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-1TRNG I HC NHA TRANG KHOA CNG NGH THC PHM

Bi ging CC PHNG PHP HIN I NG DNG TRONG PHN TCH THC PHM

Bin son: TS.GVC. HONG TH HU AN

NHA TRANG, 10/2012

Chng 1. GII THIU CHUNG V

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K THUT PHN TCH THC PHM


I. S CN THIT PHI NH GI CHT LNG THC PHM Hin nay, ngnh cng nghip thc phm nc ta ang pht trin nhanh chng. X hi cng pht trin, yu cu ca ngi tiu dng i vi cht lng thc phm ngy cng cao. Vic nh gi ng cht lng thc phm s gip c s sn xut pht hin nhng xc nh c nguyn nhn khng m bo phm cht lng (sai st trong khu s dng nguyn liu, qui trnh ch bin thc phm, trong cc thao tc trong sn xut,) iu chnh kp thi qu trnh sn xut nhm nng cao cht lng sn phm. V vy, vic nh gi cc ch tiu cht lng thc phm l cng tc rt cn thit trong cng nghip ch bin thc phm. t bit Vit Nam hin nay, trong tnh trng cc c s sn xut cha tun th nghim ngt cc quy nh lut php v an ton thc phm, vic kim sot cht lng thc phm l vn ln t ra i vi cc c quan qun l an ton v sinh thc phm. II. KHI QUT V CC PHNG PHP PHN TCH NH LNG NG DNG TRONG PHN TCH THC PHM Cc phng php phn tch nh lng c chia lm 2 nhm ln: II.1. Phng php phn tch ha hc (phng php phn tch c in): da trn vic s dng phn ng ha hc gia cht cn phn tch vi thuc th no : aX + bR = cP + dQ Phng php phn tch ha hc gm: II.1.1. Phng php phn tch th tch (phng php chun ): II.1.1.1. Nguyn tc: o th tch VR ca dung dch chun R R: dung dch chun (c nng NR bit chnh xc) cn dng VR (ml); NR phn ng va vi mt th tch chnh xc VX ca mu phn tch (cha cht X cn xc nh) theo phn ng tng qut: aX + bR = nP + d Q Da vo cc th tch V X, VR; NR c th tnh c hm lng cht X c trong mu phn tch.
Nx = VR .N R VX

X: dung dch phn tch Vx ml; Nx

II.1.1.2. Phn loi phng php phn tch th tch:

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a) Phng php chun acid baz (phng php trung ha): * Nguyn tc: da trn vic s dng phn ng trung ha A1 + B2 = B1 + A2 (A1/B1 v A2/B2 l 2 cp acid - baz lin hp) b) Phng php chun kt ta: * Nguyn tc: da trn vic s dng phn ng to thnh hp cht kh tan gia cht cn xc nh X vi thuc th to ta R (dung dch chun): nX + mR = XnRm Thng dng nht l php o bc: xc nh cc halogenur (Cl -, Br-, I-, SCN-) bng dung dch chun l AgNO3 (phng php Mohr; phng php Fajans; phng php Charpentier-Volhard) c)Phng php chun phc cht: * Nguyn tc: da trn vic s dng phn ng to thnh mt phc cht bn, khng mu gia cht cn xc nh (X) vi dung dch chun l mt thuc th to phc R no : nX + mR = [XnRm] (phc cht) Thng dng nht l phng php chun complexon: s dng dung dch chun l complexon III (mui dinatri ca EDTA; EDTA) chun cc cation kim loi. d) Phng php chun oxy ha kh * Nguyn tc: Da trn vic s dng phn ng oxy ha kh: aOx1 + bKh2 = cKh1 + dOx2 (Ox1/Kh1 v Ox2/Kh2 l 2 cp oxy ha kh lin hp) Cc php chun oxy ha kh thng dng l: - Php o permanganat: dng dung dch chun l KMnO4 - Php o Dicromat: dng dung dch chun l K2Cr2O7 - Php o Iod Thiosulfat: dng dung dch chun l I 2 chun cht kh (chun trc tip; chun ngc) hay dng dng dch chun Na 2S2O3 (chun gin tip cht oxy ha). II.1.1.3. u nhc im v phm vi ng dng ca phng php chun : - u im: nhanh, kh chnh xc (sai s khong 0,11%), dng c n gin, r tin - Nhc im: gii hn nh lng cao (c 10 4 M). - Phm vi ng dng: xc nh cc cu t a lng (hm lng 0,1 100%)

II.1.2. Phng php phn tch khi lng

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* Nguyn tc: Chuyn cu t cn phn tch X c trong mu nghin cu vo trong thnh phn ca mt cht thnh phn ha hc xc nh (gi l dng cn, k hiu: C). Da vo khi lng dng cn C (m c, gam), khi lng mu phn tch (a, gam), cng thc phn t ca dng cn, c th tnh c hm lng cht phn tch X trong mu nghin cu. * u nhc im: - u im: chnh xc kh cao (0,1%). - Nhc im: mt nhiu thi gian - Phm vi ng dng: t dng (ch dng khi yu cu kt qu phn tch c ng cao). II.2. Phng php phn tch vt l ha l (phng php phn tch hin i; phng php phn tch cng c) II.2.1. Nguyn tc chung: da trn vic s dng cc thit b phn tch o cng ca mt i lng vt l no lin quan n cu t phn tch, t xc nh hm lng ca cu t phn tch. II.2.2. Phn loi cc phng php phn tch cng c: II.2.2.1. Phng php phn tch quang hc : da trn s tng tc gia cc tiu phn cht phn tch vi bc x in t. Cc phng php phn tch quang hc thng dng trong phn tch thc phm gm: - Phng php quang ph hp th phn t UV-Vis (UV-Vis Spectrometry) - Phng php quang ph pht x nguyn t (AES: Atomic Emission Spectrometry) - Phng php quang ph hp th nguyn t (AAS: Atomic Absorption Spectrometry) b) Phng php phn tch in ha : da trn nhng hin tng lin quan n tnh cht in ha ca mu kho st. Cc phng php in ha thng dng trong phn tch thc phm bao gm: * Phng php o th (Potentiometry): - Phng php trc tip: Nguyn tc: o th ca in cc ch th i vi ion cn xc nh. ng dng: o pH; nh lng cc cation v anion khc nhau trong dung dch V d: nh lng Na+; K+; Ca2+; Pb2+; NO3-, F-; Cl- , CN-,

- Phng php chun in th:

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Nguyn tc: o s thay i th ca in cc ch th i vi ion cn xc nh trong qu trnh chun . V ng chun , t xc nh chnh xc im tng ng. ng dng: - Chun chn lc cc ion (ngay c khi dung dch c mu) - Cho php chun c cc cu t a lng v vi lng (nng ln hn 10 5 10 6 M) * Phng php phn tch cc ph (Polarography): Nguyn tc: Nghin cu s ph thuc ca cng dng khuch tn (gy ra do s khuch tn cc cation trong dung dch n catod) khi tng dn in th p t ln catod (gi l sng cc ph). nh lng cc cation da vo chiu cao sng cc ph. c) Phng php phn tch sc k: Nguyn tc: Tch hn hp cu t phn tch da trn kh nng tng tc khc nhau ca cc cu t i vi pha tnh (pha ng yn) v pha ng (pha kh hay lng, di chuyn lin tc qua pha tnh) ri nh lng cc cu t tch ra nh phng php phn tch ha l (o quang; o dn din; ch s khc x,) Cc phng php phn tch sc k thng dng trong phn tch thc phm l: - Sc k bn mng - Sc k lng hiu nng cao - Sc k kh
PHNG PHP PHN TCH NH LNG

PP HA HC (PP PT c in)

PP VT L - HA L (PP PT cng c/ PP PT hin i)

PT TH TCH Chun acid - baz Chun phc cht Chun kt ta Chun oxy hakh

PT KHI LNG

PT QUANG: QP phn t QP nguyn t - Hp th - Pht x

PT IN HA o th o dn in o in lng in khi lng Cc ph /Volt-Amper

PT SC K Sc k in di

Hnh 1.1. Phn loi cc phng php phn tch cng c II.2.3. u - nhc im ca phng php phn tch cng c

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u im: - chnh xc cao - Gii hn nh lng thp, do c th s dng lng mu nh v phn tch cc cu t vi lng v vt. - chn lc cao, do vy c th phn tch cc mu c thnh phn phc tp - Tc phn tch nhanh nh h thng thit b c t ng ha Nhc im: - Thit b t tin - Ngi phn tch cn c chuyn mn su s dng hiu qu thit b ng dng: dng nh lng cc cu t vi lng (hm lng c 0,01- 0,1%) hay cu t vt (hm lng < 0,01%) trong cc mu thc phm, dc phm, mi trng, sinh hc, Ngoi ng dng trong phn tch nh lng, mt s phng php phn tch vt l v ha l cn c dng trong nghin cu cu trc vt cht nh: - Phng php quang ph hp th t ngoi - kh kin (ph UV-Vis) : Nhn bit phn t cht hp th nh hnh dng ph hp th v cc nh hp th c trng. - Phng php quang ph hng ngoi (ph IR): Xc nh s c mt v v tr ca cc nhm chc trong cc phn t hp cht hu c da vo cc tn s dao ng c trng cho nhm chc xut hin trong ph IR. - Phng php ph khi lng (ph MS): Bn ph phn t thnh cc ion. Xc nh phn t lng v cu trc phn t da vo khi lng v in tch (t s m/z) ca mnh ion phn t v cc mnh ion c to thnh. - Phng php ph cng hng t ht nhn (ph NMR) : t mu phn tch (kh tinh khit) cha phn t cht nghin cu cu trc vo trong mt t trng ngoi, kch thch cc ht nhn nguyn t (thng l ht nhn 1H hay 13C) trong phn t bng mt bc x v tuyn c tn s thch hp xy ra s chuyn ht nhn ln trng thi c mc nng lng t cao hn (gi l s cng hng t ht nhn). Xc nh s c mt ca cc nhm chc c trong phn t da vo cng v v tr ( chuyn dch ha hc) ca cc tn hiu cng hng c trng cho cc nhm chc ny. - Phng php ph nhiu x tia X : Ghi ph nhiu x tia X ca cht rn tinh th. Xc nh cu trc mng li tinh th cht rn da vo cc ch s c trng. III. PHN LOI CC THNH PHN HA HC TRONG THC PHM NGUYN TC CHN PHNG PHP PHN TCH THC PHM C th phn loi cc ch tiu cht lng thc phm thnh cc nhm sau da theo hm lng ca cu t cn phn tch trong mu:

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a) Cc thnh phn a lng: l cc thnh phn ha hc c bn c trong nguyn liu ch bin thc phm hay cc thnh phn khc c hnh thnh trong qu trnh ch bin, bo qun thc phm nh lng cc thnh phn a lng ny c th dng cc phng php phn tch ha hc (phng php chun ; phng php phn tch khi lng). V d: - Xc nh m thi (NH3); N tng s bng phng php Kjeldahl; xc nh acid amin tng s bng phng php chun formol,(chun acid baz) - Xc nh hm lng mui n bng phng php Mohr (chun kt ta) - nh lng Ca2+ , Mg2+ trong cc m thc vt; xc nh cng ca nc, bng phng php chun complexon (chun phc cht) - Xc nh glucid (ng kh, saccharose, ng tng, tinh bt,) bng phng php Bertrand hay phng php Xanh Metylen; xc nh etanol bng php o dicromat; nh lng vitamin C, SO2 bng php chun Iod Thiosulfat ( chun oxy ha kh) - Xc nh hm lng nc (hay cht kh tng s), khong tng s, lipid tng s bng phng php phn tch khi lng. b) Cc thnh phn vi lng: thng l cc ph gia c a vo trong qu trnh ch bin thc phm. V d: Cht chng vi sinh vt; chng oxy ho, cc cht chng ng vn; cht iu chnh m; cht lm trong; cht lm c v to gel; cht iu chnh chua; cht iu v; cht ngt nhn to; cht to mi; cht to mu; cc enzyme, Cc ph gia thc phm thng c nh lng bng phng php phn tch cng c thng thng (trc quang so mu; sc k bn mng; GC; HPLC,) c) Cc thnh phn vt: bao gm cc c t c trong thc phm do nguyn liu ch bin khng m bo cht lng, hoc b thi nhim t cc thit b sn xut/bao b trong qu trnh ch bin v bo qun, hoc c hnh thnh do cc phn ng sinh ha xy ra trong qu trnh ch bin thc phm khng ng cch, V d: Kim loi nng (Pb, Hg, Cd, As, Se, Cu, Sn, Cr,); c t do vi sinh vt tit ra (aflatoxin); d lng thuc tr su; d lng khng sinh; d lng cht kch thch tng trng; c cht gy ung th (3-MCPD), Cc c t thc phm thng c bng phng php phn tch cng c c nhy cao (GF-AAS; ICP-MS; GC-MS; LC-MS,). Trong cc chng sau s gii thiu mt s phng php phn tch hin i ng dng trong nh lng cc ph gia v c t thc phm.

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Chng 2. PHN TCH THC PHM BNG PHNG PHP QUANG PH HP TH PHN T UV-Vis
y l phng php phn tch c ng dng rt rng ri trong phn tch thc phm. Phng php ny da trn hin tng hp th nh sng bi cc phn t cht nghin cu. II.1. S hp th nh sng bi cc phn t: Chiu mt chm bc x n sc c bc sng thch hp i qua mt h ng nht ( th kh hay dung dch) cha cc phn t cht nghin cu. Nu nng lng ca bc x s dng tng ng vi nng lng cn cung cp chuyn di cc electron trong phn t t trng thi c bn (mc nng lng thp nht: E 0) ln trng thi kch thch (c mc nng lng cao hn: E*) th s xy ra hin tng hp th nh sng:

E*
h = E = E* - E0

E0
Hnh 2.1. iu kin xy ra hin tng hp th nh sng

Hnh 2.2. Cc kiu chuyn di electron c bn trong phn t hp cht hu c Khi phn t b kch thch bi bc x UV hay Vis, c th xy ra mt s cc kiu chuyn di electron trong phn t cht hu c khi b kch thch: Chuyn di *: xy ra nhng hp cht c lin kt n, i hi nng lng ln nht, ng vi vng UV xa ( < 190 nm) Chuyn di *: rt quan trng, xy ra nhng hp cht c lin kt bi v c cu trc ni i lin hp (cc hp cht thm). Chuyn di ny ng vi bc

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x c bc sng khong 200 nm. Mch lin hp phn t cng di th bc sng hp th cng chuyn v vng sng di. Chuyn di n * hay n *: lin quan ti nhng phn t cha cp electron khng phn chia trong cc d t N, O, xy ra vng gn 300 nm. Nhng bc sng hp th trn thay i ng k khi c mt cc nhm th trong phn t. Cng mt mc nng lng in t, trong phn t cn c nhiu mc nng lng dao ng v quay. Do , ph hp th in t ca phn t kh rng (l ph m, khng phi ph vch) Trong phng php quang ph hp th phn t UV-Vis s dng di bc x thuc vng t ngoi gn ( = 200 400 nm) hay vng kh kin ( = 400 800 nm). II.1. C s l thuyt ca phng php quang ph hp th phn t UV-Vis - nh lut Bouguer Beer - Lambert Chiu mt chm nh sng n sc (cng I 0) vung gc vi mt cuvet (chu o) ng dung dch cha cc phn t cht nghin cu. Trn ng i ca tia sng, c th xy ra cc hin tng sau: - s phn x nh sng trn b mt cuvet - s hp th nh sng bi vt liu lm cuvet - s hp th nh sng bi cc phn t c trong dung dch Kt qu l cng nh sng sau khi i qua dung dch s gim i. Nu xem rng khng xy ra s phn x nh sng trn b mt cuvet (s dng c b mt rt nhn) v s hp th nh sng bi vt liu lm cuvet (chn vt liu thch hp) th: I0 = IA + IT trong : IA: cng bc x b hp th IT: cng bc x sau khi i qua dung dch -

l I0 () I
A

I
T

Dung dch hp th (nng C) S hp th nh sng tun theo cc nh lut ca Bouguer (1729), Beer (1760) v Lambert (1852):

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gim cng nh sng sau khi i qua dung dch t l vi b dy lp dung dch nh sng truyn qua v nng cht hp th: IT = I0.e- k C l hay:
lg I0 =Cl IT

( : h s t l)

nh gi cng hp th nh sng ca dung dch, ngi ta dng cc i lng sau: - truyn sut (transmitance, k hiu l T): l t s gia cng tia l (I T) v cng tia ti (I0)
T = IT I0

T% =

IT .100% I0

- hp th (absorbance ; k hiu l A) hay cn gi l mt quang (optical density, k hiu l OD):

A = .l.C
Vy : A = - lg
IT = - lg T I0

H thc A = .l.C thng c gi l biu thc ton hc ca nh lut LambertBeer. c gi l h s hp th, n ch ph thuc vo bn cht ca phn t cht hp th, vo bc sng tia ti, dung mi s dng v nhit . Nu [l] = cm, [C] = mol/lt th [] = lt.mol 1.cm 1 c gi l h s hp th mol (hay h s tt phn t gam) Nu [l] = cm, [C] = g/lit th [] = lt.g 1.cm 1 c gi l h s hp th ring. i vi mt dung dch cht hp th kho st, khi b dy dung dch khng i (c nh b dy cuvet) th quan h A - C l tuyn tnh: A = k.C. y chnh l c s ca php nh lng bng phng php quang ph hp th phn t UV - Vis. II.2. Mt s lu khi s dng phng php quang ph hp th phn t UV - Vis: 1/ H s hp th ph thuc vo bc sng nh sng n sc s dng th biu din s ph thuc ca vo bc sng c gi l ph hp th ca cht nghin cu (Hnh II.2). Ph hp th ca cc phn t cht hu c trong vng UV-Vis thng l mt di rt rng gy ra bi rt nhiu chuyn di ca cc electron gia cc mc nng lng rt gn nhau. Ph hp th ca cc cht thng c mt hay vi cc i. Bc sng ti xy ra s hp th cc i c gi l bc sng hp th cc i (wavelength of maximum absorbance ; k hiu l max). Gi tr max ph thuc

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bn cht ca phn t cht hp th, y l mt trong cc c trng nhn bit mt hp cht. tng nhy v chnh xc ca php nh lng lng bng phng php o quang UV Vis, cn o hp th ca dung dch bc sng ng vi s hp th cc i ca cht nghin cu.

