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Mass Analyzers: Cromatografia Líquida Acoplada À Espectrometria de Massas (Sequencial)

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0% found this document useful (0 votes)
131 views19 pages

Mass Analyzers: Cromatografia Líquida Acoplada À Espectrometria de Massas (Sequencial)

cvngjhjkhjkhjkh

Uploaded by

Gus Kar
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Universidade Federal de Santa Maria (UFSM)

Mass Spectrometry
Departamento de Química
Campus da UFSM 97105-900 Santa Maria - RS
Prof. Dr. Renato Zanella
e-mail: rzanella@base.ufsm.br

Cromatografia Líquida Acoplada à


Espectrometria de Massas (Sequencial)

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)

3 4
Main Components of the LC-MS Triple Quadrupole Configuration
Q0 Q1 Q2 Q3
Spray Chamber

Ion Optics Mass Filter Detector


RF only Scanning RF only Scanning
RF/DC Collision Cell RF/DC

QqQ
Å Å
ÅÅ Å
• Q1 and Q3 are standard mass filter quadrupoles.
Å

– The can scan masses sequentially (e.g. 50 to 500 amu)


– The can be used to select a single mass.
• Q2 is an RF only quadrupole that is in a gas filled chamber.
– Q2 is the “collision cell” where mass fragmentation occurs.
– Q2 does not filter ions. It accepts all ion sent to it by Q1 and
passes all ions formed by collision to Q3 to be sorted.
5 6

Cromatograma de padrões de 226 pesticidas obtidos em 2 minutos.

Medida simultânea de íons produzidos no modo MRM.


Ao realizar medidas dos íons (MS/MS automático ou íons específicos) no modo MRM,
espectros de massa do íon precursor podem ser obtidos durante uma transição MRM. A
estrutura do composto pode ser simultaneamente confirmada pelo espectro do produto 500 transições MRM em um segundo
resultante do íon devido à alta seletividade durante o MRM e sem a interferência de coeluição
com outras substâncias.
7 8
Q0 Q1 Q2 Q3

RF only Scanning RF only Scanning


RF/DC Collision RF/DC 100,000
Cell
API-Electrospray

10,000

Molecular Weight
APCI
1000 FAB
Particle Thermospray
Beam
GC/MS

MRM = monitoramento de múltiplas reações


(Multiple Reaction Monitoring)
Nonpolar Very polar
MRM é também chamado de SRM (Selected Reaction Monitoring)
9 10

Mass Spectrometry Mass Spectrometry

Terms and Definitions

Molecular ion / precursor ion


Ion formed by ionization of the analyte species

Fragment ions / product ions


Ions formed by the gas-phase dissociation of the molecular ion

Relative Abundance
Relative Abundance is a measure of the relative amount of ion
signal recorded by the detector
11 12
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

Electron Impact Chemical Ionization GC


(low picomole)
Advantages Disadvantages
• Well-Established • Parent Identification
• Fragmentation Libraries • Need Volatile Sample
• No Supression • Need Thermal Stability
• Insoluble Samples • No Interface to LC
• Interface to GC • Low Mass Compounds
• Non-Polar Samples (<1000 amu)
• Solids Probe Requires
Skilled Operator
15 16
Electrospray Ionization (ESI)
Chemical Ionization (Eletronebulização)
(low picomole)
Advantages Disadvantages
• Parent Ion • No Fragment Library
• Interface to GC • Need Volatile Sample
• Insoluble Samples • Need Thermal Stability
• Quantitation Difficult
• Low Mass Compounds
(<1000 amu) (up to 6 kV)

• Solids Probe Requires


Skilled Operator
17 18

Electrospray: Types of Ions Formed


Electrospray
Ionization (ESI) • ESI can operate in either + or - mode.

