apci - unicamp
TRANSCRIPT
![Page 1: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/1.jpg)
APCI Atmospheric Pressure Chemical Ionization
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APCI: Mecanismo
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Interface
Probe Movement
Corona Needle
Vaporizer
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Compostos Orgânicos
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Organometálicos
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Intermediários neutros, transientes and lábeis : APCI !!
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APCI
![Page 9: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/9.jpg)
Características
• Injeção em solução
• Dessolvatação térmica
– Compostos termolábeis podem decompor
– Temperatura afeta muito o espectro
• Qualquer solvente pode ser utilizado
![Page 10: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/10.jpg)
Acoplamento com LC
• Introdução em solução permite o
acoplamento com cromatógrafos
• Fluxo compatível (10 – 1000 µl)
• Solventes compatíveis c/ qualquer
cromatografia
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Exemplos
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Determinação de 27
drogas em 25 minutos !!
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APPI
Atmospheric Pressure Photoionization
• Introdução de amostra por fluxo
líquido
• Uso de Uma lâmpada de UV para
PI
• “APCI” sem descarga corona
• Seletividade para vários
compostos (principalmente
Aromáticos / Apolares)
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APPI: Mecanismo de Ionização
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Lâmpadas de UV
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E se hv ≤ IE ?
D = Dopante
S = Solvente
M = Analito
Principais Dopantes: Tolueno e Acetona
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APPI: Mecanismos
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APPI
benzo[a]pireno
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APPI vs APCI
APPI favorece a ionização de compostos apolares e menos básicos
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APPI: Influência do solvente
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Hidrocarbonetos
• Não ionizam por APCI (nem por ESI ..)
• Possuem PA < solventes
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Eletrospray John Fenn – 1989
Prêmio Nobel em Química 2002 !
Yamashita, M.; Fenn, J.B., J. Phys. Chem. 88 (1984) 4451.
Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M.; Fenn, J.B., Anal. Chem. 57 (1985) 675.
Fenn, J.B.; Mann, M.; Meng, C.K.; Wong, S.F.; Whitehouse, C.M., Science 246 (1989) 64.
"Electrospray Ionization for Mass Spectrometry of Large Biomolecules,“
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Whitehouse, C.M.; Dreyer, R.N.; Yamashita, M.; Fenn, J.B., Anal. Chem. 57 (1985) 675.
Cyclosporin A é um peptídeo
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Eletrospray (ESI)
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Solução
ÁCIDO
BASE
(HCOOH)
(NH4OH)
+
contra-íon ?
solvente ?
repulsão
eletrostática !
Fase Gasosa ?
ESI
— ESI
Princípios de ESI
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ESI: Princípio Geral
Uma solução da amostra em pH ácido ou básico (ou
“neutra” de um sal) é submetida a um spray eletrolítico
sob pressão atmosférica. Um fino spray (aerosol) se
forma (cone de taylor) na presença de um alto campo
elétrico de +4000V (ou – 4000V). O contra-íon é
oxidado (ou reduzido) e formam-se gôtas com excesso
de carga positiva (ou negativa). O solvente evapora, e o
volume das gôtas é reduzido, e as gôtas se
subdividem. Eventualmente, devido a alta repulsão
entre os íons de mesma carga, ou se formam gôtas
contendo apenas um íon (modelo CRM) ou íons
evaporam (são “ejetados”) das gôtas para a fase gasosa
(modelo IEM de evaporação de íons).
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Mecanismo de Dessorção
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Nuvem de ESI
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Sonda de ESI
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ESI e NanoESI
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ESI: Moléculas Orgânicas
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ESI: Moléculas Orgânicas
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Complexos Organometálicos
O
O
O
Ru
Ru
Ru
O
O
O
O
O
O
O
O
O
O
N
N
N N
903 904 905 906 907 908 909 910 911 912 913 914 915 916 917 918 919 920 921 922 923m/z0
100
%
913.0
912.0
911.0
910.0
909.0
908.0
907.0
906.0
905.0
904.0
914.0
915.0
916.0
918.0917.0
919.1
620 640 660 680 700 720 740 760 780 800 820 840 860 880 900 920 940m/z0
100
%
913.0
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Polímeros
C9H19 O(CH2CH2O)n H
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ESI Glu-Fibrinopeptídeo (1568.9) GFP
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900
%
0
100
TEST4 7 (0.144) TOF MSMS 785.80ES+ 234785.46
GFP
m/z785 786 787 788
%
0
100
TEST4 7 (0.144) Sm (Mn, 2x3.00) TOF MSMS 785.80ES+ 204785.46
785.97
786.47
786.95
(1568.9 + 2 ) / 2 = 785.46
(1569.9 + 2 ) / 2 = 785.96
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Íons Multicarregados:
Padrão Isotópico
+1 = 1/1 = 1.00
+2 = 1/2 = 0.50
+3 = 1/3 = 0.33
+4 = 1/4 = 0.25
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Íons Multicarregados B-Gal 10 fm/uL
m/z560 570 580 590 600 610 620 630 640 650 660 670 680
%
0
100
hilo08 83 (2.432) 1: TOF MS Survey ES+ 302671.41
567.38
567.63
668.70567.85
617.44
575.82
604.17576.81 587.29
626.92
653.41627.90
671.90
672.42
672.91
B-Gal 10 fm/uL
m/z567 568
%
0
100
hilo08 83 (2.432) 1: TOF MS Survey ES+ 228567.38
567.12567.63
567.85
568.11
568.39
B-Gal 10 fm/uL
m/z668 669 670
%
0
100
hilo08 83 (2.432) 1: TOF MS Survey ES+ 103668.70668.37
668.18667.36
669.04
669.37
669.68
670.10 670.38
B-Gal 10 fm/uL
m/z671 672 673 674
%
0
100
hilo08 83 (2.432) 1: TOF MS Survey ES+ 302671.41
671.90
672.42
672.91
673.40
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ESI de Proteínas
(Macromoléculas)
teste
m/z650 700 750 800 850 900 950 1000 1050 1100 1150 1200
%
0
100
Mioglobina 891 (16.772) Cm (889:907) TOF MS ES+ 1.48e3804.9
768.3735.0
704.4
676.3
650.3
845.1
889.5
938.9
994.1
1056.1
1126.5
1206.9
Mioglobina: (16881 Da) : (16881 + 20) / 20 = 845.1
(16881 + 21) / 21 = 804.9
(16881 + 22) / 22 = 768.3
teste
m/z804 805 806
%
0
100
Mioglobina 891 (16.772) Cm (889:907) TOF MS ES+ 1.48e3804.9
(M + x) / x = m/z1
(M + x +1) / (x +1) = m/z2
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ESI: Complexos
Urease da Helicobacter pylori
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1.05 MDa !