(nm)

ma
Hnh 2.3. Ph hp th phn t UV-Vis x 2/ hp th c tnh cng tnh: hp th ca mt hn hp cc cht bng tng hp th ca tng cht hp th c trong h: A 1+2++ n = Ai = A1 + A2 +.....+ A0 Do , hp th ca mt dung dch bng tng hp th ca cht tan v dung mi. A dung dch = Acht hp th + A dung mi Trong thc t, dung dch khng ch cha cht hp th cn nghin cu X v dung mi m c th c mt s cc thnh phn khc (cc cht l, tc nhn iu pH mi trng, thuc th to phc,...). Dung dch cha tt c cc cht nh trong dung dch phn tch ngoi tr cht phn tch c gi l dung dch nn (blank solution). V vy, mt cch tng qut c th xem rng: A dung dch = Acht hp th + A dung dch nn o c hp th gy ra bi ring cht hp th X, cn pha ch dung dch nn hiu chnh my (a hp th nn v gi tr 0). Khi , hp th gy ra ch do cht hp th s ng bng gi tr hp th ca dung dch em o: 3/ nh lut Lambert Beer ch ng trong iu kin: - Bc x s dng phi n sc - Dung dch o phi trong sut v ng nht. - Khng c s tng tc gia cc phn t cht hp th. V vy, thng ch p dng nh lng cc cc dung dch c nng hp hn 10 2 M.

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4/ Trong nhiu trng hp, thng s dng bc x kh kin nh lng cc cht (phng php trc quang - so mu: photocolorimetry). Mun vy, dung dch cht hp th phi c mu m v bn. Trong thc t, a s dung dch cha cu t cn nh lng (X) khng mu hay c mu rt nht. Khi , tng nhy ca php phn tch bng phng php trc quang - so mu, cn chuyn X v dng phc c mu m bng phn ng vi mt thuc th R thch hp: X + HR XR + H+ php phn tch t kt qu chnh xc, c nhy v chn lc cao, cn lu cc im sau: - Phn ng to phc phi xy ra mt cch nh lng (phn ng xy ra hon ton chuyn ton b cht X vo trong phc mu). Mun vy, nn dng d thuc th to phc HR. Tuy nhin, lng thuc th to phc cng cn m bo sao cho phc to ra c thnh phn n nh. - Phc XR phi d tan. Nu phc to thnh km tan trong dung mi nc th c th chit phc ny sang dung mi hu c thch hp. V d: chit phc dithizonate ca Pb2+, Cu2+, Zn2+ sang dung mi CCl4; chit phc [Co(SCN)4]2- sang dung mi alcol isoamylic, Phc XR to thnh c mu cng m cng tt (h s hp th phi ln, c 103 tr ln) - Phc XR phi kh bn mu. i vi cc phc khng bn mu, cn tin hnh o trong khong thi gian thch hp sao hp th ca phc t gi tr n nh v cc i. - pH dung dch nh hng nhiu n hp th ca dung dch phc mu. Do , phi o trong khong pH sao cho hp th ca phc XR n nh v cc i. - nh lut Lambert Beer thng ch ng trong mt vng nng no ca cht nghin cu. Nguyn nhn l do vng nng thp A ca cht nghin cu xy ra s phn ly phc mu mnh hay s thay i bn cht phc mu do tng tc gia phc mu vi phn t dung mi; cn nng ln c th xy ra s lin hp phn t lm thay i bn cht cht hp th, dn n s thay i h s hp th . Do , cn tin hnh nh lng trong vng nng trong xy ra s tuyn Hnh 2.4. Khong tuyn tnh ca nh lut Lambert - Beer tnh A C.

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- Nng cc ion cn trong dung dch khng c vt qu gii hn cho php trnh nh hng n hp th ca dung dch. Nu cn, phi loi b cc ion cn bng cc bin php trc khi tin hnh o quang (v d: che, chn bc sng o thch hp, dng thuc th chn lc,...) - S thay nhit cng c th lm thay i bn cht ca phn t cht mu, v nh hng n cng mu ca phc. Do vy, cn to tin hnh to phc v o hp th dung dch trong khong nhit thch hp, sao cho hp th ca phc ln nht v n nh nht. - loi tr nh hng ca cc cu t cn n hp th dung dch, thnh phn nn ca dung dch nghin cu v cc dung dch chun phi cng ging nhau cng tt. II.3. Cu to ca quang k UV-Vis II.3.1. Cc b phn chnh ca quang k UV-Vis Quang k gm cc b phn chnh sau y (hnh 2.3) a) Ngun sng (light source): l cc loi n c kh nng pht ra cc bc x UV/Vis. * n halogen (hay n Tungsten / n Wolfram, k hiu: W): bng n bng thy tinh, bn trong cha kh tr v hi Iod; dy tc bng wolfram; pht ra bc x kh kin hng ngoi gn ( = 380 2500 nm) * n Deuteri (k hiu: D): bng n bng thch anh, bn trong cha hi thu ngn p sut thp 2 -3 torr, pht x nh to ra h quang in. Di bc x pht ra nm trong vng t ngoi gn ( = 180 375 nm) * n Xenon: pht ra bc x ch yu trong vng kh kin v mt phn bc x t ngoi gn. n Xenon hin i c th pht ra di bc x rt rng, bo gm c vng UV Vis v IR gn ( = 190 1100 nm) b) H thng thu knh (lens): cho php tp trung chm sng v hng tia sng theo ng i nht nh. c) B phn n sc (monochromator): l cc knh lc mu, lng knh hay cch t nhiu x cho php phn tch chm bc x a sc pht ra t cc ngun sng thnh bc x n sc. d) Chu o (cuvet): cha dung dch o, c th lm bng thy tinh (nu o trong vng Vis) hay thch anh (nu o trong vng UV). Cng c loi cuvet bng polymer dng o trong vng Vis nhng ch nn dng 1 ln. e) Detector: thng l t bo quang in hay t bo quang dn cho php chuyn nh sng thnh tn hiu in. f) B phn khuch i (Amplificator) : khuch i tn hiu in.

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g) B phn x l v hin th tn hiu (Signal Processing and Readout): cho php x l tn hiu v hin th kt qu o t ng trn mn hnh.

Hnh 2.5. Cc b phn chnh trong quang k UV Vis II.3.2. Phn loi cc thit b o quang: a) Phn loi theo bn cht ca b n sc: Ty theo bn cht v c tnh ca b n sc, ngi ta chia cc my o quang UV-Vis thnh cc loi sau: - My so mu (colorimeter): s dng b n sc l cc knh lc mu; cho n sc km ( = 10 50 nm) - Quang k (photometer): to ra bc x n sc bng cc lng knh; n sc cao hn ( = 5 - 10 nm) - Quang ph k (spectrophotometer): l loi my thng dng nht hin nay, trong bc x n sc c to ra bng lng knh hay cch t nhiu x nn cho n sc cao ( = 1- 2 nm). b) Phn loi theo s quang hc ca thit b Chia lm 2 loi: * My 1 chm sng (single beam): nh sng sau khi qua b n sc c i theo 1 ng duy nht n cuvet cha dung dch o. Cch vn hnh: - M my. - Chn n (nu my c 2 n) - Chn bc sng o. Ch 15 -30 min ri bt u o. - Dng mn chn en (shutter) ngn ton b nh sng chiu vo cuvet. iu chnh %T = 0 %. - a cuvet cha dung dch nn ngn ng mu. iu chnh %T = 100% (hay: hp th A = 0). - a dung dch o vo. c kt qu o.

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Hnh 2.6. S quang k UV-Vis 1 chm sng * My 2 chm sng (dual beam / double beam): nh sng sau khi qua b n sc c tch thnh 2 chm tia song song y ht nhau (1 chm i qua ngn cha dung dch nn; 1 chm i qua ngn cha dung dch mu) Cch vn hnh: - M my. - Chn n (nu my c 2 n) - Chn bc sng o. Ch 15 -30 min ri bt u o. - Dng mn chn en (shutter) ngn ton b nh sng chiu vo cuvet. iu chnh %T = 0%. - a 2 cuvet cha dung dch nn vo c 2 ngn ng mu. iu chnh %T = 100% (hay: hp th A = 0). - Cho dung dch mu vo cuvet trong ngn cha dung dch mu. c kt qu o H thng 2 chm sng t tin hn h 1 chm sng nhng cho kt qu chnh xc hn.

Hnh 2.7. S quang k UV-Vis 2 chm sng Lu : chnh xc ca php o hp th


dA .100% ph thuc vo thit b o quang A

v vo bn cht ca phn t cht hp th. Khi hp th ca dung dch qu cao (A > 1) cng bc x i n detector rt nh, do cn khuch i tn hiu ln rt nhiu ln o c nn sai s ln. Ngc li, nu hp th qu nh (A < 1) th nh

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hng ca nhiu nn n tn hiu o s ln. V vy, sai s tng i ca php nh lng


dC .100% nh thng ch nn o hp th trong khong A = 0,1 1,0. C

Hnh 2.8. S ph thuc ca sai s


dC .100% vo hp th vi cc loi C

detector khc nhau: a) photovoltaic detector; b) photomultiplier detector

II.4. Cc cch nh lng trong phng php quang ph hp th phn t UV-Vis Cc phng php ny u da trn vic p dng nh lut Lambert-Beer:

A = .l.C
II.4.1. Phng php trc tip: o hp th Ax ca dung dch nghin cu ri tnh nng cht hp th theo cng thc:
Cx = Ax .l

trong : l - b dy cuvet s dng; : h s hp th ca cht nghin cu bc sng s dng (tra cu ti liu) Phng php ny cho kt qu km chnh xc (do h s hp th ph thuc rt nhiu vo iu kin o) do t c s dng. Tuy nhin, nu khng cn nh lng chnh xc nng cht hp th m ch cn theo di quy lut bin i hm lng cht nghin cu trong mt qu trnh x l no th c th p dng c. II.4.2. Phng php so snh Gi s cn nh lng cht X trong dung dch phn tch (nng CX) Khi , theo phng php ny, cn pha ch mt dung dch chun cha cht nghin cu X (nng C0). To mu cho c dung dch phn tch 1 (cha cu t X cn xc nh) v dung dch chun trong iu kin y ht nhau. o hp th ca 2 dung dch trong dung dch mu v dung dch chun trong cng iu kin. Gi AX v A0 l hp th ca dung dch phn tch v chun. Theo nh lut Lambert - Beer: A0 = lC0
1

hay cn gi l dung dch mu (sample)

Cx =

Ax C0 A0

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Ax = lCx II.4.3. Phng php ng chun Pha ch mt dy chun ca cht nghin cu X vi nng tng dn theo cp s cng: C1, C2, ..., Cn (n = 5 8) To mu cho cc dung dch chun v dung dch mu ri o hp th trong cng iu kin. Dng ng chun A C (dng phng php bnh phng ti thiu). T , xc nh nng cht hp th trong dung dch mu bng phng php ni suy tuyn tnh.

Hnh 2.9. Phng php ng chun Ghi ch: - Thng dng cc phn mm xc nh phng trnh ng chun, t tnh c nng cht nghin cu. - Trong thc t, ng chun c th khng i qua gc ta v c dng: A = aC + b Khi , tnh nng cht hp th trong dung dch mu bng cng thc :
Cx = Ax b a

- p dng c phng php ny, nng ca dy chun v ca dung dch mu phi nm trong vng tuyn tnh ny phi nm trong vng nng tuyn tnh ca nh lut Lambert Beer. - Phng php ny ch cho kt qu ng nu mu c thnh phn nn n gin (ch cha t cu t hay khng c cu t nh hng). II.4.4. Phng php thm i vi trng hp mu c thnh phn nn phc tp (cha nhiu cu t nh hng n php o hay cha bit c cu t no c trong nn mu) th nn nh lng bng phng php thm. Trong phng php ny mu chun thm c pha ch bng

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cch ly chnh dung dch phn tch lm nn v thm vo lng xc nh dung dch chun ca cht phn tch. C 2 trng hp p dng phng php thm: a) Phng php thm mt mu chun Gi s cn xc nh nng cht X trong mt mu phn tch (c nng Cx). Pha ch dung dch chun thm bng cch thm mt lng chnh xc (CX) ca cht nghin cu vo dung dch phn tch. Vy, nng cht X trong dung dch chun thm s l: C0 = Cx + CX To mu ri o hp th ca dung dch chun v dung dch mu trong iu kin y ht nhau. Gi A0 v Ax ln lt l hp th ca dung dch chun v dung dch mu, ta c: Ax = lCx A0 = lC0 = l (Cx + C)

Cx =

Ax C A0 Ax

b) Phng php ng chun thm Pha ch mt dy n dung dch chun thm bng cch ly mt lng mu nht nh ri thm nhng lng xc nh C1, C2, ..., Cn ca cht phn tch (lng thm tng theo cp s cng). To mu v o hp th ca cc dung dch chun v mu trong cng iu kin. Dng ng chun thm: A = f (C).

