Phn A: L THUYT

Phn A:L THUYT1. KHI NIM V PHNG PHPPhng php quang ph t ngoi kh kin, hay cn gi l phng php quang ph hp th, hay phng php o quang da trn kh nng hp th chn lc cc bc x ri vo dung dch ca cht nghin cu trong mt dung mi nht nh.

Cc bc sng cc i hp th c trng cho tng cht, hoc t l hp th gia cc bc sng l c s ca vic nh tnh. hp th cc bc x ph thuc vo nng ca cht nghin cu trong dung dch cn o l c s ca php nh lng. Ngoi ra, vic o quang trong mt iu kin quy nh v dung mi, nng , bc sng cn c th lm c s cho php th tinh khit.Phng php o quang ngoi kh nng phn tch cc cht trong dung dch n cht tinh khit, n cn gip phn tch cc cht trong dung dch hn hp nhiu cht, nh s h tr ca phn mm x l vi tnh2. I CNG V NH SNG V QUANG PH HP TH2.1. nh sng:nh sng l mt loi bc x in t. nh sng va c bn cht sng va c bn cht ht. Mt s tnh cht ca nh sng ph thuc vo bn cht sng, mt s tnh cht khc ph thuc vo bn cht ht.Hnh 1: Bc x in t theo hng XM- Vect tE- Vect in- Do nh sng c bn cht sng nn n c c trng bi mt trong cc i lng sau:- bc sng, di sng(trong vng t ngoi kh kin hay dng n v l nm;1nm = 10-9m)- Tn s(s dao ng trong mt giy)C- Vn tc ng sngC= -------Hnh 2: di sng- Trong chn khng nh sng c tc 3,0 x 108m/s- Mt tia sng c th coi nh mt chm cc ht gi l lng t nh sng hay photon. Mi tia sng mang mt nng lng nht nh ng vi cc photon mang nng lng E.E = h.= h.(c/)h: hng s Planck (h = 6,62.10-34JS)2.2. Quang ph hp thPhng php quang ph hp th da vo hin tng hp th nng lng ca cc bc x nh sng chiu vo cht nghin cu. Khi chiu nh sng vo mt cht n s lm thay i nng lng ca nguyn t, phn t.2.2.1. Nng lng ca phn t:Ep= Et+ Ee+ E+ ErTrong :Ep- nng lng ca phn tEt- nng lng tnh tinEe- nng lng in tE-nng lng dao ngEr- nng lng quayNu chiu tia t ngoi kh kin vo phn t n s lm thay i thnh phn Eenn c gi l ph in t.Nu chiu tia hng ngoi vo phn t n s lm thay i trng thi dao ng ca phn t (E) nn c gi l ph dao ng.2.2.2. S hp th nng lng ca phn tMi phn t ca mt cht c mt s trng thi nng lng xc nh v gin on. Trng thi nng lng thp nht l trng thi c bn. Khi hp th nng lng, phn t chuyn sang trng thi nng lng cao hn gi l trng thi kch thch.M + hM*Phn t trng thi kch thch th khng bn vng, c xu hng nhanh chng quay v trng thi nng lng ban u, gi l s phc hi hay quay tr vM*M+ h(dng bc x)M*M+ h(calo) (dng nhit)Phn t thng trng thi kch thch rt ngn 10-9n 10-6giy.Hnh 3: S cc mc nng lngE0,E1,E2,l cc mc nng lng in t.- ng vi mi mc nng lng in t c nhiu mc nng lng dao ng v0,v1,v2.- ng vi mi mc nng lng dao ng c nhiu mc nng lng quay ro, r1,r2.S hp th nng lng ca phn t c tnh cht c trng. Mun chuyn t mc nng lng E0ln mc nng lng E1cn mt bc x c nng lng:E = h= E1 E0Ch nhng bc x c nng lng ng vi khong cch ca cc mc nng lng trong phn t mi c hp th.C nhng s chuyn mc nng lng d xy ra v cng c nhng s chuyn mc nng lng kh xy ra. iu c ngha l c nhng bc x c hp th mnh, c nhng bc x c hp th yu v cng c nhng bc x khng c hp th.2.2.3. Ph hp th phn tPhng php hp th phn t l phng php phn tch da vo nng lng ca nh sng b hp th bi phn t ca cht nghin cu.Ph chnh l mt dy cc i tng nghin cu c sp xp theo mt trt t nht nh.Quang ph theo ngha hp l ch kho st hp th phn nh sng nhn thy, nhng theo ngha rng l kho st c ngoi vng nh sng nhn thy bao gm nh sng hng ngoi v t ngoi.Mi phn t c mt s mc nng lng nht nh cn gi l c trng thi nng lng xc nh. Khi phn t hp th nng lng ca nh sng th trng thi nng lng ca phn t cng thay i t mc nng lng thp ln mc nng lng cao. S hp th nh sng l c trng v chn lc.i vi cc phn t trong thc t c nhiu vi mc nng lng gn nhau to nn kh nng hp th cc bc x c bc sng gn nhau. V th ph hp th phn t khng phi l ph vch m l ph di hin di dng cc nh hp th.

