bài giảng phân tích huỳnh quang và phát quang

HUNH QUANG V HA

PHT QUANG

(Fluorescence and chemiluminescence)

NI DUNG

PHN B CHNG TRNH

C CU IM

NI DUNG BI GING

PHN B CHNG TRNH STT Chng S tit

1 i cng v ph phn t 3

1.1. C s l thuyt ca phng php ph phn t

1.2. C s l thuyt ph pht quang

1.3. Quy tc chn lc trong ph phn t

2 Phng php hunh quang v ln quang 17

2.1. Cc qu trnh xy ra trong hin tng hunh quang v ln quang

2.2. Ph hunh quang v cc tnh cht c bn

2.3. Hin tng tt hunh quang

2.4. S pht quang ca dung dch

2.5. Cu trc ca hp cht hunh quang

2.6. ng dng phng php ph hunh quang

3 Ha pht quang 11

3.1. C s l thuyt ca phng php ha pht quang

3.2. ng dng ha pht quang trong ha phn tch

Tiu lun Thng k

(20%)

Gia k

(20%)

T lun

Cui k

(60%) T lun

C CU IM

Chng 1. i cng v ph phn t

C s l thuyt ca phng php ph phn t 1.1.

1.2 C s l thuyt ph pht quang

Quy tc chn lc trong ph phn t 1.3.

bi (): s trng thi lng t ca phn t khc nhau v

nh hng spin electron tng.

i vi cc h phn t c tng tc spin orbital nh th

cc trng thi ch khc nhau bi nh hng spin u c

cng nng lng.

= 2S + 1 S: tng spin phn t

=1: trng thi n bi (singlet);

=2: trng thi bi hai (dublet);

=3: trng thi bi ba (triblet);

Mt s khi nim c bn

1.1. C s l thuyt ca phng php

ph phn t

Nng lng phn t: E = Eel + Evib + Erot

Mc nng lng

quay (Equay)

Mc nng

lng dao ng

(Edaodong)

Mc nng lng

in t (Eel)

Eelectron > Edaodong > Equay

Mt s khi nim c bn


ph phn t

Nng lng phn t: E = Eel + Evib + Erot

Ph electron Ph dao ng

(IR)

Ph quay (vi

sng)

Mc nng

lng quay

(Erot) Mc nng lng

dao ng (Evib)

Mc nng

lng in t

(Eel) E0

E1

Mt s khi nim c bn


ph phn t

Cc bc chuyn in t trong phn t

Khi nhn nng lng, mt electron orbital lin

kt hoc khng lin kt c th chuyn ln cc orbital

phn lin kt cn trng mc nng lng cao hn.

Khi xy ra cc bc chuyn in t sau:

*;

*;

n *;

n *;

Bc chuyn mc N V

Bc chuyn mc N Q

Bc chuyn mc N R

Mt s khi nim c bn


ph phn t

Cc bc chuyn in t trong phn t

*

*

n

E

*

*

n *

n *

C s l thuyt phng php ph phn t

h2

Khi nguyn t, phn t hp th photon

th chuyn trng thi t trng thi c bn

ln trng thi kch thch c mc nng

lng cao hn. Ph hp thu

Nng lng do nguyn t, phn t hp

thu c th gii phng di dng bc x

in t, t trng thi kch thch v trng

thi ban u. Ph pht x


Pht quang m cc

Pht quang nhit

Quang pht quang

Phn loi hin tng pht quang


Pht quang ha hc

Pht quang sinh hc

Pht quang in v pht quang

ma st

=> Mi dng pht sng trn gi l s pht quang (Luminescence)

S pht x trong vng thy c nhit thp hn 5000C gi

l hunh quang v pht quang ha hc c nhiu ng dng vo mc

ch phn tch.

Gin nng lng Jablonski

S0: trng thi c bn;

S1: trng thi kch thch c bi

n;

T1: trng thi kch thch c bi 3;

A: hp thu (adsorption);

F: hunh quang (fluorescence);

P: ln quang (phosphorescence)


1.2.1. Gin nng lng Jablonski


1.2.1. Gin nng lng Jablonski


--> Qu trnh pht bc x

~~> Qu trnh khng pht bc x

IC Internal conversion s chuyn ha ni phn t

ISC Intersystem Crossing kt hp ni phn t, t S1 T1

IEC Internal and External conversion s mt nng lng di dng nhit

SR(VR) Relaxation of vibration Hi phc do dao ng

Qu trnh hp thu nng lng (A): Khi hp thu

sng in t, phn t b kch thch in t chuyn t

trng thi So S1 hoc S2 (t=10-16-10-15 s).

Trng thi ny khng bn, electron c xu hng

gii ta nng lng hp thu tr v trng thi c

nng lng thp, bn vng hn. S gii ta nng lng

c th thc hin theo nhiu cch ty thuc vo cu trc

phn t.

1.2.2. Cc qu trnh gii ta nng lng phn t


Qu trnh lm mt hot tnh nng lng trng

thi kch thch c th khng km theo s pht proton h:

IC (chuyn ha mi phn t) nng lng cho qu trnh

chuyn v ni phn t (S2 S1);

VR (s phc hi do dao ng) mt nng lng do dao

ng v do s quay (S1(v) S1(0); T1(v) T1(0)) ;

IEC (s chuyn ha di dng nhit) chuyn nng

lng cho cc phn t xung quanh (dung mi) di dng

nhit;



Qu trnh lm mt hot tnh nng lng trng

thi kch thch c th khng km theo s pht proton h:

ISC (kt hp ni phn t) chuyn nng lng gia cc

mc c bi khc nhau (S1(v=0) T1(v=1,2,3), khi

tha mn 2 iu kin:

- Nu trong phn t ng cong th nng S1 v T1 ct

nhau, khi

E = ES1-ET1 < 10 Kcal

- Mc dao ng v=0 ca S1 bng vi v cao ca T1.



Qu trnh lm mt hot tnh nng lng trng thi

kch thch c th km theo s pht proton h:

F (pht x hunh quang) bc x pht ra khi thc hin

bc chuyn S1(v=0) S0(v=0,1,2), thi gian thc hin

bc chuyn 10-9-10-6 s;

P (pht x ln quang) bc x pht ra khi thc hin bc

chuyn T1(v=0) S0(v=0,1,2,3); thi gian thc hin bc

chuyn 10-4-10-2 s;

DF (hunh quang tr)



i vi phn t c tm i xng (quy tc la chn theo

tnh chn l, quy tc Laporrte):

Bc chuyn t g u hoc u g: c php;

Bc chuyn t g g hoc u u: b cm;

Chuyn mc c bi khc nhau b cm, v d chuyn

mc singlet triplet b cm do bi;

Chuyn mc cc phn t khng c tm i xng ph

thuc vo tnh i xng ca trng thi u v trng thi

cui;

1.3. Quy tc chn lc trong ph phn t

Chng 2. Phng php hunh quang

v ln quang

2.1. Cc qu trnh xy ra trong hin tng hunh quang v

ln quang

2.2. Ph hunh quang v tnh cht c bn




2.6. ng dng ca hin tng hunh quang

Intersystem

crossing

absorption fluorescence phosphorescence

S0

T1

S1

Hunh quang Ln quang

S0 + h S1(v);

2.1. Cc qu trnh xy ra trong hin tng hunh

quang v ln quang

S1(v) S1(0);

S1(0) S0(v) + h;

S0 + h S1(v);

S1(0) T1(v);

T1(v) T1(0);

T1(0) S0(v) + h;

S1(v) S1(0);

Hunh quang tr S0 + h S1(v);

S1(v) T1(v);

T1(v) S1(0);

S1(0) S0(v) + h;

Intersystem

crossing

absorption delay

fluorescence

S0

T1

S1


quang v ln quang

So snh ph hunh quang v ph ln quang

Ging: cng bn cht, u l qu trnh pht x.

Khc:

thi gian sng (thi gian tn ti ca mt trng thi kch

thch).

- ca bc x ln quang khong 10-4-10-2 s;

- ca bc x hunh quang ch khong 10-9-10-6s, v vy

bc x hunh quang tt ngay khi tt nh sng kch thch.

mi quan h gia ph hunh quang, ph ln quang vi

nh sng kch thch: ex < em (F) < em (P)


quang v ln quang



quang v ln quang


A

phenanthrene


quang v ln quang

So snh ph hunh quang v hunh quang tr

Ph hunh quang tr c thnh phn ph nh ph hunh

quang, nhng c thi gian pht quang nh hin tng ln

quang.

Nguyn nhn ca hin tng hunh quang tr l do trc

khi bc x photon phn t trng thi triplet mt thi gian.


quang v ln quang


Ph hunh quang: hm s phn b nng lng do cht hunh

quang bc x theo tn s hoc theo bc sng.

Hnh dng: ging ph hp thu, cng thuc phn b Gauss.

Ph hunh quang c xc nh bi thnh phn, cu trc ca

tm hunh quang (nhm mang mu C=C, C=O, C=S, -N=N-

) v b nh hng bi mi trng ngoi.


Qui lut Kashi: Dng ca ph hunh quang khng ph

thuc vo nh sng kch thch.

F

S0

S1

F: S1(v=0) S0(v)

A A F

nm

Ph hp thu A (UV), A (VIS) v ph

hunh quang ca fluorescein K: h s hp thu tng i

A


F

S0

S1

A

F A

.10-12, s-

1 Tnh i xng ca ph hunh quang v

ph hp thu ca rodamin

Qui lut Levshin: Khi cc mc dao ng trng thi kch thch v

trng thi c bn c cu to nh nhau v khong cch nng lng

ca cc mc tng ng nhau th ph hunh quang v ph hp thu

i xng gng vi nhau qua ng vung gc vi trc tn s v i

qua giao im ca hai ph .


