Mass Analyzers: Cromatografia Líquida Acoplada À Espectrometria de Massas (Sequencial)
Mass Analyzers: Cromatografia Líquida Acoplada À Espectrometria de Massas (Sequencial)
Mass Spectrometry
Departamento de Química
Campus da UFSM 97105-900 Santa Maria - RS
Prof. Dr. Renato Zanella
e-mail: rzanella@base.ufsm.br
Técnicas de Ionização e
Analisadores de Massas
Detector/
Liquid Data
Ionization Mass Analyzer
Chromatography Collection
2011 •Very important! •ESI •Triple Quadrapoles
•Many columns •APCI •Ion-Traps
•Many solvent systems
•APPI •Hybrids 2
Mass Analyzers
• Double Focusing Magnetic Sector
• Quadrupole Mass Filter
• Quadrupole Ion Trap
• Linear Time-of-Flight (TOF)
• Reflectron TOF
Em LCMS/MS (MRM) a intensidade do pico do analito é baixa se comparada a uma análise
LCMS realizada no modo SIM (Single Ion Monitoring). No entanto, o LCMS/MS fornece maior • Fourier Transform Ion Cyclotron Resonance
sensibilidade que o LCMS devido à melhora significativa dos níveis de ruído com consequente
aumento da relação sinal/ruído. (FT-ICR-MS)
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Main Components of the LC-MS Triple Quadrupole Configuration
Q0 Q1 Q2 Q3
Spray Chamber
QqQ
Å Å
ÅÅ Å
• Q1 and Q3 are standard mass filter quadrupoles.
Å
10,000
Molecular Weight
APCI
1000 FAB
Particle Thermospray
Beam
GC/MS
Relative Abundance
Relative Abundance is a measure of the relative amount of ion
signal recorded by the detector
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Ionization Techniques Electron Impact GC
Gas-Phase Methods
• Electron Impact (EI) GC
• Chemical Ionization (CI)
Spray Methods
• Electrospray (ESI)
• Atmospheric Pressure Chemical Ionization (APCI)
• Atmospheric Pressure Photo-Ionization (APPI)
• New dual sources (ESI/APCI) or (APCI/APPI)
Desorption Methods
• Matrix-Assisted Laser Desorption Ionization (MALDI)
• Fast Atom Bombardment (FAB) 13 14
Positive mode:
– Best suited to basic drugs that form a stable HCl salt.
• [M+H]+ is the primary ion formed
• [M+nH]n+ and [M+Na+]+ can also be formed.
Negative mode:
Typical ions produced by electrospray ionisation:
– Best suited to acidic drugs that form stable Na salts.
[M+H]+ protonated molecule
Positive mode: [M+Na] +, [M+K] + … adducts • [M-H]-, [M-nH]n- and [M+I-]-
[M+CH3CN+H] + protonated, + solvent adducts
[M-H] - deprotonated molecule
Negative mode: 19 20
[M+HCOO -] -, … adducts
API-Electrospray Ionization Increasing fragmentation with increasing cone voltage.
Atmospheric Pressure Ionization The fragmentation obtained by in source fragmentation is
useful for confirming a peak identity, getting structure
Electrospray Ions
information, improving selectivity, building libraries…
Nebulizer (gas Heated nitrogen drying
shown in red) gas
-4000 V
Solvent spray
+Å
+
Å Å
Å Å Å Å Å Å Å Å Å Å Å Å Å
Dielectric capillary
entrance
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Seleção da fonte:
APCI • Experiência do laboratório e o tipo de instrumento;
(high femtomole) • ESI é mais frequentemente utilizada;
• Facilidade do uso;
Advantages Disadvantages • Robustez;
• Parent Ion • Need Volatile Sample • Seletividade para mais compostos
• Insensitive to Salts • Need Thermal Stability ESI : • técnica preferida e menos propensa a degradação térmica
• Interface to HPLC • trabalha numa faixa mais ampla de compostos
• Can use Normal Phase
Solvents • APCI: • Máxima sensibilidade no modo íon positivo para
compostos neutros;
• Handles High Flow Rates
• Para compostos de íon negativo que requer
captura de elétron para ionização (nitratos)
LC-MS/MS SRM chromatograms of a standard solution (100 μg L-1; 1000 μg L-1 for Naphth,
Fluor)
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Photoionization APPI Source
Drugs
[A-m]+ + m
[A-
Chemical MeOH, AcCN,
weapons chloro-solvents
Fragmentation
S Aromatics
H 2O, CO 2, O 2 , N2
A+
IP Ionization potential
Energy [eV]
IP Benefits of Photoionization
† Ionizes wide range of compounds (e.g.,
non-polars, electronegative cpds, etc.)