.3. Quang ph t ngoi kh kin2.3.1. Mu sc ca nh sng:Tu theo bc sng m nh sng c chia thnh tng vng :-Vng t ngoi 185 400 nm-Vng kh kin 400 760 nm

Ring trong vng kh kin (mt nhn thy c) c nhiu mu, mi mu ng vi mt chm tia c bc sng gn nhau:760 630 nmCam630 600 nmVng600 570 nmLc570 500 nmLam500 450 nChm450 430 nmTm430 400 nmChm tia sng n sc l chm c mt bc sng duy nht.2.3.2. S hp th nh sng v mu sc ca vtS ph nhau ca cc tia sng:Chiu hai tia sng (v d: tia vng v tia lam) vo mn nh (trng), iu chnh cng thch hp ca hai tia, s c lc thu c nh sng trng (khng mu). Ta ni hai tia (mu) trn ph nhau (hay b sung nhau).Cc cp mu ph nhau:Lc vng-TmVng-LamDa cam-Lam lc-Lc lam ta-LcChng ta bit rng nh sng t nhin khi phn tch ra cha rt nhiu mu sc, nhng li khng mu v cc tia ph nhau tng i mt.Cc cht hp th nh sng mt cch chn lc:C cc tia sng vi bc sng nht nh c hp th mnh hn cc tia khc. V th trong phn nh sng cn li phn x n mt ta th cng ca nhng tia ph vi nhng tia b hp th tr nn mnh hn v ta cm thy mu ca nhng tia .V d: nu mt vt c mu th hp th mnh mu xanh lc v ngc liTm li, mu ca vt v mu b vt hp th l hai mu ph nhau.2.3.3. S hp th v cu trc phn t- Cc cht hu c c dy ni n: - C H;- C-C- c cc in t trong lin kt n l in t+ h*S chuyn dch in t t mc nng lngln*yu cu mt nng lng tng ng vi cc bc x c bc sng130nm .Cc cht c dy ni i hoc ba C = C ; CC trong cc lin kt ny c c lin kt+ h*S chuyn dch in t t mc nng lng*hp th mc nng lng thp hn ng vi185nm.Tm li: Gia cu trc ho hc v quang ph hp th c mi quan h cht ch. Ch c nhng phn t no c nhng in t d b kch thch mi hp th nng lng nh sng chuyn t trng thi c bn sang trng thi b kch thch. l cc phn t c cha nhng ni i, ni i lin hp, nhn thm hay nhng d tN, S, O. Mi cc i hp th trn quang ph u c ngun gc l s chuyn trng thi ca in t trong phn t.2.3.4. nh lut Lambert BeerChiu mt chm tia n sc c cng I0qua dung dch c chiu dy 1. Sau khi b hp th, cng chm tia cn li I . truyn qua T = I / I0. hp th A = - lg T = lg( I0/ I) hp th A (mt quang A ) ca dung dch t l thun vi nng C ca dung dch theo biu thc :A = k . l .CTrong : - k l h s hp thph thuc vo cu to ca cht tan trong dung dch.- l l chiu dy lp dung dch+ Trng hp C tnh theo mol/l v l tnh bng cm, ta c k =Do A =.l.Cc gi l h s hp th phn t v A =khi C = 1mol/l v l = 1cmc trng cho bn cht ca cht tan trong dung dch ch ph thuc bc sng nh sng n sc. nhn mnh c khi ngi ta vit:A =.l.C+ Trng hp C tnh theo phn trm (kl/tt) c biu th bng gam trong 100 ml dung dch, l theo cm, k c gi l h s hp th ring hoc h s tt ring, k hiu E.A = E.l.CA = Ekhi C = 1% v l = 1cmh shay dng trong m t cu trc, tnh cht quang ph ca cc cht hu c, cn trong phn tch kim nghim ta hay dng E.Cc iu kin p dng nh lut:- nh sng n sc: Khi bc sng thay i cc h s hp th cng thay i. Mt chm tia cng n sc th nh lut cng ng.- Cng mt dung dch nhng o trn cc my khc nhau c th thu c cc tr s A khc nhau. C nhiu nguyn nhn nhng trc ht l do tnh n sc ca nh sng.- Khong nng thch hp: Do nhiu nguyn nhn vt l (s phn x, s khuch tn nh sng), ho hc (s phn ly, nh hng ca lc ion) m nh lut Lambert Beer ch ng trong mt gii hn ca nng . V vy, khi xy dng phng php nh lng cn phi kho st k trc tm khong nng .- Cc yu t ho hc khc: Lm th no cht hp th nh sng khng b bin i bi cc phn ng ho hc trong dung dch. V vy, pH dung dch, s c mt cc cht l c kh nng phn ng vi cht cn o hoc gy nhiu (cn tr hay tng cng) s hp th nh sng ca cc cht cn o u phi tnh n.