Qui lut Stoke - Lommel: Ph hunh quang b dch chuyn v pha

bc sng di (cn ca ph) so vi ph hp thu (do mt nng

lng bi dao ng VR(SR)).

= max(em) - max(ex) => s dch chuyn Stocke


Cc trng hp khng tun theo qui lut Stoke - Lommel

Bc x cng hng: Bc x pht ra

t S1(v=0) S0(v=0) c cng di

sng bng vi bc x hp thu.

Hunh quang tng nhy (antistocke)

c em < ex: dng hunh quang ny

c thy trong cc dung dch cha mt

s hydrocarbon a vng trong hn hp

cht kh.

F

S0

S1

F: S1(v=0) S0(v)

A

Bc x cng hng

2.3.1. Hiu sut hunh quang Hin tng tt pht quang l hin tng lm gim hiu sut ca

nng lng pht quang.

Hiu sut pht quang nh gi s bin i hiu dng gia Eex sang

Eem. C 2 loi hiu sut pht quang:

Hiu sut nng lng (NL): l t s nng lng hunh quang ca

cht kho st Eem v nng lng nh sng kch thch Eex.

Hiu sut lng t (LT ): l t s gia s lng t hunh quang Nem

v s lng t nh sng kch thch (hp thu).

1ex

emNL

E

E

1ex

emLT

N

N


2.3.1. Hiu sut hunh quang

Mi lin h gia hiu sut nng lng v hiu sut lng t

)(

)(

)(

)(

..

..

TBex

TBem

LT

TBexex

TBemem

ex

emNL

hN

hN

E

E

Trong ex(TB), em(TB) l tn s hiu dng trung bnh ca nh

sng kch thch v nh sng pht xa.

Khi kch thch bng nh sng n sc th: ex(TB) = ex, em(TB) = em

em

exLT

ex

emLTNL

Trong iu kin thun li th NL~ 1.

Thc t em> ex LTNL


2.3.1. Hiu sut hunh quang

S ph thuc ca hiu sut hunh quang vo nh sng kch thch

nh lut Babilov (nh lut tt hunh quang): khi tng bc

sng kch thch th hiu sut hnh quang tng, sau s khng thay

i trong khong bc sng no ri cui cng s gim r rt khi

tng bc sng kch thch.

0.2

0.2

0.6

0.8

1.0

200 400 600 , nm

S ph thuc ca hiu

sut hunh quang vo

bc sng nh sng

kch thch


2.3.2. Cc yu t nh hng n hiu sut

hunh quang - tt pht quang

Tt pht quang c th do nhiu nguyn nhn khc nhau:

- Cc qu trnh chuyn ha ni phn t;

- Do s tng tc gia cc phn t.

S chuyn ha ni phn t t S1 S0 khi ng cong S1

ct S0:

S tn mt nng lng theo cc mc dao ng v quay

(di dng nhit).

c trng cho cc phn t c cu trc mm (c nhiu

nhm quay t do), v d thuc nhum azo,

triphenylmetan.




S hnh thnh cc cht c tnh pht quang thay i

Trng hp 1: ng th nng S1 ct T1 => F gim,

P tng.

Phn t cha electron khng ghp cp (gc t do) c

kh nng phn ng cao to hng lot cht ha hc mi c

hot tnh quang hc khc cht hunh quang ban u (c

th cho cht hunh quang c cng mnh hn nhng

cng c th to thnh cht v hot th hiu sut hunh

quang gim hoc tt hunh quang).




nh sng kch thch: Khi ex ngn (nh sng kch thch c

nng lng ln) phn t cht hunh quang c th b phn li,

in hnh nh:

Lin kt C C c th t khi s dng ex ~ 200nm;

Lin kt C N c th t khi s dng ex ~ 250nm

Trong ph hunh quang thng ex 250nm.




Mt hot tnh hunh quang do va chm khng n hi vi cc

phn t xung quanh. Nng lng ca trng thi kch thch ny s

bin thnh nng lng dao ng ca phn t va chm vi n.

Hiu ng tt ny ph thuc nhiu nguyn nhn:

Nhit ;

nht;

Nng ca chnh cht hunh quang;

Cc cht l;

nh hng ca oxi: do s che ph che ph ca vn ph hunh

quang v vn ph hp thu mc d khng c v chm n hi.



2.4.1. nh hng ca pH

Ph hunh quang v ph hp thu ca dn proton ha v dng

khng proton ha ca cc acid v baz hu c khc nhau.

S thay i pH dung dch c th lm thay i mu v cng

hunh quang ca chng.

ng dng: + ch th hunh quang acid baz;

+ xc nh hng s phn li trng thi kch thch

bng cch so snh ph hunh quang v ph hp thu khi thay

i pH dung dch.


2.4.1. nh hng ca pH

Ph hp thu ca

Acridin

Ph hunh quang

ca Acridin

Cacridin = const = 4.10-6 g/ml; 1 pH = 7; 2 pH = 6; 3 pH = 5; 4 pH = 4

Khi gim tnh acid ca dung dch acridin mu hunh quang

thay i t mu xanh l cy trong mi acid (hunh quang ca dng

ion acridin) sang mu tm trong mi trng kim (hunh quang ca

phn t acridin). Dng lm ch th acid baz

2.4.1. nh hng ca pH quinin

Q(OH)2

HQ mu xanh da tri

Q(OH)+ Q2-

HQ mu tm khng pht HQ

Kt qu nghin cu cho thy cng hunh quang ca

quinin t l vi nng ion pht quang.

Da vo ph hunh quang c

th xc nh hng s phn li

ca quinon.

S ph thuc ca mu hunh quang

p-nitrophenol c dng ging s ph

thuc ca bc phn li vo pH


2.4.1. nh hng ca pH

trnh nh hng ca pH cn tin hnh phn tch hunh

quang iu kin nht nh sao cho trong dung dch tn

ti:

dng ion;

dng phn t;

dng ion v dng phn t vi mt t l xc nh

bng cch s dng dung dch m.

Cch loi tr nh hng ca pH


2.4.2. nh hng dung mi

Hp cht hu c hunh quang c th chia lm 2 nhm:

o Hidrocacbon ;

o hp cht cha N, O (c tnh acid hay baz, moment lng cc).


nh hng ca dung mi th hin qua:

nht: u nh hng n 2 nhm cht HQ

Khi nht cao s va chm lm gim hot tnh HQ

gim hiu sut hunh quang cao.

nht (kh) < nht (lng) < nht (rn)

cht kh khng th hin tnh ln quang, cht lng cho

ln quang c thi gian ngn hn cht rn.


Hin tng solvat ha:

hin tng solvat ha cng mnh th xc sut

chuyn nng lng cho cc phn t khc cng cao

v kh nng xut hin hiu ng tt hunh quang

cng ln.

S tng solvat ha c th lm ph hunh quang dch

chuyn v pha sng di.




pH dung dch: cc dung mi khc nhau c pH khc

nhau nh hng n kh nng phn li ca cht HQ

thuc nhm 2.

tng tc gi dung mi v cht tan:

tng tc lng cc (tng tc Debai);

tng tc gia cc h in t (tng tc London);

tng tc ha hc.




Tng tc Debai v London gy ra hin tng solvat ha

cho tt c cc hp cht HQ v lm cho ph hunh quang dch

chuyn v pha bc sng di (tuy khng ng k) nhng cu

trc ph khng thay i. Hin tng solvat ha cng mnh

(ch s khc x ca dung mi cng ln) xc sut chuyn

nng lng cho cc phn t cng cao hiu ng tt hunh

quang cng ln.

tng tc ha hc: i vi cht c nhm hidroxyl, cacboxyl;

amin to lin hp do lin kt hidro.



Cc cht hu c c in t bt nh v.

Mt s ion v c, c bin dng tng:

F-< NO3-


2.5.1. Cc hp cht hunh quang hu c

2.5.2. Cc hp cht phc hunh quang



L cc hp cht thm hoc d vng c ni i tip

cch, phn t c tnh i xng, phng



iu kin cn thit mt hp cht c kh nng pht x

hunh quang: phn t ca hp cht phi c cu trc cng.



C ch ca qu trnh pht x

C ch ca s pht bc x c th gii thch theo s sau:

Phn t b kch thch, hnh thnh cu trc nh cc hoc

chuyn sang c ch gc c bo v, trnh va chm vi cc

phn t ca dung dch bao quanh cu trc khng bn s

phng bc x hunh quang tr v trng thi u. Ngc li

hunh quang s tt.

Trng thi u Trng thi kch thch

CH N CH N+ -

CH N. .



nh hng ca nhm th

Khi gn nhm th alkyl thay i rt t;

Khi gn nhm th (OH, OR, NH2) tng hiu sut hunh

quang;

Nhm c in t khng lin kt (c bc chuyn n *)

tt hunh quang;

Khi gn cc nguyn t c s th t ln trong phn t cht

HQ hay dung mi thng lm tng ln quang, gim hunh

quang. V d hiu ng tng trong dy: F



Cc hp cht thm c cu trc cho php chuyn ng

quay tng phn ring bit trong phn t khng pht hunh

quang nhit thng. Nhng tnh hunh quang s xut hin

khi chuyn ng quay v dao ng b dng li:

H thp nhit ;

Hp ph ln cht mang rn (Al2O3, SiO2,);

To thnh cc phc chelat.


