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Materi LCMS Pak Tri PDF
Materi LCMS Pak Tri PDF
Materi LCMS Pak Tri PDF
Lingkup Materi
• Pengantar HPLC
• Pengantar MS
• Ion Source
• Mass Analyser
• Analisis Kimia dengan LC-TQD MS
• Analisis Kimia dengan LC-HRMS
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Pengantar HPLC
Kromatografi
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Kromatogram
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12
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Detektor
• UV - Ultraviolet
• ED - Electrical Conductivity
• RI - Refractive Index
• FD - Fluorescence
• MS - Mass Spec
• Mass to charge ratio (m/z)
• Allows specific compound ID
• Several types of ionization techniques
• ELSD - Evaporative Light Scattering Detector
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LC-MS LC-MSMS
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Pengantar MS
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What is resolution in MS
• Resolving power
m
R m (FWHM)
m
m 10%
m 500
• Quadrupole MS R 833
m 0.6
m 500
• Orbitrap MS R 100 ,000
m 0 .005
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mmeas mtrue 6
m / z 10
mtrue
500.1 500.0 6
• Quadrupole MS m / z 10 200ppm 100 mDa error
500
23
95
85
T : FT MS {1,1} + p ESI Full ms [100.00 -15 00.00]
5 1
210
80
75
200
190 2 1
70
180
170
m/z 707.21625
65 160
150
C, N, O, H
60
Relative Abundance
140
130
55
120
50 110 707.21625
C 30 H 31 O 11 N 10
100
45
150 38
90
40 80
723.19019
70
C 30 H 29 O 13 N 9
35
60
30 50
40
50 12
25 30
20
15
20
10 649.21008
C 27 H 33 O 13 N 6
695.20386
C 27 H 33 O 15 N 7
785.2348 6
C 30 H 45 O 15 N 10 10 3
0
650 700 750 800
10
5
m/z
128.07596
5 3
127.10697 127.41898 127.54161
0
127.0 127.2 127.4
m /z
127.6 127.8 128.0 2 2
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R = 100,000
R = 20,000
289.1390 100 289.1424
100
289.1022 289.1329 289.1547
90
90 289.0514
80 80
289.1215
70
70 289.1424
289.1021 289.1329
60 289.0514 60
Relative Abundance
Relative Abundance
50 289.1215 50
40 40
30 30
20 20
10 10
0 0
289.00 289.05 289.10 289.15 289.20 289.00 289.05 289.10 289.15 289.20
m/z m/z
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28
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30
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32
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Ion Source
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Hard Soft
• GC-MS • LC-MS
• Electron impact
• Ion molecular
• Fragmentation
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Soft Ionization
• Several common modes differing by method of ion formation:
– Electrospray (ESI)
– Atmospheric Pressure Chemical Ionization (APCI)
– Atmospheric Pressure Photo-Ionization (APPI)
– New dual sources (ESI/APCI) or (APCI/APPI)
- Matrix Assisted Laser Adsorption (MALDI)
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Electrospray (ESI)
Counter electrode
Capillary
FLOW
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Liquid
Flow
Positively
Charged
Taylor Cone Droplets
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40
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• Liquid flow is forced through a narrow capillary to give it a high linear velocity
• The APCI Probe heater combined with nebulizer gas then vaporises the liquid
flow
• The solvent and analyte vapour passes though the corona discharge region to
produce gas phase ions
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Mass analyser
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• Low Resolution
• Quadrupole (Mass Filter, Ion
Trap)
• Quadrupole Tandem
Type Resolving Power
• High Resolution (FWHM)
• Time-of-Flight (Linier TOF, FT-ICR-MS 1,000,000
Reflectron)
FT-Orbitrap 100,000
• Double Focusing Magnetic
High-Res-TOF 60,000
Sector
TOF 10,000
• Orbitrap
Quadrupole / 10,000
• Ultra high resolution IonTrap in
• ICR ion cyclotron resonance UltraZoom mode
Quadrupole / 1,000
Iontrap
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Quadrupole
• Suitable for Quantitation and known compound confirmation Good
linearity
• Structural information
• Single Quad
• Mass confirmation
• Get molecular ion
• NO fragmentation
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MS/MS
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Quadrupole
Advantages Disadvantages
• Inexpensive • Low Resolution (<4000)
• Easily Interfaced to Many • Low Accuracy (>100ppm)
Ionization Methods • MS/MS requires multiple
analyzers
• Low Mass Range (<4000)
• Slow Scanning
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• orbitrap Orbitrap 2
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Aplikasi MS
Qualitative
• Identification of unknown : HRMS or TOF
Quadruple : MS Scan (limited only m/z information)
• Confirmation of targeted compound:
Quadrupole : MS Scan, SIR, MS/MS, MRM
Quantitation
• Quadrupole SIR (LCMS)
• Quadrupole SIR, MRM (LC-MSMS)
• HRMS : (SIM, PRM)
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LC-MS/MS
Confirmed as a
compound if m/z of ion
molecular and 2 m/z of
daughter ion confirmed
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60
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70
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Scan Mode
• Full scan (R=70,000, Max: 140,000)
Metabolomics / Lipidomics / Small molecule screening / Intact protein
Targeted quantitation
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90
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Workflow of Proteomics
Functional Annotation/Pathways/Overrepresentation
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DDA Method
MS1 Top N peaks from MS1 Selected
1st MS2
2nd MS2
3rd MS2
4th MS2
...