3. MY QUANG PH T NGOIKH KIN (UVVIS)

3.1. S cu toMy o quang ph UV-Vis l mt thit b dng o hp th hoc truyn qua ca dung dch gm c cc b phn c bn sau:- Ngun sng: cung cp cc bc x in t- B phn tn sc: c nhim v chn t ngun bc x mt bc sng c trng- B phn ng mu o- B phn detectordng o cng tia bc xNgoi ra cn c cc b phn khc nh lthu knh hoc gngc nhim v chuyn tip cc tia sng qua thit b3.2. Cu to chi tit v tnh nng3.2.1. Ngun sngMt ngun sng l tng l ngun sng to ra cc bc x c cng khng i trn ton b khong bc sng, vi nhiu thp v n nh trong khong thi gian di. Trn thc t ngun sng tho mn tt c cc iu kin trn l khng tn ti.C hai loi ngun sng hay c s dng trong my quang ph UV-Vis:3.2.1.1. n h quang deuterium:Sinh ra cng lin tc trong vng UV v cung cp cng c th dng c trong vng Vis (hnh 4).n h quang deuterium hin i c nhiu thp do c mt b phn hn ch nhiu trn ton b thit b hot ng. Theo thi gian, cng cc tia sng pht ra t n h quang deuterium gim dn. n loi ny c thi gian s dng khong 1000 gi (tc l thi gian m cng ca n gim cn mt na so vi ban u).-Hnh 4: Cng tia sng ca n h quang deuterium3.2.1.2. n halogen tungstenSinh ra cng tt trn mt phn vng UV v ton b vngVis(hnh 5).Hnh 5 : Cng tia sng ca n halogen tungstenLoi n ny c nhiu rt thp v dao ng t v c thi gian s dng khong 10.000 gi.a s cc my quang ph dng o trong khong UV Visc cha hai loi n. Trong cc thit b ny hoc l c b phn chn ngun bt gia hai loi n khi cn hoc nh sng pht ra t hai ngun c trn vi nhau to ra mt ngun c nht.Ngoi ra cn mt ngun sng khc l n xenon to ra cng lin tc trn ton b vng UV-Vis (hnh 6). Tuy nhin, v nhiu t n xenon rt ln so vi hai loi n trn cho nn n xenon ch c dng trong cc php o phn x khuych tn, cng cao ca tia sng c quan tm hng u.Hnh 6 : Cng tia sng ca n xenon3.2.2. B phn tn scB phn tn sc to ra cc bc sng khc nhau t cc tia sng c tn sc cc gc khc nhau. Khi c kt hp vi mt khe ra thch hp, b phn ny c th c dng chn ra mt bc sng c trng (chnh xc hn v c di sng hp) ca tia sng t ngun lin tc. B phn tn sc c hai loi: lng knh v cch t, thng c dng nhiu trong cc my quang ph UV-Vis.-Lng knh to ra nh sng nhiu mu nh cu vng t nh sng mt tri. Nguyn tc ny c s dng trong my o quang ph. Lng knh cu to n gin v r tin, nhng gc tn sc khng tuyn tnh (hnh 7a). Hn na gc tn sc li ph thuc vo nhit . V vy, a s cc my quang ph hin i c b phn cch t thay th cho lng knh. B phn ny c lm bng thu tinh. V pha trn c cc rnh rt hp, kch thc ca cc rnh ny theo th t nh bc sng ca nh sng c tn sc. Cui cng, mt lp nhm c ph ln trn to ra mt ngun phn x. Tia sng chiu vo cch t c phn x li cc gc khc nhau, ph thuc vo bc sng.-Cch t to ra gc tn x tuyn tnh khng ph thuc vo nhit . Tuy nhin, chng phn x tia sng cc th t khc nhau, nn c hin tng gi ln nhau (hnh 7b). Chnh v vy, ngi ta phi s dng h thng lc chc chn rng ch cc tia sng phn x cn thit n c detector. Mt cch t lm va tn x ng thi hi t tia sng.B n sc ho gm mt khe ti, mt b phn tn sc v mt khe ra. L tng nht l tia i ra khi b n sc ho l tia n sc. Tuy nhin, trn thc t tia i ra lun l mt di sng i xng. rng ca di chiu cao gi l rng di ca thit b.Hnh 7 : B phn tn sc3.2.3. B phn ng mu oCc o phi hon ton trong sut tt c cc bc sng v bt k s hp th no t cc cng lm gim khong hp th tuyn tnh ca mu o. V d, thit b c th cho gii hn trn ca khong tuyn tnh l 2,5 AU nhng nu cc o hp th 1 AU th khong cn li cho mu ch l gia 1 v 2,5 AU tc l cn 1,5 AU.Nhng cc r tin nht c lm bng plastic. Cc cc ny khng th dng vi tt c dung mi v hp th mnh di 300nm. Cho nn khng thch hp vi vic o vng ny. c tnh ca tng loi cc ph thuc vo nh sn xut.Cc thu tinh t hn mt cht so vi cc plastic, chng bn hn. Cc thu tinh hp th mnh vng di 320 nm bi vy khng thch hp o vng ny.Cc thch anh trong sut cho n tn di 210nm. Cc o tt nht hin nay l cc silica tng hp vi cht lng cao, c th trong sut n di 190nm. Hnh 8 cho thy tnh cht hp th ca cc cc o c lm bng cc nguyn liu khc nhau.Hnh 8 : c tnh truyn qua canguyn liu lm cc oB dy ca cc o c th t 0,1 n 10 cm nhng hay dng nht l cc c b dy 1 cm. i khi cc cc dy hn cng c dng i vi nhng cht c nng thp hoc c hp th km.3.2.4. DetectorDetector c nhim v chuyn tn hiu nh sng thnh tn hiu inMt detector l tng s cho p ng tuyn tnh trn mt khong rng, vi nhiu thp v nhy cao.Cc my quang ph thng cdetector l ng nhn quang hoc detector diode quang.