V d: pht HQ khi hp ph ln cht mang rn (Al2O3, SiO2,):

V d: phc chelat hunh quang

2, 2 - diazobenzen Aldehidsalicyliden

(salicyaldehid)

salicyliden 2 aminophenol


2.5.2. Cc hp cht phc hunh quang V d: phc hunh quang

dn xut oxy hoc amino ca antraquinon

Al3+, B3+, Be2+,..

8-Hydroxyquinonlin

Al3+, CN-, F- Ga3+, In3-,..

Rodamin 6G

In, Re, Ta, Tl

Calcein

Ca2+



Cc yu t nh hng n hiu sut pht quang

ca phc hunh quang

nh hng ca bn cht

dung mi: Cc dung mi

thng s dng trong

phng php hunh quang

l cc dung mi hu c,

dung mi c nht cao,

cng hunh quang cao.

DMF: dimetylformamid;

DMSO: dimetylsunfaxid;

DIOX: dioxan;

EtOH: etanol.



ca phc hunh quang

nh hng ca pH:

S ph thuc cng hunh quang cc phc gia

semicarbazolsalicyden aldehid (SS) vi ion kim loi chit trong iso-

pentanol vo nng HClO4



ca phc hunh quang

nh hng ca ion kim loi:

Nhng ion kim loi c lp v e ngoi cng bn vng 2,8,18 e

nh Be(II), Mg(II), Zn(II), Al(III), Sc(III), Ga(III) cho chelat

hunh quang c cng t l vi th ion ha ca kim loi.

1 - formylhidrazen

2 - semicarbazen

Kim loi nh cho cng HQ cao

hn kim loi nng

nghch t (ln

quang mnh hn).



ca phc hunh quang

nh hng ca ion kim loi:

Cc ion kim loi chuyn tip: Cr(III), Fe(III), Cu(II), thng

khng cho chelat hunh quang ngc li cn lm tt hunh quang

ca nhng cht khc.

Hiu ng tt tng theo bn ca phc cht:

Mn(II), Fe(III) < Co(II), Ni(II) < Fe(III),Cu(II), Cr(III)

Nguyn nhn lm tt HQ:

CH N+ -

Fe2+

Fe3+

e eCH N



ca phc hunh quang

nh hng ca ligand

Hiu sut cng cao khi ligand to vi ion kim loi chelat nhiu

vng.

CH N

MO O

CH N

MO

Salicyal-o-aminophenol Salicyal-o-aminobezyl

2.6. ng dng ca hin tng hunh quang

2.6.1. u, nhc im ca phng php hunh quang

2.6.2. Nguyn l ca php phn tch hunh quang

2.6.3. S my hunh quang v nguyn tc vn hnh

2.6.4. Xc nh cc hp cht hu c hunh quang t nhin

2.6.5. S pht hunh quang da trn phn ng ha hc

2.6.6. ng dng ph hunh quang trong phn tch th tch

2.6.1. u, nhc im ca phng php hunh quang

Phng php ph hunh quang c s dng rng ri nh c

nhiu hp cht hunh quang t nhin v cc hp cht hunh

quang c to thnh trong cc phn ng ho hc.

u im so vi ph hp thu:

nhy cao hn 10-1000 ln, gim nhiu nn;

Kh nng xc nh t ppb n ppm;

Tnh chn lc cao hn;

C th xc nh trong dung dch c hoc c mu;

Tuy nhin: Xc nh trong dung dch cn lu n yu t mi

trng: pH, nht, hm lng oxy ho tan, cc ion l,v

d b mi mt khi quang st ph hunh quang.


I0, 1 I, 1

IF, 2

l Ia

S m t nguyn l ca php o

hunh quang

i vi mt cht hp thu quang:

lCI

I0lg

lC

I

I 100

Ia lng nh sng b hp thu:

)101(00lC

a IIII

Hiu sut hunh quang:

a

F

I

I )101(0

lC

aF III


Khai trin theo Maclaurin:

!3

ln

!2

ln

!1

ln1

3322 axaxaxa x

!3

)303,2(

!2

)303,2(

!1

303,2101

32 lClClClC

B qua v C nh

ClIIF 303,20

Trong : Io - cng ca nh sng kch thch;

- hiu sut hunh quang, c trng cho tng hp cht;

- h s hp thu mol ca hp cht hunh quang, l/mol.cm;

l - chiu dy lp dung dch, cm;

C - nng cht kho st, mol/l.


ClIIF 303,20

Nhn xt: IF ph thuc vo:

bn cht ca cht (, , to);

vo ngun sng ban u (I0);

nng cht hunh quang (C).

Trong iu kin xc nh, khi gi cc iu kin th nghim khng

i:

IF = K.C vi K = .Io..l

dng nh lng cc cht theo ph hunh quang.


1

2

3

4

5

6

7

9

10

1 n xenon;

2 thu knh;

3 gng lm;

4,9 cch t nhiu x cong;

5 mng thch anh;

6 dung dch mu;

7,9 t bo quang in;

S quang hc ca my quang ph hunh quang (Spectrofluorometer)


S quang hc ca my hunh quang dng knh lc ( Fluorometer)

Lamp

Sample aperture

disk

Shutter

Primary fillter

Mirror

Reference

aperture disk

Reference

photomultiplier

Sample

Secondary

filter Sample

photomultiplier

2.6.4. Xc nh cc hp cht hu c hunh

quang t nhin

c nhy cao trong phn tch hunh quang, cn:

Chn nh sng kch thch ph hp;

Chn pH;

Dung mi.


quang t nhin

Xc nh hm lng vt ca cc cht hunh quang t nhin:

Nhiu cht l c cht mi trng hoc c cht thc

phm: mycotoxin, phycotoxin v cc hp cht a vng thm,

cc amin nh histamin, phenylamin,

Cc vitamin: A, B2,

Dc phm: amphetamin, barbituric acid (trong thuc ng),

barbiturat, morphin v cc dn xut, quinin v quinidin,

salicylic acid v cc salicylat.


quang t nhin

Xc nh Aflatoxin (mt h ca c t vi nm mycotoxin)

Gii thiu chung v aflatoxin: Aflatoxin l nhm c t nguy

him c to ra t qu trnh trao i cht ca loi nm

Aspergillus flavus trn nhiu loi nng sn thc phm nh u

tng, u phng, bp, go, u nnh l cht c v n gy

ra:

Bnh t huyt trng

Ri lon qu trnh tng hp ARN, ADN

Ung th

Xc nh Aflatoxin

Aflatoxin tn ti ch yu cc dng cu trc sau:


Xc nh Aflatoxin

Tnh cht ca aflatoxin:

Trong tt c cc Aflatoxin k trn th B1 xut hin nhiu nht

vi c tnh cao nht, v vy c xem l i tng chnh trong

vic xc nh Afl.

Afl ha tan tt trong dung mi phn cc yu nh CHCl3,

CH3COCH3, CH3OH, benzene, ACN Khng ha tan trong n -

hexan, ete etylic, ete du ho.

Afl u c tnh hunh quang t nhin khi kch thch di

sng 365 nm, Afl B1 cho hunh quang xanh tm, loi Afl G cho

nh sng xanh lc.


Xc nh Aflatoxin

Cc c tnh l ho ca aflatoxin


Aflatoxin Cng thc

nguyn

Trng

lng

phn t

Hunh

quang

di tia

UV

Bc sng hunh quang khi kch thch

bng tia 365nm

em (nm) trong

Metanol ACN Clorofor

m Aceton

Afl B1 C17H12O6 312 Xanh lam 430 416 415 422

Afl B2 C17H14O6 314 Xanh lam 430 419 415 420

Afl B2a C17H14O7 320 Xanh lam

Afl M1 C17H12O7 328 Xanh lam 428

Afl M2 C17H14O7 330 Xanh lam 430

Afl M2a C17H14O8 346 Lc

Afl G1 C17H12O7 328 Xanh lc 450 440 435 448

Afl G2 C17H14O7 320 Xanh lc 450 437 435 448

Afl G2a C17H14O8 346 Xanh lc

Xc nh Aflatoxin


Tiu chun v sinh an ton lng thc, thc phm theo s Q

46/2007/Q-BYT

STT Tn c t vi nm Sn phm

Gii hn nhim

ti a chophp

g/kg

1 Aflatoxin B1 Thc phm 5

2 Aflatoxin tng s Thc phm 15

3 Aflatoxin M1 Sa v cc sn

phm t sa 0.5

Xc nh Aflatoxin


Hu ht cc phng php xc nh Afl u da trn tnh hunh

quang. C 2 k thut thng c s dng:

Phng php ph hunh quang xc nh tng;

Sc k bn mng kt hp vi thit b o m hoc sc

k lng hiu nng cao vi u d hunh quang FLD.

Xc nh Aflatoxin


Cc bc cn thc hin trong mt quy trnh phn tch

1. Chun b mu phn tch

Lm ng nht mu bng cc dng c thch hp;

Lng mu xc nh Afl tu vo chng loi thc phm (

~ 10 100g).

2. Chit xut mu

Chn la dung mi tu thuc vo tnh cht ho hc ca Afl;

Lng dung mi chit gp ~ 5 ln khi lng mu;

C th dng my xay vi tc cao ~ 3 pht, hoc my lc

30 pht;

Lc hoc ly tm tch b.

