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KHI PH & LC-MS

Mass Spectrometry & LC-MS


Phm gia Hu
H ni 07-08-2009
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Cng c PT hay dng nht hin nay.
K thut cung cp thng tin v:
1. Khi lng v cng thc phn t 1 hp cht
2. MS cng vi NMR v IR cho php nghin cu cu trc phn
t
3. Thnh phn nh tnh v nh lng ca mt hn hp.
4. My MS chuyn cc Phn t thnh cc ion th kh v tch
chng theo t s m/z v ghi li cng tng ion, cho mt
ph khi.
5. PT c mu rt nh, dng rn, lng, kh ca cc cht PTL
nh c (mt s Da) ti cc protein (c trn 300 000 Da).
(Dalton (Da) = amu)
Khi ph (MS) l g?
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Khi ph (MS) l g?
Nguyn l c bn l
s to thnh cc ion,
cc ion nyc tch hay lc theo
t l m/z (khi lng/in tch) ca chng,
v c pht hin.
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Cc thnh phn mt my MS
Ngun ion PT khi
Detector
ion
Ph Khi
m/z
S
i
g
n
a
l

I
n
t
e
n
s
i
t
y
Np mu
R R
L L
K K
Chn khng
cao
X X l l v v l l u u d d
li li u u
Trnh ion PT va chmvi cc
phn t trong khng kh
C

n
g

n
h
i

u
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1. Ngun ion ho
Bin i cht PT, to thnh cc ion phn t
(molecule) v cc ion mnh (fragment)
Hai k thut chnh hay dng:
Bn ph electron (Electron Impact - EI):
to ion dng chmelectron nng lng
cao: phng php cng
Ion ho ho hc (chemical ionization -CI)
to ion thng qua phn ng vi cc ion
thuc th: phng php mm
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V d: Electron bn ph methanol:
to cation gc methanol, mt ion phn t:
CH
3
OH + 1 e
-
CH
3
OH
+.
+ 2 e-
Ch mt s (~0.01%) phn t cht PT (M) c
ion ho
Phn ln cc ion phn t b ph thnh cc mnh
nh hn (cation, gc, phn t trung ho nh
hn)
1.1. Bn ph electron (EI)
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+
+
Si t
Khu vc
Gia tc i on
PT
khi
Chm electron
1.1. Bn ph electron (EI)
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Ch cc mnh tch
in c pht hin
M
e
-
2e
-
M
+
Mu
Cation gc
/ Ion phn t

Bn ph / ion ho
[M]
70 eV
F
1
+
NF
1
F
2
+
Mnh
cation gc
F
2

[Mnh Con]
+
Phn mnh
F
1
+
F
1

Mnh
trung ho
+
+
Mnh
cation
Mnh
cation
Mnh
gc
Gc
Cc LK d gy l cc LK yu nht, to cc mnh bn
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Ph MS ca
pentobarbital,
bn ph
bng electron
Ion phn t M+., c m/z 226, mt gn ht.
To thnh cc mnh m/z 197, 156, 141, 112, 98, 69, and 55,
Cc pic ny l du vt cc mnh trong cu trc ca phn t M.
Pic ln nht trn ph MS l pic c b n (coi nh c cng 100%) . Cng pic khc tnh theo
% ca pic c bn.
Pic c bn
Molecular ion peak M
+
.
Pic ion phn t
Khi l ng phn t =226
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1.2. Ion ho hc (CI) PHNG PHP mm
To t mnh.
Dng tha thuc th dng kh nh CH
4
.
Chmelectrons (100200 eV) chuyn CH
4
thnh cc SP nh CH
5
+
CH
5
+
l cht cho proton mnh, phn ng ho hc vi
cht phn tch to ra phn t proton ho MH
+
, c nhiu trn ph
MS ion ho ho hc dng methan
3 5 4 4
4 4
2
CH CH CH CH
e CH e CH
. .
.





MH CH M CH
4 5
(M+1) peak
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Pic ca ion phn t MH+ m/z 227 (M+1) l pic ln th nh.
t cc pic ca mnh so vi ph EI
Ph ion ho ho hc ca
pentobarbital
Ion ho
Ho hc
C

n
g

n
g

i
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2. B Phn tch Khi
Tri timca my khi ph, c nhimv tch
cc ion c tr s m/z khc nhau thnh tng phn
ring bit.
Phn loi:
B phn tch t,
B phn tch t cc (Quadrupole)
B phn tch by ion t cc (Ion trap)
B phn tch thi gian bay (TOF)
Sau y l b phn tch t
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m/z =B
2
r
2
e/2V
Focused ion M
+
Ion M
+
Ionizing
voltage
2.1 B phn tch t
Sample
inlet
Low vacuum
Detector
Magnetic field
B
Focusing slits
Accelerating
voltage V
Where m =mass,
e =electron charge,
z =#of charges/ion
r =radius of curvature
Increase B
Radiusr
z e B
mV 2
r
2