- ng nhn quang c chc nng t hp cc tn hiu chuyn i qua vi giai on khuych i trong thn ca ng. Bn cht ca nguyn liu lm cathode l xc nh nhy ca ph. Mt nhn quang in n to ra nhy tt trn ton b khong UV Vis. Loi detector ny sinh ra nhy cao mc nh sng thp. Tuy nhin, trong lnh vc p dng phn tch quang ph, nhy cao thng i i vi nng thp dn n hp th thp. pht hin chnh xc s khc nhau rt nh gia mu trng v mu th, detector phi c nhiu thp mc cng cao.- tng kh nng , diode quang c s dng trong cc my o quang ph. Trc kia, diode quang c nhy thp trong khong UV thp, nhng vn ny c hiu chnh trong cc detector hin i. Gii hn pht hin xp x 170-1100 nm i vi detector s dng silicon. Cc my quang ph hin i c mt dy cc detector diode quang thay v mt detector.-H quang hc : Cc thu knh hoc cc gng lm c dng chuyn tip v hi t tia sng qua thit b. Cc thu knh n gin th r tin nhng b sai lch v mu, l v cc tia sng c bc sng khc nhau khng c hi t cng ng mt im trong khng gian. Tuy nhin, vi thit k cn thn, s sai lch v h mu ca cc thu knh c th trong h quang hc c th c dng loi tr ln nhau v mt h quang hc c hiu qu c th c to thnh vi cc hp phn n gin v r tin ny. Cc gng lm khng t bng thu knh v hon ton khng c s sai lch v h mu. Tuy nhin, b mt bng nhm th d dng b n mn, do vy t hiu qu3.3. Phn loi my o quang ph3.3.1. My quang ph mt chm tiaC hai loi: quang ph thng thng v diode array3.3.1.1.My o quang ph thng thngTia sng a sc t ngun c hi t vo khe ti ca b phn n sc ho, n s truyn mt cch chn lc mt di hp ca tia sng. Tia sng ny sau i qua b phn o mu n detector. hp th ca mu c xc nh bng cch o cng nh sng n detector ca mu trng v so snh n vi cng nh sng n detector sau khi i qua mu thHnh 9 : S ca my o quang ph mt chm tia thng thngNh cp trn, a s cc my o quang ph c hai ngun n, mt n deuterium v mt n tungsten, v dng hoc l ng nhn quang hoc gn y hay dng hn l diode quang lm detector.Thit k ny l thch hp vi vic o hp th mt im ca ph. N khng thch hp trong vic o cc thnh phn khc nhau cc bc sng khc nhau hoc trong vic thu ph ca mu. thc hin qu trnh ny cc my o quang thng thng, cc phn ca b phn n sc ho phi quay cho nn ny sinh vn l s khng lp li v mt c hc i vi thit b. Hn na qu trnh thu s liu rt chm.3.3.1.2.My o quang ph diode array- nh sng a sc t ngun c i qua mu o v c hi t vo mt khe ti ca b a sc ho.- B phn a sc ho tn sc nh sng vo chui diode, trn mi diode o mt di hp ca ph. rng di ca tia sng c pht hin bi mt diode lin quan n kch c ca khe ti ca b a sc ho v kch c ca diode. Mi diode c chc nng nh l mt khe ra ca b n sc ho.Hnh 10 : S ca my quang ph diode array- V tr tng i ca mu v b phn tn sc l ngc so vi my quang ph thng thng, cu hnh ny c a ra l ngc v h quang hc.- hn ch cc phn ng quang ho c th xy ra, mt b phn ng c dng ngn tia sng t ngun pht ra cho ti lc o. Khi qu trnh o c bt u, b phn ng s t ng m v tia sng i qua mu n chui diode. S khc nhau v cng ca nh sng n detector khi o mu trng v mu th c ni n phn my quang ph thng thng.- My quang ph diode array bi vy o rt nhanh do cc s liu c thu ng thi, lp li ca bc sng tt v c tin cy cao.Hnh 11 ch ra h quang hc ca my o quang diode arry hin i HP 8453.Hnh 11: S my HP 8453Cu hnh mt chm tia ny c s ti thiu ca phn h thng quang thu c hiu qu cao nht v cha 1024 diode c th o t 190-1100 nm vi phn gii cao.- My mt chm tia thng r v c h quang hc n gin v bi vy nhy cao.- My quang ph diode array c bit thch hp vi mt chm tia v ph thu c rt nhanh v v c thi gian o gia mu trng v mu th c hn ch. Hn na cht ni chun c th c dng loi s dao ng ca n.- Trong my quang ph mt chm tia, mu trng v mu th c o lin tip nhau, vi khong thi gian cch nhau vi giy khi o im v mt vi pht nu ta o c di ph. S dao ng ca n c th xy ra v gy ra s sai s ln trong qu trnh o.3.3.2. My quang ph hai chm tia- My hai chm tia c pht trin loi s thay i ca cng n gia cc ln o mu trng v mu th. loi ny mt b phn ng ngt nh sng c t trn ng i ca h quang hc gn ngun sng. B phn ng ngt ny chuyn ng ca tia sng gia ng n mu trng v mu th ri n detector. N s quay mt tc sao cho vic o lin tip gia mu trng v mu th s xy ra vi ln trong mt giy, bi vy s hiu chnh uc s thay i ca cng n trong thi gian o (hnh 12).- So snh vi my mt chm tia, my hai chm tia c h quang hc phc tp hn, nn nhy gim. c nhy cao phi o trong thi gian di.- Theo l thng, n nh cao ca my hai chm tia l l do chnh trong vic thit k ca my quang ph hiu nng cao. Tuy nhin, vi nhng tin b gn y v n v thit k in m tng c n nh ca loi mt chm tia v dn n s quay tr li ca my mt chm tia. My mt chm tia c nhy cao v d s dng

