advantage of the fact that the combination of
elements contained in a molecule have a very specific, non-nominal molecularweight:
– Carbon has a mass of 12.0000– Hydrogen has a mass of 1.0078– Oxygen has a mass of 15.9949– Nitrogen has a mass of 14.0031
Mass accuracy depends on resolutionHigher resolution allows for better mass accuracy
• Accurate mass measurements take advantage of the fact that the combination ofelements contained in a molecule have a very specific, non-nominal molecularweight:
– Carbon has a mass of 12.0000– Hydrogen has a mass of 1.0078– Oxygen has a mass of 15.9949– Nitrogen has a mass of 14.0031
Mass accuracy depends on resolutionHigher resolution allows for better mass accuracy
Mass AccuracyMass Accuracy• Accurate mass measurements take advantage of the fact that the combination of
elements contained in a molecule have a very specific, non-nominal molecularweight:
– Carbon has a mass of 12.0000– Hydrogen has a mass of 1.0078– Oxygen has a mass of 15.9949– Nitrogen has a mass of 14.0031
Mass accuracy depends on resolutionHigher resolution allows for better mass accuracy
• Typical mass accuracy capability for various MS types:
Mass AccuracyMass Accuracy
• Typical mass accuracy capability for various MS types:
Mass AccuracyMass Accuracy
Source: Metabolomics Fiehn’s lab
Major accurateMajor accurate--massmass analyzersanalyzers for Life Sciencefor Life ScienceMajor accurateMajor accurate--massmass analyzersanalyzers for Life Sciencefor Life Science
OrbitrapOrbitrap Mass Analyzer:Mass Analyzer: Principle of OperationPrinciple of Operation
qm
kz /
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
OrbitrapOrbitrap Mass Analyzer:Mass Analyzer: Principle of OperationPrinciple of Operation
)/ln(2/2
),( 222mm RrRrz
kzrU
z
φHyper-logarithmic potential distribution:
“ideal Kingdon trap”
r
12
2
R
Rmz 2
2
R
Rmzr
Makarov A. Anal. Chem. 2000, 72, 1156-1162.
Characteristic frequencies:• Frequency of rotation ωφ
• Frequency of radial oscillations ωr• Frequency of axial oscillations ωz
Many Ions Generate a Complex “Transient”Many Ions Generate a Complex “Transient”
FourierTransform
Time Domain
FourierTransform
Mass Spectrum
Many Ions Generate a Complex “Transient”Many Ions Generate a Complex “Transient”
FourierTransform
Frequency Domain
FourierTransform
Mass Spectrum
• Accurate mass measurement is the experimentally determined massmeasured to an appropriate degree of accuracy and precision ( Gross, J.Am. Soc. Mass Spectrom.,1994 )
• Accurate mass measurements narrow down the list of possibleformulae for a particular molecular weight
• Mass spectrum and analyst complete the picture:– Isotope distributions indicate/eliminate elements ( e.g. Cl, Br, Cu )
– User-supplied info eliminates others ( e.g. no F, Co )
– Suggested formula has to make chemical sense: (C6H28O2 is not reasonable noris Cl3H2Co4 )
Mass Accuracy: I.D. of unknown compoundsMass Accuracy: I.D. of unknown compounds
• Accurate mass measurement is the experimentally determined massmeasured to an appropriate degree of accuracy and precision ( Gross, J.Am. Soc. Mass Spectrom.,1994 )
• Accurate mass measurements narrow down the list of possibleformulae for a particular molecular weight
• Mass spectrum and analyst complete the picture:– Isotope distributions indicate/eliminate elements ( e.g. Cl, Br, Cu )
– User-supplied info eliminates others ( e.g. no F, Co )
– Suggested formula has to make chemical sense: (C6H28O2 is not reasonable noris Cl3H2Co4 )
• Accurate mass measurement is the experimentally determined massmeasured to an appropriate degree of accuracy and precision ( Gross, J.Am. Soc. Mass Spectrom.,1994 )
• Accurate mass measurements narrow down the list of possibleformulae for a particular molecular weight
• Mass spectrum and analyst complete the picture:– Isotope distributions indicate/eliminate elements ( e.g. Cl, Br, Cu )
– User-supplied info eliminates others ( e.g. no F, Co )
– Suggested formula has to make chemical sense: (C6H28O2 is not reasonable noris Cl3H2Co4 )
Mass Accuracy: I.D. of unknown compoundsMass Accuracy: I.D. of unknown compounds
• Accurate mass measurement is the experimentally determined massmeasured to an appropriate degree of accuracy and precision ( Gross, J.Am. Soc. Mass Spectrom.,1994 )
• Accurate mass measurements narrow down the list of possibleformulae for a particular molecular weight
• Mass spectrum and analyst complete the picture:– Isotope distributions indicate/eliminate elements ( e.g. Cl, Br, Cu )
– User-supplied info eliminates others ( e.g. no F, Co )
– Suggested formula has to make chemical sense: (C6H28O2 is not reasonable noris Cl3H2Co4 )
• The effect of mass accuracy and molecular weight on thenumber of potential chemical formulae.
Mass accuracy for chemical formulae IDMass accuracy for chemical formulae ID
• The effect of mass accuracy and molecular weight on thenumber of potential chemical formulae.
Mass accuracy for chemical formulae IDMass accuracy for chemical formulae ID
Generate Formula fromGenerate Formula from MonoisotopicMonoisotopic MassMass
Right click on massThen select
Generate formula form mass
0.549 ppm Mass Accuracy
Generate Formula fromGenerate Formula from MonoisotopicMonoisotopic MassMass
10 ppb
100 ppb
1000 ppb
Elemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic MassElemental Composition Generates by Monoisotopic Mass
80
100
120
140
160
No o
f For
mul
ae
Chemical element NoC 50H 100O 10N 10Cl 10
Elemental Composition StatisticsElemental Composition Statistics
0
20
40
60
80
1 1.5 2
No o
f For
mul
ae
Mass Accuracy (ppm)
22 55 77
147147
Elemental Composition StatisticsElemental Composition Statistics
5 10 50Mass Accuracy (ppm)
1616
3030
Compound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern MatchCompound ID confirmation through Isotopic Pattern Match
UHPLC with QUHPLC with Q ExactiveExactive Mass SpectrometerMass Spectrometer
http://planetorbitrap.com/
UHPLC with QUHPLC with Q ExactiveExactive Mass SpectrometerMass Spectrometer
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