Hnh 2.10. Phng php ng chun thm Nng cht nghin cu trong mu phn tch c th c xc nh bng cc phng php sau:

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- Cch 1: Ko di th v bn tri, ct trc honh ti u, l Cx. - Cch 2: T O k 1 ng ph song song vi th. T Ao k 1 ng thng song song trc honh, ct ng ph ti M. T M k vung gc trc honh ti Cx. - Cch 3: Dng phn mm EXCEL xc nh c phng trnh ng chun thm c dng: A = aC + b. C th chng minh c rng: CX = b/a II.4.5. nh lng mt hn hp cu t bng phng php o quang UV Vis Xt mt dung dch cha 2 cu t (I) v (II), c cc i hp th tng ng l 1 1 v 2. Gi A1 v A2 ln lt l hp th ca dung dch cho o bc sng 1 v 2 vi cuvet c b dy l = 1 cm. Da vo tnh cht cng tnh ca hp th, ta c:
(I ) ( II ) A1 = 1 .C ( I ) + 1 .C ( II ) (I ) ( II ) A 2 = 2 .C ( I ) + 2 .C ( II )

(I ) ( II ) ( II ) (I ) Cc h s hp th 1 ; 1 ; 2 v 2 c th c xc nh bng cch o

hp th ca cc dung dch chun ca cc cu t (I) v (II) ln lt cc bc sng 1 v 2. Gii h phng trnh 2 n trn s tnh c nng ca cc cu t (I) v (II) trong dung dch. Da trn nguyn tc ny, c th nh lng cc hn hp nhiu cu t. Hin nay, mt s my o quang hin i ci t cc phn mm cho php nh lng ng thi hn hp 8 cu t. II.5. nh gi phng php o quang UV Vis: - ng v chnh xc: ng ca phng php o quang UV-Vis ph thuc vo 3 yu t: thit b, ha cht, phng php phn tch v ngi phn tch. Trong iu kin l tng (xem rng s dng thit b v ha cht tt nht, phng php ng ; ngi phn tch c thnh tho) th kt qu phn tch c th t chnh xc ( lch chun tng i) khong 0,5%. - Gii hn pht hin (LOD : limit of detection): Gii hn pht hin (LOD) ca mt phng php phn tch l nng nh nht c th cho tn hiu ghi nhn c. i vi phng php trc quang, theo nh lut Lambert - Beer :
C min = Amin max .l

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i vi a s cht hp th, ta c: max 103 l.mol 1 .cm 1 . Nu dng thit b o hon thin nht (vi chnh xc ca thang o hp th l 0,001), ch v cuvet c b dy 1 cm, th:
LOD = C min 10 3 = 10 6 M 3 10 .1

- chn lc: khng cao do cng mt thuc th to phc mu c th tc dng vi mt s ion khc nhau to nn mu sc gn ging nhau. tng chn lc ca php phn tch trong mt s trng hp phi tch chit loi b cu t cn tr. II.6. M s ng dng ca phng php quang ph hp th phn t UV-Vis trong phn tch thc phm Do thit b tng i r tin, thao tc kh n gin, nhanh chng nn phng php ny c ng dng rng ri trong phn tch ni chung v phn tch thc phm ni ring. Nh s dng cc thuc th to phc mu, phng php cho php nh lng a s cc ion v c (Fe2+, Cu2+, Pb2+, Zn2+, Sn2+, ) v mt s hp cht hu c (dung dch protein, peptid, acid amin, acid nuleic,) hm lng vi lng. II.6.1. nh lng protein: 1) Phng php Biuret : Nguyn tc: Da vo phn ng gia protein v Cu+2 trong mi trng kim to phc mu xanh tm c hp thu cc i bc sng 540 nm. Phng php ny c s dng i vi dung dch mu phn tch c hm lng protein tng i ln (khong 20 2.000 g/ml)

Phc ng (II)-biuret a) Phng php Lowry: Hu ht cc protein u cha Tyrosin v Tryptophan. Hm lng ca nhng acid amin ny ty thuc vo loi protein. V vy, nhng protein cng mt loi vi nhau c hm lng cc acid amin ny ging nhau.

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Khi cho protein tc dng vi thuc th Folin s to thnh mt phc cht c mu xanh c trng (max = 750 nm). Cng mu ca phc ny t l vi hm lng Tyrosin v Tryptophan (v cng l hm lng protein). V th, c th o cng mu ca phc ny xc nh hm lng protein (dng phng php ng chun). b) Phng php Bradfort: Da phn ng to mu xanh ( max = 595 nm) gia protein vi thuc th Coomassie (Coomassie Brilliant Blue G-250: c mu cam ) trong mi trng axit Cng mu t l vi nng protein trong dung dch. nh lng protein trong dung dch bng phng php ng chun.

II.6.2.nh lng acid amin bng thuc th ninhydrin Ninhydrin (2,2-Dihydroxyindane-1,3-dione) l thuc th c mu vng. Phn ng ninhydrin l vi acid amin c dng nh tnh v nh lng axit amin, imino axit v amin. Nguyn tc : Khi un nng ninhydrin vi amin bc 1 trong mi trng kim s thu c sn phm c mu tm m (max = 570 nm). Phn ng qua cc giai on sau:

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Phc mu tm (max = 570 nm) Cc acid amin, protein v peptid u cha nhm chc amin bc 1. Tuy nhin, phn ng decarboxyl ha (phn ng u tin) ch xy ra khi c mt -amino acid t do nn ch c th dng phn ng ny nh tnh hay nh lng dung dch acid amin t do. hp th ca sn phm to ra ph thuc tuyn tnh vo s lng nhm amin t do nn c th dng phng php trc quang so mu (dng ng chun) nh lng hm lng acid amin t do. Dung dch protein hay peptid c th c nh lng bng cch thy phn trong mi trng kim to thnh amino acid ri nh lng nh trn (chun b mu trng l mu cha dch protein hay peptid cha b thy phn) II.6.3. nh lng mt s kim loi nng a) Xc nh Cu2+ bng phng php trc quang-so mu vi thuc th natri dietyldithiocacbamat (NaDDC; DDC) Nguyn tc: Trong mi trng kim yu (pH = 9 9,2), ion Cu2+ to vi thuc th DDC phc cng cua (chelate) c mu nu kh tan trong nc nhng tan tt trong cc dung mi hu c nh CHCl3, CCl4. Trong CHCl3 phc c mu nu nh vng (max = 430 nm). Cng mu ca phc Cu-DDC sau khi chit t l vi nng Cu2+ trong mt khong kh rng. Do , nh lng Cu2+ bng thuc th DDC, ngi ta dng phng php chit trc quang nh sau: Cho Cu2+ to phc vi DDC trong mi trng NH4OH kim c mt amonicitrat v complexon III che cc ion cn. Chit phc Cu-DDC sang CHCl 3 ri o quang 430 nm. b) Xc nh Zn2+ bng phng php chit - trc quang vi thuc th Dithizone (DTZ) Dithizon (tc Diphenylthiocacbazone) l thuc th hu c c kh nng to vi nhiu ion kim loi cc phc dithizonat kh tan trong nc nhng tan tt trong cc dung mi hu c nh CHCl3, CCl4. DTZ tan trong CCl3 to thnh dung dch c mu

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xanh, khng cn tr vic o quang cc phc to thnh. Do , ngi ta thng dng phng php chit trc quang vi DTZ xc nh chng. Nguyn tc: Trong mi trng pH = 4 7, Zn 2+ to vi DTZ mt phc c mu km tan trong nc nhng tan tt trong CCl4. pH ny cc ion Cu2+, Cd2+, Pb2+, Co2+, Bi3+, Hg2+, Ag+ cng to phc vi DTZ. trnh nh hng ca cc ion ny, tin hnh to phc Zn-DTZ pH = 5 c d SCN - v CN-. Loi cc cht c kh nng oxy ha DTZ (Cl2, Br2, I2, H2O2) bng cch un si dung dch. Mt s hp cht v c gy c dung dch CCl4 c loi bng cch v c ha mu bng H2SO4, HNO3, H2O2. o hp th ca phc Zn-DTZ 530 nm. c) Xc nh Pb2+ bng phng php chit - trc quang vi thuc th Dithizone Nguyn tc: To phc Pb-Dithizonate (K hiu: Pb-DTZ) trong mi trng pH = 8 9 c mt CN che cc ion kim loi khc. Chit phc Pb-DTZ sang dung mi CCl 4 ri o hp th 520 nm. Lu : - Trong mi trng ph = 8 9, Sn 2 v Bi3+ cng b chit cng vi Pb-DTZ nn trc phi chit cc phc Sn-DTZ v Bi-DTZ trong mi trng acid (Pb-DTZ khng b chit, cn li trong pha nc). Tch Sn2+ v Bi3+ trc nh sau: Thm hidrazin vo dung dch mu nc, un nng kh Sn 4+ v Sn2+ v kh cc cht khc. ngui, thm dung dch natri tactrat vo v chnh pH v 2,5 3 bng dung dch axit tactric. Tin hnh chit nhiu ln, mi ln bng 5 ml dung dch Dithizone 0,1% trong CHCl3 cho n khi pha hu c vn gi nguyn mu xanh ca Dithizone. Cui cng, lc pha nc vi ln vi CHCl 3 n khi pha hu c khng cn mu xanh. Bng cch ny c th loi b c Hg2+, Ag+, Cu2+. - Nu trong mu nc cha lng ng k cc cht hu c, cn phi v c ha chng bng cch cho vo vi ml HNO3 c v HClO4 c ri c mu n kh, sau tm t bng HNO3 v ha tan bng nc ct. - Cc cht c kh nng oxy ha c kh trc bng hidrazin. d) Xc nh Cd bng phng php chit - trc quang vi thuc th Dithizon Nguyn tc: To phc Cd-Dithizonate trong mi trng kim mnh c mt tartrat ri chit sang dung mi CCl4. o hp th ca dung dch phc to thnh (c mu ) bc sng 515 nm. Loi cc ion cn (Ag+, Ni2+, Co2+, Cu2+) bng cch chit chng bng Dithizone trong mi trng acid trc khi xc nh Cd2+.

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Chng 3. PHN TCH THC PHM BNG PHNG PHP QUANG PH NGUYN T
III.1. PHNG PHP QUANG PH HP TH NGUYN T (AAS: Atomic Absorption Spectrophotometry) III.1.1. Nguyn tc phng php : Khi chiu mt chm bc x n sc c bc sng thch hp qua mt m hi nguyn t th cc nguyn t s hp th mt phn nng lng ca chm bc x ny v chuyn t trng thi c bn sang trng thi kch thch. l hin tng hp th nguyn t.

E*
h = E = E* - E0

E0
S hp th nguyn t ch xy ra nu nh nng lng ca cc photon trong chm bc x s dng ng bng hiu cc mc nng lng kch thch v nng lng c bn ca bc chuyn di trong nguyn t : E* - E0 = h = hc/ Do , mi nguyn t ch c th hp th mt s bc x c tn s xc nh c trng cho cu trc electron ca nguyn t . Da vo tn s ca bc x b hp th, c th nh tnh cc nguyn t c trong mu phn tch. Ngoi ra, do hin tng hp th, cng ca nh sng sau khi qua dung dch s b gim i. hp th ph thuc vo s lng cc nguyn t gy ra hin tng hp th, tc l ph thuc vo nng nguyn t v on ng nh sng i qua m hi nguyn t. Thc nghim cho thy gia hp th A v nng nguyn t hp th trong mu phn tch c mi mi quan h sau: A = - lg (I/ I0) = k.a.Cb (*) trong : A : hp th I0, I : cng tia ti v tia l k : h s hp th, c trng cho bn cht ca nguyn t hp th a : hng s, ph thuc vo cu to thit b v iu kin phn tch C : nng cc nguyn t hp th c trong m hi nguyn t b : h s ph thuc vo nng cc nguyn t hp th c trong m hi (nu C nh th b = 1 ; nu C ln th 0 < b < 1) H thc (*) chnh l c s ca php nh lng bng phng php AAS.

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III.1.2. S cu to v nguyn l hot ng ca thit b AAS S cu to thit b AAS c trnh by hnh 3.1, gm nhng b phn chnh sau y:

Hnh 3.1. S cu to ca thit b AAS - Ngun sng (Light Sorce) - B phn iu bin (Modulator ; Chopper) - B phn nguyn t ha (Atomization ; Atomizer) - B n sc (Monochromator) - Detector - B phn x l v hin th tn hiu (Signal Processor/Readout) Nguyn l hot ng: Chm bc x n sc pht ra t ngun pht x c cng mnh, c trng cho nguyn t phn tch s c iu bin v chiu qua m hi nguyn t c to ra bng cch cung cp nhit cho mu phn tch. Khi , chm bc x ny s b hp th mt phn bi cc nguyn t ca nguyn t cn nh lng. Bc x sau khi b hp th s c phn tch bi mt b phn n sc, ri qua b phn ghi nhn, khuch i, x l v hin th tn hiu di dng cc vch hp th. Da vo v tr vch hp th c trng v cng tn hiu hp th c th nh tnh v nh lng cc nguyn t c trong mu phn tch.

Hnh 3.2. S hp th v ph hp th nguyn t

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III.1.3. Cc thnh phn chnh b phn chnh trong thit b AAS II.1.3.1. Ngun sng: cho php to ra cc bc x nhy, c trng i vi nguyn t cn phn tch. Chm tia ny phi c cng n nh, khng b nhiu lan bi cc yu t vt l v c th iu chnh c. Hin nay cc thit b AAS c th dng 3 loi ngun sng nh sau : a) n cathod lm (HCL: hollow cathode lamp): l loi ngun pht bc x n sc c dng sm v ph bin nht trong cc thit b AAS. y l 1 bng n bng thy tinh hay thch anh, bn trong cha kh tr c tinh khit cao (Ne, Ar, N2) di p sut rt thp (5 15 mmHg). Trong n c mt cathod lm lm bng nguyn t cn o c tinh khit rt cao (99,99%) v mt anod l mt si dy kim loi tr v bn (Pt, W). C hai in cc c gn cht trn b v catt phi nm ng trc xuyn tm ca n. Khi t 2 cc ca n di mt hiu th cao, cc nguyn t kh tr s b ion ha, sinh ra cc cation bn ph cathod ang nng , lm cho mt s nguyn t trn b mt cathod b ha hi, chuyn ln trng thi kch thch. Khi tr v trng thi c bn, cc nguyn t ny s pht ra cc bc x c trng cho nguyn t lm cathod. Mt b phn n sc s cho php chn lc trong chm bc x pht ra ny ny nhng tia c tn s c trng nht.

Hnh 3.3. Cu to n HCL Cc n HCL c cu to nh m t trn l cc n n, mi n ch phc v cho phn tch mt nguyn t. Hin nay cc hng ch to c cc n kp i (Ca + Mg; Cu + Mn; K + Na ) , kp ba (Cu + Pb + Zn) hay kp su nguyn t (Cu + Mn + Cr + Fe + Co + Ni), trong cathod ca n l hp kim ca cc nguyn t v thnh phn ph hp sao cho cng pht x ca cc nguyn t l gn tng ng nh nhau, khng nh hng ln nhau. Cc n kp thng c nhy km hn cc n n tng ng v ch to cng kh hn n n nn n nay cng ch c mt s loi m thi. b) n phng in khng in cc (EDL: Electrodeless Discharge Lamp) n EDL l mt ng thch anh chu nhit, di 15 18cm, ng knh 5 6cm, bn trong cha kh tr (Ne, Ar, N2 c p sut vi mmHg) v lng nh nguyn t cn

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phn tch (kim loi hay mui d bay hi ca nguyn t ) vi nng nht nh ph hp to ra chm tia pht x ch bao gm mt s vch ph nhy, c trng ca nguyn t phn tch. n ni vi mt cun cm ng bng ng ni vi ngun in cao tn. Khi n lm vic, di tc dng ca nng lng cao tn cm ng, n c nung nng , kim loi hay mui kim loi trong n c ha hi v b nguyn t ha, cc nguyn t t do c sinh ra s b kch thch v pht ra ph pht x ca n. chnh l vch pht x nhy, c trng ca kim loi cha trong n.

Hnh 3.4. Cu to n EDL So vi n HCL, n EDL cho nhy cao hn, n nh hn, bn hn v vng tuyn tnh ca php o cho mt nguyn t cng rng hn so vi n HCL. Tuy nhin s n EDL c sn xut t hn (hn 30 n) v gi thnh li cao hn. c) n ph lin tc c bin iu (Continous Sources): c pht trin gn y v c s dng nhiu hin nay trong cc thit b AAS. y l cc n pht bc x lin tc nhng c bin iu v lc giao thoa nh mt h thng quang hc to ra chm bc x n sc. Loi n ny c u im l d ch to, r tin v c bn tng i cao, ch cn mt n c th xc nh ng thi hay lin tip nhiu nguyn t trong cng mt mu phn tch m khng phi thay n. Nhc im l c n sc v nhy km hn cc n HCL hay EDL. III.1.3.2. B phn iu bin (modulator = chopper): cho php loi tr cc bc x nhiu gy ra bi s pht x ca m hi nguyn t trong ngn la. y l mt gng xoay c 2 phn h v rn cho php ngt chm sng khi i n detector khi n p vo mt rn ca gng. Khi chm sng do n pht ra b ngt th tn hiu n detector ch do chm tia sinh ra do hin tng pht x ca cc nguyn t trong mu (Pe): Tn hiu 1 = Pe Khi chm sng do n pht ra khng b ngt th tn hiu n detector s gm tng tn hiu ca chm tia sau khi b hp th c truyn qua mu (P) v chm tia pht ra do hin tng pht x ca cc nguyn t trong mu (Pe): Tn hiu 2 = P + Pe

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Do , hiu 2 tn hiu ny s cho gi tr cng bc x truyn qua mu sau khi b hp th: Tn hiu iu chnh = Tn hiu 2 - Tn hiu 1 = (P + Pe) Pe = P

B iu bin (Chopper) Hnh 3.5. B iu bin v nguyn l hot ng II.1.3.3. B phn nguyn t ha (atomization device): cho php to ra mt m hi ng nht cha cc nguyn t t do trng thi c bn (hn ch ti a hin tng ion ha) t dung dch cn o. Hin nay c mt s k thut nguyn t ha sau: a) K thut nguyn t ha bng ngn la (FAAS: Flame Atomic Absorption Spectrophotometry): H thng nguyn t ha bng ngn la bao gm: * Bung phun sng (Nebulizer): trong dung dch o c phn tn thnh sol kh cha cc git cht lng nh li ti c kch thc kh ng nht. Dung dch mu c ht vo bung phun nh mt dng kh p sut cao gm bi kh t. Khi ra khi u ng ht, cht lng b p vo mt bu thy tinh (glass impact bead) to ra aerosol. Cc ht ln s b loi b v thi ra nh mt h thng cn c bit. Hnh 3.6. Bung phun sng Bung phi trn v u t: Sol kh c hnh thnh s c phun trc tip vo ngn la (i vi loi n t ton din) hay c dn vo mt bung phi trn vi kh t v cht oxy ha (i vi loi n t trn trc) ri t chy. Khi , cc ht dung mi b bay hi hay bc chy. Di tc dng nhit ca ngn la, cc phn t mui to thnh s b phn ly thnh cc nguyn t t do. n t l mt khe hp, c chiu di c 10 cm, c t dc theo ng i ca chm sng ti. C th thay i cng hp th ca mu bng cch iu chnh chiu cao ca n.