4. NG DNG TRONG PHN TCH, KIM NGHIM

4.1. nh tnh4.1.1. S dng cc cc i hp thCc cc i hp th l c trng nh tnh ca cc cht nn hay c dng nh tnh.V d: Dung dch trong ncVitamin B12c cc cc i 278 nm, 361 nm v 550 nmChloramphenicol c cc i 278 nmDexamethanson c cc i 240 nm4.1.2. S dng cc cc i hp thkt hp vi dng phCho bit mc tinh khit ca mt cht :V d: Vitamin B12(theo USP 24)A361/A278t 1,70 n 1,90A361/A550t 3,15 n 3,40Penixilin V (theo BP 2003)A3060,36A2740.564.2.nh lngNhng vn trnh by y nhm gip cho vic tm hiu, nhn xt v nhng qui trnh nh lng c sn tin ti t xy dng nhng qui trnh kim nghim.4.2.1. Chn cc iu kin nh lng4.2.1.1. Chn bc sngTa thng chn bc sng ng vi cc i hp th ln nht.- Khi ng chun c dc ln nht. Cng mt sai sA sai sC nh nht.- Timax, sai s bc sng t nh hng.4.2.1.2.Chn khong nng thch hpKhong nng trong quan h gia hp th v nng l tuyn tnh.Mt vn khc c lin quan, c ghi trong mt s dc in l nng phi c chn sao cho hp th thu c ri vo khong vng ti u l 0,2 0,8 v cng gn 0,43 cng tt.iu ny c rt ra khi nghin cu s phthuc gia sai s tng i dA/A v gi tr hp th.Nu c iu kin ta vn nn tun theo yu cu ny, mc d c th vi my o hin i vi khuch i logarit, ch th hin s th cc vn va nu thay i4.2.1.3. Chn cc iu kin lm vic khc c kt qu ng, cht cn nh lng phi hp th nh sng ti vng my c th o c v khng b cn tr bi cc cht khc. Nhng trng hp nh dung dch tim B12, thuc nh mt Chloramphenicol 0,4%, cc iu kin va nu tho mn d dng. Tuy nhin, nhiu trng hp phi p dng cc bin php khc.Chitcht cn kim nghim khi tp ri mi nh lng c (v d: chit Vitamin B1bng dung dch HCl 0,1N khi cc t dc trong vin).Lm cc phn ng mu(i khi kh phc tp)V d: Dexamethason hp th cc i ti 240 nm, nhng bc sng ny nhiu cht hu c khc cng hp th (nht l trong cc ch phm). V vy, ngi ta dng phn ng vi Xanh Tetrazolium trong mi trng Tetramethyl amoni hydroxyd, sn phm mu o bc sng 485 nm.nh hng ca pHTrong dung dch nc, pH c nh hng rt ln n bc sng hp th cc i (max) cng nh hp th cc i ca dung dch (Amax). (Hnh 13)Khi pH thay i thmaxca dung dch s chuyn dch v Amaxcng thay i. Numaxchuyn v bc sng di th gi l s chuyn (bathocromic shift). Numaxchuyn v bc sng ngn th gi l s chuyn xanh (hypsocromic shift).nh hng ca dung miC nhiu dung mi trong sut thch hp cho vng kh kin v phn ln vng t ngoi. Cc dung mi phi tinh khit. Phn ln cc cht mch thng no u l nhng dung mi thch hp cho vng t ngoi. Khi chn dung mi trn c s ho tan th dung mi khng phn cc tt hn dung mi phn cc v cc dung mi khng phn cc cho cc nh hp th nhn hn.Bnh thng nc v cc alcol u c th l dung mi cho vng t ngoi, nhng nu cn etylic c ln aldehyd th s b hp th rt mnh cho nn phi lu v tinh khit ca dung mi.Hnh 13: nh hng ca pH i vi ph t ngoi ca PhenobacbitoneA: trong HCl 0,1M; B: trong m borat 0,05M (pH = 9,2);C: trong NaOH 0,5M (pH = 13)4.2.2. Cc phng php nh lng4.2.2.1. Phng php s dng E l phng php s dng trc tip nh lut Lamber Beer, ch yu di dng:A = E.l . Ckhi l = 1cmC =Mun p dng phng php ny cn phi c my quang ph hp th (vi b n sc ho dng lng knh hay cch t), ng thi my phi c chun c v bc sng ln hp th. chun ho v bc sng c th dng nhng knh chun nh knh Holmium hay Dydimium vi dng ph v cc tr s cc i xc nh. chun ho v hp th cng c th dng cc knh lc chun km theo my hay dng cc dung dch chun c th chun c c bc sng ln hp th. Hay dng hn l dung dch K2Cr2O7trong H2SO40,01N.4.2.2.2. Cc phng php s dng cht chunNh phng php ng chun, phng php so snh v phng php thm u i hi phi c cht chun. Trong mt s dc in lun lun qui nh phi lm so snh vi chun. Nhng chun ny phi c sn xut v cung cp bi nhng c quan c trch nhim.4.2.2.3. Chun o quangS thay i v hp th ca dung dch c th c dng xt v s thay i nng ca mt thnh phn trong qu trnh nh lng. hp th t l trc tip i vi thnh phn hp th. th biu din s ph thuc gia hp th v th tch dung dch chun (trong trng hp phn ng l hon ton) bao gm 2 ng thng m giao im ca 2 ng ny chnh l im tng ng.Hnh 14 : Cc dng ca chun o quang.(a) ng biu din ca s chun m ch c dung dch chun c s hp th, nh trong trng hp nh lng As3+bng dung dch Bromat Bromid.