PHNG PHP

KHI PH
Bi Vn Hiu - NguynThi Ngc
Lp: 10B CNSH - KH
NI DUNG CHNH
I. Tng quan v khi ph
1. Khi ph l g?
2. Nguyn tc hot ng v nguyn l chung.
II. S kt hp gia khi ph cc phng php
khc
1. Sc k kh kt hp vi khi ph
2. Sc k lng kt hp vi khi ph
3. Kt hp in di hai chiu-khi ph
III. Cc phng php ion ha hin i
IV. Xc nh protein s dng khi ph
TNG QUAN V KHI PH
KHI PH L G?
L phng php nghin cu cc cht bng
cch o, phn tch chnh xc khi lng phn
t ca cht da trn s chuyn ng ca
cc ht mang in hay ion trong mt in
trng hoc t trng nht nh. .
K thut ny c nhiu ng dng, thng
c kt hp vi mt s sinh hc phn t
khc nh:
Khi ph kt hp vi sc k kh.
Khi ph kt hp vi sc k lng.
Khi ph kt hp in di

My khi ph

L mt thit b dng cho phng php ph
khi, cho ra ph khi lng ca mt mu,
tm ra thnh phn ca n.
C th ion ha mu v tch cc ion ca n
vi cc khi lng khc nhau v lu li thng
tin da vo vic o c cng dng ion

. Mt khi ph k thng thng gm 3 phn:

Phn ngun ion
Phn phn tch khi lng
Phn o c.
Cu to:

B phn a mu;
Ngun ion ho;
H thng bm chn khng;
B phn tch khi o t l khi lng/in
tch ca phn t dng ion ho (m/z);
B phn o tn hiu xc nh s lng
cc ion mi gi tr m/z;
H thng phn tch s liu, bao gm my
tnh cu hnh cao vi cc phn mm
chuyn dng.
S cu to h my khi ph vi cc kh nng lp
t khc nhau
Theo tnh nng b ghi, ngi ta chia cc my
khi ph thnh hai loi:
My khi ph k ghi bng knh nh. Tn
hiu ph c ghi bng knh nh dng
vch c en khc nhau.
My khi ph k: cc tn hiu ca chm
ion c ghi di dng xung in bng
cc dao ng k in t nhiu knh, hoc
a vo my tnh in t,tn hiu s c
a ra di dng bng s hoc th thch
hp.
Ngy nay trong phn tch khi ph ngi ta
dng cc my khi ph k.


Nguyn l hot ng ca my khi ph

Nguyn l hot ng ca my khi ph

Mu cht cn phn tch s c chuyn thnh
trng thi hi, sau mi bt u qu trnh
o khi ph
o c c tnh ca cc phn t c th,
my khi ph s chuyn chng thnh cc
ion,kim sot chuyn ng ca chng bi cc
in t trng bn ngoi
Qu trnh c thc hin trong mi trng
chn khng

Trong khi p sut kh quyn vo khong 760
mmHg, p sut mi trng x l ion thng t
10
-5
n 10
-8
mmHg (thp hn mt phn t ca
p sut kh quyn).
Ion sau khi c to thnh s c phn tch
bng cch gia tc v tp trung chng thnh mt
dng tia m sau s b un cong bi mt t
trng ngoi.
Cc ion sau s c thu nhn bng u d
in t v thng tin to ra s c phn tch v
lu tr trong mt my vi tnh.
II. S kt hp khi ph v cc
phng php khc
1. Khi ph kt hp vi sc k kh
(Gas Chromatography Mass Spectometry)
Phng php Sc k kh kt hp vi Khi
ph(vit tt l GC-MS hoc GCMS) l mt
phng php mnh m vi nhy cao c
s dng trong cc nghin cu v thnh phn
cc cht trong khng kh.
Bn cht GC-MS l s kt hp ca Sc k kh
(Gas Chromatography) v Khi ph (Mass
Spectometry). Ngng pht hin ca phng
php ny l 1 picogram.
Cu to ca GC-MS
- Sc k kh (GC): Phn tch hn hp ha cht thnh mt
mch theo tng cht tinh khit
- Khi ph (MS): ) Xc nh nh tnh v nh lng