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Hnh 3.7. Bung phun sng, bung phi trn v u t trong n t trn trc

ngn cn hin tng ti kt hp thnh cc phn t (recombination) v hin tng ion ha (ionization), s kch thch nguyn t bi nhit ca ngn la to nn s pht x, cn chn nhit ngn la thch hp cho vic nh lng nguyn t cho bng cch thay i bn cht kh t v cht oxy ha cng nh iu chnh lu lng v t l ca chng. t c nhit ngn la c 2300 0C, ngi ta thng dng hn hp khng kh acetylen vi lu lng acetylen l 1,6 m/s. Mun c nhit ngn la cao hn (khong 3000C) th dng hn hp C2H2 + O2 hay C2H2 + N2O (Bng 3.1)

Hnh 3.8. Cc giai on nguyn t ha bng ngn la H thng nguyn t ha ngn la c u im l lp li tt, d ti u ha iu kin nguyn t ha mu. Nhc im l nhy thp do hiu sut nguyn t ha km. Nguyn nhn l do a s cc ht aeerosol to ra kh ln c th t chy bng kh t (95% mu khng th i n ngn la) v s pha long mu ng k bi kh t. Ngoi k thut nguyn t ho bng ngn la nh trn, c th dng cc k tht nguyn t ha khng ngn la.

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b) K thut nguyn t ha khng ngn la: ra i sau k thut nguyn t ha bng ngn la, nhng pht trin rt nhanh v hin nay ang c ng dng rt ph bin. u im ca k thut ny l cho php nh lng mt cch chn lc, c nhy rt cao (c ppb), nh lng c cc nguyn t cu to nn cc hp cht rt bn nhit. m k thut FAAS khng cho php (Bng 3.2). V vy, k thut ny thng dng phn tch cc vt kim loi, c bit l khi xc nh cc loi mu y hc, sinh hc, dc phm, thc phm *Nguyn tc v cc giai on ca qu trnh nguyn t ha mu khng ngn la Nguyn tc: s dng k thut nguyn t ha nhit in (ETA: electrothermal atomization), trong qu trnh nung nng, ha hi v nguyn t ha mu phn tch xy ra rt nhanh (60 80 giy) trong mt ng graphit nh (GF: Graphite furnace) hay trong mt thuyn tantan (Ta) nh. Ngun nng lng thng dng l dng in c cng dng rt cao (50 600 A) v th thp (< 12V) hay nng lng ca dng cao tn cm ng (ICP). Di tc dng ca cc ngun nng lng ny, cuvt cha mu s c nung nng tc khc, ha hi v nguyn t ha mu to ra cc nguyn t t do trng thi hi c kh nng hp th bc x n sc to ra ph hp th nguyn t ca n. Nh vy, h thng nguyn t ha mu theo k thut khng ngn la gm c: - Bung nguyn t ha: l cuvt cha mu phn tch. - Ngun nng lng nung nng l graphit n nhit nguyn t ha mu. - B iu khin ci t chng trnh v kim sot qu trnh nguyn t ha mu.

Hnh 3.9. L graphite Qu trnh nguyn t ha mu: xy ra theo 3 giai on k tip nhau: sy kh mu, tro ha luyn mu, nguyn t ha mu. * Sy kh mu: phi m bo dung mi ha tan mu bay hi nh nhng v hon ton, khng lm bn mu, mt mu. Nhit v thi gian sy kh ca mi mu ph thuc vo bn cht ca cc cht trong mu v dung mi ha tan n. Thc nghim

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cho thy rng khng nn sy kh qu nhanh nhit qu cao. Ni chung, nhit sy kh ph hp vi a s cc mu v c trong dung mi nc vo khong t 100 150oC trong thi gian t 25 - 40 giy vi lng mu bm vo cuvt nh hn 100 l. Cc mu hu c trong dung mi hu c phi sy nhit thp hn v tc tng nhit cng phi chm hn. * Tro ha luyn mu: l giai on tip theo t chy cc hp cht hu c v mn c trong mu sau khi sy kh, ng thi cng nung luyn mu mt nhit thun li cho giai on nguyn t ha tip theo t hiu sut cao v n nh. Giai on ny nh hng rt nhiu n kt qu phn tch nu chn iu kin tro ha khng ph hp. Thc nghim cho thy mi nguyn t u c mt nhit tro ha luyn mu ti u, l nhit m s tro ha luyn mu ti nhit hoc nh hn n th cng vch ph hp th l khng i, cn nu tro ha nhit ln hn th cng vch ph b gim v khng n nh (iu ny c th l gii l do mu b phn hy mt). Nhit tro ha ti u ca mi nguyn t ph thuc vo bn cht, dng hp cht m nguyn t tn ti, k c matrix mu (nn mu). Ni chung, cc nguyn t bn nhit v tn ti dng hp cht bn nhit thng phi tro ha luyn mu nhit tng i cao. V d: Si c nhit tro ha ti u l 1100oC; Ni: 1000oC, Pb: 600oC Ngoi ra, tc tng nhit trong qu trnh tro ha luyn mu cng rt quan trng v nu tc tng nhit qu ln s lm bn mu. Thng thng, ngi ta dnh 1/3 tng thi gian tro ha luyn mu c dng gia nhit t nhit sy mu n nhit tro ha (tc khong 60oC 100oC/giy); cn 2/3 thi gian cn li l gi nhit khng i chn luyn mu. i vi nhng mu ca hp cht hu c hay trong dung mi hu c th giai on ny cng phi cn thn trnh s bay hi trc ca nguyn t phn tch dng hp cht c kim. Ni chung, vi mi loi mu, trong mi loi dung mi u phi kho st pht hin nhit tro ha, tc tng nhit cho ph hp.

* Nguyn t ha mu: l giai on quyt nh cng vch ph v b nh


hng bi hai giai on trn. Giai on ny c thc hin trong thi gian rt ngn (thng t 3- 6 giy, i khi 8 - 10 giy) nhng tc gia nhit rt ln (1800 oC 2500oC/giy hay tc ti a m my c th cho php) t ngay tc khc nhit nguyn t ha. Trong cc ti liu km theo thit b AAS thng gii thiu ngi s dng tham kho v nhit sy mu, nhit tro ha luyn mu v nhit nguyn t ha mu. Tuy nhin, khi phn tch cc mu c th nn da vo cc gi tr kho st hoc kim tra li.

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K thut ha hi lnh (CV-AAS: Cold Vapour AAS)

Mt s nguyn t c th c nguyn t ha bng cch s dng phn ng ha hc to thnh hp cht d bay hi nhit thng. V d: As, Se, Sb, Bi, Ge, Sn, Te, Pb to thnh hydrur d bay hi khi phn ng vi NaBH4 trong mi trng acid. Sau , hi hydrur c mt dng kh tr dn n ngn la hay mt cuvet thch anh c t nng t trn ng i ca chm sng ca h thng o mu (k thut Hydride Gas AAS = HG-AAS)

Hnh 3.10. S b hydrur ha kim loi Thy ngn c th c xc nh bng phng php ha hi lnh (Cold Vapour AAS = CV-AAS), trong n c kh v Hg kim loi (dng hi) SnCl 2 sau hi Hg cmt dng kh tr dn n mt cuvet ng mu khng t nng t trn ng i ca chm sng ca h thng o mu.

Hnh 3.11. S b ha hi thy ngn

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Bng 3.2. Gii hn pht hin ca mt s nguyn t bng cc k thut F-AAS v ET-AAS

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III.1.4. Ti u ha iu kin nh lng bng phng php AAS chn iu kin ti u cho vic nh lng mt nguyn t trong mt i tng mu phn tch no , cn qua cc bc kho st sau: a) Chn phng php nguyn t ha mu : ty thuc vo nng nguyn t cn phn tch c trong mu. - K thut FAAS cho php xc nh nhanh hn v c chnh xc cao hn nu hm lng nguyn t cn xc nh trong mu ln hn nhiu so vi gii hn pht hin ca k thut ny. - K thut ETA-AAS c nhy cao hn nn thng p dng cho cc nguyn t kh bay hi, khi phn tch vt nguyn t hay vi lng mu nh b) Chn bc sng phn tch v rng vch ph : nhy ca vch ph AAS ca mt nguyn t ph thuc vo nng ca nguyn t trong mu v bc sng phn tch. Mi nguyn t c mt nng c trng (characteristic concentration) ng vi mt bc sng xc nh, l nng cht phn tch cho hp th bng 0,00436 (ng vi %T = 99%). V d : Bc sng v nng c trng ca Cu

Vic chn bc sng phn tch c chn la ty thuc vo nng nguyn t phn tch trong mu v sao cho t nhy cao nht, vch ph t b cn tr bi cc yu t vt l (pht x nn ca ngun sng) v ha hc (s c mt ca cc nguyn t khc).

c) Chn phng php x l mu phn tch: K thut FAAS v ETA-AAS iu i


hi mu dng lng hay dung dch. Do , mu rn cn c ha tan trong dung mi thch hp hay v c ha bng HNO3, H2SO4, hay HClO4 (dng bp in, l vi sng). Mu lng c th phn tch trc tip hoc c pha long bng dung mi hoc c chit - lm giu cht cn phn tch nu nn mu cha cc thnh phn cn tr vic phn tch. V d: Chit v lm giu Cd2+, Co2+, Cu2+, Fe3+, Pb2+, Ni2+, Zn2+ bng dung dch ammonium pyrrolidine dithiocarbamate trong methyl isobutyl ketone (MIBK)

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d) Ti thiu ha cc yu t nh hng quang ph v ha hc * Ti thiu ha nh hng quang ph: - Nu vch hp th ca nguyn t cn chng lp vch hp th ca nguyn t cn phn tch chng th chn bc sng phn tch sao cho khng b nh hng. - S hp th do cc phn t c trong ngn la (do s nguyn t ha cha hon ton) hay s tn x bi cc ht kch thc ln (tp hp nguyn t) c th xy ra. c bit, s hp th v tn x bi cc phn t hydoxid hay oxid ca cc cht c trong nn mu xy ra mnh < 300 nm. Trong nhng trng hp ny, truyn sut km v lm tng biu kin s hp th ca mu. Nu thnh phn nn ca mu bit th c th loi tr nh hng ny bng cch phn tch mu trng, chun b cc mu chun c thnh phn nn ging mu phn tch. Nu thnh phn nn khng bit r th c th hiu chnh s hp th nn (s hp th gy ra do nn mu) bng cch tng nhit ngn la phn ly cc oxyt, hydroxid (v d: tng lu lng nhin liu cht oxy ha, iu chnh t s nhin liu: cht oxy ha, thay i t hp nhin liu - cht oxy ha). Bin php khc l hiu chnh nn bng n pht x lin tc D2. * Ti thiu ha nh hng ha hc: Vic nh lng mt s nguyn t rt phc tp do nh hng ca cc yu t ha hc trong qu trnh nguyn t ha. - nh hng ca dung mi: Nu dng dung mi l nc th qu trnh nguyn t ha xy ra chm do tn nhiu nng lng ha hi nc, do php o km nhy. nng cao nhy ca php o, thng dng dung mi MIBK (methyl isobutyl keton). Khi , qu trnh chuyn ha xy ra nhanh hn do dung mi ny d ha hi v phn ng t chy cht hu c sinh ra mt lng nhit ng k, lm tng nhy php o ln t 2 - 6 ln so vi dng dung mi nc. - nh hng ca s ion ha: nhit cao, nhng nguyn t c nng lng ion ha thp (v d: cc kim loi kim hay kim th) ch yu tn ti trng thi ion. V vy, khi nh lng nhng nguyn t ny, cn thm vo mu o mt lng d mui ca cc nguyn t d b ion ha hn (thng dng mui K+, Cs+). - nh hng ca dng anion lin kt: Mt s anion (nh PO4 3-, SO42-,) c th to vi ion kim loi cn phn tch cc hp cht bn nhit nn kh b ngyn t ha. Trong trng hp ny, cn thm vo dung dch phn tch mt lng d cht to phc bn d bay hi vi ion kim loi cn phn tch (gi l tc nhn bo v: protecting agent) hay thm cc mui ca cc nguyn t khc c th kt hp vi cc anion trn nhng hp cht bn nhit (gi l tc nhn phng thch: releasing agent).

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Nhng mui c thm vo vi mt lng d nhm n nh s nguyn t ha cc nguyn t nh trn c gi chung l cht m ha - quang ph ( spectrochemical buffer). V d: - Khi nh lng Pb c th thm EDTA vo dung dch o to phc bn PbY 2 nhm trnh to thnh cc mui bn nhit Pb(NO3)2, Pb3(PO4)2, PbCO3,... - Khi nh lng Calci thm mt lng tha LaCl 3 vo dung dch Ca2+ to thnh LaPO4 nhm trnh to hp cht bn nhit Ca3(PO4)2. e) Chn phng php nh lng C th dng 1 trong 3 cch sau: Phng php ng chun: Trong thc t, khi nh lng bng phng php AAS, ng chun A = f (C) ch tuyn tnh trong mt khong nng kh gii hn. Trong mt s trng hp, cn phi xy dng ng chun khng tuyn tnh c dng bc 2 hay bc 3 (c t nht 8 im chun).

a) tuyn tnh

b) tuyn tnh mt phn c) khng tuyn tnh Hnh 3.12. Mt s dng ng chun AAS

Khi s dng phng php ng chun ny cn lu pha ch mu chun c tnh cht nguyn t ha ging nh mu phn tch. Nu khng, cn iu chnh thnh phn nn mu chun cho tng ng bng cc cht m quang ph. Phng php kp chun: p dng khi nng cht phn tch trong mu o A nm ngoi vng tuyn tnh ca ng chun Trong phng php ny, ch dng 2 mu chun c nng nguyn t cn phn tch l C1 v C2 rt gn vi nng c trong mu phn tch (Cx). Khi , c th xem l xy ra s tuyn tnh A C trong khong nng (C1; C2) c th tnh nng cht phn tch trong mu theo cng thc: C A A C1 Cx C2
C x = C1 +
x 1

A2 A1

.(C 2 C1 )

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Phng php thm: C 2 k thut ng chun v kp chun u i hi thnh phn nn ca mu chun v mu phn tch phi tng t nhau. i vi mu cha bit r thnh phn nn hay c nhng cht cn tr phc tp n vic phn tch th nn dng phng php thm. Trong phng php ny s dng ngay chnh dung dch mu phn tch lm nn v thm vo nhng lng khc nhau ca cht phn tch (tin hnh tng t trong phng php trc quang so mu). Tuy nhin, k thut ny cng ch p dng c khi ng chun thm l tuyn tnh. III.1.6. nh gi phng php AAS Phm vi p dng: Phng php AAS thch hp cho vic nh lng cc cu t vt v siu vt (nu dng k thut ETA). Bng cch pha long mu, phng php AAS cng c th p dng phn tch cu t vi lng. ng: Khi ti thiu ha cc nh hng quang hc v ha hc, ng c th t c 0,5 5%. Sai s h thng khi dng k thut ETA-AAS thng ln hn khi dng k thut FAAS. chnh xc: Vi hp th ln hn 0,1 0,2 th lch chun tng i ca phng php AAS c 0,3 1% i vi k thut FAAS v l 1 5% vi k thut ETAAAS. nhy: nhy ca k thut FAAS b nh hng nhiu bi thnh phn v v tr ngn la ti quan st s hp th. Thng thng, nhy c ti u ha bng cch ht mu chun v iu chnh cc iu kin o (t s nhin liu-cht oxy ha, tc phun sng, chiu cao ca n t) sao cho t hp th ln nht. Vi k thut ETA-AAS, nhy chu nh hng bi cc giai on sy kh mu v tro ha mu trc khi nguyn t ha. nhy cng ph thuc vo nn mu: nhyc th gim do cc nh hng ha hc. C th tng nhy bng cch thm vo dung dch mt alcol, ester hay ceton hay dung mi hu c no . chn lc: Do b rng vch ph hp th hp nn phng php AAS c chn lc rt tt. V vy, phng php AAS c th p dng phn tch trn 60 nguyn t khc nhau vi nng di mc ppm. Thi gian, chi ph v yu cu thit b: K thut FAAS cho php phn tch nhanh (c th 250 350 ln o/gi khi dng thit b t ng), cn k thut ETA-AAS ch t 20 30 ln o/gi). III.1.7. ng dng ca phng php AAS trong phn tch thc phm