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Ion Beam
Detector
Magnetic Field
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2.2 B phn tch t cc
(Quadrupole)
Mt s ion c t s m/z xc
nh cng hng vi th xoay
chiu xc nh c th i thng qua
khong khng n detector. Trong
khi .
Cc ion khc khng s c qu o
khng n nh, va chm vi cc
cc v b gi li .
Bng cch thay i th xoay chiu p vo cc cc, cc ion c t s m/z khc
nhau c th vt qua khong khng n detector.
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2.2 B phn tch t cc
By ion (Quadrupole Ion trap)
By ion t cc hot ng theo nguyn l ca b phn tch
khi t cc; ch c mt imkhc l cc ion c lu gi
v a dn ra khi by
Bng cch thay i th xoay chiu p vo cc cc, cc
ion c t s m/z khc nhau c th vt qua khong khng
n detector.
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2.3 B phn tch t cc Chp Ba (Triple Quadrupole)
Ba b t cc ni tip nhau:
Q1: cc ion c tch
Q2: Vi p sut cao, cc ion b phn lydo va chmvi kh tr c mt
nh nit, argon, heli. B Q2 to ra phn ly do va chmchng b phn
mnh tip to ra cc ion nh hn, ion con (daughter ions).
Q3: lmnhimv tch cc ion con




Detector
Q
1
2
Q
3
Q
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phn gii ca my khi ph
l kh nng ca my c th phn bit c 2 pic c
khi lng gn nhau m1 v m2. phn gii R l:
R = m 2 / (m 2 - m 1)
Hai pic cnh nhau c coi l tch ring khi phn chng lp gia
hai pic nh hn 10% pic nh.
B phn tch t hi t n c tr s R khong 5000.
Cn c my vi phn gii cao hn phn bit cc
ion c khi lng gn nhau.
V d: phn bit 2 ion c m1=280,2881 v m2=280,2768
myMS cn c R=25 000
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2.3 B phn tch Thi gian bay (TOF: Time Of Flight)
Nguyn tc:
Dng b phn gia tc ion y ion bay ti
detector, o thi gian bay
Thi gian bay c chun ha theo t l
m/z
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2.3 B phn tch Thi gian bay (TOF: Time Of Flight)
u im:
phn gii rt cao (>10.000)
chnh xc rt cao ( 2- 5 mDa)
ng dng:
Xc nh cht mi (xc nh cng thc)
Yu cu xc nh khi lng chnh xc
Xc nh c tp
o t l cc pic ng v
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Xc nh tp
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3. Detector
Nhn electron (electron multiplier):
Tc ng ca mt ion ln b mt cc
dynode khc to ra cc electron
ngy cng nhiu electron.
1 10
6
(hnh bn)
Nhn quang (photomultiplier):
Cc electron to ra theo cch trn va
chm vi mt b mt pht quang
to ra photon. Cc photon ny
c thu nhn v s lng ca
chng t l vi cng tn hiu
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WS 2005
My MS t cc chp 3 (Trip Quad)
TSQ
(Thermo)
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Waters Micromass Q-Tof Premier
1
2
3
4
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Waters Micromass Q-Tof Premier
1. Phun: Z-spray sch hn
2. T-Wave = ion optics = ion guide: to thnh
hnh ng hng ion vo gia, t mt mt
ion trn ng i
3. T-Wave th hai: cho MS
2
4. TOF: La chn V mode/ W mode
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GC-MS
Tch bng GC Xc nh bng MS
C th vin ph chun
Mass spectrometer acts as a detector for GC
Interface
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LC-MS
Tch bng LC Xc nh bng MS
Kh khn: loi nh hng ca dung mi v cc
thnh phn trong pha ng; khng c th vin ph
ghp ni gia LC v MS:
giao din ion ho ho hc p sut thng APCI
(atmospheric pressure chemical ionisation)
giao din phun in ESI (electrospray)
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ion ho ho hc p sut thng APCI
Nit c dng phun sng pha
ng to thnh mt sol kh hay kh dung (aerosol) ca
kh nit v cc git nh dung mi .
Sol kh ny dc a qua mt khu vc
c t nng. y dung mi b loi v cc
cht c ion ho nh cc phn ng ion-phn t p
sut kh quyn, cc electron v cc ion s cp c to
ra do phng in.
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APCI: Atmospheric Chemical Ionization
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giao din phun in ES
Cc git dung dch thu nh v tng in tch.
Cc phn t dung mi khng tch in b loi
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Ion ha bngphun in ESI
(Electrospray Ionization)
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LC-MS
Tch bng LC Xc nh bng MS
Kh khn: loi nh hng ca dung mi v cc
thnh phn trong pha ng; khng c th vin ph
ghp ni gia LC v MS:
giao din ion ho ho hc p sut thng APCI
(atmospheric pressure chemical ionisation)
giao din phun in ES (electrospray)
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V d ng dng LC-MS
Pht hin tp cht trong cc thuc tng hp,
LC: pha ng ACN-amoni ethanoat, C18
giao din phun in.
Sc TIC: ion m ra gii pht th 17,7 v cc pic
tp ra trc v sau .

MS: Cht ra 8,35 ph cho ph khi c cc pic


thuc ion (M+H)+ xut hin m/z = 225 v 227 theo t
l 3:1 cho thy s c mt ca nguyn t clo. Cht tpl
quinazolin c 3 nhmth.
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V d ng dng LC-MS
Sc TIC
MS pic pht
8,35
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Trnh by d liu
Dng TIC (Total Ion Chromatogram)
Dng SIM (Single Ion Monitoring)
m/z xc nh
Tt c
Tt c
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Trnh by d liu
Dng TIC (Total Ion Chromatogram)
Dng SIM (Single Ion Monitoring)
Dng MRM (Multiple Reaction Monitoring)
m/z xc nh Tt c m/z xc nh
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