Xc nh Aflatoxin



3. Lm sch mu: Dng chit pha rn hoc chit lng - lng.

Chit pha rn (sc k ct), cht nhi ct phi hp ph mnh Afl:

Silicagel hoc silicagel kt hp C18; Florisil (MgSiO3); Ct i lc

min dch (gi Afl theo c ch to khng nguyn khng th).

Ct sc k i lc min

dch cha khng th

khangs Afla

Dch chit mu

cha Afla

Khng th tch v

gi Afla trong ct

Cho metanol qua

ct ra gii Afla

S hot ng ca ct i lc min dch

Xc nh Aflatoxin



Chit lng - lng: Tu vo mc ch loi b tp cht, chn 2

dung mi khng trn ln sao cho dung mi ny cho Afl tan tt, cn

li dung mi kia tan tt tp cht.

4. Lm giu mu: Trong k thut sc k thng dng c quay hoc

c quay di lung kh nit.

5. Pht hin v xc nh

Da trn tnh cht vt l hoc ho hc ca cht phn tch chn

la k thut o thch hp.Vi Afl thng s dng:

Sc k lp mng (nh tnh hoc bn nh lng);

Sc k lp mng kt hp vi my o m (Densitometer);

Quang ph hunh quang;

Sc k lng hiu nng cao u d hunh quang

Xc nh Aflatoxin

Phng php ph hunh quang xc nh tng Aflatoxin

Chit xut Afl bng CHCl3 hoc MeOH 70%;

Lm sch tp cht qua ct silicagel hoc oxid nhm;

Ra ct bng n-hexan v ete etylic;

Ra gii Afl bng Cloroform MeOH (97:3) vi ct silicagel hoc

acetonitril (9:1) qua ct florisil;

C mu. Ho tan cn trong MeOH;

Thm dung dch iod hoc nc brm tng nhy cho Afl B1 v G1.

+ H2O I2

CH3OH

ex = 365 nm em = 425 nm 81

Xc nh Aflatoxin

Phng php Sc k lp mng

iu kin s dng:

Sc k mt chiu

Cho nhiu trng hp. Tit kim thi gian;

Dng mt dung mi khai trin: Cloroform aceton (9:1);

Cn th nghim khng nh: Phun H2SO4 50% (v:v), Afl B1 v G1

cho hunh quang mu vng di nh sng UV.

Sc k hai chiu

Cho hm lng thp hoc mu cha nhiu tp cht;

Hai dung mi khc nhau chy vi hng vung gc;

DM1: ete etylic metanol - nc (94 4.5 1.5) loi bo;

DM2: cloroform aceton (9:1) tch Afl.

Xc nh Aflatoxin

Phng php Sc k lp mng

Sc k hai chiu Sc k mt chiu

1 2

3

1 2 3

B

A

C

M

F2

F1

S

La

ufrich

tun

g

Laufrichtung

1 2 3 M

Lau

frichtu

ng

Xc nh Aflatoxin

Sc k lp mng kt hp vi my o m

o cng nh sng phn x chiu bng tia UV-Vis;

o cng nh sng hunh quang kch thch bng tia UV-Vis.

Nguyn tc: nh lng Afl bng cch so snh cng hunh

quang ca vt mu v vt chun trn bn sc k. Hm lng Afl

c tnh g/kg hoc mg/kg

S dng ex = 365 nm v em = 443 nm. Dng cng thc:

m

s

I S V YC

I W X

Im : Cng hunh quang ca vt mu;

Is : Cng hunh quang ca vt chun;

X: Th tch dung dch mu chm ln bn mng (l);

Y: Th tch dung dch chun chm ln bn mng (l);

V: Th tch cui ca dch chit mu, k c nhng ln pha

long;

S: Nng Afl ca dung dch chun (g/ml);

W: Khi lng mu tng ng vi th tch dch chit khi

tin hnh lm sch qua ct (g).

Xc nh Aflatoxin

Sc k lng hiu nng cao u d hunh quang (HPLC-FLD)

Do Afl B2 v G2 khng cho hunh quang mnh trong pha thng, B1

v G1 khng cho hunh quang mnh trong pha o

Nhm nng cao kh nng hunh quang ca Afl trong pha o, cn to

dn xut cho B1 v G1:

Nu dng iod, to dn xut sau ct

Nu dng trifluoroacetic acid (TFA), to dn xut trc ct.

+ H2O TFA

toC

Th t tr khi khng to dn xut : G2, G1, B2, B1

tr khi to dn xut vi TFA : G1, B1, G2, B2

Cng hunh quang ca cc dn xut tng ln r rt

ex = 365 nm em = 455 nm / ACN

Quy trnh xc nh Afl trong bp, lm sch bng ct

min dch VI CAM

~ 25 g bp + 5g NaCl + 125 ml MeOH 70%

xay tc cao 2 pht, lc

50 ml dch lc 1 + 100 ml nc

trn u, lc

75 ml dch lc 2 (~ 5g mu), cho ln ct

tc 1-2 git/gy

Ra 2 ln, mi ln 10 ml nc

tc 2 git/gy

Ra gii Afl bng 3ml MeOH, tc 1 git/gy

C dch cha Afl trn ni cch thu n cn + 200 l TFA

Ho tan mu bng ACN n 1 ml

Tim mu vo HPLC -FLD

65o C, 5 pht

86

Xc nh cc hp cht a vng thm (PAH)

Gii thiu: PAN l nhng hydrocarbon c t 2 vng benzen tr ln, c

ngun gc thin nhin v hot ng ca con ngi.

Ngun t nhin: du th, than bn, than

T hot ng ca con ngi:

Cng ngh cracking du c xc tc, tinh luyn than

Sn xut hc n, nha ng

Pht in dng du FO

Kh thi ca phng tin giao thng, t than, t du

Chy rng, t rc, s chy ph liu

Ch bin thc n: chin, xo, nu, nng

Khi thuc l

PAH l nhng cht c c kh nng gy ung th, bin i gen, nhim mi

trng nghim trng. PAH c tm thy trong t, nc, khng kh v

trong thc phm.


Cng thc ca 16 PAH


Tnh cht l ho: Cht rn iu kin thng, tan nhiu trong cc dung

mi hu c. H s c tnh ca PAH

PAN H s c

tnh (TEF)

PAN H s c

tnh (TEF)

naphthalene 0,001 Chrysene 0,01

Flourene 0,001 Benzo(b)flouranthene 0,1

Acenaphthene 0,001 Benzo(k)flouranthene 0,1

Phenathrene 0,001 Benzo(a)pyrene 1

Anthracene 0,01 Dibenz(a,h) antharancene 1

Flouranthene 0,01 Benzo(ghi)perylene 0,01

Pyrene 0,001 Indenol(1,2,3-cd)pyrene 0,1

Benz(a)abthracene 0,1


Xc nh hydrocarbon a vng thm trong khng kh bng

HPLC-FLD

Khng kh c cha PAH do khng kh b nhim bi kh thi

phng tin giao thng. Chng tn ti trong cc ht bi.

Bi th: c kch thc > 10 m cha naphthalene,

acenaphthene, fluorene v phenanthrene.

Bi tinh: kch thc < 10 m, l bi c kh nng xm nhp vo

ng h hp. Cc PAH c nhiu vng (>4) t tn ti trong bi

th m ch yu trong bi < 10 m (PM10)



HPLC-FLD

1. Ly mu:

Ly mu bi trong khng kh bng my bm ly mu lu lng cao

s dng b tch bi tch chng thnh 2 phn theo kch thc ht.

Bi th ly bng giy lc si thu tinh, c chia rnh

Bi PM 10 ly bng giy lc Whatman GF/A, kch thc 20.3*25.4

mm

Thi gian ly mu (tu theo p lc bm v hm lng PAH)

Mu c bo qun trong giy nhm, trnh nh sng, gi trong bnh

ht m 24 gi, cn khi lng mu, gi lnh to < 5oC


HPLC-FLD

2. Quy trnh chit v lm sch

Lm sch qua ct Almina/silicagel

(8g / 8g gim hot 5% nc)

ra ct bng a/ 20 ml n-hexan

b/ 20 ml n- hexan + DCM (95-5)

c/ 40 ml n-hexan + DCM (80-20)

Dung dch ra gii + 0.5 1ml DMSO

c quay n bay ht n-hexan

Chuyn phn DMSO vo l ng mu bit khi lng, trng 3 ln

bng ACN mi ln 1 ml, cn trc khi y np.