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Phng php AAS c dng ph bin phn tch vt kim loi trong nhiu loi thc phm. Sau y gii thiu nguyn tc phn tch mt s kim loi nng trong thc phm bng phng php AAS. a) Xc nh As: * Dng k thut HG-AAS Nguyn tc: Mu c v c ho trong l ph mu vi sng ci t chng trnh theo tng i tng. Sau , As (V) c kh v As (III) v phn ng vi NaBH4 to thnh hp cht hydrua arsen (AsH3): 3BH4- (dd) + 3H+ (dd) + 4H3AsO3 (dd) 3H3BO3 (dd) + 4AsH3 (k) + 3H2O (l) Sau , AsH3 c dn ti mt cuvet ch T bng thch anh nguyn t ha v o ph hp th ca As. iu kin o my Vch ph phn tch As Cng dng n HCL ca nguyn t Arsen Khe o Chiu cao u t Kh ngn la Tc dn mu Tc dn thuc th (NaBH4, HCl 5 M) Thi gian o 193,70 nm 10 mA (80% I Max) 1 nm Ch auto (5 - 6 mm) Khng kh nn & Acetylen & Ar 5 6 mL/pht 2 3 mL/pht 5 giy

* Dng k thut GFAAS Nguyn tc: Mu c v c ho trong l ph mu vi sng c ci t chng trnh theo tng i tng. Xc nh hm lng arsen trong dung dch to thnh bng o ph hp th nguyn t l graphit. iu kin o my Vch ph phn tch arsen Cng dng ca n HCL ca As Khe o Chiu cao u t Kh mi trng Loi cuvt Mi trng dung dch Lng mu np (l) Chng trnh nhit cho l Graphite 1 2 193,70 nm 10 mA (80%IMax) 1,0 nm Ch auto Argon High density graphite HNO3 2 % 20 T (oC) 150 250 Time Heat mode 20 10 Ramp Ramp Flow rate 0,1 0,1

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3 4 5 6 7

600 600 600 2200 2500

10 10 3 2 2

Ramp Step Step Step Step

1,0 1,0 0,0 0,0 1,0

c) Xc nh hm lng Se * Dng k thut HG-AAS Nguyn tc: Mu c v c ha trong l vi sng theo tng i tng. Cc ion Se(IV) v Se(VI) phn ng vi NaBH 4 trong mi trng axt sinh ra hp cht Selen hydrua (SeH4 v SeH6), cc hp cht ny c dn vo cuvet thch anh nguyn t ho thnh Se v o ph hp th ca Se. iu kin o my Cng dng ca n HCL ca Se Vch ph o Khe o Chiu cao n t (Burner Head) Kh ngn la: Acetylen - Khng kh nn Tc dn mu Tc dn thuc th Tc dn HCl (5 M) Thi gian o 23 mA (70% IMax) Se-196,0 nm 1,0 nm 16 mm 2 L/phut 5~6 ml/ph. 2~3ml/ph 2~3ml/ph 3 giy

* Dngk thut GFAAS Nguyn tc: Mu c v c ho trong l ph mu vi sng c t chng trnh theo tng i tng. Xc nh hm lng selen trong dung dch to thnh bng o ph hp th nguyn t l graphit iu kin o my Vch ph o selen Cng dng ca n HCL ca Se Khe o Chiu cao o Kh mi trng Loi Cuvt Mi trng dung dch Lng mu np (l) 196,0 nm 20 mA (80% IMax) 1,0 nm Ch auto Argon High density Graphite HNO3 2 % 20

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Chng trnh nhit cho l Graphite 1 2 3 4 5 6 7

T (oC) 150 250 600 600 600 2200 2400

Time Heat mode 20 10 10 10 3 2 2 Ramp Ramp Ramp Step Step Step Step

Flow rate 0,1 0,1 1,0 1,0 0,0 0,0 1,0

d) Xc nh hm lng Hg * Dng k thut CV-AAS Nguyn tc: Mu c v c ho trong l ph mu vi sng c t chng trnh theo tng i tng. Hg(II) c chuyn v Hg kim loi bng SnCl 2. Hi Hg bay ln c a ti h thng quang ph hp th nguyn t o ph. * iu kin o my Vch ph o thy ngn Cng dng n HCL ca Hg Khe o Chiu cao u t Thi gian o 253,70 - nm 4 mA (80 % IMax) 1 nm Ch auto (16 mm) 5 giy

III.2. PHNG PHP QUANG PH PHT X NGUYN T (AAS : Atomic Emission Spectrometry) III.2.1. Nguyn tc phng php: da trn hin tng pht x nguyn t nh sau Khi cung cp mt nng lng (thng l di dng nhit) cho mt m hi cha cc nguyn t t do, cc electron trong nguyn t s chuyn t mc nng lng c bn (E) ln mc nng lng kch thch (E* ): M0 + E M* Sau 108s, chng li quay tr v trng thi c bn ban u v gii phng mt nng lng di dng bc x in t c tn s : M* M0 + h trong : h = E* - E

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Hnh 3.13. S pht x nguyn t Nu thu v phn ly chm bc x ny thnh cc bc x n sc, s thu c ph pht x nguyn t ca nguyn t b kch thch. Da vo hin tng ny, ngi ta xy dng phng php quang ph pht x nguyn t, cho php nh tnh v nh lng hn 60 nguyn t khc nhau. - nh tnh: Ph pht x ca mt nguyn t gm cc vch pht x c trng nguyn t . Nh , c th nhn bit s c mt ca cc nguyn t c trong mu phn tch. Thng thng, nhn bit s c mt ca mt nguyn t no trong mu thng da vo s c mt hay vng mt ca cc vch phn tch hay vch cui cng ca nguyn t nghin cu, l cc vch rt nhy v c trng cho nguyn t cho (chng l mt hay vi vch cn li trong quang ph ca nguyn t khi hm lng ca nguyn t kho st trong mu phn tch gim xung n mt gii hn no ). - nh lng: Cng ca vch pht x t l vi s nguyn t trng thi kch thch v do ph thuc vo nng nguyn t pht x. Lomakin tm c mi quan h ny nh sau: I = kCb trong : k l hng s thc nghim, ph thuc vo iu kin ha hi mu v kch thch nguyn t. Nu c nh cc iu kin ny th k = const b: hng s ph thuc vo bn cht nguyn t, bc sng ca vch pht x v nng ca nguyn t trong mu. i vi mi nguyn t trong iu kin o xc nh th tn ti mt gi tr nng ti hn C0 trong khi Cx < C0 th ng biu din I C l tuyn tnh (tc b = 1x) v khi Cx > C0 th ng biu din khng tuyn tnh (b < 1). Nng Hnh 3.13. Quan h gia cng vch pht x v nng nguyn t trong mu C0 gi l nng gii hn ca cng tuyn tnh.

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Da vo quan h I C, c th xc nh c nng nguyn t pht x trong mu bng cch o cng vch pht x c trng ca nguyn t . nh lng, ngi ta thng dng phng php kp chun, phng php ng chun hay phng php thm tng t nh trong phng php AAS. III.2.2. S thit b EAS v nguyn l hot ng : S thit b EAS (Hnh 3.14) cng tng t nh thit b AAS, ch khc l khng c b phn u tin l ngun pht x.

Hnh 3.14. S thit b AES Nguyn l hot ng ca thit b AES : Cc hp cht trong mu phn tch c ha hi, nguyn t ha v chuyn sang trng thi kch thch bng mt ngun nhit lng kch thch Q (v d: ngn la n kh, ngun h quang in, ngun tia la in cao tn, ngun plasma hay ngun laser) : MeX (rn, dd) + Q MeX (hi) MeX (hi) + Q Me (hi) + X (hi) Me (hi) + Q Me* (hi) Me* (hi) Me (hi) + h Nguyn t b kch thch sau s pht x khi quay v trng thi c bn : Bc x pht ra s c phn tch bng mt h thng n sc v chn lc bc x c tn s c trng cho nguyn t cn o i vo t bo quang in v b phn khuch i tn hiu, cui cng c hin th trn thit b o. III.2.3. Phn loi loi thit b AES Thng phn loi thit b AES da trn ngun nng lng c s dng ha hi, nguyn t ha v kch thch nguyn t. a) Thit b quang ph pht x nguyn t ngn la (FAES: Flame Atomic Emission Spectrometer):

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Trong thit b ny ngun nng lng s dng kch thch nguyn t l ngn la n kh c to t s t chy cc kh t (axetylen, propan,...) vi cht oxy ha (khng kh, oxy, H2, N2O,...). Nhit ca ngn la n kh ph thuc vo bn cht v t l cc kh s dng, lu lng kh t v thng t khong 1700 3000 0C. Nhit ny kh thp, ch kch thch cc nguyn t kim loi kim v kim th. Bng 3.3. Nhit ca mt s hn hp kh t

Cu to ca ngn la gm 3 phn: - Phn ti: st ming n, c nhit thp - Phn gia: gn nh khng mu, nhit cao nht. Do , nn a mu vo v tr ny - Phn trn cng (v v ui ca ngn la); mu vng, nhit thp nht, ti y ra nhiu qu trnh ph. Do , thit b phi c ch to sao cho phn ny nh nht v trnh a mu vo phn ny hn ch qu trnh ph. H FAES cu to n gin, r tin, cho ngn la n nh nn kt qu thng c chnh xc tt. Tuy nhin, do c im ch to ra nhit thp nn thit b ny ch dng phn tch cc km loi kim (Li, Na, K, Rb, Cs) v 1 vi kim loi kim th (Ca, Mg) vi nhy c 1 10 g/ml.

Hnh 3.15. Cu to v nhit cc vng ca ngn la n kh

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b) Thit b quang ph pht x nguyn t dng h quang in v tia la in : s dng cc ngun kch thch sau: - H quang in (arc): dng s phng in lin tc gia 2 in cc (kim loi hay graphite) lm bay hi v kch thch nguyn t. Nu mu l thanh kim loi th n c dng lm 1 in cc phng in Nu mu khng dn in th n c nghin vi bt than ch v nhi vo phn rng bn trong in cc graphite.

Hnh 3.16. Cc loi in cc to h quang in - Tia la in (spark): dng xung in kch thch. Hin nay, thng thay ngun ny bi ngun laser hay plasmar. Loi thit b ny hin nay t dng do nhy khng cao, km n nh. Thng ch dng nh tnh v bn nh lng. c) Thit b quang ph plasma cao tn cm ng ghp khi ph (ICP-MS) (ICP-AES: Inductively-Coupled Plasma Mass Spectrometer) Ngun kch thch l mt ngun cao tn cm ng (ICP) c to ra bi my pht cao tn (pht sng v tuyn, thng c cng sut 1-5 kW, tn s 27 MHz). Ngun ny c kh nng to ra nhit rt cao (ln n 10.0000K), do c kh nng lm bay hi dung mi, ha hi mu, kch thch v ion ha cc nguyn t. Khi , mu c chuyn sang trng thi plasma trong cha cc cation v electron. Hnh 3.17. Ngun ICP

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Nguyn l hot ng ca thit b ICP-MS nh sau: n plasmar gm c cc ng thch anh ng tm. Mu c phun thnh aerosol v c dng kh mang (Ar hay hn hp He - Ar) dn vo phn bn trong ca ng. Phn v bn ngoi ca ng c lm mt bi kh mang. Mt my pht cao tn sinh ra mt dng din dao ng trong mt cun cm ng (induction coil) bao quang ng thch anh. Cun cm ng ny to ra mt t trng; t trng ny li sinh ra mt dng in dao ng trong cc ion v electron ca kh mang. Cc ion ny li va chm vi cc nguyn t khc trong kh mang v mu, kch thch chng chuyn thnh cc ion to ra plasmar. Cc ion sau c a vo detector khi ph qua mt khe hp (bn trong detector c chn khng rt cao). Cc ion sau c tng tc v tp trung thnh mt dng ion bng mt h thng cc thu knh tnh in (electrostatic lenses, l cc nam chm to ra t trng nh hng ng i ca cc ion). Dng ion sau c phn tch bi mt b lc khi t cc (quadrupole mass filter). B lc ny s chn lc cc ion da trn t s khi lng/in tch ca chng (m/z) sao cho ch c cc ht ng vi gi tr m/z cho mi c th i vo detector nhn ion t phn cui ca ng t cc, to ra dng in, c ghi nhn v th hin di dng cc peak trong ph khi lng. Mi ion c mt gi tr m/z c trng cho khi lng v in tch ca n m/z, nh c th nhn bit s c mt ca nguyn t trong mu phn tch. Cng ca peak tn hiu ph thuc vo nng ion trong mu, iu ny cho php nh lng nguyn t c trong mu da vo cng vch ph MS (dng mu chun)

Hnh 3.18. S thit b ICP-MS

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III.3. So snh phng php AAS v EAS : Cc phng php AAS v EAS cho php nh tnh v nh lng khong 60 nguyn t (gm tt c cc kim loi v mt s nguyn t khng kim loi nh B, Si, P, ..) thuc nhiu i tng phn tch khc nhau (hp kim, kim loi, khong vt, cc mu sinh hc, ...). i vi a s nguyn t, sai s ca phng php FAAS thng vo khong 0,6 -3%. Cc phng php AAS khng ngn la thng cho sai s di 2%. So vi phng php AES c in (dng ngun kch thch l ngn la n kh, h quang, tia la in), phng php AAS c u im l: - nhy cao hn nhiu ln (AES: c 1 ppm; AAS : c 0,1 0,005 ppm). - chnh xc v chn lc cao do t vch ph hn (AES cho rt nhiu vch ph, c th nhm ln khi phn tch) - iu kin kch thch ph tng i d dng Tuy nhin phng php AAS c mt s hn ch: - khng p dng c cho C, P, cc halogen, ... - thng phi ha tan mu trc khi o lm ko di thi gian phn tch v khng th xc nh ng thi nhiu nguyn t (mi nguyn t cn mt n kch thch tng ng) Trong phn tch thc phm, cc kim loi nng nh Fe, Ni, Co, Cu, Mn, Cr, thng c phn tch bng phng php FAAS. Cc c t d bay hi nh Pb, As, Sb, Hg... thng c chit bng dung mi hu c ri nh lng bng phng php AAS ha hi lnh (CV-AAS). i vi a s nguyn t, sai s ca phng php FAAS thng vo khong 0,6 -3%. Cc phng php AAS khng ngn la thng cho sai s di 2%. Hin nay, nh kh nng to ra c nhit rt cao cho php ion ha hu ht cc nguyn t v nn mu, thit b ICP-MS l mt cng c AES rt hu hiu phn tch nguyn t (ngay c cc nguyn t bn nhit, p dng cho nhiu nn mu khc nhau). Phng php ny cho nhy kh cao (0,1 10 ng/ml), lp li tt, sai s nh, vng tuyn tnh rng, tc phn tch ln, nh hng ca nn mu khng c hoc rt t. Tuy nhin, thit b ICP-MS rt t tin nn vn cha ph bin bng cc thit b AAS.

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Chng 4. PHN TCH THC PHM BNG K THUT SC K


IV.1. LCH S PHNG PHP SC K Phng php sc k c pht minh vo nm 1906 bi Tswett - mt nh sinh vt hc ngi Nga. Khi cho hn hp dch chit sc t ca l cy trong ha tan trong eter du m (cha chlorophyll v cc xanthophyll) hp ph ln u ct cha CaCO3 nghin mn ri cho dung mi eter du m lin tc chy qua ct, Tswett nhn thy rng trong qu trnh di chuyn t trn xung di ct hn hp trn (lc u nhn thy c mu lc) dn dn c tch ra thnh nhng di mu khc nhau (lc, lc lam, vng). Nh vy, bng cch ny phn tch hn hp sc t l thnh cc phn on cha cc sc t ring bit. Tswett gi phng php tch ny l phng php sc k v tp hp cc di mu tch ra trn ct l sc k .

