PHNG PHP

KHI PH
NHM 7 _ DH07SH
NI DUNG CHNH
Tng quan v khi ph
Khi ph l g?
Nguyn tc hot ng ca khi ph
S kt hp vi cc phng php
khc - ng dng
Sc k kh kt hp khi ph
Sc k lng kt hp khi ph
in di kt hp khi ph
TNG QUAN V KHI PH
KHI PH L G?
l phng php nghin cu cc cht, bng
cch o chnh xc khi lng phn t cht
, da trn in tch ca ion; dng thit b
chuyn dng l khi ph k.
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

S HNH THNH V
NGUYN TC HOT NG
S ion ho
nghin cu cc cht bng phng php khi
ph, cc phn t cht nghin cu phi dng
kh hoc hi, phi c ion ho bng cc
phng php thch hp(va chm in t ,bng
trng in t ,ion ho hc,chiu x bng cc
photon.)



Phng php ion ho bng
va chm in t

Trong bung ion ho, cc in t pht ra t
cathode lm bng vonfram hoc reni, bay v
anode vi vn tc ln.
Cc phn t cht nghin cu trng thi hi
s va chm vi in t trong bung ion ho,
c th nhn nng lng in t v b ion ho.

Ion ho bng trng in t

Ti bung ion ho t cc mi nhn l b
phn pht t trng di dng dy dn mnh
(2.5 m) hay cc li mnh
t in p vo cc mi nhn. cho mt
trng in t c gradien 10
7
-10
10
V/cm
cc in t s b bt khi phn t cht nghin
cu do hiu ng ng hm nn khng gy s
kch thch,vn gi nguyn trng thi c bn,
do cc vch ph s rt mnh.

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:
My gm mt a ng mu, my bn laser,
mt ng trn o chiu in cc lin tc v
detector.
Bt k my khi ph no cng c bn khi
chc nng sau y:
H thng np mu
Bung ion ho
B phn tch
B ghi tn hiu

Da vo b phn phn tch m ngi ta chia cc
loi my khi ph nh sau:


B phn tch t.
B phn tch t cc.
B phn tch theo thi gian
B phn tch cng hng ion cyclotron.

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
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.
ng dng ca my khi ph trong phn tch
protein

protein c tinh ch ri ct bng enzyme
pepsin . Enzyme pepsin ct polypeptide ti
nhng im nht nh trn chui (ging
enzyme gii hn acid nucleic).
Ta c th d on c cc mnh
(fragments) polypeptide sau khi b ct

a mu x l pepsin vo a v cho my
chy. Laser ion ho cc fragments lm cho
chng tch in dng v bt ra bay vo ng.

ng ny c chiu di nht nh, 4 pha gn 2
loi in cc (+) v (-)

ng c th xoay trn, do cc cc in i
chiu lin tc lm cho cc mnh polypeptide
khng bm c vo thnh m bay theo chiu
xon c
Vn tc bay ca 1fragment ph thuc 2 yu t
in tch (z)
khi lng (m)

My khi ph o c thi gian, bit trc
qung ng => tnh c vn tc => xc
nh c ch s m/z ca fragment.
Cc tn hiu c pht hin bi detector v
khuch i, cui cng biu din trn th
dng cc nh (peak). Mi nh tng ng 1
fragment.

Vi my MS/MS (tandem MS) th y mi l
ln MS th nht, cho php hin th cc
fragment ca mt polypeptide b ct bng
pepsin. Ln MS th 2 cho php khng nh
chc chn 1 fragment nht nh nh h thng
lc.
Figure : Schematic of a MALDI-TOF Mass Spectrometer
Phn tch kt qu:
V protein bao gm 20 amino acid c khi
lng khc nhau do khi lng ca mt
trnh t nh cng ni ln c trnh t ca
n. (Gi s Valin c khi lng l 3,
methionin l 5 th mt fragment c khi
lng l 8 s c trnh t Valin Methionin
hoc Methionin Valin).

S kt hp cc phng php sinh hc
phn t khc_ ng dng

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 (0.000000000001
gram).
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.

Mt s loi GC-MS khc
1.3.1 My sc k kh kt hp khi ph vi
phn gii cao(HRGC-HRMS) chuyn
phn tch DIOXIN.
My c th phn tch c hm lng siu
vt Dioxin ph hp vi tiu chun EPA 1613
ca M phn gii 10.000, gii hn pht
hin mc 0,5ppt.

My c th t n phn gii 80.000,
nh lng c ng thi 17 ng phn
PCDDs / PCDFs trong mu vt mc gii
hn pht hin 0,01ppt
My cn c chc nng t ng kim tra
phn gii sau mi 12 gi v t ng dng
phn tch nu pht hin phn gii gim
thp hn gii hn cho php.

ng dng

phn tch Dioxin v Furan trn nhiu lnh vc
v mi trng : t, bn trm tch, nc sng
ngi, nc ngm
v thy hi sn : tm, c, mc
v nng sn thc phm : go, u, c ph,
sa
c bit l quy trnh phn tch nh lng
Dioxin trong mu bnh phm : m m, huyt
thanh, gan
My sc k kh kt hp vi khi ph nhiu
ln(GC/MSn)
Chc nng
Nhn danh, xc nh cu trc cc hp cht
hu c tng hp v t nhin tng i d bay
hi
nh lng vi lng v a lng.
Kh nng phn tch
D lng ha cht c nng ppb tr ln
c t, d lng thuc tr su, dit c, d
lng khng sinh
Ph gia cng nghip, hng liu, du
i tng mu
Thuc tr su, thuc dit c;
Thc phm, nng sn thc phm;
Dc phm, hng liu, tinh du;
Hp cht t nhin khc;
Sn phm cng nghip.


Sc l lng kt hp khi ph(LS/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