Phn tch bng HPLC - FLD

Chit Soxhlet vi DCM to = 40oC, 24 gi

Chit lng - lng

a/ 30 ml mu + 15 ml DMSO

b/ 150 ml nc + 100 ml n-hexan

Xc nh hydrocarbon a vng thm trong khng kh bng HPLC-FLD

STT Tn PAH LOD (ng/ml) LOQ( ng/ml)

1 Naphthalene 0.010 0.034

2 Fluorene 0.058 0.190

3 Acenaphthene 0.036 0.120

4 Phenanthrene 0.051 0.170

5 Anthracene 0.020 0.065

6 Fluoranthene 0.019 0.063

7 Pyrene 0.024 0.081

8 Benzo(a) Anthracene 0.018 0.061

9 Chrysene 0.018 0.061

10 Benzo(b) Fluoranthene 0.041 0.140

11 Benzo(k) Fluoranthene 0.009 0.029

12 Benzo(a) Pyrene 0.015 0.051

13 Dibenzo (a,h) Anthracene 0.017 0.057

14 Benzo(g,h,i) Perylene 0.032 0.110

15 Indeno(1,2,3-cd) Pyrene 0.075 0.250

2.5. Xc nh cc hp cht hunh quang da

trn cc phn ng ho hc

Cc tc cht thng gp to ra hp cht hunh quang

o Phtaldialdehid (OPA)

o Phenylen diamine (OPDA)

Resorcinol

To cc sn phm ng vng phn t c cu trc cng

2.6.5. Xc nh cc hp cht hunh quang da

trn cc phn ng ho hc

2.6.5.1. Tc cht l aldehid dng nh lng amin

V d: Xc nh Histamin bng ph hunh quang

Nguyn tc

Chit xut Histamin trong mu hi sn bng

tricloroacetic acid (TCA). Lm sch qua ct cationit. To

phn ng vi OPA trong mi trng kim. o cng

hunh quang ca mu v chun ex = 336 nm v em =

450nm. Tnh hm lng Histamin c trong mu.


trn cc phn ng ho hc


V d: Xc nh Histamin bng ph hunh quang

Phn ng


trn cc phn ng ho hc


Ha cht:

- dung dch chun gc histamin 100 g/ml: pha t

histamin dihydroclorid trong dung mi tricloacertic acid

10%.

- dy chun C =1; 3; 5; 7; 10 g/2ml: ht th tch cn

thit dung dch chun gc vo bm 100 ml ri nh mc

bng HCl 0,2M.


trn cc phn ng ho hc


Cch tin hnh

a) chit xut mu: cn khong 10 g mu vi chnh

xc 1 mg cho vo thit b Ultra Turrax lm ng

nht, thm 80 ml tricloacetic acid 10% chit trong 1-2

pht. Sau lc dch chit qua giy lc vo bm 100 ml

ri nh mc bng tricloacetic acid 10% n vch.


trn cc phn ng ho hc


Cch tin hnh

b) lm sch dch chit: ly 250 l di ln ct cationit

Amberlit CG 50. Ra ct bng m pH ~ 4-6 (khong

120 ml), b nc ra. Ra gii histamin bng HCl 0,2M

vo bm 25 ml cho ti vch mc (dung dch A).


trn cc phn ng ho hc


Cch tin hnh

c) o cng hunh quang:

- ly 2 ml dung dch A + 1 ml NaOH + 0,1 ml OPA 0,1%.

Lc k, yn 3 -5 pht ri thm 2 ml HCl 0,7M, lc u ri o

IF.

- dung dch so snh: chun b tng t, thay 2ml dung dch

A bng 2ml nc ct.

- dy chun: ly 2ml dung dch chun thc hin ging nh

trn.


trn cc phn ng ho hc


Tnh ton kt qu:

Hm lng Histamin trong 100 g mu c tnh theo cng

thc:

m

CgmgH

500.)100/(

C: nng histamin c tnh t ng chun, g/2ml;

m: khi lng ca mu, g;

500: h s pha long, chuyn i t g ra mg v 100 g mu.


V d 2. Xc nh thuc tr su (TTS)

Thuc tr su b (TTS) carbamate c iu ch t acid

carbamic v c s dng rng ri cho vic bo v cy trng

khi s ph hoi ca cn trng.

L ester ca acid carbamic (NH2 COOH)

Do chng km bn trong mi trng t nhin, nn c s

dng rng ri dit cn trng, su b.

C th xc nh ng thi nhiu carbamat qua phn ng to

dn xut theo php o bng ph hunh quang hoc bng HPLC

FLD trc ct.

Hoc ring l tng carbamate bng HPLC FLD phn ng

sau ct (post column)



Cc loi thuc tr su thng dng



Phn ng



Nguyn tc:

Chit xut cc carbamate bng dung mi hu c, lm

sch, lm giu (nu cn). Tim mu vo my HPLC, thc

hin phn ng sau ct. o carbamate em = 450nm vi

ex =340nm.


Cc thng s cho phn tch bng HPLC v to dn xut

Pha tnh: ct Alltech carbamate 250m x 4.6mm, 5 m hoc tng ng

Pha ng: Hn hp nc-MeOH-ACN. Chng trnh gradient.

Thi gian Lu lng Nc% MeOH% ACN %

0

4,00

4,10

16,10

19,10

1,5

1,5

1,5

1,5

1,5

88

88

68

30

88

12

12

16

35

12

0

0

16

35

0

Th tch tim mu: 20 L Nhit ct phn tch: 30oC Nhit ng phn ng: 95oC Lu lng NaOH 0.05M: 0.5 mL/pht Lu lng OPA/Me: 0.5mL/pht


ng chun

Dng ng chun c khong nng ca tng

carbamate t 0.02 100ng (R2 = 0.9960).

Dng ng chun ny tnh hm lng carbamate c

trong mu.


a) Xc nh TTS Carbamate trong nc

Trng hp nc ung hoc nc sch: vic chun b

mu n gin ch cn acid ho bng HCl v pH = 3. Tim

mu vo my.

Trng hp nc b mt hoc nc thi: tu thuc vo

hm lng v mc tp cht. Vic chun b mu cn

thc hin theo mt s cc bc


Quy trnh chun b mu nc sng

250 mL nc, chnh pH = 2 3 bng HCl

lc

Dch lc cho qua ct SPE C18 c thch

nghi ho bng MeOH v nc

Dch ra gii cho bay hi n kh

nh mc bng 2mL MeOH

Tim 20 L vo HPLC - FLD

ra ct bng 10mL nc

Ra gii bng DCM : n hexan (7:3) ~ 20mL

V d 2. Xc nh thuc tr su (TTS) Quy trnh chun b mu rau

50 g rau

Cho dch qua ct SPE C18

Dch chit c quay bt MeOH (cn khong 40 mL)

25 mL dch sau ly tm, thm nc n 50 mL

Tim 20L vo HPLC

Phn cn li pha long bng nc ct n 100 mL

C quay ui DCM, n-hexan n kh

Tch chit bng MeOH bng my xay c tc cao

Ln u 100 mL MeOH Ln sau (50 x 2) mL MeOH

Ly tm

Ra ct bng 10 mL nc

Ra gii bng 20 mL DCM : n-hexan (7:3)

Ho tan cn bng 2 mL MeOH

2.6.5.2. Tc cht l o Phenylen diamin (OPDA)

Cho sn phm hunh quang vi diceton hay o-quinon.

- - oxo acid cho dn xut hunh quang khi ngng t

cht ny vi o phenylen diamin trong mi trng H2SO4.

NH2

NH2

+C

C

O

O

OH

R

OH

RN

N

Hydroxy quinoxalin HQ


Cho sn phm hunh quang vi diceton hay o-quinon.

Vi acid ascorbic (Vitamin C)

Oxi ho acid ascorbic (AA) n dehydroascorbic (DHAA).

Cht ny phn ng vi OPDA cho sn phm quinonxalin

nh lng AA bng phc hunh quang.

ex = 350 nm

em = 430 nm


Vi tocophenol (Vitamin E)

Vitamin E b oxi ho trong mi trng HNO3 n quinon.

Sn phm ny ngng t vi OPDA cho dn xut phenazin

hunh quang.


Vi Alloxan

CHO

NH2

+CH2

CO

OH

CH3

OH

N CH3


Selenit SeO32- cng cho hp cht hunh quang vi OPDA

hoc dn xut ca OPDA

2,3 diaminonaphtalen ex = 372 nm

em = 590 nm trong toluen isopentanol


Tc cht l etylendiamin(en)

Vi Epinephrine


Tc cht l etylendiamin(en)

Vi Norepinephrine

- 2H

- 2H2O

- H2O

2.6.5.3. Tc cht l phenol

Cho phn ng Pechman vi polycarboxylic acid no khng

no hoc c mang nhm OH.

Acid malic HOOC CH2 CHOH COOH phn ng vi

resorcinol trong mi trng acid m c to thnh

umbelliferon. Trong giai on u to thnh formyacetic

acid, sau l giai on ng vng.


Acid malic


Acid citric phn ng vi resorcinol


Phn ng xc nh phenol

acetoacetat

2.6.5.4. Phn ng to phc hunh quang

Nhng thuc th thng dng xc nh cc ion kim loi:

Alizarin Garnet R

1-amino-4-hydroxyanthraquinone

Bezoin

Calcein

8 hydroxy quinolin

Morin

Salicylidene-o-aminophenol

Salicyaldehid semicarbazone

Resorcinol

Rodamin B


V d 1: 8 hydroxyquinolin cho phn ng vi ion Mn+

Vi Al3+ cho phc trishydroxyquinolinat nhm chit

bng CHCl3 pH = 5.7 vi ex =365nm, em= 520nm c

LOD = 0.1 g/mL.

Ion nh hng Ga3+


V d 1: 8 hydroxyquinolin cho phn ng vi ion Mn+

Xc nh ion F- da trn kh nng lm tt hunh

quang ca phc hydroxyquinolinat Al (III) theo phn ng.


V d 2: Xc nh CN-

Ion CN- chim Pd2+ trong phc Pd(HQS)2 cho 8

hydroquinon-5-sulfonic(HQS) t do.