Hnh 4.1. Th nghim tch hn hp sc t l cy ca Tswett

Phng php ny sau b lng qun trong mt thi gian di. Mi n nm 1931 v v sau, nh nhng cng trnh ca Vinterstin, Lederer, Izmailov, Shraiber, Synge, Martin,... nghin cu c s l thuyt ca qu trnh tch sc k v pht trin cc thit b ghp u ra ca ct sc k vi nhng detector cho php nh tnh v nh lng cc cu t tch ra th phng php ny mi c ch . T n nay, phng php sc k pht trin mnh m v tr thnh phng php rt hiu nghim nh tnh v nh lng cc cu t trong mu thuc nhiu i tng phn tch khc nhau (v c, hu c, sinh hc, thc phm, mi trng,...) IV.2. MT S KHI NIM LIN QUAN N QU TRNH TCH SC K Pha tnh (stationary phase): l pha rn, xp, ng yn, gm cc ht mn, c nhi vo mt ct (bng thy tinh kim loi) hay l mt pha lng c ph hay gn ln b mt mt cht mang rn no . Pha ng (mobile phase): l pha lng hay kh c di chuyn lin tc qua pha tnh di tc dng ca mt trng lc no nh trng lc (v d: trong php sc k ct c in), lc mao dn (nh trong sc k giy, sc k bn mng), hoc nh p sut (sc k lng cao p, sc k kh,...)

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Ra gii (elution): l qu trnh tch cc cu t phn tch c lu gi trn pha tnh bng cch cho mt pha ng di chuyn lin tc qua pha tnh. IV.3. NGUYN TC CHUNG CA PHNG PHP SC K IV.3.1. C s l thuyt ca phng php sc k: Mc d c ch tch c th khc nhau i vi tng phng php sc k, nhng cc qu trnh sc k u tun theo nh lut phn b Nernst: mt nhit nht nh, t s nng ca mt cu t A no trn pha tnh v pha ng l mt hng s
[ A] S = K D = const [ A] M

trong : [A] S v [A]M ln lt l nng cu t A no trn pha tnh v pha ng KD l hng s phn b, ph thuc vo nhit v bn cht ca mi cu t, bn cht ca pha tnh v pha ng. Vy, cu t no c i lc vi pha tnh cng ln so vI pha ng th gi tr K D cng ln v s b gi li trn pha tnh cng mnh. S khc nhau v hng s phn b KD ca cc cu t trong mt hn hp i vi mt h pha tnh v pha ng no chnh l c s cho php tch chng ra khi nhau bng phng php sc k. IV.3.2. Gii thch s tch hn hp cu t trong qu trnh sc k: Khi cho hn hp mu phn tch ln u ct sc k, s xy ra s lu gi cc cu t trn pha tnh. Trong qu trnh sc k, khi cho pha ng di chuyn qua pha tnh, cc cu t trong hn hp phn tch s di chuyn dc theo pha tnh cng vi pha ng nhng vi tc khc nhau ty thuc vo hng s phn b ca n gia pha tnh v pha ng: cu t no c i lc vi pha tnh cng ln (tc KD cng ln) s b lu gi cng cht v di chuyn vi tc cng chm, v ngc li. Nu hng s phn b K D ca cc cu t phn tch khc nhau ln th hn hp nghin cu dn dn s c tch thnh cc vng ring bit, mi vng tng ng vi mt cu t. Nu u ra ca ct sc k c ghp ni vi mt detector c kh nng ghi nhn cc tn hiu c trng no ca cc cu t phn tch (nh hp th nh sng, chit sut, dn in, cng pht hunh quang, ...) v biu din s thay i ca nng cht phn tch theo thi gian ra gii, ta s thu mt sc k cha cc peak (mi tn hiu). Nu qu trnh phn tch tt (cc cu t c phn tch hon ton) th mi peak s c trng cho mt cu t trong hn hp. Ngoi ra, din tch peak s t l vi nng cu t ny trong dung dch phn tch. Da vo , c th nh tnh v nh lng cc cu t c trong hn hp phn tch.

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Hnh 4.2. S tch hn hp cu t trong qu trnh sc k v sc k IV.3.3. Cc i lng c bn trong sc k - Ti u ha qu trnh sc k IV.3.3.1. Cc i lng c bn trong sc k : a) Kh nng lu gi ca mt cu t trn mt h sc k c c trng bng cc i lng sau * Thi gian lu (tR: retention time): l thi gian cn thit mt cu t no i t u ct n cui ct v c pht hin bi detector. Ty thuc bn cht ca pha tnh v pha ng c trong h sc k m mi cu t s b lu gi khc nhau v do c thi gian lu tR khc nhau. Trn sc k (Hnh 4.3), thi gian lu ca mt cu t (tR) l khong thi gian t lc bt u bm mu (t0 = 0) n lc cu t ra khi ct sc k v c pht hin bng detector (ng vi lc xut hin nh ca peak sc k tng ng) Nu hn hp sc k cha mt cu t hon ton khng b lu gi trn ct th n s i ra khi ct ngay cng vi pha ng v cho mt peak u tin. Thi gian lu ca cu t ny rt nh, c Hnh 4. 3. Sc k ca hn hp 2 cu t gi l thi gian cht (k hiu l tm). vi mt cu t khng b lu gi Thi gian cht chnh l thi gian cn thit pha ng i t u ct n detector nn:

- 50 tm = L u

trong : L - chiu di pha tnh u : tc tuyn tnh trung bnh ca pha ng nh gi thi gian lu thc s ca mt cu t trong pha tnh phi dng thi gian lu hiu chnh tR: tR = tR tm * Tha s dung lng ca mt cu t i trn pha tnh c nh ngha bi cng thc:
ki =
'

mS C .V V = S S = KD S mM C M .VM VM

vi: mS, mM v CS, CM: khi lng v nng cu t i trong pha tnh v pha ng VS, VM - th tch ca pha tnh v pha ng KD = CS/CM : hng s phn b ca i gia pha tnh v pha ng C th chng minh c rng:
ki =
' ' t Ri t m t R = tm tm

Vy, c th tnh c k' ca mt cu t da vo sc k . Gi tr k' ti u cho qu trnh tch sc k: k= 1- 5 Vi hn hp phn tch phc tp c th chp nhn: k = 1 - 10 b) Hiu nng ca ct sc k : Hiu nng ca mt ct sc k l kh nng tch cc cu t trong hn hp ra khi nhau, n c c trng bi cc i lng sau: * S a l thuyt: Theo l thuyt a (Martin v Synge, 1942), mi cu t A trong hn hp sc k s di chuyn lin tc trn pha tnh theo nhng bc rt nh, trong mi bc nh vy thit lp rt nhanh mt cn bng phn b ca A gia pha tnh v pha ng: AS AM Mi phn pha ng a thm vo s lm chuyn dch cn bng trn v tch mt phn cu t A trong pha tnh chuyn sang bc tip theo, ti li thit lp mt cn bng phn b mi. Mi bc chuyn di ng vi mt cn bng phn b c thit lp gia pha tnh v pha ng c gi l mt a l thuyt. Qu trnh c tip din lin tc, kt qu cu t A s c phn b trn mt s a theo phn b Gauss, trong nng A t s cc i ng vi cc a b gia.

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Nh vy, mt ct sc k c chiu di L c tng tng chia thnh N a l thuyt c cng cao H c nh s t 1 n N (H c gi l chiu cao ca mt a l thuyt ). Vy:
H = L N

Hiu nng tch ca ct sc k cng cao nu N cng ln (hay H cng nh). iu ny cng c th hin trn sc k : Hiu nng tch ca h thng sc k cng tt th cc peak sc k thu c cng nhn, hp, khng b gin rng. C th tnh c s a l thuyt da vo sc k thu c theo cng thc:
t t N = 16 r = 5,54 r W
2 2

W: rng chn peak : rng ng vi mt na chiu cao ca peak phn gii ca ct (resolution - k hiu Rs)
Rs = 2(t r 2 t r1 ) W2 + W1

Rs 1: cc peak c tch ri nhau phn gii c. (Tt nht l Rs 1,5. Khi , c th

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nh lng chnh xc cc cu t ) Rs < 1: cc peak b chng ln ln nhau phn gii khng tt kh nh lng chnh xc

Hnh 4.4. Cc v d v s phn gii cc peak sc k * chn lc (selectivity k hiu ):

tr2 t r1

' '

k 2' = ' k1

vi: k'1, k''2 v t'R1, t'R2: tha s dung lng v thi gian lu hiu chnh ca hai cu t (1 v 2) c peak nm cnh nhau trn sc k . > 1 chn lc tt (phn bit c cc cu t 1 v 2) < 1 chn lc khng tt kh phn bit c cc cu t 1 v 2 phn gii ca ct ph thuc vo N, k' , :
Rs = k' 1 1 ' 4 1 + k N

(*)

Trong sc k kh, k' thng rt ln (t 30 - 50), nn:


Rs = 1 1 N 4

IV.3.3.2. Ti u ha qu trnh sc k: ngha l chn iu kin sc k nh nhit , pha tnh (bn cht pha tnh, kch thc ht, xp, chiu di ct), pha ng (bn cht pha ng, tc , lu lng pha ng, ch ra gii,...) sao cho t c phn gii tt nht cn th c vi thi gian chp nhn c. Thng thng, thay i cc thng s trn nh sau: - Thay i k' v : bng cch thay i nhit (c bit hay dng trong sc k kh) hay thay i thnh phn pha ng (hay dng trong sc k lng) Thay i N bng cch thay i mt hay vi thng s nh: chiu di ct sc k (L), tc pha ng (u), chiu cao a l thuyt H (thay i kch thc ht pha tnh, nht pha ng, thay i b dy lp film mng trng trn pha tnh.

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IV. 4. Phn loi cc phng php sc k Cc cch phn loi c s dng nhiu nht l: a) Phn loi theo trng thi tp hp ca pha ng y l cch phn loi c s dng rng ri nht - Sc k kh (vit tt GC: Gas Chromatograhy): pha ng trng thi kh - Sc k lng (vit tt LC: Liquid Chromatograhy): pha ng trng thi lng - Sc k siu ti hn (vit tt SFC: Supercritical Fluid Chromatograhy): pha ng l mt cht lng trng thi siu ti hn (v d: CO2 500C, p sut 150 bars) b) Phn loi theo phng tin tch: - Sc k ct (Column Chromatograhy): pha tnh c np vo ct (thy tinh, kim loi) hay c trng thnh bn trong ca ct mao qun. - Sc k phng (Plannary Chromatograhy): pha tnh c trng ln b mt ca mt bn phng (v d: sc k giy, sc k bn mng) c) Phn loi theo c ch tch: da trn bn cht tng tc gia cht sc k vi pha tnh v pha ng. - Sc k hp ph (Adsorption Chromatography): da trn kh nng hp ph khc nhau ca pha tnh (rn) i vi cc cu t trong hn hp sc k. - Sc k phn b (Partition Chromatography): s dng kh nng phn b (hay tan) khc nhau ca cc cu t i vi pha tnh (lng) v pha ng (lng). - Sc k trao i ion (Ionic Chromatography): da trn kh nng trao i ion khc nhau ca cc ion trong hn hp sc k vi cc ion linh ng trn b mt pha tnh - Pha tnh y l cc cht hp ph trao i ion (thng l cc nha trao i ion tng hp) - Sc k loi tr (Size-Exclusion Chromatography): l phng php tch da trn s khc nhau v kch thc phn t ca cc cu t phn tch. IV.5. CC PHNG PHP SC K N GIN (c thm) IV.5.1. Sc k ct (CC: Column Chromatography) y l phng php sc k c in nht, c Tswett p dng ln u tin vo nm 1903. IV.5.1.1. Phng tin tch: l cc ct hnh tr bng thy tinh, kim loi hay hp kim, cht do... u di c kha (tng t mt burette), ng knh khong 1- 2 cm tr ln. T l gia chiu di L v ng knh d ca ct phi theo t l thch hp t hiu qu tch t u. Pha tnh c cu to t cc ht mn c nhi vo ct sc k.

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IV.5.1.2. K thut trin khai sc k ct: thng dng phng php ra gii (elution) Chun b ct: Lt mt lp bng thy tinh dy 2 - 3 mm vo u di ca ct ri nhi pha tnh vo ct (nhi kh hay nhi t; ch khng lm nt ct). u trn ca ct pha tnh cng lt mt lp bng thy tinh trnh lm xo trn pha tnh khi ra gii. Cho dung mi tr thch hp i qua ct thm t pha tnh. Np mu: Mu c c c cho ln u ct pha tnh. - Ra gii: Cho pha ng lin tc i qua ct, hn hp sc k s c tch thnh cc vng khc nhau trn ct v ln lt ra khi ct. tng hiu qu tch v rt ngn thi gian sc k, c th tng dn Hnh 4.5. Chun b ct sc k vi pha tnh l silicagel lc ra gii ca pha ng s dng (thay i phn cc ca pha ng). nh tnh v nh lng cc cu t trong hn hp sc k, u ra ca ct, ngi ta hng cc phn on ng vi tng cu t tch c ri em xc nh bng cc phng php phn tch thch hp. IV.5.1.3. ng dng v hn ch ca phng php sc k ct: ng dng: dng tch v iu ch cc cu t t mt hn hp Hn ch: Thi gian phn tch kh di Tn nhiu mu, pha tnh v dung mi pha ng. Kh nng tch khng hiu qu, nht l i vi cc hn hp mu phc tp IV.5.1.4. Cc phng php sc k ct a) Sc k ct hp ph: Pha tnh: l cc cht hp ph nh cellulose, tinh bt, ng, Na 2CO3, CaCO3, Ca3(PO4)3, MgCO3, MgO, silicagel, florisil, alumin (Al2O3), C hot tnh. Silicagel v alumin vi c ht 50 230 m (tc 70 290 mesh) l cc cht hp ph thng dng dng tch hn hp cc cht khng phn cc c bn sn trn th trng - Pha ng: l dung mi c tinh khit cao, tr vi cht tan v cht hp ph, c kh nng ha tan tng i tt cc cht nghin cu trong hn hp phn tch.