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90
80
70
elativeAbundance
60
50
644.30328
z=3
40 554.29718
R
z=1
843.43579
30 455.33417 z=?
z=1 690.34387
z=? 983.57703
20 z=1
10 1136.45715
z=2 1322.60962 1502.32751 1685.00903 1803.28552
z=? z=? z=? z=?
0
400 600 800 1000 1200 1400 1600 1800 2000
m/z
ZhouLei_eGC1_5ul_FullMSddMS2 #19592 RT: 79.21 AV: 1 NL: 1.13E5
T: FTMS + p NSI d Full ms2 538.96@hcd30.00 [100.00-1670.00]
159.11290
z=1
100
90
80
392.21429 701.40942
60 z=1
z=1
100
50 515.33044
200.13957
90 z=1
z=1
40
80 343.16104
z=?
30
70 801.37189
301.12952 ZhouLei_eGC1_5ul_FullMSddMS2z=1 #19594 RT: 79.22 AV: 1 NL: 2.13E4
20 401.28763
RelativeAbundance
60 909.46985
10 1835.21204 2093.78516
z=?
100 z=? z=?
50
0
200 400 90
600 800 1000 1200 1400 1600 1800 2000 2200
40 175.11888
294.18137 m/z
z=1 z=?
80 764.40491
30 494.25977 z=1
z=?
20 70
809.45410
RelativeAbundance
z=?
607.34601 1094.54468
10 60 z=? 776.39569
z=?
z=?
0 50
200 400 600 800 1000 1200 1400 1600
m/z
40 1257.58215
z=?
934.51245
276.13403 z=?
30
z=? 604.29236
444.83582 z=?
20 z=?
93
10
0
200 400 600 800 1000 1200 1400
m/z
50mi n
60 14.38
43.05
50
59.37 65.99
40 15.67
29.59 59.04
41.23 49.66 83.51
30 69.88 75.88 142.88
13.68 16.54 54.91 61.54 144.16
20.98 132.72
20 90.30 98.53
27.56 37.73 107.84 112.14 142.15
13.02 126.13
10 119.25
0.22 10.45
0
0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150
Time (min)
90
80
Relative Abundance
70
60
50
40
30
20
448.23917 580.79761 716.35876 816.42767 895.47113
10 z=2 1075.56592 1160.58887 1298.63977 1660.04175 1784.01672 1906.63831
z=2 z=2
z=1 z=1 z=1 z=1 z=1 z=? z=? z=?
0
400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000
m/z
94
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Peptide sequence
350
y₅⁺
300 543.31464
Intensity [counts] (10^3)
250
[M+2H]²⁺+H
200 b₆²⁺-H₂O, b₃⁺ 739.39655
284.12430
y₁₂²⁺
150
645.79779 y₇⁺ y₁₀⁺
y₁⁺ y₂⁺ y₈⁺ y₁₁⁺
b₄⁺ 763.39838 1078.50464
100
147.11324 260.19751 892.44025 1193.53577
399.15060 y₅⁺-H₂O y₉⁺ y₁₂⁺
50 525.30170 1021.48047 1290.59949
0
200 400 600 800 1000 1200 1400
m/z
95
Targeted SIM
96
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97
304.2773
m/z
98
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S/N (spectrum)
20
0
195.0877
3000
Caffeine
N=20741.58 NL: 1.12E8
100
[190.10-200.10]
80 2000
Relative Abundance
60 Lowest detected
40
SIM (10amu) signal/scan 1000
99
PRM
100
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TERIMA KASIH
102
51