Sau cho HQS phn ng vi Mg2+ to thnh

Mg(HQS)2 hunh quang pH = 9.6 9.8 m HBO2/NaOH

2.6.5.4. Phn ng to phc hunh quang Phng php hunh quang chn lc xc nh

mt s ion kim loi Ion Thuc th ex, nm em, nm nhy,

g/ml

Ion cn tr

Al3+ Alizarin granet R 470 500 0,007 Be, Ca, Cr, Co,

F-, NO3-

F- Phc hunh quang

Al3+ vi Alizarin

granet R (tt hunh

quang)

470 500 0,001 Be, Co, Cr, Cu,

Fe, Ni, Th, Zn

B4O72- Benzoin 370 450 0,04 Be, Sn

Zn2+ Benzoin - Xanh lc 10 Be, B, Sb

Cd2+ 2 (o

hydroxyphenyl) -

benzoxazole

365 Xanh

chm

2 NH3

Li+ 8 - hydroxyqunolin 370 580 0,2 Mg

Sn4+ Flavanol 400 470 0,1 F-, PO43-, Zn


nh lng vitamin B1

Vitamin B1 (Thiamin) l hp cht khng hunh quang,

d tan trong nc, c cu trc ion.


nh lng vitamin B1

Khi b oxy ho bi K3Fe(CN)6 trong mi trng kim

to thnh thiochrom hunh quang mu xanh.

ex = 365 nm ; em = 436 nm


nh lng vitamin B1

Nguyn tc: Chit xut vitamin B1 trong mi trng acid,

thu phn bng enzym diastase c thiamine t do. Lm

sch qua ct cationit, oxy ho n thiochrome hunh quang

mu xanh.

Hm lng vitamin B1 c tnh bng cch so snh vi lng

vitamin bit trc.


nh lng vitamin B1

Cch tin hnh:

a) Chit xut Vitamin B1 trong mu

- Dung dch phn tch: Cn W g mu (cha ~330 g thiamin

trong 1 g thuc, 50 g thc n c b sung vitamin) un vi 60

mL H2SO4 0.05 M trn ni cch thu ang si trong 1h.

- Dung dch so snh (thm chun): Thc hin tng t nhng

c thm 40 mL dung dch chun 1 g/mL.


nh lng vitamin B1

Cch tin hnh:

b) Thu phn bng enzym

Lm ngui cc dung dch a n toC phng.

iu chnh bng dung dch acetat n pH 4 4.5

Thm 5mL enzym diastase v sy m trong t sy 20 pht

45oC

Lm ngui n nhit phng, chuyn vo bnh nh mc 200

mL, thm nc n vch, lc. B nc lc u ~10mL


nh lng vitamin B1

Cch tin hnh:

d) S oxy ho

Ly 5 mL(hoc 10 mL nu mu ngho vitamin B1) vo phu

chit 100 mL.

Thm 3 mL dung dch oxy ho + 15 mL iso BuOH, lc mnh ~

2 pht.

Nu b c, thm 1mL EtOH.

Lm kh lp dung mi hu c bng mt lng nh Na2SO4

khan.


nh lng vitamin B1

Cng thc tnh

1

1

( ) 100( 1/100 ) x B

B B

I IB gVitB g

I I W

: Lng vitamin B1 thm vo mu (g);

Ix: Cng HQ ca mu phn tch;

IB1: Cng HQ ca mu thm chun;

IB: Cng HQ ca mu trng;

W : Khi lng cn;

Nc ct ng vi im O ca trc tung.

2.6.6. ng dng ph hunh quang trong ha

phn tch

2.6.6.1. Phm vi ng dng v nguyn tc

Phm vi ng dng:

Lm ch th cho hu ht mi php chun th tch: acid

baz, complexon, kt ta.

Da trn nguyn tc: thay i tnh cht hunh quang ti

im tng ng ca s chun .


phn tch

2.6.6.2. Ch th acid baz

L acid hu c hoc baz hu c c dng khng ion ho

v dng ion ho ph thuc vo pH ca dung dch.

Cc dng ny c mt electron khc nhau tnh

cht hp thu v pht x khc nhau

Khi pH bin i im tng ng, ch th s chuyn

t dng ny sang dng khc km theo s thay i tnh

cht hp thu v pht x.


phn tch

2.6.6.2. Ch th acid baz

u im:

Khong i mu hp.

Chun c trong dung dch c, c mu.

s i mu mnh, d quan st, s dng hn hp dung

mi: nc MeOH, nc EtOH, nc aceton.

Nhc im:

Mi mt.

Cn thc hin chun ni ti.

Mt s ch th hunh quang trong chun acid - baz

Tn ch th Cng thc Khong pH i

mu

Thay i hunh

quang

Metyl

umbelliferone 0.0 2.0 lc xanh lt

Benzoflavin 0.3 1.7 vng lc

Esculin 1.5 2.0 KHQ xanh

chm

1,5-Naphtylamin

sulfamide

Nc 1

Nc 2

2.0 4.0

9.5 13.0

Tng cng

hunh quang

vng cam

Vng cam - lc

137


mu

Thay i hunh

quang

Acid salicylic 2.5 3.5 Tng cng HQ

chm

Fluorescein 4.0 5.0 Tng cng

xanh lc

Eosin

(tetrabromo-

fluorescein)

2.5 4.5

Tng cng

hunh quang

vng cam

Erythrosine

(tetracodofluoresc

ein)

3.0 4.2

Tng cng

hunh quang

lam



mu

Thay i hunh

quang

Naphtol (2-

naphtol) 7.0 8.5

xanh lt - chm

tm

Quinin nc 1 C20H24O2N2 5.0 6.1 chm tm

Quinin nc 2 C20H24O2N2 9.5 10.0 tm - KHQ

SS acid

10.0 12.0

tm - lc

Napthionic acid 11.5 14.0 Xanh tm - xm

139


2.6.6.3. Ch th complexon

nh ngha, c im:

Ch th mu hunh quang kim loi l nhng cht c tnh

hunh quang thay i (xut hin hay bin mt) khi to

phc vi ion kim loi c s dng lm ch th im tng

ng trong php chun complexon.

u im: cho cc dung dch c hoc c mu

Nhc im:

trnh nh sng tp, cn thc hin ni ti.

Mi mt, c bit khi ch th km tinh khit.


Mt s ch th hunh quang kim loi thng dng

Fluorescein complexon (Calcein hay Fluoresxon)

Danh php ho hc: (x x bis N,N dicarboxylmethyl)

amino methyl fluorescein

Fluorexon (calcein) - l sn phm ngng t gia

fluorescein vi formaldehid v iminodiacetic.




Cch pha ch th Calcein

Ch th calcein thng c pha vi nng 0,1% trong

dung dch NaOH 0,001M hoc dng di dng rn t l

1:100 vi KNO3 hoc NaNO3.




C s s dng calcein trong chun complexon

Calcein pht hunh quang cc i pH ~ 5. Khi tng pH

cng HQ gim, n pH = 11 12 (khi thm KOH vo

dung dch ti nng 0,25M) HQ tt hn.

Chun mt s kim loi kim th: Khi pH = 11 12

nu thm vo dung dch nhng kim loi kim th Ca2+, Sr2+,

Ba2+ th hunh quang mu vng lc li xut hin. Khi cc ion

kim loi ny to phc ht vi EDTA th hunh quang tt.




nh lng Cu2+: Trong khong pH = 4 10 hunh

quang ca calcein t do b tt bi ion Cu2+. C th xc nh

ion Cu2+ pH trn v sau im tng ng khi ton b ion

Cu2+ c chun ht bng EDTA nh sng hunh quang

ca Calcein t do li xut hin.




nh lng Co2+, Cr3+, SO42-: bng k thut chun

ngc, lng d EDTA c chun li bng dung dch Cu2+,

ti im dng chun hunh quang ca calcein t do b tt

do Cu2+ d.



Umbellicomplexon v xanthocomplexon

ch th xanh Calcein




ch th xanh Calcein

Tnh cht: l cht khng mu, t tan trong nc nhng

tan tt trong mi trng baz yu, nh CH3COONa.

Trong mi trng acid v trung tnh (pH = 4 -10): c hunh

quang mu xanh mnh, vi ex = 370 nm, em = 445 nm.

Trong mi trng kim mnh: pH > 10 hunh quang yu,

v tt hn pH =12.

Nn c gi l ch th xanh Calcein




ch th xanh Calcein

pH = 4 10: cc cation ca Mn, Fe, Co, Ni, Cu, Hg, Bi lm tt

hon ton hunh quang ca xanh calcein. Cc cation Zn2+,

Cd2+ lm tt mt phn, cn cc ion kim loi kim th khng

nh hng.

pH =12: s c mt ca cc ion kim loi kim th lm cho

hunh quang xut hin li (tr Mg2+). Cc ion Mg2+, Cd2+ v

Pb2+ ch cho hunh quang yu.



tng chnh xc trong php chun , c th dng

thm mt cht hunh quang khc:

Chun Ca bng EDTA, ch th Calcein pH = 12 thm

vi git 0.01% acridin. im cui chuyn t HQ vng lc

xanh r.

Tng t thm rodamin B hay fluorescein, khi dng

xanh Calcein lm ch th. im cui chuyn t HQ xanh

sng HQ hng gch.



Nhm ch th ny c hnh thnh trong qu trnh

tng hp cc ch th oxi ha kh. Chng l nhng dn

xut ca benzidin.

Benzidin complexon

CH CH

X

Y

NN

CH2COOH

CH2COOH

HOOCH2C

HOOCH2C

X: -OCH3, Y: -H

X: -OC2H5, Y: -H C hunh quang xanh chm

khi pH = 4 -10, HQ tt khi c mt

Cu2+.



ng dng:

nh lng cc ion ca Bi, Cd, Ce, Fe(II), Fe(III), Hg, Ind,

La, Ni, Pb, Th, Ta, Sn, Zr, bng k thut chun

ngc vi lng d EDTA c chun bng dung dch

CuSO4.