(b) ng biu din ca s chun m sn phm to ra c s hp th nh trong trng hp nh lng Cu2+bng EDTA.(c) ng biu din ca s chun khi dung dch chuyn thnh sn phm khng hp th(d) ng biu din ca s chun mt dung dch c mu to ra sn phm khng mu bi mt dung dch chun c mu.(e),(f) ng biu din ca s chun khi ta thm lin tip cc dung dch chun to ra lin tip hai phc cht c hp th khc nhau.u im: C th dng nh lng o th tch cc dung dch c mu m khng tm c ch th mu thch hp4.2.2.4. Phng php chit o quang (hay phng php acid mu)+ Nguyn tc:Chn mt cht acid mu thch hp cho tc dng vi cht base hu c trong nhng iu kin thch hp, theo c ch:xA-m+yB(H+)n=Ax-mBy(H+)n(acid)(base)(cp ion)+ Cc yu t nh hng:-pH: mi acid mu to cp ion vi base hu c mt pH nht nhV d:+ Helianthin thng pH 4,4 4,6+ Xanh bromothymol thng pH 6,6 6,8pH c nh hng rt ln ti s chit cp ion vo pha hu c.- Dung mi: Cn la chn dung mi thch hp chit trit cp ion to thnh m khng cho cht acid mu sang. Thng thng s dng cc dung mi: dichloroethan, chloroform, dichloromethan Trong mt s trng hp c th cho thm mt s cht c kh nng phn cc cao vo dung mi solvat ho cp ion to thnh d dng cho vicchuyn chng sang pha hu c.- Ngoi ra,nng thuc th, s ln chit, thi gian chit, khong tuyn tnh, s bn vng ca cp mu ion, cng rt cn c lu .+ Cc v d:nh lngChlorpheniraminmaleatPhenylpropanolaminhydrocloridAtropin sulfatAcid muHelianthinXanh bromothymolLc bromocresolpH4,57,05,6Dung miChloroformChloroformChloroformo A 4222nm4162nm416 nm4.2.2.5. nh lng hn hpa) Phng php ton ph:Do mt quang c s cng tnhnh lng ng thi hai thnh phn c ph hp th chng ln nhau.Ta c th o hp th ca dung dch hn hp ti t nht hai bc sng khc nhau trong s chnh lch v hp th ca hai thnh phn cng ln cng tt. (Hnh 15)Hnh15: Bc sng c chn nh lng hai thnh phn( bc sng c nh du bng mi tn).(k1/k2)1= max(k1/k2)2= maxNu gi A1v A2l hp th ca dung dch c cha hai thnh phn 1 v 2 ti cc bc sng1v2ta c:A1=C1+C2;A2=C1 +C2Cc h s,,vc th xc nh c bng cc php o ring bit cc dung dch ca cc thnh phn nguyn cht. Cn hai nng C1v C2cha bit, ta c th tnh c bng cch gii h 2 phng trnh trn.Phng php ny v nguyn tc c th em p dng cho cc hn hp n thnh phn. o mt quang n bc sng v gii h phng trnh c dng:A1=C1+C2+ .......+CnA2=C1 +C2+.........+Cn..........................................................An=C1+C2+.........+CnGii h phng trnh ny ta c cc nng phi tm C1, C2, ..., Cn.Thc t ch p dng tho mn cho cc hn hp 2 hoc 3 thnh phn v hai l do chnh sau: Tng tc lin phn t c th lm sai lch nh lut Beer, lm cho h s k khng cn l h s trung thc na. Hn na cc sai s ca dng c v cc s liu mt quang s tng ln gp bi khi gii h phng trnh, lm cho sai s ln. Gn y, ngi ta c d kin khc phc tr ngi ny bng phng trnh phc tp hn, trong cc php o mt quang cc bc sng ln hn n nhiu ln v cch tt nht l gii h phng trnh bng my tnh.b) Phng php ph o hm bc haiPhng php ph o hm l s chuyn dng ca ph hp th sang ph o hm bc mt, bc hai hoc bc cao hn. Ph o hm bc mt l th ca gradient ng cong hp th (tc ca s thay i hp th vi di sng, dA/d). i vi di sng ph o hm bc hai l th ca cong ca ph hp th (d2A/ d2). Nu hp th tun theo nh lut Lambert Beer, o hm bc hai ti bt k di sng no () cng lin quan ti nng bi phng trnh sau:d2A=d2A(1%, 1 cm)cdd2d2Trong :A: hp th bc sngA(1%, 1cm): hp th ring bc sngc: Nng ca cht hp th biu th di dng % (kl/tt)d: B dy ca lp cht hp th tnh bng cm.4.3. Mt s ch trong thc nghim- Truc khi o mu, phi bt my trc 15 pht cho my n nh- Mu em o phi bo m trong- Cc o phi tt, thng cho php sai s v dy ca cc l0,01 mm.- thu c kt qu chnh xc, nn s dng cng cc o vi mu chun v mu th- t cc o phi thng ng trong khoang o mu, nn lun lun hng cng mt mt ca cc o v pha nh sng.- Cc o phi oc trng ra t nht 3 ln bng dung dch nh o.- Cc phi c dng cn thn trnh xc, trnh li vt tay trn b mt ca cc o, v du t vt tay c th gy ra s hp th ln.- Khi o mu c chun b trong dung mi d bay hi th phi dng np y trong qu trnh o mu, trnh sai s v nng .- i vi nhng mu yu cu nghim ngt v nhit th phi c b phn iu nhit trong sut qu trnh o.