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

Dielectric capillary
entrance

Evaporation Rayleigh Coulomb Evaporation Analyte Ion


Limit Explosion

21 22

Electrospray Ionization (ESI) ESI


(low femtomole to zeptomole)
ESI = Ionização por eletronebulização à pressão atmosférica Advantages Disadvantages
1º) Bombeamento da amostra através de uma agulha nebulizadora; • Parent Ion • No Fragmentation
• High Mass Compounds • Need Polar Sample
2º) Aplicação de uma voltagem entre a agulha e o cone que a rodeia;
(>100,000 amu) • Need Solubility in Polar
3º) Formação de uma nuvem de gotículas carregadas; • Thermally Labile Solvent (MeOH, ACN, H2O,
Acetone are best)
Compounds (<0º C)
4º) Evaporação do solvente devido à presença de um dispositivo aquecido; • Sensitive to Salts
• Easy to Operate
5º) Aumento da densidade de cargas na superfície das gotículas; • Supression
• Interface to HPLC
6º) Repulsão entre as cargas até os íons evaporarem das gotículas. • Zeptomole sensitivity
with nanospray
23 24
Atmospheric Pressure Atmospheric Pressure Chemical Ionization (APCI)
Chemical Ionization (APCI)
Atmospheric Pressure Ionization APCI = Ionização Química à Pressão Atmosférica

1º) Bombeamento da amostra através de um capilar;

2º) Vaporização do solvente e da amostra, por um


dispositivo aquecido;

3º) Ocorre “descarga elétrica em coroa”;

4º) Ionização do solvente da fase móvel;

5º) Os íons formados reagem com as moléculas do analito.

Solvent molecules (S) being protonated by the corona (SH+), then


25 26
reacting with the analyte molecule (M) to give the protonated form MH+.

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)

APCI : N-metilcarbamatos, compostos neutros


27 28
Principais Diferenças entre APCI e ESI
• Mecanismo de Ionização:
Ionisation Techniques
– Enquanto ESI tem voltagem aplicada à ponta do spray, APCI apresenta um nebulizador
aquecido e pneumaticamente assistido. A voltagem (~3 kV) é aplicada a uma agulha de metal ESI characteristics
na saída do spray. Soft ionization method, provides molecular weight information.
– A descarga Corona ioniza as moléculas do solvente que por sua vez ionizam os analitos por Suitable for analyzing large bio- or synthetic polymers.
transferência de prótons formando [M+H]+ ou [MH]-
Suitable for analyzing polar and even ionic compounds.
• Vazão da FM: Less fragmentation.
– APCI tolera vazões na ordem de 0,2 a 2,0 mL/min, enquanto que ESI opera no máximo até APCI characteristics
1,0 mL/min.
– ESI é melhor indicado para fluxos tão baixos quanto 5 μL/min, compatível com Provides molecular weight information.
acoplamentos capilares (μLC ou CZE). Suitable for analyzing less polar compounds compared to ESI.
Increased fragmentation compared to ESI.
• Fragmentação:
– Devido ao uso de aquecimento, APCI pode produzir alguma fragmentação, enquanto que
ESI pode até formar alguns íons pseudo-moleculares. MALDI characteristics
– APCI não produz cargas múltiplas, portanto não é adequado para compostos de alta MM. Soft ionization method, provides molecular weight information.
Suitable for analyzing very large bio- or synthetic polymers.
• Sensibilidade:
– Sem ser uma regra, APCI tende a render melhor sensibilidade para solutos menos polares. Suitable for analyzing polar and even ionic compounds.
29 Less fragmentation. 30

Atmospheric pressure photoionization (APPI)


ESI e APCI
The photoionization mechanism is simplified under vacuum conditions: photon
absorption by the analyte molecule, leading to electron ejection, forming a
molecular radical cation M.+
Priority pollutant PAHs in surface and drinking water by LC/APPI/MS-MS
Two specific transitions per compound were selected for simultaneous monitoring in SRM mode.

LC-MS/MS SRM chromatograms of a standard solution (100 μg L-1; 1000 μg L-1 for Naphth,
Fluor)

31 32
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

33 34

Direct APPI vs. Dopant-assisted APPI Which is Best ionization source?


Direct APPI
• It depends on the exact application.
M + hv ® M+ + e- Analyte molecule M is ionized to a molecular
radical ion M+. (If analyte ionization potential • Increasing polarity and molecular weight and thermal
is below photon energy)
instability favors ESI.
M+ + S ® MH+ + S[-H] In the presence of protic solvents, M+ may • Most drugs of abuse are highly polar and are easily
abstract a hydrogen atom to form MH+.
analyzed using ESI.
• High molecular weight proteins also require ESI
Dopant APPI
• Lower polarity and molecular weight favors APCI or APPI.
D + hv ® D + e + - A photoionizable dopant is delivered in large
• Lower background, but compounds must be more
concentration to yield many D+ ions.
thermally stable.
D+ + M ® MH+ + D[-H] D+ ionizes analyte M by proton or electron
D+ + M ® M+ + D transfer.