Benzidin complexon



Stiben

X: -H, Y: -H

X: -SO3H, Y: -H

C hunh quang xanh chm khi pH = 4, HQ tt

khi c mt Cu2+.

CH CH

XY

NN

CH2COOH

CH2COOH

HOOCH2C

HOOCH2C

XY

nh lng ion kim loi bng k thut chun ngc.



8 oxyquinolin v 8 oxyquinolin sulfonic

N

OH N

OH

SO3H

8 oxyquinolin

8 oxyquinolin sulfonic



8 oxyquinolin v 8 oxyquinolin sulfonic

ng dng:

nh lng Zn2+ pH = 10 (m ammoni) cho hunh

quang vng lc m. Ti im cui ca s chun Zn2+

bng EDTA, hunh quang s tt.



3 hydroxy 2 naphtoic acid


OH

COOH

Tnh cht: dung dch cha 3 hydroxy 2 naphtoic

acid pht HQ xanh lc. Khi thm Al3+ dung dch pht HQ

xanh chm.



ng dng:

nh lng Al3+;

nh lng ng thi Al3+ v Fe3+ bng EDTA.



Quy trnh nh lng hn hp Al3+ v Fe3+

vi 3 hydroxy 2 naphtoic acid

10,00 mL

Mu cha Al3+ v Fe3+

2 - 3 git ch th

Chun Al3+ bng EDTA di

n t ngoi n HQ chm

HQ lc.

Ghi V(EDTA), mL

Chnh pH = 2

un nh n 500C

Chnh pH = 3 (m glycin)

Chun Fe3+ bng EDTA n

khi dd mt mu xanh chm ca

phc cht st vi ch th

Ghi V(EDTA), mL

2.6.6.4. Ch th hp ph

L hin tng hp ph cc cht mu ln b mt ht

keo mang in tch v lm mu sc b bin i. Nu s

bin i xy ra gn im tng ng th c th dng

lm ch th xc nh im cui ca s chun .

V cc cht ny l acid hay baz nn kh nng s

dng lm ch th hp ph ph thuc vo pH mi trng.

2.6.6.4. Ch th hp ph V d: Trong chun xc nh cc Hal,v d Cl- bng AgNO3

s dng Fluorescein (H2Fl) lm ch th.

H2Fl H+ + HFl- Ka1 = 10

-5

Kt ta AgCl l cc ht keo

Trc im tng ng, cc ht keo mang in tch m

[AgCl]m.nCl- hay [AgCl]m.nCl

-.(n-x)Na+.xNO3-

HFl- mang in tch m khng b hp ph.

Sau im tng ng, cc ht keo mang in tch dng

[AgCl]m .Ag+ hay [AgCl]m .Ag

+.(n-x)NO3-.xNa+

HFl- b hp ph bi ht keo [AgCl].Ag+.HFl-

Khi b hp ph ion ny s b bin dng(mt in t thay

i) lm cho mu sc thay i HQ vng lc gch hng, mt

hunh quang

2.6.6.4. Ch th hp ph

Tn ch th Ion cht

chun

Ion xc

nh Mu HQ

Bengali hng (3,6 diclo 2,4,5,7

tetraiodofluorescein) (0.5% trong nc) Ag+ I- Hng tm

3,6-diclorofluorescein (0.1% trong 60-70%

EtOH hoc 0.1% dng mui trong nc) Ag+

Cl-, Br-,

SCN-,

I-

tm-xanh tm

Hng- tm

Vng lc-cam

Eosin (tetrabromofluorescein) (0.5% trong

dung dch 60-70% EtOH) Ag+

Br-, I-,

SCN Cam thm

Erythrosine 0.5% trong nc Pb2+ MoO42- Cam m

Fluorescein (0.1% trong EtOH) Ag+ Cl-, Br-,

SCN- Vng lc hng

Phenosafranine (0.1% trong nc) Ag+ Cl-

Br-

Tm hng

tm xanh da

tri

Chng 3. Ha pht quang

3.1. C s l thuyt ca phng php ha pht quang

3.2. ng dng ha pht quang trong ha phn tch


ha pht quang

3.1.1. nh ngha v phn loi pht quang ha hc

Pht quang ho hc (PQHH): l s pht sng t mt

phn ng c in t trng thi kch thch chuyn v trng thi

c bn.

Phm vi ng dng: Phn ng ho pht quang khng

nhiu nhng rt c bit v mt s phn ng pht quang quan

trng l cc hp cht mi trng: NOx,SOx,O3 trong kh quyn.


ha pht quang

3.1.1. nh ngha v phn loi ha hc

V d 1: Xc nh NO theo phn ng:

NO + O3 NO2 + O2

NO2* NO2 + h ( = 600 2500 nm) tp

trung 1200 nm.

Khong nng NO c th c xc nh: 1ppb 10

ppm.

Phng php cho php xc nh hm lng NO trong

kh quyn t mt t ln 20 km.


ha pht quang


V d 2: Xc nh NO2 trong khng kh theo phn ng phn

hy 7000C :

NO2 NO + O

Hoc cho dng nguyn t Oxi kh NO2:

NO2 + O NO + O2

Cho php xc nh hn hp NO v NO2.


ha pht quang


Pht quang sinh hc (PQSH): l s pht nh sng t

mt phn ng ho hc c s xc tc bi enzym trong c th

sng.

V d s pht sng ca om m:

Luciferin


ha pht quang


Pht quang in ho (PQH): l phn ng pht

sng do cc phn ng in ho xy ra ti in cc.


ha pht quang

3.1.2. C ch ca qu trnh pht quang ha hc

C ch n gin cho mt phn ng pht quang ho hc:

A + B I* + C

I* I + h

Trong :

I* l dng trung gian c trng thi kch thch in t, trng

thi ny km bn v pht ra bc x h tr v trng thi

ban u.


C 3 c ch pht quang thng gp:

Phn ng (I) (II): pht quang trc tip.

Phn ng (III): pht quang gin tip.


Cc phn ng pht quang ha hc v pht quang sinh

hc thng lin quan n s tch hay phn mnh cc

lin kt O-O trong hp cht peroxide hu c, c bit

l cc peroxide vng thm.

c th t nhy cao nht:

mt phn ng pht quang ha hc phi c kh nng

sinh ra mt photon nh sng.

Mi hp cht pht quang ha hc hoc nhm pht

quang ha hc c th to ra khng qu mt photon

nh sng.

(CL) 1.


-CO2


iu kin chung mt phn ng ho hc phi

tho mn cho s pht quang

Phi cung cp nng lng to ra trng thi kch

thch v vy phn ng phi pht nhit, sao cho:

-G hc/ex

Trong :

G: s bin thin nng lng t do ca phn ng (

Kcal/mol);

ex: bc sng kch thch ca cht pht bc x.

i vi phn ng pht quang ha hc trong vng kh kin

th: -G = 40 70 Kcal/mol.


iu kin chung mt phn ng ho hc phi

tho mn cho s pht quang

Phi c cch thun li a phn t ln trng thi kch

thch v pht quang ho hc.

S pht x photon phi l mt qu trnh kh hot u i

cho s pht quang ho hc trc tip.

i vi s pht quang gin tip th hiu sut v nng

lng chuyn t trng thi kch thch n cht nhn v

hiu sut hunh quang ca cht nhn phi tt.

3.1.3. Hiu sut ca qu trnh pht quang

ha hc

Hiu sut lng t ca qu trnh pht quang ha

hc CL c nh ngha l:


ha hc

i vi phn ng pht quang ha hc trc tip:

FlexChemCL

chem: Hiu sut ca phn ng ha hc, tng ng vi sn phm to

thnh. Gi tr chem thng bng 1.

ex: Hiu sut ca qu trnh to thnh dng kch thch in t trong

mt phn ng v c gi tr nm trong khong 0-1. ex biu th cho

hiu sut chuyn ha nng lng.

Fl: Hiu sut lng t hunh quang l xc sut ca trng thi kch

thch pht ra mt photon bng cch pht bc x hunh quang ch

khng phi bng cch phn r theo cc qu trnh khc. Fl thng c

gi tr t 0-1, Fl c gi tr nh nht l 0,1.


ha hc

i vi phn ng pht quang ho hc gin tip:

ETFlexChemCL

ET : hiu sut lng t truyn nng lng, biu th kh nng

chuyn i trng thi kch thch ca phn t cn xc nh thnh

trng thi kch thch ca cht nhn (acceptor), thng c gi tr ~1.

Fl : hiu sut lng t hunh quang ca cht nhn cng c gi

tr 0-1.


ha hc

Cng pht quang ha hc:

ng cong l thuyt biu din

cng pht quang theo thi gian v - th tch ca hn hp

pht quang ho hc.

- vn tc ca

phn ng

[C]cm3s-1


ha hc

Cng pht quang ha hc:

Phn tch bng k thut phng php hunh quang

b chi phi bi:

tnh cht ca phn ng c s dng;

tnh cht pht quang ca cht pht bc x

phi kim sot cn thn cc iu kin thc hin phn

ng.


ha hc

Cc yu t nh hng n cng pht quang ho hc

p sut: i vi pha kh, p sut trong ng phn ng

phi duy tr p sut thp bng bm ht chn khng

trnh mt nng lng do va chm.