Bi thc hnh:nh lng Paracetamol trong vin nn bng phng php o quang(theo DVN III)

1. Nguyn tcTrong cng thc cu to ca paracetamol c cc lin kt i lin hp, do vy, n c kh nng hp th nh sng t ngoi. Ngoi ra, paracetamol cn ho tan trong kim. V vy, s dng dung dch trong kim nh lng paracetamol bng o quang ti bc sng cc i hp th 257nm.Tnh kt qu theoA(1%, 1cm); ly 715 l gi tr A(1%, 1cm) .2. Tin hnhCn 20 vin, tnh khi lng trung bnh vin, nghin thnh bt mn. Cn chnh xc mt lng bt vin tng ng khong 0,15 g paracetamol, cho vo bnh nh mc 200 ml, thm 50 ml dung dch natri hydroxyd 0,1N (TT), 100 ml nc, lc 15 pht thm nc n vch. Lc u, lc qua giy lc kh, b 20 30 ml dch lc u.Ly chnh xc 10 ml dch lc cho vo bnh nh mc 100 ml. Thm nc va n vch, lc u.Ly chnh xc 10 ml dung dch trn cho vo bnh nh mc 100 ml, thm 10 ml dung dch natri hydroxyd 0,1N ri thm nc n vch, lc u. o hp th ca dung dch bc sng 257 nm, dng cc dy 1cm. Mu trng l dung dch natri hydroxyd 0,01N. Tnh hm lng paracetamol (C8H9NO2) theo A(1%, 1cm); ly 715 l gi tr A(1%, 1cm) bc sng 257 nm3. Tnh kt quLng paracetamol c trong vin tnh theo khi lng trung bnh vin c xc nh theo cng thcX(gam) =Atx 200 xm /715 x mtTrong :At: hp th ca mu th715: Gi tr A(1%, 1cm) bc sng 257 nmm: Khi lng trung bnh ca vin (gam)mt: Khi lng bt vin dng th (gam).

PHNG PHP QUANG PH

Phng php quang ph ng dng trong ngnh kim nghim thuc l mt s phng php phn tch s dng ph quang hc: Ph hp th t ngoi, kh kin, hng ngoi; Ph hp th nguyn t; Ph hunh quangthuc v cc phng php phn tch quang hc.Cc phng php phn tch quang hc da trn vic nghin cu s tng tc ca bc x nh sng trn cht kho st hoc s pht racc bc x nh sng di mt tc ng ha l no .V d: Khi chiu nh sng vo mt dung dch, c hin tng khc x hoc quay mt phng nh sng phn cc, hoc c th c s hp th quang hoc pht hunh quang.V vy kt qu nghin cu cho php ngi ta a ra cc phng php phn tch quang hc khc nhau.1 - Phng php quang ph hp th UV-VIS ( phng php o quang).Da trn kh nng hp th chn lc cc bc x ri vo dung dch cht nghin cu, hp th ny ph thuc vo nng cht tan.2 -Phng php quang ph hp th IR (quang ph hng ngoi).Khi phn t cht nghin cu b chiu tia hng ngoi, cc nhm nguyn t ca n s hp th nng lng v thay i trng thi dao ng, to nn mt nh hp th c trng trn ph IR, bc sng xut hin nh v tp hp cc nh l c s ca vic nh tnh.3 - Phng php quang ph hunh quang.Da trn kh nng pht ra cc bc x nhn thy c bc sng xc nh v c cng ph thuc vo hm lng ca cht nghin cu , khi n b mt chm tia t ngoi chiu vo.4 - Phng php quang ph hp th nguyn t.Da trn kh nng hp th cc bc x c trng chiu vo khi cht nghin cu c nguyn t ha trong ngn la hoc trong l. Da vo phn nng lng bc x b hp th c th nh lng c cc nguyn t.5 - Phng php o phn cc.Da trn s o gc quay ca mt phng nh sng phn cc i qua dung dch nghin cu.6 - Phng php o khc xDa trn s o chit sut ( gc khc x) ca nh sng i qua cht nghin cu.Ngoi ra cn cc phng php phn tch quang hc khc nho c, o quang ngn la, o ph pht x nguyn t, o ph pht x PlasmaTrong chng ny chng ti ch gii thiu cc phng php quang ph c ng dng nhiu trong kim nghim thuc, l :Phng php quang ph UV-VI S;Phng php quang ph hunh quang.