† Predominantly parent ion signal
† Minimum fragmentation
† Minimum solvent signal
† Minimum ion suppression
Solvent (S) Analyte (A)
† Signal linear with concentration
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http://www.noble.org/PlantBio/MS/ion_tech_main.html 39 40
Analisadores
Data Acquisition Modes
SIM vs SRM Double-Focusing Magnetic Sector
Three Acquisition Modes: Advantages
• Very High Resolution (60,000)
Ø Scanning • High Accuracy (<5 ppm)
Complete spectra are repeatedly measured between two
• 10,000 Mass Range
extreme masses
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http://www.asms.org
Analisadores Analisadores
Analisador Quadrupolar (Q) How Does a Quadrupole Mass Filter Work?
• Analisador de varredura
• Conjunto de 4 pólos de sinais opostos, que alternam sinais de radiofreqüência entre Filtering High Mass Negative Rods
*
pares, permitindo que apenas um íon atinja o detector de cada vez +
M
M+
M+
*
Filters out all m/z values except the one it is set to pass
43 Obtains a mass spectrum by sweeping across the entire mass range 44
MS/MS with Triple Quadrupoles Analisadores
MS/MS with Triple Quadrupoles Analisadores
The analyser of a “triple quad” instrument consists in two quadrupoles, separated by a collision
cell. Such a configuration is often referred as a "tandem in space" instrument.
Precursor ions and product ions are created and analysed in different physical spaces.
A triple quad instrument can be used in various ways, which are represented on the next figure.
The firsts two experiments are in fact using the triple quad in a single quadrupole mode
Analisadores Analisadores
CID = Dissociação induzida por colisão
• Os íons são acelerados para aumentar a energia cinética e
passar em uma câmara com o gás de colisão.
Usa-se, geralmente: Ar para o Triplo Quadrupolo
He para a Ion Trap
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Analisadores Analisadores
Advantages Disadvantages
• Inexpensive • Low Resolution (<4000)
• Easily Interfaced to • Low Accuracy (>100 ppm)
Many Ionization • MS/MS requires multiple
Methods analyzers
• Low Mass Range (<4000)
• Slow Scanning
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MS/MS
Analisadores
Ion-Trap Analyzer Ion-Trap Analyzer
MS
Advantages Disadvantages
Advantages Disadvantages • High Resolution (>20,000 in some models) • Low Resolution for MS/MS
• Extremely High Mass Range (>1 MDa) • Low Resolution (4000) • High Accuracy (<5ppm)
• Fast Scanning • Low Accuracy (>200 ppm) • 10,000 Mass Range
• MS/MS not possible • Fast Scanning
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Sistema Q-TOF/MS Analisadores
If we look for the molecular mass of the chloridazon pesticide, we can find various values in the spectra
1.221.6379: this is the average mass. It is based on the average atomic masses.
2.221: this is the nominal mass, calculated on the nominal mass of the most abundant isotopes
3.221.0278: this is the exact (or monoisotopic) mass, based on the exact mass of the most abundant natural
isotopes
The value is slightly different from the expected 222.0278 and 219.0278 because these spectra were
obtained with a quadrupole instrument, which does not provide sufficient mass resolution and mass
61 accuracy for obtaining the exact mass. 62
The next smaller peaks correspond to the C13 and Cl37 isotopes.
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Carbamate analysis by LC/MS
Column: Ultra Carbamate, 100 x 4.6 mm, 3 µm Mass Spectra
Conc.: 100 µg/L each
Mobile Phase: A: 90:10 water:methanol + 10 mM ammonium formate
B: 10:90 acetonitrile:methanol + 10 mM ammonium formate
Gradient: 90:10 A:B to 10:90 A:B from 0-15 min.
Flow-rate: 0.25mL/min
Mode: ESI+ Base Peak: most abundant
ion in spectrum
[M + H]+
abundance
Psuedomolecular ion
or Quasi-molecular ion
A+1
A+2
{ from naturally
occurring isotopes
mass/charge
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Development of Methods for the determination of cyanotoxins in surface
and drinking water by using SPE and LC/MS-MS
Figures of Merit for Mass Analyzers
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Yoneyama K et al. Plant Cell Physiol 2010;51:1095-1103
Sistemas de LC-MS
Water soluble vitamins
LC-MS/MS
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