S c mt ca cc cht l: nu cht l c kh nng pht

quang s gy sai s dng, ngc li s lm tt pht

quang.

Khong cch gia b trn mu n ni nhn nh sng

(ng nhn quang): cng ngn cng tt.


ha hc

u im so vi hunh quang

o trc tip nh sng pht ra.

Khng nh hng bi hin tng tn x ca nn

nhy hn.

Tn hiu phn tch phn bit c vi tn hiu pht

quang ca dung dch nn v khng s dng ngun nh

sng bn ngoi a phn t ln trng thi kch thch.

3.2. ng dng ha pht quang trong ha

phn tch

3.2.1. Phm vi ng dng

- Xc nh hm lng cc cht bng cch o cng

pht quang;

- Lm ch th.


phn tch 3.2.2. u v nhc im ca phng php ha

pht quang Nguyn tc

Da vo vic o cng bc x pht ra hoc b dp tt t mt

phn ng pht quang ho hc.

u im

- nhy cao gii hn pht hin thp;

- chn lc cao;

- Khong tuyn tnh theo nng rng;

- Thit b n gin: khng cn ngun kch thch bn ngoi, ch

cn u d n sc (ng nhn quang), khng cn bt c b lc

no.


phn tch

3.2.3. Thit b o trong ha pht quang

Nhng loi thit b o lng trong pht quang ho hc

a: H tnh; b: H thng dng chy; c: o lng 2 pha trn b mt rn; A: Cht

phn tch; R: Thuc th; L *: Loi pht quang c to ra trng thi kch thch.


phn tch


c chnh xc cao:

- H thng phi kn, trnh nh sng lt vo.

- Cng nh sng o c ch t phn ng pht

quang gia cht phn tch v thuc th, nu c ngun

nh sng khc lt vo th phi loi b bng b lc t

trc detector.

- Bung mu thit k sao cho cng gn u d cng tt

trnh sai s do tn hiu pht ra thng yu.


phn tch


Phng php nh lng bng ha pht quang:

- o cng tn hiu pht quang cc i (da vo kho

st cng tn hiu theo thi gian t lc bt u cho

n lc kt thc phn ng gia thuc th v cht cn

phn tch).

- Xy dng ng chun tng quan gia tn hiu v

nng cht chun.

- Xc nh nng cht phn tch da vo ng chun.


phn tch


nhy

Gii hn

pht hin

Hiu sut phn

ng ha pht

quang

- Dung mi;

- pH dung dch;

- Nng ;

- tinh khit;

3.2.4. Xc nh hm lng cc cht bng phng php

ho pht quang

Danh php ho hc:

5-amino-2,3-dihydrophtalazine-1,4-dione

Cng thc ho hc: C8H7N3O2

Khi lng phn t: 177.16 g/mol

Tnh cht:

Khng tan trong nc, tan trong dung

dch kim

Nhit nng chy: 318 320oC

L thuc th cho phn ng pht quang

ho hc

Gii thiu v thuc th Luminol


ho pht quang

Phn ng pht sng gia Luminol vi hydroxyperoxide

Cht oxi ho: H2O2, KMnO4, K2CrO4, KIO4, K3[Fe(CN)6], ClO-, BrO-

Nhng kim loi: Ag(II), Co(II), Cu(II),Cr(III), Fe(II), Fe(III), Mn(II),

xc tc lm tng cng pht quang.


ho pht quang

Phn ng pht sng gia Luminol vi hydroxyperoxide

Cc cht hu c: c th to phc vi cc ion kim loi ni

trn, hoc lm tng hay c ch phn ng pht quang ca

Luminol vi cht oxi ho.

u c th nh lng c nh amino acid, fructoza,

galactoza, glucoza, cc thio, thuc dit c, thuc st trng,

dn xut ca benzen c cha nhm NO2, -NH2, -OH,


ho pht quang

Xc nh ion kim loi da trn kh nng xc tc cho phn

ng pht quang ca Luminol vi hydroxyperoxide

Nguyn tc: Da vo vic o hiu s cng nh sng pht

ra khi c ion kim loi xc tc v khi khng c xc tc trong

cng iu kin. T nh lng ion kim loi ny.


ho pht quang

Xc nh ion kim loi da trn kh nng xc tc cho phn

ng pht quang ca Luminol vi hydroxyperoxide

Phn ng:

S phn ng ca Luminol

H2O2 khi c ion kim loi lm

xc tc


ho pht quang

V d 1: Xc nh Cr(III) trong nc trn h thng tim

dng chy

Nguyn tc:

Cr(III) c xc nh da vo kh nng xc tc ca n

ln phn ng pht quang gia luminol v H2O2 trong mi

trng kim (pH ~11), lm tng tc phn ng v khi gi

nguyn cc thng s khc th nng Cr(III) s tuyn tnh

vi cng pht sng ca phn ng.

Loi tr nh hng ca cc ion gy nhiu bng EDTA

nh vo phc gia Cr(III) vi EDTA bn v mt nhit

ng nhng chm v mt ng hc ti nhit phng.


ho pht quang


dng chy

H thng dng chy xc nh Cr(III) bng phn

ng gia Luminol vi H2O2


ho pht quang


dng chy

Cc iu kin ti u cho s xc nh Cr3+

- C[Luminol] = 1,5.10-3 M vi tc dng 3,5 ml/pht;

- C [H2O2] = 2.10-3 [M] vi tc dng 1,5 ml/pht;

- Nc vi cht n nh 5.10-6M EDTA tc dng 11,1 ml/pht;

- pH = 11,9 - m NaHCO3/Na2CO3

- Khong tuyn tnh 0,5 100 ng/ml c R2 = 0,997 vi

LOD = 0.191 ng/ml LOQ = 0.500 ng/ml


ho pht quang


dng chy

Cc iu kin ti u cho s xc nh Cr3+

S xc nh Cr3+ b nh hng bi cc cu t sau nu nng

Cr3+ l 1 g/L.

Nhng ion Mc chp nhn

CaII, F-, Cl-, I- >10 mg/L

MnII, HgII, NiII, ThII, ZnII, UVI, MoVI >1 mg/L

MgII, CrVI, AlIII, BaII, VV, InII, FeII, FeIII 1 mg/L

CeVI 0,2 mg/L

CoII 10 g/L


ho pht quang

V d 2: Xc nh paracetamol bng phng php pht

quang ho hc trn h thng tim dng chy

Danh php ho hc:

N-(4-Hydroxyphenyl) acetamide

Cng thc ho hc: C8H11NO2

Khi lng phn t: 129 g/mol

Tnh cht:

max trong EtOH l 250 nm ( =

13800)

Nhit nng chy: 169 170.5 oC

Dc tnh: h nhit, gim au


ho pht quang



NGUYN TC:

Hexacyanoferrate (III) c s dng l mt cht ng oxy ho vi

H2O2 ln h Luminol lm tng cng pht quang ca dung dch.

Paracetamol l cht kh, khi tc dng vi hexacyanoferrate s lm

gim nng ca cht ny dn n lm gim cng pht quang

ca h Luminol. Da vo gim cng hunh quang nh lng

c paracetamol trong mu.


ho pht quang



H thng tim dng chy xc nh paracetamol vi u d pht

quang ho hc


ho pht quang



Cc thng s ti cho s xc nh:

Nng Luminol: 0.002 M, tc dng 2.8 mL/pht

Nng H2O2: 0.02 M

Nng K3[Fe(CN)6]: 0.002 M

pH: 11 (m NaHCO3 , Na2CO3)


ho pht quang



Tn hiu ca 10 mu nn


ho pht quang



Tn hiu theo nng Paracetamol chun


ho pht quang



ng chun ca Paracetamol

LOD 0.35 ppm

LOQ 1.08 ppm

3.2.2.2. Ch th pht quang ho hc

Hin tng pht quang c s dng lm ch th

trong php chun complexon xc nh im tng

ng da trn s xut hin hay bin mt nh sng pht

quang.


Mt s ch th ha pht quang trong chun complexon


Ch th Luminol

H Luminol ion kim loi xc tc H2O2 pht quang

trong mi trng kim. Khi c mt EDTA to phc bn vi

ion kim loi xc tc ca h pht quang ha hc, th h

khng pht quang na. Cn c vo du hiu ny kt thc

php chun .

u im:

Ch th pht quang cho php chun trong dung dch c,

c mu vi hm lng rt nh ca ion kim loi


V d: Chun complexon Cu(II) dng Luminol lm

ch th

Cch thc hin:

a. Chun bng Trilon B

Chun trong dung dch xc nh cha Cu(II), 10 mL

Luminol 0.01%, 2mL H2O2 3%, m NH4Cl/ NH3 pH =

10. Dung dch pht sng trong ti.

Chun bng Trilon B, ngng pht sng ti im tng

ng.



ch th

Cch thc hin:

b. Chun Trilon B bng dung dch Cu(II)

Dung dch bao gm: Trilon B, Luminol, H2O2 m

NH4Cl /NH3. Chun bng dung dch Cu(II) n khi

dung dch bt u pht sng.



ch th

Cch thc hin:

c. Chun ngc

Nhng kim loi khng phi l xc tc cho h Luminol,

H2O2 nu nhng kim loi ny to phc vi EDTA bn hn

phc CuY2- th c th dng php chun ngc bng dung

dch Cu(II) nh: Fe(III), Ni(II), Hg(II),Pb(II), Sc(III), In(III),

Th(IV)

Documents

bài giảng phân tích huỳnh quang và phát quang