This is PI-initiated APCI


35 36
Fast Atom Bombardment (FAB) FAB
(nanomole)
Advantages Disadvantages
• Parent Ion • No Fragment Library
• High Mass Compounds • Solubility in Matrix (MNBA,
(10,000 amu) Glycerol)
• Quantitation Difficult
• Thermally Labile
Compounds (R.T.) • Needs Highly Skilled
Operator
• Relatively Low Sensitivity

37 38

Matrix-Assisted Laser Desorption Ionization


(MALDI) MALDI
(low femtomole)
Advantages Disadvantages
• Parent Ion • No Fragment Library
• High Mass Compounds
(>100,000 amu)
• Wide variety of matrices
• Thermally Labile • Quantitation Difficult
Compounds (R.T.)
• Easy to Operate

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

Ø Selected-Ion Monitoring Disadvantages


If the analysis aims at detecting target compounds of known • Very Expensive
spectral characteristics with maximum sensitivity • Requires Skilled Operator
• Difficult to Interface to ESI
• Low resolution MS/MS without
Ø Selected-Reaction Monitoring multiple analyzers
Detection of selected reactions, based on the decomposition
reactions of ions that are characteristic of the compounds to be analysed

41 42
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

Filtering Selected Mass Positive Rods

M+

Filtering Low Mass Positive Rods

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

- The first quad (Q1) is set to pass one ion of interest


- The second quad (q2) is set to pass all ions and is flooded with a collision gas to fragment the
parent ion present (collision induced dissociation or CID)
- The third quad (Q3) obtains a daughter ion spectrum by sweeping across the desired mass range
45 46

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

• o uso da fonte CID tem desvantagens importantes, como a dificuldade


prática de funcionamento na voltagem ótima de fragmentação para todos
compostos em um método multirresíduo.

• As desvantagens podem ser resolvidas trabalhando com MS/MS Sequencial


pelo uso do Triplo Quadrupolo ou sistemas Íon Trap porque a voltagem usada
pode ser mais baixa ou semelhante para todos os compostos e o número de
fragmentos produzidos pode ser controlado por um bom ajuste do MS/MS
para especificar a fragmentação e produzir espectros simples.

47 48
Analisadores Analisadores

Quadrupole Mass Analyzer/Filter

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

49 50

MS/MS
Analisadores
Ion-Trap Analyzer Ion-Trap Analyzer

MS

• Acúmulo de íons no interior de um campo quadrupolar 3D;


• Liberação controlada de íons de diferentes massas;
• Permite realizar MSn
51 52
Analisadores Analisadores

Quadrupole Ion Trap Espectrômetros de Massas em Sequência (MS/MS)

Advantages Disadvantages • A informação é aumentada com a combinação de dois MS;


• Inexpensive • Low Resolution (<4000) • A técnica permite a medida de fragmentação de um pico
• Easily Interfaced to • Low Accuracy selecionado em um espectro de massa que produz o espectro de
Many Ionization (>100ppm) massas do íon precursor;
Methods • Space Charging • Podem ser de dois modos:
• MS/MS in one analyzer Causes Mass Shifts
• Consecutivos no espaço usando dois espectrometros separados;
• Low Mass Range
(<4000) • Sucessivos no tempo usando a mesma massa no sistema mais
de uma vez (ion trap)
• Slow Scanning
53 54

Linear Time-of-Flight (TOF) Analisadores


Reflectron Time-of-Flight (TOF)
Analisadores

• íons são produzidos em pulsos e acelerados em alta velocidade


por um campo elétrico num tubo de deslocamento.
• a distância da origem até o detector é fixa,
• o tempo que o íon leva para atravessar o analisador é inversamente proporcional a
sua velocidade

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
55 56
Sistema Q-TOF/MS Analisadores

Combinação de um MS Quadrupolo com um analisador TOF O ESPECTRO DE MASSAS

• Espectro de massas = diagrama da intensidade relativa


QUADRUPOLE MS TOF MS

dos picos em função das razões massa-carga.