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ESI de Vírus
Intacto
![Page 42: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/42.jpg)
Fatores
de ESI
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Fatores de ESI
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Fatores de ESI
50% CH3OH + 0.1% Ác. Acético
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ESI ESI
![Page 46: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/46.jpg)
ESI 5% ACN 50% ACN 95% ACN
1700V
1900V
2100V
2300V
2500V
3100V 30 µm Tip @ 500 nL/min
0.1% Formic Acid
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Adutos com Cátions
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MALDI
Ionização e Dessorção a LASER
Auxiliada por Matriz
Franz Hillenkamp
Michael Karas
Koichi Tanaka
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MALDI: Princípio
ION FORMATION IN MALDI MASS SPECTROMETRY Renato Zenobi and
Richard Knochenmuss, Mass Spectrometry Reviews, 1998, 17, 337–366
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MALDI: Princípio
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Nuvem de plasma (Plume)
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Mecanismo de Ionização
Energia do Laser UV ~ 4 eV
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Mecanismo de Ionizção
• Ionização Multifotonica
• Interconversão térmica
• Dessorção de íons formados
• Reações íon-molécula
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MALDI: Íons Monocarregados
10000 12000 14000 16000 18000 20000 22000 24000 26000 28000m/z0
100
%
Protmix 6 (0.102) Sm (Mn, 2x5.00); Sb (1,30.00 ); Cm (1:10) TOF LD+ 3.60e312417.97
17016.75
12630.54
24042.51
17224.64
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MALDI: Sensibilidade
J Am Soc Mass Spectrom 2001, 12, 1055–1063
42 Zeptomols (25000 Moléculas) de substância P !!!
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LASERS
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Matrizes
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MALDI: Características
• Forma íons monocarregados
• Sensível
• Rápido para amostras individuais
• Mais Tolerante a contaminantes
• Problemas com massa baixa
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Ions Monocarregados
JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 35, 1–12 (2000)
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Polímeros
PEG 10KDa
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Proteínas
?
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MALDI – Resolução Espacial
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MALDI – Resolução Espacial
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MALDI - Resolução Espacial
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Imageamento
por MALDI
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Técnicas de Ionização
Ambiente
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Técnicas de Ionização
Ambiente • Técnicas de ionização derivadas das
técnicas tradicionais que permitem a
análise de amostras sem preparo ou
separação
• DESI e DART são as duas principais (e as
primeiras) técnicas
• Uma enorme lista de métodos (> 80) já
foram descritas
Monge, María Eugenia; Harris, Glenn A.; Dwivedi, Prabha; Fernández, Facundo M. (2013). "Mass Spectrometry:
Recent Advances in Direct Open Air Surface Sampling/Ionization". Chemical Reviews 113 (4): 2269–2308
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Desorption Electrospray Ionization
(DESI)
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DESI - Fonte
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DESI – Alcalóides de Flores
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DESI – Carotenóides
Tomate
DESI – Carotenóides
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DESI - Peptídeos
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DESI - Proteínas
![Page 75: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/75.jpg)
DESI - Fármacos
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DESI - Explosivos
![Page 77: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/77.jpg)
DESI - Imageamento
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DART
• Direct Analysis in Real Time
• Desenvolvido pela JEOL
• Baseado na Ionização de Penning
• Método de Ionização em condição
ambiente
![Page 80: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/80.jpg)
DART
Robert Cody
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Ionização de Penning
Nitrogênio ou Neônio
Negativo
Hélio
![Page 82: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/82.jpg)
DART
![Page 83: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/83.jpg)
Explosivos
![Page 84: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/84.jpg)
Alimentos
![Page 85: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/85.jpg)
Fármacos & “Fármacos”
![Page 86: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/86.jpg)
Urina
![Page 87: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/87.jpg)
Plantas
![Page 88: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/88.jpg)
Polímeros
![Page 89: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/89.jpg)
Fungicidas
![Page 90: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/90.jpg)
DART
• Análise ambiente
• Comparável à DESI
• Não há relatos de massas altas
![Page 91: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/91.jpg)
Comparação
![Page 92: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/92.jpg)
Comparação
![Page 93: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/93.jpg)
Abrangência da Espectrometria de Massas
ESI APCI
MALDI
EI Polaridade
Ma
ssa
ESI
![Page 94: APCI - Unicamp](https://reader033.vdocuments.site/reader033/viewer/2022042718/626762036edc1843de46a857/html5/thumbnails/94.jpg)