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- Ra gii: Qu trnh ra gii c thc hin theo th t tng dn kh nng gii hp. i vi pha tnh phn cc, s ra gii xy ra theo th t tng dn phn cc ca dung mi pha ng. Ngc li, i vi pha tnh khng phn cc, s ra gii xy ra theo th t gim dn phn cc ca pha ng Trt t tng dn phn cc ca cc dung mi thng dng nh sau: Ether du m < hexan < cyclohexan < CCl4 < benzen < toluen < diclorometan, CHCl < dietyl ete < acetat etyl < aceton < pyridin < propanol < etanol < metanol < nc < acid acetic. - K thut tch: Thng thc hin theo phng php i xung (cho pha ng chy t trn xung di ct di tc dng ca trng lc hay dng p lc). b) Sc k ct phn b : Pha tnh l mt pha lng (nc hay dung mi hu c) c hp ph trn b mt cht mang rn hay lin kt ha hc vi cht mang (ph bin nht l bng phng php ghp nhnh trn nhm silanol ca silicagel) Pha tnh phn cc sc k pha thng (NP: normal phase). V d: diatomite, silicagel, oxyt nhm, Pha tnh khng phn cc hay km phn cc sc k pha ngc (RP: reversed phase). V d: RP-8, RP-18, Chn pha tnh: cn c trn bn cht hn hp cu t cn tch sao cho tng tc gia pha tnh vi cht cn tch khng mnh qu (cht s b gi qu cht kh ra gii, thi gian phn tch qu di) cng khng yu qu (cht gn nh ra khi ct ngay phn gii s km) Chn pha ng theo cc nguyn tc sau: + phi ha tan tt cu t cn tch + ty theo bn cht pha tnh: Pha tnh phn cc (NP) pha ng l dung mi hu c t phn cc n phn cc trung bnh Pha tnh khng phn cc hay km phn cc (RP) pha ng l dung mi phn cc n phn cc trung bnh + c lc ra gii trung bnh + thnh phn pha ng phi khng b thay i trong qu trnh sc k + c nht cng thp cng tt V d:

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Pha tnh l H2O /silicagel tch hn hp acid amin pha ng l phenol

c bo ha nc Pha tnh l ct C18 (khng phn cc) tch cc carotenoid, pha ng c th l metanol, acetonitril hay hn hp MeOH H2O, acetonitril H2O, c) Sc k ct trao i ion (vit tt IC: Ionic Chromatography): Pha tnh l cc cht trao i ion (ionit) bao gm: Ionit v c: gm ionit v c t nhin (nhm zeolit, t st, glauconit,) v ionit v c tng hp (gm cc alumosilicat nh zeolit, permutit,) Ionit hu c: gm ionit hu c t nhin (cellulose, than bn,) v ionit hu c tng hp (tc nha trao i ion). Loi ionit c s dng nhiu nht l cc loi nha trao i ion tng hp, bao gm hai loi l cationit v anionit Cn bng trao i ion: Cn bng trao i cation trn nha cationit: Gi s dng nha sulphonat RSO3- H+. Khi cho nha tip xc vi dung dch cha cation Mn+ s xy ra phn ng trao i: nRSO3- H+(R) + Mn+ (dd) (RSO3- )nMn+(R) + nH+(dd) hay vit n gin: nH+(R) + Mn+(dd) Mn+(R) + n H+(dd) Hng s trao i ion ca nha c nh ngha nh sau:
K
M nH

[ M R ][ H dd ] n = [ M dd ][ H R ] n
[M R ] [ M dd ]

H s phn b DM ca cation Mn+ gia hai pha l:


DM =

DM c trng cho i lc ca nha i vI cation M n+: Cation c i lc vi cationit cng ln th b hp ph trao i cng mnh, do DM cng ln. Mt khc:
M D M = K nH

[ H R ]n [ H dd ] n

i lc ca nha i vi cation ph thuc vo pH ca dung dch tip xc: mi trng cng acid cng th i lc ca nha i vi cc cation cng km Cn bng trao i trn nha anionit: Tng t, cn bng trao i anion trn nha anionit (loi amonium) xy ra nh sau: nRNH3+OH (R) + A n (dd) nRNH3+An-(R) + nOH (dd) hay: nOH (R) + A n (dd) An -(R) + nOH (dd)

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Hng s trao i ion ca anionit v h s phn b ca anion A cng c nh ngha tng t nh i vi nha cationit. Cc giai on ca qu trnh sc k trao i ion :

n-

gia hai pha l

- Chun b ct: Ct sc k trao i ion dng trong phng th nghim thng l cc ct bng thy tinh, c t l ng knh v chiu di ln hn 10, u di c kha (ging nh burette). trnh ionit lm nghn ct, phn di ct c lt bng bi thy tinh hay bng thy tinh dy 3-10 mm. Nha ionit sch c ngm trng trong nc v rt vo ct cng vi nc trnh xut hin bt kh bao quanh ht ionit. Lng ionit ch nn chim khong 2/3 th tch ct. Pha trn lp ionit lt mt lp bng thy tinh trnh xo trn ct khi rt dung dch kho st vo. Hp thu ion trong dung dch phn tch ln ionit: Cho dung dch kho st ln ct ionit vi vn tc thch hp (2 - 3 ml/pht). Th tch dung dch c ly chnh xc v tnh ton sao cho cc ion cn trao i c hp th ht trn nha (lng ionit nn ly tha so vi lng tnh ton) Gii hp ion b hp thu trn ionit Tch cc ion b hp thu khi ionit bng cc dung dch thch hp (nh dung dch acid c nng khc nhau, hoc dung dch cc hp cht hu c c kh nng to phc vi ion cn tch...) Ra tip ionit bng cc dung dch thch hp ui ht dung dch gii hp cn st trong cc khe h ca cc ht ionit. Ti sinh ionit Ti sinh cationit: cho dung dch HCl 2 - 4% chy qua ct (i vi nha RH), hoc dung dch NaCl 2 - 4% (i vi nha RNa) Ti sinh anionit: cho qua ct dung dch NaOH 2 - 4% (i vi nha RNH2) hay dung dch HCl, NaCl 2 - 4 % (I vi nha RCl) Cui cng, ra li bng nc ct ui ht dung dch ti sinh nm trong ct. d) Sc k loi tr (vit tt SEC: Size Exclusion Chromatography): cn gi l sc k ry phn t, sc k gel, hay lc qua gel, thm qua gel. Nguyn tc: Trong sc k loi tr, pha tnh l nhng loi vt liu rn, c kh nng ngm dung mi (nc hay dung mi hu c) v trng ln to thnh b khung gel c xp cao vi kch thc cc l xp xc nh. Khi cho dung dch cc phn t cht tan qua ct, phn t no c kch thc nh hn kch thc l xp ca gel s chui su vo cc l ny v b gi li; phn t no c kch thc ln hn l gel s chy qua nhng khe h gia cc ht xp ca gel. Do ,

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khi cho pha ng i qua ct, cc phn t ln hn kch thc l xp ca gel s ra khi ct nhanh hn, cn cc phn t nh s i ra chm hn.. Do vy, th t ra gii trong SEC s theo chiu gim dn kch thc phn t. Nu kch thc phn t gn ging nhau th th t ra gii s theo chiu gim dn phn t lng. Nh vy, c ch tch da trn s khc bit v kch thc v khi lng phn t ca cc cu t c trong dung dch. Do , phng php ny cn c gi l sc k ry phn t (Molecular Sieve Chromatography).

Hnh 4.5. Tch bng sc k loi tr Cc loi gel v ry phn t: Ry hu c: dextran mng (tc sephadex), polyacrylamit, polyvinylacetat, polystyrol,..). u im ca ry hu c l c kch thc l xp a dng v khng c hot tnh hp ph, nhng nhc im l trng n mnh gy nn cht ct. Ry v c: cc zeolit tng hp, thy tinh xp, silicagel xp cao, Ry v c khng c nhc im trn nhng c kh nng hp ph mnh nn phi kh hot tnh b mt mi s dng. Yu cu quan trng i vi chn la cc ry phn t l kch thc l xp ca gel phi bng hay ln hn kch thc cc phn t cn tch. Nu hn hp cn tch cha cc phn t c kch thc thay i trong mt vng rt rng th tch bng cch ghp nhiu ct lin tip, mi ct cha mt loi ry phn t c kch thc l xp khc nhau. Pha ng (dung mi ra gii): phi ha tan cc cu t trong hn hp cn tch c kh nng lm trng gel tng thch vi detector s dng Pha ng a nc: tch cc polymer sinh hc c phn t lng ln (nh polysaccharid, protein) khng lm bin tnh chng

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Pha ng hu c: tch cc phn t hu c tng hp (v d: toluen tch cao su, polystyrol ; chloroform tch silicon, nha epoxy, polyeter bo, ) K thut thc nghim: - Thit b: c th dng ct sc k thng thng (phng php c in) hay thit b sc k lng cao p (phng php hin i) phn tch.

Hnh 4.6. S thit b sc k loi tr hin i - Tin hnh: Trong phng php c in, dng ct sc k thy tinh di 20 200 cm, ng knh 5 50 mm. u di ct lt mt lp bng thy tinh ngn khng cho gel lt xung. Np gel vo ct di dng va: trn mt lng thch hp gel hay ry phn t vi dung mi, lc u cho n ht bt kh v to thnh va; rt va vo ct, va rt va g nh. M kho cho dung mi chy qua ct (lu lc no cng phi gi li mt t dung mi trn gel khng kh khng lt vo lm nt ct), Dng micropipet np mu vo ct (lng mu ph thuc vo hiu nghim ca ct v nhy ca detector) ng thi vn cho dung mi chy qua ct. Khi a mu vo th t bnh hng (c khc vch th tch) cui ct. Thng thng, ngi ta hng c 0,5 1,0 ml cho mt phn on (tng ng vi mt c kch thc phn t) SEC l phng tin rt hiu qu : - tinh ch cc dch chit sinh hc trc khi phn tch sc k (v d: loi cc phn t ln nh chlorophyll, cc cht mn, , ch gi li cc phn t nh) - tch v xc nh s phn b phn t lng ca cc polymer t nhin (polysaccharid, protein, polypeptid, nucleotid) v polymer tng hp.

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SC K BN MNG (TLC: Thin Layer Chromatography) Pha tnh: l cc ht cht mn cht rn (kch thc c 0,5 - 0,25 m) c trng thnh lp mng (dy 100 - 200 m) trn b mt ca mt bn thy tinh, nhm hay nha polyester. Cc cht dng lm pha tnh c th l: silicagel, oxyt nhm, CaCO3, cellulose, tinh bt, sephadex, hay nha trao i ion, hoc cc dng silicagel c bin tnh ho hc. Trong mt s trng hp, c th trn vi thch cao hay tinh bt lm cht kt dnh. C ch tch c th l hp ph, phn b, trao i ion hay ry phn t ty vo bn cht ca pha tnh v cht sc k (tng t nh trng hp sc k ct) Pha ng: vic chn la pha ng cng tng t nh trng hp sc k ct tng ng Qu trnh thc hin gm cc giai on: Chun b bn mng: c th dng loi bn mng bn sn trn th trng hay t chun b. Dng bt ch k 2 vch di song song dc theo 1 cnh ca bn (cch mp bn khong 0,5 1 cm) nh du tuyn xut pht v tuyn dung mi. Np mu: dng mt mao qun thy tinh chm mu ln bn v tr tuyn xut pht sao cho to thnh vt mu c ng knh c 1 - 3 mm. kh. Trin khai sc k: t bn chun b vo mt bung trin khai, tc l mt chu thy tinh c np y kn, cha sn pha ng (mc pha ng phi nm di tuyn xut pht). bo ho kh quyn trong bung trin khai bng pha ng, c th dng mt ming giy lc tm pha ng p ln thnh bnh. Di tc dng ca lc mao dn, pha ng s i ln qua pha tnh ko theo hn hp phn tch v tch chng thnh cc vt (Nu tch tt th mi cu t s ng vi mt vt trn). Khi pha ng di chuyn va n tuyn dung mi th ly bn ra, nh du cc vt cu t v cho dung mi bay hi. K thut trin khai Sc k mt chiu: dung mi di chuyn theo mt chiu nht nh, gm 2 loi: trn Sc k xuyn tm: Mu c chm vo tm ca mt bn mng hnh trn ri c t trong bnh trin khai (cng hnh trn, nh bnh ht m). Pha ng c dn n tm bn mng nh mt si bc v y hn hp sc k i t tm bn ra ngoi theo cc vnh trn ng tm. Sc k i ln: pha ng di chuyn ln trn do lc mao dn nh m t

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Sc k gradient: tch hiu qu hn, ngi ta tin hnh sc k nhiu ln theo cng mt chiu, trong thay i dn dn phn cc ca pha ng.

Hnh 4.7. Sc k bn mng i ln

Hnh 4.8. V d v tch hn hp lipid n gin v phospholipids trong huyt thanh bng k thut TLC 1 chiu hiu nng cao CE: cholesterol esters; TG: triacylglycerols; FFA: acid bo t do; C: cholesterol; PE: phosphatidylethanolamine; PC: phosphatidylcholine; SPH, sphingomyelin; LPC: lysophosphatidylcholine Sc k hai chiu: Mu sc k c chm 1 gc ca mt bn mng hnh vung v ln lt trin khai theo hai chiu vung gc vi nhau hai h dung mi khc nhau sao cho cc vt cu t tch ra phi nm phn bn trong ca bn K thut ny cho php tch tt hn hn hp phn tch nhm trnh nhm ln trong nhn bit cc vt cht.

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Hnh 4.9. Sc k bn mng hai chiu Hnh 4.10. V d v tch hn hp lipid phc tp bng k thut TLC 2 chiu MGDG: monogalactosyldiacylglycerols; DGDG: digalactosyldiacylglycerol; SQDG: sulfoquinovosyldiacylglycerol; DPG: diphosphatidylglycerol; PG: phosphatidylglycerol; PE: phosphatidylethanolamine; PI: phosphatidylinositol; PS: phosphatidylserine; PC: phosphatidylcholine.

Cch pht hin vt cu t sau khi sc k i vi cc cu t c mu: c th nhn bit bng mt thng d dng - i vi cc cu t khng mu: pht hin bng cch phun thuc th hin mu thch hp (dung dch I2, H2SO4 c; ) thch hp hay thuc th c kh nng to vi cht phn tch mt hp cht c kh nng pht hunh quang di tc dng tia t ngoi. nh tnh v nh lng nh tnh:

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Mi cu t c c trng bng i lng tha s gi (Rf: retention factor) tng ng vi qung ng di chuyn ca n trn bn mng v c nh ngha nh sau:
Rf = x x0

vi: x khong chy ca vt cu t nghin cu, tnh t tuyn xut pht n tm vt cht x0 - khong chy ca dung mi

Hnh 4.11. Xc nh Rf ca cu t phn tch

Rf ph thuc vo bn cht ca tng cht, bn cht ca pha tnh v pha ng. Do , v nguyn tc, c th nhn bit cc cht trong hn hp sc k da vo vic so snh gi tr Rf xc nh bng thc nghim v gi tr c cng b. Tuy nhin, Rf cn thay i theo iu kin sc k (nhit , m,...) nn tt hn l dng phng php ng sc k mu v cht chun trn cng mt bn mng. nh lng: Tin hnh ng sc k mu v chun (c nng cc cht cn nh lng bit chnh xc). Sau khi pht hin cc vt cht, c th nh lng bng cc cch sau: o din tch cc vt bng my o din tch (densitometer): din tch vt cht t l vi hm lng ca n trong mu :
CM S = M CC SC

Do , c th nh lng bng phng php so snh hay phng php ng chun Co cc vt cht mu v chun tng ng v ha tan bng dung mi thch hp v nh lng chng bng cc phng php phn tch cng c no (v d: trc quang) ng dng Phng php ny thng dng nhn bit cc cu t trong mt hn hp hay kim tra tinh khit ca mt hp cht tch c. u im ca phng php ny l: nhanh, n gin phn gii kh tt, c kh nng tch v nhn bit ng thi nhiu cu t c lp li v chn lc cao

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Ngy nay, nh ghp ni vi cc thit b cho php nhn bit v nh lng cc cu t tch c (v d: TLC-HPLC), phng php ny tr nn rt hiu nghim trong vic phn tch cc mu sinh hc. IV.6. PHNG PHP SC K HIN I IV.6.1. Sc k lng hiu nng cao (HPLC : High performance Liquid Chromatography) tng hiu qu tch ca phng php sc k ct, cn tng s a l thuyt ca ct bng cch gim kch thc cc ht pha tnh (xung khong 3 - 5 m). Tuy nhin, khi ct c nn rt cht nn phi dng h thng bm cao p y pha ng i qua ct. Kt qu l hnh thnh mt k thut sc k mi: k thut sc k lng cao p (High pressure liquid chromatography) hay chnh xc hn l sc k lng hiu nng cao 2(High performance liquid chromatography), vit tt l HPLC.

a) Thit b HPLC

Hnh 412. S thit b HPLC n gin Thit b HPLC gm nhng b phn quan trng sau: h thng bm cao p, ct sc k, detector, h thng thu nhn v x l tn hiu. - H thng bm cao p: c th l mt bm hay nhiu bm dng bm pha ng t bnh cha vo ct . Cc loi bm cao p hin nay c ch to vi k thut hin i nhm m bo tc dng ca pha ng n nh lin tc. C 2 ch bm l ng p (constant pressure) v ng dng (constant flow)3. Ty thuc vo ch ra gii, c th dng mt hay nhiu bm .
2

gi l sc k lng hiu nng cao chnh xc hn v hiu qu tch v phn tch cao ca h thng HPLC khng phi quyt nh bi p sut cao m do nhiu yu t : cu to pha tnh nhi ct, h thng detector, h thng bm mu, h thng bm cao p, 3 Thng s dng ch constant flow hn

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+ Ra gii isocratic: pha ng c thnh phn khng i (c chun b sn trong cc chai ng dung mi) ch cn dng mt bm + Ra gii gradient: thnh phn pha ng thay i trong qu trnh ra gii. C 2 trng hp : - Gradient p sut thp (low pressure gradient): cc dung mi t cc bnh cha c ht v trn vi nhau p sut thp trong b trn dung mi (mixer) theo t l xc nh (t l ny thay i trong qu trnh sc k theo mt chng trnh lp sn, gi l chng trnh gradient dung mi), sau mi c bm qua ct bng mt bm cao p - Gradient p sut cao (high pressure gradient): mi dung mi trong thnh phn pha ng c ht vo cc bm cao p ring r, sau mi c trn vi nhau p sut cao trong b trn dung mi theo t l xc nh (theo chng trnh gradient dung mi) ri i qua ct sc k cn nhiu bm Lu : trnh lm hng ct sc k v xy ra hin tng nhiu nn,.trc khi bm dung mi pha ng vo ct sc k, cn kh cc bt khi ha tan trong pha ng (degass) bng cch siu m, ht chn khng, hay sc kh He (helium purge) qua dung mi. trnh s xm nhp ca khng kh, nn gi dung mi di kh quyn He. - B phn bm mu (injector): c kh nng bm lng mu c 0,1 100 l vi lp li tt (sai s khng qu 0,2%) v phi lm vic di p sut cao n 7000 psi. Mun vy, thng dng valve bm mu (injector valve) hay b phn bm mu t ng (autosampler) Loi valve ny c 6 cng, vi 1 vng cha mu c th tch xc nh tu chn (gi l loop, c nhiu loi 10, 20, 50, 100 l,). Mt nm xoay cho php chuyn i valve t v tr np mu vo vng cha mu (LOAD) sang bm mu vo ct (INJECT): + khi nm xoay v tr LOAD (Hnh 4.13, a), np mu vo (dng syringe Hamilton), mu s c cha trong loop (th tch mu tha s c thi ra ngoi). + Np mu xong, lp tc xoay nm vn ca valve (xoay cng chiu kim ng h) sang v tr INJECT (Hnh 4.13, b) - ng thi bm nt START h thng bt u tnh thi gian phn tch. Khi mu s c bm cao p y qua ct sc k cng vi pha ng.

