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 400C cho ti 3200C. 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.

MALDI
TOF Mass map

Gel-separated protein Sequence database

Tandem

or

mass spectrometry Tandem mass spectrum

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.