1. No category

Liquid Chromatography
Mass Spectrometer
液相層析質譜儀
speaker: 應用化學系 黃立心
date: 2015/4/9
2
LC-MS of 貴重/共同儀器中心
設備
 LC-QqQ MS
 液相層析-三段四極柱式質譜
 LC-Q-TOF MS
 液相層析-四極柱-飛行時間式質譜
游離方式
 ESI 電灑游離、APPI 大氣壓力光游離
 田家炳光電大樓 617 室
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 chromatography
 separation (+ enrichment)
 mass spectrometry
 separation + detection + identification
 isotope analysis
 qualitative analysis ; quantitative analysis
Why are they combined together ?
LC - MS 強調儀器硬體上的連接
LC / MS 強調方法、技術上的結合
4
Part I
Mass Spectrometer
 basic concepts
 ion sources
 mass analyzers
 tandem MS
used in LC-MS
5
Mass spectrometer overview
vacuum
gas phase
ions
sample
inlet
ion
source
ion sorting
by m/z
mass
analyzer
ion
detector
data
system
mass spectrum
6
Ion & spectrum
 mass to charge ratio , m/z
 single charged, multiple charged
 mass of atom/molecule
 accurate mass & monoisotopic mass
 average mass & nominal mass
 mass resolution
 parent ion
daughter (product, fragment) ion
7
8
原始質譜圖
A = C41H66N10O14S2
relative intensity
100
mass
986.42
987.42
988.42
989.42
990.42
991.42
50
relative
abundance
100
50.1
21.1
6.9
1.9
0.4
B = C41H77N15O11S
mass
987.56
988.57
959.57
990.57
991.57
992.57
relative
abundance
100
50.7
17.7
4.9
1.1
0.1
0
985
986
100
987
B988
989
990
991
單一同位素質譜圖
992
993
994
993
994
A
保留訊號峰
A = 986.42
B = 987.56
50
0
985
986
987
988
989
990
m/z
991
992
9
Ion sources used in LC-MS
 ionization
 high-energy exciting or attach other ion to the sample
 hard or soft ionization
 properties of sample : mass , stability…
what kind of data
you want ??
 other concerns
 continuous sample introducing
 large amount of solvent  large amount of vapor
 operate under atmospheric pressure
10
Electrospray ionization 電灑游離 , ESI
1 atm
vacuum
1.5 - 2 kV
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ESI
needle tip
min. flow rate :
~20 nL/min
sampling
cone (to
analyzer)
solvent of high polarity :
water, ACN (acetonitrile, CH3CN), methanol…
with volatile acid (H+ source) :
formic acid, acetic acid…
12
 ESI :
M + nH+  [M + nH]n+
 multiple charged ions
 advantage?
disadvantage?
 very soft
 van der Waals complex
ion is possible !!
 sample
 small to macro molecules
 high polarity
sample : 17 kDa protein
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Atmospheric pressure photoionization
大氣壓光游離 , APPI
霧化器
analyzer
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 energy of photon
solvent  dopant

sample or dopant
dopant ion react with sample
 ionization of sample
enough to ionize