PHN TCH, KIM NGHIM THUCBNGQUANG PH HUNH QUANG

1. KHI NIM V PHNG PHPPhng php quang ph hunh quang(Fluorescence spectrophotometry)l mt trong cc phng php phn tch quang hc. Cc phng php phn tch quang hc da trn vic nghin cu s tng tc ca bc x nh sng trn cht kho st hoc s pht ra cc bc x nh sng di mt tc ng ha l no .V d: Khi chiu nh sng vo mt dung dch, c hin tng khc x hoc quay mt phng nh sng phn cc, hoc c th c s hp th hoc pht hunh quang.V vy kt qu nghin cu cho php ngi ta a ra cc phng php phn tch quang hc khc nhau.- Phng php quang ph hp th ( phng php o quang).- Phng php quang ph hunh quang.- Phng php quang ph hp th nguyn t.- Phng php o phn cc.- Phng php o khc x...Phng php hunh quang da trn nguyn tc: Khi chiu mt chm tia t ngoi (bc x kch thch) vo mt s cht, cc cht ny pht ra bc x nhn thy c bc sng xc nh ty thuc tng cht v c cng ph thuc vo hm lng ca chng. Khi ngng chiu bc x kch thch, nu bc x pht quang tt ngay th s pht quang ny gi l hunh quang, nu bc x pht quang cn tn ti mt lc th gi l ln quang. Trong bi ny chng ti ch cp ti hunh quang.Trong kim nghim thuc, quang ph hunh quang c ng dng nh tnh, nh lng cc dc cht c kh nng pht hunh quang hoc to c dn cht pht hunh quang. Tuy khng ph bin bng quang ph hp th, nhng quang ph hunh quang c nhy cao hn hng trm ln v c th phn tch chn lc cc cht trong hn hp.2. I CNG V QUANG PH HUNH QUANG2.1. S hnh thnh quang ph hunh quang2.1.1. Khi nim v hin tng pht quangHin tng pht quang ni chung l tnh cht ca cc cht c kh nng pht quang di cc tc nhn kch thch khc nhau. Cc tc nhn c th do ma st, do nhit, do hiu ng cc phn ng ha hc, do tc nhn chiu x ca cc bc x in t... (c gi l: nhit pht quang, in pht quang, ha pht quang, sinh pht quang... ). Khi tc nhn kch thch l cc tia t ngoi hoc phn c bc sng ngn ca nh sng kh kin ta c hin tng pht quang quang hc hay cn gi l hunh quang.2.1.2. S to thnh ph hunh quang - C ch ca s pht quangTheo thuyt lng t, mi ht s cp (nguyn t, ion, phn t) c mt h thng duy nht cc trng thi nng lng. Trng thi nng lng thp nht gi l trng thi c bn (S0). Khi b kch thch, nh c hp th photon nh sng th nng lng ca photon s c truyn sang ht v ht s c chuyn sang mt nng lng cao hn gi l trng thi kch thch S1; S2...( mi trng thi phn t c th tn ti cc phn mc nng lng khc nhau 0; 1; 2; 3... ) sau mt thi gian rt ngn (10-610-9giy ) cc ht trng thi kch thch s tr v trng thi c bn ban u theo mt trong cc hin tng sau y:-Hin tng bt hot(Deactivation; Vibrational relaxation):Cc phn t mc nng lng cao ca trng thi ny tr v mc nng lng thp nht ca trng thi v gii phng ra nng lng di dng nhit nng do va chm gia cc phn t lm tng nhit mi trng, gi l hin tng phc hi khng bc x hay l s "th gin" phc hi dao ng sau khi b kch thch (thi gian phc hi < 10-12giy).-Hin tng chuyn ni(Internal conversion):Cc nguyn t t mc thp nht ca trng thi kch thch v trng thi c bn gii phng ra nng lng bng cch bc x cc photon nh sng, gi l s phc hi c bc x, nh sng bc x ny chnh l hunh quang (ko di 10-910-6giy).-Hin tng vt ni h (Intersystem crossing):Cc phn t trng thi kch thch S1khng chuyn v trng thi c bn S0m chuyn sang trng thi siu bn T1, ri mi t T1chuyn v S0v bc x cc photon nh sng lm cho qu trnh pht quang xy ra chm gi l hunh quang chm hoc ln quang (10-310 giy)Cc hin tng ny c biu din s hnh 1.Hnh 1. Cc hin tng quang hcxy ra sau khi cc ht s cp b kch thchTrong hin tng pht quang, mt phn nng lng ca nh sng kch thch b tiu tn chuyn thnh nhit nng do hin tng bt hot v vy nng lng nh sng kch thch bao gi cng ln hn nng lng nh sng bc x, hay ni cch khc, bc sng nh sng bc x lun di hn bc sng ca nh sng kch thch, to ra s dch chuyn cc i pht quang v pha bc sng di hn so vi cc i hp th, gi l s dch chuyn Stock. Cht no c dch chuyn Stock cng ln th php o hunh quang cng chnh xc (v t b nh hng ca ph hp th). V d: Xem hnh 2.

Hnh 2: Ph kch thch EX v ph bc x y EM ca Griseophunvin.Cc i EX = 297 nm. Cc i EM = 416 nm.2.2. Cc i lng c trng ca ph hunh quangHiu sut hunh quang(K hiu lF): L c trng vt l ca mt phn t, cho bit kh nng hunh quang ca mt chtS photon pht xF=0