KỸ THUẬT PHÂN TÍCH SINH HÓA HIỆN ĐẠI

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Khi nim Quang

ph k l mt thit b dng o s lng nh sng mt mu hp th c khi cho tia sng i xuyn qua mu v o lng cng ca nh sng n u d (detector).

Nguyn tcsng l mt dng photon, i din bi cc bng tm.Khi mt photon gp mt phn t mu phn tch, mu s hp thu photon. S hp thu lm gim s lng photon ca tia sng, do lm gim cng ca tia sng. Cc ngun nh sng c thit lp phng 10 photon cho mi giy. Cc photon chuyn ng v c hp thu khi tia sng qua cc khe cha cc mu. Tia

Cu toC cc b phn chnh sau: Ngun bc x: Ngun bc x t ngoi: n hidro v n teri l mt ngun bc x lin tc nh s chuyn cc phn t kh vo trng thi kch thch.

Nng lng hp th hai dng: - Nng lng ng hc ca cc nguyn t hidro - Nng lng photon ca bc x t ngoi Ngun bc xa hng ngoi: Nhn c t mt vt rn tr b un nng t mt dng in. Khi b un nng n 1500C gia hai in cc s bc x mt nng lng trong vng t 1 4 m.

B phn n sc Chc

nng: Phn bc x thnh cc sng thnh phn c di khc nhau. Di y l s n gin mt my n sc n.

v tr D l cch t, C , E l cc gng phn x Bc x sau khi i qua khe vo B (input) s n gng ri n cch t D, chm bc x s b tch ra cc thnh phn n sc. Cc nh sng n sc s c chnh hng bi gng E v i ra ngoi qua khe ng ra F(output)

S bc x n sc

sng tn x Raman thng yu nn s b nh sng nhiu ny che lp. gii quyt vn ny ngi ta dng my n sc nh

*My n sc i

i.

S my n sc i

My n sc ba My

n sc ba c kh nng kh nh sng nhiu mnh hn my n sc i. N cho php quan st c cc di Raman gn st vch Rayleigh.

S my n sc ba

tect tect

c nhim v bin i nng lng bc x thnh tn hiu in, trong cc my so mu quang in hay dng t bo quang in hay nhn quang in. tect ca bc x cn phi tc ng ln bc x trong mt vng rng ca bc sng; phi nhy vi bc x c cng nh v phn ng nhanh vi bc x cho tn hiu in c th d dng khuych i ln. Quan trng nht l lm sao cho tn hiu nhn c t l thun vi cng ca chm sng n, c th nh sau:

S = I.k + dTrong : - S: l tn hiu ca tect trong cc n v cng dng, in tr hay hiu in th. - k: l s o nhy ca tect trong cc n v ca tn hiu in trn mt n v cng bc x. - d: cn gi l dng ti (dark current), l mt tn hiu hng nh i vi tect khi khng c bc x.

Gi tr ca d thng khng ln, v nguyn tc cc my vi tect c dng ti c trang b mt in k cho php nh mt tn hiu ngc li dn n gi tr d bng khng.S I kk Trong : S, S0: l cc tn hiu in ca tect khi bc x i qua dung dch phn tch v dung dch so snh (dung dch trng). I

S

0

Thay

cc phng trnh ny vo phng trnh nh lut Lambe - Bia ta c: I 0 S0 lg lg D I S

Mt s t bo quang inT

bo quang in lp chn: c cu to t in cc ng hay st trn c a vo vt liu bn dn, b mt ca cht bn dn c ph mt lp mng trong sut t vng bc hay ch. C ch: chiu mt chm sng th mt s electron trong lp cht bn dn c mt nng lng ln vt qua bn chn v thm nhp vo mng kim loi

Nu ni mng vi bn theo mt pha khc ca lp bn dn bng si ch bn ngoi v nu in tr khng qu ln th xut hin dng in, dng in ny thng l ln hoc c th c khuych i o bng mt in k hay mt microampe k. Nh vy lc ca dng in t l vi cng ca bc x chiu vo t bo quang in, ni chung cc dng c t 10 n 100A.

T bo quang in chn khng T bo quang in bao gm mt catt na hnh ng v mt ant dy c t gia mt bnh chn khng. B mt lm ca catt c ph mt lp vt liu nhy nh sng v pht ra electron di tc dng ca bc x

S t bo quang in vi hiu ng quang in ngoi

1- Ch anot; 2- Catot nhy quang; 3- Acquy; 4- in tr; 5- B phn ch th

Trong cc my UV-VIStct l cc t bo quang in vi hiu ng quang in ngoi hay cc nhn quang in. B mt ca catot v thnh phn khng khc b mt catot ca t bo quang in pht ra cc electron khi chiu sng

S nhn quang in

Cc yu t nh hng n kh nng hp th UV-VIS: nh hng ca cu trc phn t cht tan Nhm mang mu: Cc nhm mang mu lm cho phn t c kh nng hp th cc bc x c bc sng di hn trong UV-VIS khong trn 200nm. T nhng c tnh ca cc nhm mang mu ny ta s tnh c cc i hp th ca phn t.

tr mu L nhng nhm cht khng c kh nng hp th UV-VIS nhng c nh hng n cc nhm mang mu. nh hng n cng hp th v lm chuyn dch cc i hp thNhm

V tr khng gian V tr cc lin kt i nh hng rt ln n s hp th ca cc h lin hp. c bit l c th lm thay i v tr cc i hp th v h s hp th phn t. cc hng lin kt khc nhau, cc nhm mang mu v tr mu c nhng cc i hp th khc nhau.

hng ca mi trng Dung mi Dung mi c kh nng hp th cc bc x UVVIS. Do khi chn dung mi chun b dung dch o ph UV-VIS phi tnh n tan ca cht cn nghin cu v kh nng hp th ca chnh dung mi. S dng cc dung mi khc nhau c th lm sai khc v tr cc i trn ph hp th ca cc cht.

nh

Tng tc gia cc lng cc: Khi s dng cc dung mi phn cc nh nc v cc cht tan cng phn cc th s xy ra tng tc gia cc lng cc. N s lm cho cc i hp th dch v phi bc sng di hoc pha bc sng ngn.

Lin kt Hydro: Cc dung mi c lin kt hydro nh ethanol c th tc ng ln cc e n trong phn t. V ngc li cc dung mi c cha cc e n cng tc dng ln phn t cht tan c lin kt hydro. iu ny lm cho cc i dch chuyn v pha bc sng ngn. pH: pH c th lm thay i cu trc phn t cht tan t thay kh nng hp th ca phn t cht .

Nng v cc tng tc khc trong dung dch: Khi dung dch qu long hay qu c c th gy sai lch ha hc do s phn li hay to cc sn phm trng hp. Khi dung dch cht nghin cu c ln thm cc cht l hay to phc vi ion trong dung dch cng lm sai lch ha hc xy ra hoc tng cao thm.

nh hng ca thit b: Kh nng hp th UV-VIS cng b nh hng bi kh nng to nh sng n sc ca thit b. Do nu ng nhn quang hay t bo quang in qu gi th b phn pht hin tr nn km nhy, ko theo s gim n sc ca chm tia. T nh hng n kh nng hp th.

nh hng ca cc hin tng quang hc khc: Khi ta chiu chm tia qua mu o, chum tia ngoi vic truyn thng qua dung dch cn c th chu nh hng ca mt s hin tng quang hc nh phn x, khuch tnc bit l hin tng tn x do dung dch khng ng nht v quang hc. V vy cn m bo trong sut ca dung dch hn ch cc hin tng gy nh hng trn.