not enough to ionize
O2 , N2
15
 atmospheric pressure chemical
ionization , 大氣壓化學游離 , APCI
 corona 電暈放電
 inductively coupled plasma ,
感應耦合電漿 , ICP
 8,000 - 10,000 K argon plasma
 inorganic elemental analysis
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ESI
APPI
can be applied to
small & medium molecules
that insoluble in water
APCI
small molecules to macro molecules
(peptide, protein, DNA… )
that soluble in water
17
Mass analyzer used in LC-MS
 principles of analyzing ions with different m/z
 different trajectories in electric/magnetic field
 the same kinetic energy , different velocity
 motion in electric/magnetic field  induced current
 concerns
 m/z range
 resolution
 scanning or not , scanning speed
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Quadrupole 四極柱
mass “filter”
ions of appropriate m/z
can pass through
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+(U+V∙cosw t)
high-pass filter
–(U+V∙cosw t)
low-pass filter
if RF (AC) only  no filter function
20
Time of flight 飛行時間式 ,TOF
getting the
same kinetic
energy
5-30 kV
techniques for reducing
space & energy spread
m/z = 2eV(t / L)2
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 flight tube : 1 - 2 m
 reflectron , electrostatic reflector
 correcting the energy dispersion
 longer flight distance
22
Quadrupole vs TOF
Quadrupole
Time of Flight
mass range
< 4,000 ; usually < 2,000
> 100,000
resolution
usually set for unit resolution
linear: ~3000
reflector: > 30,000 ; usually ~15,000
mass accuracy
50-100 ppm
2-50 ppm
scan speed
< 4,000 Th/sec
106 Th/sec
kinetic energy of ion
low
high
accelerating voltage
~ 5-20 V
~ 5-30 kV
operating pressure
< 10-3 Torr
< 10-6 Torr
other features
continuous
pulse
compact size
large size
$
$($$)
23
 cylindrical quadrupole ion trap 圓柱形四極離子阱
 orbitrap 軌道阱
 exciting trapped ions  induced current
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Tandem MS 串聯質譜
 why tandem MS ??
 single or multiple analytes
 different operating modes
?
 precursor ion  product ion
 occur during hard ionization
 collision-induce dissociation, CID
 precursor ion
inert gas (N2, Ar,…)
 photo dissociation & other techniques
25
MS/MS operating modes
操作模式
目的
操作方法
子離子
（產物離子）
product ion
獲得母離子的結構資
訊
MS1 選擇單一母離子
MS2 掃描記錄所有子
離子
母離子
（前驅物離子）
precursor ion
尋找會產生相同子離子的母離 MS1掃描所有母離子
子
MS2選擇單一子離子
中性丟失
neutral loss
尋找會產生相同中性碎片的母 MS1和MS2維持相同質荷比
離子
差，同時掃描
選擇反應監測
selected reaction
monitoring
監測特定反應
MS1選擇單一母離子
MS2選擇單一子離子
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 product ion mode
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 other modes
precursor ion
28
selected reaction monitoring
neutral loss
29
Triple-quadrupole, QqQ
MS1
collision chamber
(AC only q)
collision gas
MS2
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Quadrupole-time-of-flight, Qq-TOF (Q-TOF)
MS1
(Q)
collision chamber
(AC only q)
MS2
(TOF)
detector
ion guide
ion guide
collision gas
precursor ion
product ions
reflectron
31
QqQ & Q-TOF
 Why there is a quadrupole in the collision chamber ?
pressure , ion guide , energy
 comparison
 operating modes : QqQ – all 4 modes
TOF can not be a filter  Q-TOF – product ion mode
 product ion sensitivity , resolution , mass accuracy : Q-TOF
 simple & easy : QqQ
 other MS/MS ? ion traps , TOF-TOF , TOF-Q
not for LC/MS
32
quadrupole or TOF ??
33
34
Part II
Liquid Chromatography
 principle of chromatography
 GC or HPLC
 HPLC , LC/MS
35
MS (even tandem MS) is not enough
 sample complexity
 multiple analytes have to be analyzed
 number , variety , concentration of analytes & matrixes
 most samples, especially “real world” samples, vary widely in
complexity
 sample – MS compatibility
 matrixes/impurities , concentration
 more information about a complex analyte
 macro biomolecules : protein, DNA, polysaccharide
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Principle of chromatography
flow of mobile phase
mobile
phase
stationary
phase
detecting
A
B
A
B
A
B
A
B
B
time
chromatograph
層析圖
37
Gas vs liquid chromatography
 GC
 volatile molecules : low molecular weight , low-polarity
+ thermal stable
 easy to couple with various ion sources of MS
 LC (HPLC , HP = high performance or high pressure)
 difficult to couple with MS
 nonpolar to ionic , small to Marco molecules
 various mechanism of separation alternatives
most versatile
technique !!
38
HPLC
sample
column
PUMP
detector
injector manual or
automatic
eluent 沖提液
(mobile phase)
separation according to :
 polarity – normal or reverse phase
 ion interaction – ion exchange
 size of molecule
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QqQ MS
mobile
phase
auto sampler
pump
HPLC
column
electrospray
ion source
40
TIC (total ion counts)
chromatograph
LC/MS/MS
time
m/z
product ion
spectra
m/z
information
of molecule
quantitation
precursor ion
spectra
41
Considerations
separation efficiency
amount of sample
recovery
sample
column
& eluent
molecular weight
ionization efficiency
soft or hard ionization
number of charge
ion
source
analyzer
flow rate
solvent of eluent
additive in eluent
42
Part III
Application Examples
 melamine 三聚氰胺 additive in food
 protein identification
 peptide mass fingerprinting 胜肽質量指紋
 peptide sequence tag 胜肽序列標籤
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44
Separating melamine from foods
 foods : complex , variety  can
to be analysis by MS directly
 melamine
 polar , slightly dissolved in water
 using reverse phase HPLC + ESI
45
food sample (eg milk powder)
operate manually
+ (water + CAN + 2.5% formic acid)
removing insolubles , proteins
HPLC separation
MS/MS analysis
on line analysis
hydrophilic
interaction
46
– H2NCN
– NH3
precursor ion
product ion
product ion
m/z = 127
m/z = 85
m/z = 68
selected reaction monitoring : m/z 127  85
quantification
47
Amino acid  peptide  protein
 amino acid
 20 essential a-amino acids with different side groups (R)
 average molecular weight ~110 Da
 peptide
 50 or fewer amino acids linked together by peptide bonds
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 protein
 > 50 amino acids
 function
 may have a few modifications on side group of amino acid
 structure
 primary structure – sequence of amino acid
▲ sequencing, identification, quantification…
 secondary , tertiary , quaternary structures
 MS
49
Protein identification
protein
 MS analysis
 Why MS data from
peptides
peptides
is usually useful than
that
from intact protein ?
50
Peptide mass fingerprinting , PMF
A
B
E
protease
(蛋白酶)
C
D E F
B
purified protein
C
A
F
D
mixture of peptides
separation is not required!!
MS
…….
…
……
……….
….
……
database search ,
identification
mass fingerprint
m/z
51
 database search setting
 result of search (extracted)
52
53
Peptide analyzed by MS/MS
x3
a1
y3 z3
b1
c1
collision condition !!
peptide : SIMAETLK
precursor ion ([M+H]+)
monoisotopic mass
= 892.5
x2
a2
y2 z2
b2 c2
x2
a3
y2 z 2
b3 c3
product ions
54
D
B
A C E
a
b g d
1
2
3
4
5
protease
(蛋白酶)
6
6
identify
multiple
proteins
3
g
LC separation
D
4
d
C
1
A
b
5
E
peptide mixture
protein mixture
database search
a
B
2
m/z
spectra of
product ions
m/z
spectra of peptide
precursor ions
MS/MS
time
chromatograph
55
 vs PMF
 proteins do NOT need to be separated
 spectrum of product ions
  sequence of peptide
 find an unique peptide  the protein can be identified
▲ multiple peptide is needed in PMF
 type & position of modification
56
 database
 by computation
 genomic data  protein  peptide  product ion of peptide
2 matched peptide
data of product ions
(computed & found)