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b) a) Hnh 4.13. Valve Rheodyne 6 cng vi cc v tr:


a) NP MU (LOAD); b) BM MU (INJECT)

Valve bm mu bng tay Rheodyne:

Hnh 4.14 . Valve bm mu bng tay H thng bm mu t ng (autosampler): t tin, thng s dng khi phn tch hng lot mu. N c kt ni vi b vi x l cho php iu khin t ng qu trnh bm mu. Dung dch mu cha trong cc l nh y kn (vial) c ht vi mt th tch xc nh bng syringe c bit v c y vo loop c th tch tng i ln (~100 l) trong mt valve 6 cng (tng t nh trong valve bm mu bng tay).

Hnh 4.15. Agilent 1100 Series Autosampler Vic chun b mu trong sc k lng cao p rt quan trng. Mu phi c chun b dng dung dch. Cc mu rn nn c ha tan trong dung mi thch hp (tt nht

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l bng dung mi pha ng), sau phi loi b cc ht rn khng tan (bng cch siu lc hay ly tm) trc khi bm vo ct trnh lm nght h thng bm mu hay ct sc k. Nhng cu t cn tr qu trnh phn tch cng cn c loi b bng cc phng php chit tch thch hp. - Ct sc k: l mt ng thng thng lm bng mt loi thp khng g c bit, di c 3 25 cm, ng knh trong t 2,6 4,6 mm (i vi ct phn tch) hay t 6,4 12,7 mm (i vi ct sc k iu ch).

Hnh 4.16. Ct sc k tiu chun Cc ht pha tnh c nhi vo ct v c gi gia 2 a xp hai u ct. i vi ct c ng knh trong 2,6 mm, kch thc ht pha tnh thch hp l 30 m. Pha tnh nhi trong ct c th c bn cht khc nhau ty thuc vo k thut tch s dng (sc k hp ph rn- lng, sc k phn b lng lng, sc k lng trao i ion, sc k ry phn t). kt qu phn tch n nh, c bit khi phn tch nh lng hay phn tch cc polymer bng k thut sc k ry phn t, ct sc k thng c gi trong l iu nhit (dao ng nhit khng qu 0,2 0C) cha khng kh nng tun hon vi tc cao. tng tui th ct sc k, trc ct phn tch c lp thm mt ct bo v (guard column) di khong 1 cm gi nhng tp cht khng mong mun v gim bt tc ng xu n ct do s thay i p sut t ngt. Lu lng pha ng khng nn vt qu vi ml/pht. - Detector: Sau khi qua ct sc k, cc hp cht trong mu phn tch s c tch thnh tng phn on v i vo detector ghi nhn tn hiu. Cc tn hiu thu c s c th hin di dng cc peak trn sc k . Cc loi detector dng trong HPLC bao gm: Detector hp th t ngoi - kh kin (UV-Vis detector): o cng hp th bc x t ngoi ca pha ng i ra khi ct sc k ( mt bc sng.hay nhiu bc sng) Loi detector ny c dng ph bin nht v d s dng, t ph thuc vo nhit v lu lng pha ng. Tuy nhin, n ch s dng cho cc cht c kh nng hp th bc x t ngoi kh kin trong vng 190 - 600 nm (pha ng s dng phi khng hp th bc x trong vng bc sng nghin cu)

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pht hin nhng cht khng hp th t ngoi, c th chuyn chng thnh dng dn xut c kh nng hp th bc x t ngoi. Detector chit sut (RI detector: Refractive index detector): o s thay i chit sut ca ca pha ng ra khi ct sc k. Detector ny vn nng nhng km nhy, khng chn lc, thng dng trong sc k ry phn t.. Detector hunh quang (FD: Fluorescence detector): o s thay i cng pht hunh quang ca cc hp cht phn tch sau khi hp th bc x t ngoi thch hp ca pha ng sau khi qua ct tch. Detector ny rt nhy v rt chn lc Ngoi ra, cn c nhng detector khc nh detector in ha (electrochemical detector), detector khi ph (mass spectrometric detector), nhng chng t thng dng hn - H thng iu khin v x l d liu : l my vi tnh vi cc phn mn chuyn dng v giao din (interface) cho php iu khin hot ng ca tng b phn trong h thng nh iu chnh ch bm pha ng (tc dng, ch ra gii), nhit ct, bc sng bc x pht ra ca detector, tnh kt qu phn tch, lu tr d liu b) Chn pha tnh v pha ng trong phn tch HPLC Vic chn pha tnh ty thuc bn cht hn hp cn tch v c ch sc k la chn v theo nguyn tc tng t trong sc k ct c in. a s trng hp l phn tch cc hp cht hu c c phn cc yu hay trung bnh c ch tch thng l sc k phn b lng lng. Khi , c th chn 1 trong 2 loi ct sc k: - ct pha thng (normal phase) nhi bi cc ht pha tnh phn cc (cu to t silicagel ghp vi cc nhm phn cc nh aminopropyle, cyanopropyl, benzyl,). Khi , pha ng phi khng phn cc (v d: hexan) hay km phn cc (hn hp hexan vi dung mi km phn cc nh CHCl3, CH2Cl2, THF, ) Cc cu t s ln lt c tch ra theo th t tng dn phn cc. - ct pha ngc (reversed phase): nhi bi cc ht pha tnh khng phn cc (cu to t silicagel gn vi cc dy nhnh khng phn cc nh C8 hay C18) Khi , pha ng phi l dung mi phn cc (v d: metanol, acetonitril) hay hn hp ca chng vi nc (v d MeOH : H2O 40 : 60; ) hay phn cc trung bnh Cc cu t s ln lt c tch ra theo th t gim dn phn cc. c) ng dng Phng php HPLC c th c dng phn tch cc t km phn cc n phn cc, cc hp cht ion, v c hay hu c vi phn t lng t 300 2000 dalton, tc l chim ti 80 % cc ng dng trong k thut sc k. V vy, phng php ny c ng dng rng ri trong nhiu lnh vc: ha hc, sinh ha, mi trng, dc

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phm, nng lng thc phm (v d: kim tra tinh khit ca dc phm, phn tch d lng v vt c t trong mi trng v thc phm,). IV.6.2. PHNG PHP SC K KH (GC: Gas Chromatography) Sc k kh l mt trong cc phng php tch v phn tch rt nhy v hu hiu, ra i vo nhng nm 1940 nhng thc s bt u pht trin mnh t nm 1952 nh cc cng trnh nghin cu ca gio s Keulemann (H Lan). Ngy nay, n l mt trong cc cng c c ng dng rng ri trong nhiu lnh vc: ha hc, ha sinh, thc phm, dc phm, mi trng, v nhiu lnh vc khoa hc k thut khc. IV.6.2.1. Nguyn tc: Trong sc k kh, s tch da trn s khc nhau v tan ca cc cu t trong hn hp phn tch trong pha tnh lng c trng ln b mt ca mt cht mang rn. tr. Pha ng l mt kh tr khng tng tc vi pha tnh v mu phn tch (v d : H 2, N2, CO2, He hay Ar, c cha trong mt bom kh hay c to ra nh mt my sinh kh), ng vai tr l mt kh mang (carrier gas) dn mu ( trng thi hi) i vo ct sc k. Qu trnh xy ra trong phn tch sc kh kh nh sau: Mu phn tch c ha hi v c chuyn vo ct sc k nh dng kh mang (c p sut t 0,2 - 4 bars). Ti xy ra qu trnh sc k, tch hn hp phn tch thnh cc cu t ring r. Sau khi ri khi ct tch, cc cu t ln lt i vo detector cc thi im khc nhau, ti chng c ghi nhn nh nhng tn hiu in. Cc tn hiu ny c khuch i ri chuyn sang my ghi v x l d liu (v sc k , tnh din tch cc peak, lu kt qu, in kt qu, ). Cng tng t nh trong phng php HPLC, da vo sc k thu c c th nh tnh v nh lng cc cu t trong hn hp phn tch nh sau : - nh tnh : da vo thi gian lu ca cu t nghin cu (so snh vi thi gian lu ca mu chun; phng php ng sc k) - nh lng: da vo din tch peak (hay chiu cao peak) ca cu t nghin cu (phng php so snh; phng php ng chun; phng php thm) IV.6.2.1. Thit b GC Hnh 4.16 trnh by s cu to (a) v hnh nh (b) ca thit b GC.

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(a) S thit b GC

(b) Thit b GC ghp ni vi my tnh Hnh 4.17. S cu to (a) v hnh nh (b) ca thit b GC Thit b GC gm cc b phn chnh sau y: + Bung bm mu (Injector) c cc chc nng sau: - ha hi mu (nu mu trng thi lng) - a mu vo u ct sc k nh dng khi mang. Mu c bm vo injector bng mt microsyringe c kim di v nhn, xuyn qua mt septum cao su v c kh mang a vo bung bay hi ( vapourisation chamber). Ti , mu s c ha hi ngay lp tc v c dng kh mang a vo u ct sc k. Nhit ha hi mu phi cao hn 50 0C so vi nhit ha hi ca cu t kh bay hi nht trong mu. t phn gii tt (cc peak ca sc k sc nhn, khng b gin rng), lng mu a vo ct khng c ln qu. i vi cc loi ct mao qun (l loi ct thng dng nht trong sc k kh hin nay) th th tch mu bm vo ch cn khong 10 3 l.

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a) b) Hnh 4.18. Microsyringe (a) v injector (b) dng trong sc k kh C th chn 1 trong 2 ch bm mu sau y : - split (chia dng): s dng khi lng mu bm vo ln qu. ch ny, mu sau khi ha hi s c chia thnh 2 dng theo t l chn la (sao cho sc k t phn gii tt), mt phn s theo kh mang vo u ct sc k, phn cn li s c kh mang y ra ngoi (qua split oulet) - splitless (khng chia dng): s dng khi lng mu phn tch nh hay va phi. + Ct sc k: Hin nay thng dng ct mao qun FSOT (Fused Silica Open Turbular Column) c ng knh trong khong 0,1- 0,35 mm v di 15 - 100 m. Ct l mt ng mao qun bng silicagel, bn trong ph mt mng mng dy 0,25 - 0,5 m (thc cht mng ny l dy nhnh hu c ghp trn b mt silicagel bi lin kt ho hc), bn ngoi ph bng mt lp polyimid. Nh vy, ct rt bn i vi cc tc nhn l - ha hc v rt do (ct di c th cun thnh vng xon, rt gn). Ngoi ra, ct mao qun cn c nhng u im sau: - hiu qu tch rt cao (cho php tch nhng hn hp rt phc tp vi phn gii cao) - lng mu phn tch t ; lu lng kh mang s dng nh

Hnh 4.19. Cu to ca ct mao qun dng trong sc k kh Trong sc k kh, nhit nh hng rt ln n hiu qu tch, do t kt qu phn tch chnh xc, ct phi c t trong mt l iu nhit (sai s nhit khng qu 0,10C). Nhit ti u ca l ph thuc vo im si ca cc hp cht

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trong mu phn tch. Nhit l thng cao hn mt t so vi nhit si ca cu t kh bay hi nht, nhng phi thp hn nhit ti a cho php ca ct khong vi chc . Lu : Nhit ct cng cao thi gian sc k cng ngn nhng hiu qu tch gim i; nhit thp c th cho kt qu tch tt hn nhng thi gian sc k ko di. Do vy, cn kho st chn nhit ti u cho qu trnh tch. Nu mu cha cc hp cht c im si rt khc nhau thi nn ra gii bng dng chng trnh nhit (nhit ct tng lin tc hay tng bc trong qu trnh tch) + Detector: c nhiu loi, vi nhy v chn lc khc nhau. Vic chn detector ch yu ph thuc bn cht mu phn tch, ngoi ra cn ph thuc bn cht kh mang s dng. Bng sau trnh by c tnh ca mt s detector thng dng.
Detector Loi Ph thuc dng khi lng (Mass flow dependent) Kh s dng nhy v chn lc Kh nng pht hin Khong tuyn tnh

Ion ha ngn la (Flame Ionization Detector : FID)

Vn nng cho hu ht cc H2 v khng hp cht hu c c nhm 100 pg kh -CH2 - ; nhy cao c trng v nhy, dng cho cc hp cht hu c cha cc nguyn t m in (N, Cl, P, ..) phn tch thuc tr su, thuc dit c,

107

Bt gi in t (Electron capture detector: ECD)

Ph thuc nng (Concentration Kh Ar dependent)

50 fg

105

Ph thuc dng Nitrogen Phosphore khi lng detector: NPD) (Mass flow dependent)

Hp cht cha N, P (phn H2 v khng tch dc phm, d lng kh thuc tr su, )

10 pg

106

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Hnh 4.20. l s detector FID. Cc cu t ra khi ct sc k c trn vi kh H2 (kh mang) v khng kh, b t chy nhit cao, to thnh dng ion v electron dn in. Cc ion v electron ny chuyn v cc bn cc tri du nm 2 pha ca ngn la (hiu th gia 2 bn cc ny khong 250 300 V) v sinh ra dng in. Dng ny c khuc i v ghi nhn thnh cc tn hiu di dng peak sc k. Hnh 4.20. Detector FID Ngoi ra, cn c nhng loi detector khc nh detector quang k ngn la (Flame Photometric Detector FPD), detector khi ph (Mass Spectrometric Detector MS) b) u, nhc im - ng dng: Phng php sc k kh l phng php hu hiu phn tch nhng hn hp phc tp trong thi gian rt ngn. So vi phng php sc k lng hiu nng cao, phng php ny c nhy cao hn, t tn km hn. Tuy nhin, phng php ny ch p dng phn tch cc hp cht d bay hi (v d cc ester, hp cht trong sn phm ha du, hng liu,) hay c kh nng chuyn thnh dn xut d bay hi (nh cc acid bo, ). i vi nhng hp cht km bn nhit (nh cc hp cht sinh hc) th khng th p dng phng php ny m phi dng phng php HPLC. Phng php GC c ng dng rng ri phn tch thc phm (v d: thnh phn hng liu, acid bo trong thc phm; thnh phn acid amin,). c bit, dng k thut GC-MS cho php phn tch d lng c t mc ppb (v d: 3-MCPD trong nc tng, nc mm, phn tch d lng khng sinh, thuc bo v thc vt,...trong thc phm.).

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TI LIU THAM KHO


Ting Vit: 1) Nguyn Th Thu Vn, Phn tch nh lng, 2004, HQG TP. H Ch Minh. 2) Phm Lun, C s l thuyt ca php o ph hp th phn t UV-Vis, 1994, HQG H Ni. 3) Phm Lun, C s l thuyt phng php phn tch ph hp th nguyn t, 1999, HQG H Ni. 4) J. H. Kenedy, C s ha hc phn tch - Ch.19-Phng php sc k, 1994, (Hong Minh Chu dch) 5) Phm Hng Vit, C s l thuyt ca phng php sc k kh, 2003, NXB. Khoa hc v K thut Ting Anh 6) L. Ebdon, E. H. Evans; A. Fisher, S. J. Hill, An Introduction to Analytical Atomic Spectrometry, 1998, J. Wiley. 7) Marvin C. McMaster, 2003, HPLC A Practical Users Guide, 2 Ed. 2007, J. Wiley 8) Harold M.; Mc. Nair; James M. Miller, Basic Gas Chromatography, 1997, J.Wiley

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