(Pico base = pico com a intensidade mais alta,


Z-spray Hexapole Quadrupole Hexapole
íon souce íon bridge collision cell arbitrariamente assinalada em 100. Em LC-MS o pico
MS-MS base geralmente é a molécula protonada e
corresponde ao íon positivo mais estável;
Reflectron

Algumas características do Q-TOF/MS

$Íon molecular = é o pico de maior razão massa-carga e


• Espectros de massas contínuos “full-scan” tornam o MS/MS Q TOF mais sensível (10 - 100 vezes) que os representa o peso molecular do composto.
Quadrupolos em sequência;
• TOF e o MS/MS Q TOF tem a mesma sensibilidade no modo scan e no modo de seleção do íon;
• A maior capacidade do MS/MS Q TOF comparado com instrumentos Triplo Quadrupolo e MS/MS Íon Trap
está na sua habilidade para determinar massas precisas nos fragmentos de íons gerados na cela de
colisão. 57 58

The API Mass Spectrum


Operating conditions
Electrospray (ESI) and APCI are both API (atmospheric pressure ionisation)
techniques and produce a soft ionisation. Thus, the MS spectra obtained with Eluent: the mobile phase must be suitable for ionisation. If it is not the case, a
API ionisation will consist mainly of the "molecular" ions, unless fragmentation small amount of modifier can be added, in order to generate the solvent gas
techniques are applied. phase ions.
The possible fragmentation techniques are in source CID (collision induced Buffers: buffers must be volatile.
dissociation), CID in the collision cell of a tandem type instrument,
fragmentation in an ion trap. This is very different from the spectra obtained
with EI (electron impact ionisation). • Tampões voláteis (ex. Formiato de amônio)
concentração do tampão, ácido ou base usado para ajustar o
pH deveriam ser o mais baixa possível (0,1%);

Substitute phosphates, sulfates, and borates with ammonium


acetate or formate, trifluoroacetic acid (TFA), heptafluorobutric acid
(HFBA), tetrabutylammonium hydroxide (TBAH)
sulfamethazine spectrum Negative ESI mass spectrum for
59 60
glucose, with chloride adducts.
Mass Definition
The mass spectrometer measures the exact mass. Looking at the below mass spectra, the most abundant
peak is at 221.95 (top) and 219.81 (bottom). These spectra are obtained with positive ionisation (top) and
Choosing Between Ionization Techniques negative ionisation (bottom). The peaks correspond to the protonated or deprotonated molecule.

General rules can be given to chose between ESI, APCI, or EI.


ESI is preferred for compounds which are ionic or very polar or thermo labile, or
with masses higher than 1000…
APCI is preferred for compounds which are not very polar.
If we take the example of pesticides, we can rule out that:
polar pesticides will be in ESI, less polar in APCI, volatiles in APCI, non polar
better done with GC/EI

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.

Benzoylurea pesticides ESI - Organophosphorus ESI +

63 64
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
65 66

Comparação entre instrumentos MS/MS Seletividade superior em LC-MS


comparada com LC-UV
Instrumento Sensibilidade Seletividade Exatidão Faixa Características
em full scan dinamica
Triplo quadrupolo baixa alta baixa alta Perda Neutra
MS/MS Ion trap alta alta baixa média MSn
LC/TOF/MS alta média alta média Massa exta e
sensibilidade
LC/QTOF/MS média alta alta média Massa exata dos
fragmentos

Instrumento Características Vantagens Desvantagens


Muito sensível em MS/MS para quase Muito caro
Q-TOF MS/MS Massa exata dos todos íons precursores selecionados Não é possível
fragmentos de íons Full scan do íon precursor perda neutra Será a técnica LC-TOF-MS a
Triplo Quadrupolo
MS/MS
Perda Neutra Sensível para quantificação no modo MRM Não dá a massa
exata
melhor solução?
Quadrupolo Ion Trap MSn Sensível no scan de fragmentos Não é possível
facilmente deduziveis Perda Neutra e
MRM

67 68
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

Type m/z range Resolving Power Cost


Double Focusing 2-5000 20,000 $$$$
Single Focusing 1-1400 1500 $$$
Quadrupole 1-4000 1000 $$
Ion trap 10-4000 1000 $$
Time of flight 1-100,000 30,000
$$$
Fourier Transform 18-10,000 >100,000 $$$$ LC/MS/MS SRM chromatograms of a standard solution of MC-RR, MC-YR, MC-LR, MC-LA and NOD (20 mg/L)
T. Triantis et al., Toxicon, 2010, 55, 979-989

69 70

LC–MS/MS analyses of strigolactones in root exudates

71 72
Yoneyama K et al. Plant Cell Physiol 2010;51:1095-1103
Sistemas de LC-MS
Water soluble vitamins
LC-MS/MS

73 74

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