A. Ca tim mu (injection port): 1 microliter
dung mi cha hn hp cc cht s c tim
vo h thng ti ca ny. Mu sau c
dn qua h thng bi kh tr, thng l
helium. Nhit ca tim mu c nng
ln 3000C mu tr thnh dng kh.
B. V ngoi (oven): Phn v ca h thng GC
chnh l mt l nung c bit. Nhit ca l
ny dao ng t 40
0
C cho ti 320
0
C.
C. Ct (column): Bn trong h thng GC l
mt cun ng nh hnh tr c chiu di 30
mt vi mt trong c trng bng mt loi
polymer c bit. Cc cht trong hn hp
c phn tch bng cch chy dc theo ct
ny.
Sau khi i qua ct sc k kh, cc ha cht
tip tc i vo pha khi ph. y chng b
ion ha.Sau khi khi ph,chng s ti b
phn lc
Da trn khi lng ,b lc la chn ch cho
php cc ht c khi lng nm trong mt
gii hn nht nh i qua.
Thit b cm bin c nhim v m s lng
cc ht c cng khi lng. Thng tin ny
sau c chuyn n my tnhv xut ra
kt qu gi l khi ph
Khi ph l mt biu phn nh s lng cc
ion vi cc khi lng khc nhau i qua b
lc.

-My tnh:

B phn chu trch nhim tnh ton cc tn
hin do b cm bin cung cp v a ra kt
qu khi ph.


Cng dng
Phn tch: GC-MS c th phn tch cc hn
hp ha cht phc tp trong khng kh hay
trong nc. y, tc c quyt nh
bi tnh bay hi. Cht no c tnh bay hi
cao s di chuyn nhanh hn cht c tnh bay
hi thp.
nh lng: GC-MS c th nh lng mt
cht bng cch so snh vi mu chun, l
cht bit trc v c nh lng chun
bng GC-MS.
Nhn dng:
Nu trong mu c mt cht l xut
hin, khi ph c th nhn dng cu trc
ha hc c nht ca n. Cu trc ca
cht ny sau c so snh vi mt
th vin cu trc ca cc cht bit.
Nu khng tm c cht tng ng
trong th vin ,ta thu c mt d liu
mi v ng gp vo th vin cu trc
sau khi tin hnh thm cc bin php
xc nh c chnh xc loi hp cht
mi ny.


2. Kt hp sc k lng a chiu-khi ph
lin tip (MDLC-MS/MS)

Phng php sc k lng ghp khi ph l
phng php c hiu l mu c phn
tch v tinh sch qua sc k, sau mu c
a qua my khi ph tip tc xc nh
khi lng nguyn t ca n.
Kt thc qu trnh ta thu c lng mu
nht nh v cu trc phn t .
ng dng
Xc nh protein
Kim tra cht lng nng sn, thc phn ch
bin, thc n gia sc v thy hi sn: c t
nm mc, vitamin, thnh phn aa, ph gia
thc phm, cht hot ng b mt, d lng
khng sinh (chloramphenicol, nitrofuran,
fluoroquinolone, h tetracycline,
sulfamide), d lng hormone kch thch
tng trng b cm, vi sinh vt c hi, mu
b cm trong thc phm (cc loi Soudan), d
lng thuc tr su.

in di kt hp khi ph
Thng c dng xc nh protein.
Phng php ny cho php ta quan st v
nhn dng s hin din tt c cc protein
cc trng thi t bo

Khi ph l phng php mang li nng sut
v chnh xc cao trong qu trnh nhn bit
nhng protein phn tch bng in di 2
chiu.
Protein c phn tch trn gel
polyacrylamide sau c a vo my khi
ph phn tch.Cc my mc ngy nay c
th t thc hin cc bc phn tch v cho
php chng ta quan st c nhng protein c
mc ln.

Sequence
database
Tandem mass spectrum
Mass map
Tandem
mass
spectrometry
MALDI
TOF
or
Gel-separated protein
Hnh: Xc nh protein thu c trong cc mu sinh hc.Protein t mu c th c
phn tch bng in di mt hoc hai chiu v protein c nhn dng bng cch s
dng MALDI-TOF v nh du khi peptide.Peptide thu c ti nhng v tr c bit
ca protein c phn tch trn gel polyacrylamide c th c phn tch bng
cch s dng khi ph tandem.D liu t khi ph tandem c th c dng to
nn 1 c s d liu

Xc nh Protein

C 2 cch chnh trong khi ph xc nh protein.
Ly du khi peptit (PMF) dng khi ca cc peptit
phn gii lm u vo tm kim trong CSDL
ca cc khi bit trc t danh sch cc protein
bit. Nu mt chui protein trong danh sch
tham kho trng khp vi gi tr th nghim th c
l do tin rng protein c tn ti trong mu gc.
Tandem MS ang tr thnh mt phng php th
nghim ph bin xc nh protein.
Phn ly do va chm (CID) c dng trong cc ng
dng chnh khi to mt tp cc phn mnh t
mt ion peptit c th. Qu trnh phn tch ch yu
da vo cc ch phm phn tch b gy lin kt
peptit. V s n gin ca vic phn tch ny, n c
th dng khi ca cc phn mnh quan st c
so trng CSDL ca cc khi bit vi mt hay
nhiu chui peptit.