-
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
1.1. C s l thuyt ca phng php
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
1.1. C s l thuyt ca phng php
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
1.1. C s l thuyt ca phng php
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
1.2. C s l thuyt ph pht quang
-
Pht quang m cc
Pht quang nhit
Quang pht quang
Phn loi hin tng pht quang
1.2. C s l thuyt ph 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. C s l thuyt ph pht quang
-
1.2.1. Gin nng lng Jablonski
1.2. C s l thuyt ph pht quang
-
1.2.1. Gin nng lng Jablonski
1.2. C s l thuyt ph pht quang
--> 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
1.2. C s l thuyt ph pht quang
-
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;
1.2.2. Cc qu trnh gii ta nng lng phn t
1.2. C s l thuyt ph pht quang
-
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.
1.2.2. Cc qu trnh gii ta nng lng phn t
1.2. C s l thuyt ph pht quang
-
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)
1.2.2. Cc qu trnh gii ta nng lng phn t
1.2. C s l thuyt ph pht quang
-
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
2.3. 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 ca hin tng hunh quang
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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
2.1. Cc qu trnh xy ra trong hin tng hunh
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)
2.1. Cc qu trnh xy ra trong hin tng hunh
quang v ln quang
-
So snh ph hunh quang v ph ln quang
2.1. Cc qu trnh xy ra trong hin tng hunh
quang v ln quang
-
So snh ph hunh quang v ph ln quang
A
phenanthrene
2.1. Cc qu trnh xy ra trong hin tng hunh
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.
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
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.
-
2.2. Ph hunh quang v tnh cht c bn
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
-
2.2. Ph hunh quang v tnh cht c bn
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 .
-
2.2. Ph hunh quang v tnh cht c bn
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
-
2.2. Ph hunh quang v tnh cht c bn
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. Hin tng tt hunh quang
-
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. Hin tng tt hunh quang
-
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. Hin tng tt hunh quang
-
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.
2.3. Hin tng tt hunh quang
-
2.3.2. Cc yu t nh hng n hiu sut
hunh quang - tt pht quang
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).
2.3. Hin tng tt hunh quang
-
2.3.2. Cc yu t nh hng n hiu sut
hunh quang - tt pht 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.
2.3. Hin tng tt hunh quang
-
2.3.2. Cc yu t nh hng n hiu sut
hunh quang - tt pht quang
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.
2.3. Hin tng tt hunh quang
-
2.4. S pht quang ca dung dch
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. S pht quang ca 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. S pht quang ca dung dch
-
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. S pht quang ca dung dch
-
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).
2.4. S pht quang ca dung dch
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.
-
nh hng ca dung mi th hin qua:
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.
2.4.2. nh hng dung mi
2.4. S pht quang ca dung dch
-
nh hng ca dung mi th hin qua:
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.
2.4.2. nh hng dung mi
2.4. S pht quang ca dung dch
-
nh hng ca dung mi th hin qua:
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.
2.4.2. nh hng dung mi
2.4. S pht quang ca dung dch
-
Cc cht hu c c in t bt nh v.
Mt s ion v c, c bin dng tng:
F-< NO3-
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
2.5.2. Cc hp cht phc hunh quang
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
L cc hp cht thm hoc d vng c ni i tip
cch, phn t c tnh i xng, phng
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
iu kin cn thit mt hp cht c kh nng pht x
hunh quang: phn t ca hp cht phi c cu trc cng.
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
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. .
-
2.5. Cu trc ca hp cht hunh quang
2.5.1. Cc hp cht hunh quang hu c
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
-
2.5. Cu trc ca hp cht hunh quang
2.5.2. Cc hp cht phc hunh quang
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.
-
2.5.2. Cc hp cht phc hunh quang
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. Cu trc ca hp cht hunh quang
-
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+
2.5. Cu trc ca hp cht hunh quang
-
2.5.2. Cc hp cht phc hunh quang
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.
-
2.5.2. Cc hp cht phc hunh quang
Cc yu t nh hng n hiu sut pht quang
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
-
2.5.2. Cc hp cht phc hunh quang
Cc yu t nh hng n hiu sut pht quang
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).
-
2.5.2. Cc hp cht phc hunh quang
Cc yu t nh hng n hiu sut pht quang
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
-
2.5.2. Cc hp cht phc hunh quang
Cc yu t nh hng n hiu sut pht quang
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.
-
2.6.2. Nguyn l ca php phn tch 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
-
2.6.2. Nguyn l ca php phn tch hunh quang
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.
-
2.6.2. Nguyn l ca php phn tch hunh quang
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.
-
2.6.3. S my hunh quang v nguyn tc vn hnh
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)
-
2.6.3. S my hunh quang v nguyn tc vn hnh
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.
-
2.6.4. Xc nh cc hp cht hu c hunh
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.
-
2.6.4. Xc nh cc hp cht hu c hunh
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:
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
-
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.
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
-
Xc nh Aflatoxin
Cc c tnh l ho ca aflatoxin
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
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
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
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
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
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
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
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
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
Cc bc cn thc hin trong mt quy trnh phn tch
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
2.6.4. Xc nh cc hp cht hu c hunh quang t nhin
Cc bc cn thc hin trong mt quy trnh phn tch
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.
-
Xc nh cc hp cht a vng thm (PAH)
Cng thc ca 16 PAH
-
Xc nh cc hp cht a vng thm (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 cc hp cht a vng thm (PAH)
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)
-
Xc nh cc hp cht a vng thm (PAH)
Xc nh hydrocarbon a vng thm trong khng kh bng
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
-
Xc nh hydrocarbon a vng thm trong khng kh bng
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.
-
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
Phn ng
-
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
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.
-
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
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.
-
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
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).
-
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
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.
-
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
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.
-
2.6.5.1. Tc cht l aldehid dng nh lng amin
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)
-
2.6.5.1. Tc cht l aldehid dng nh lng amin
V d 2. Xc nh thuc tr su (TTS)
Cc loi thuc tr su thng dng
-
2.6.5.1. Tc cht l aldehid dng nh lng amin
V d 2. Xc nh thuc tr su (TTS)
Phn ng
-
2.6.5.1. Tc cht l aldehid dng nh lng amin
V d 2. Xc nh thuc tr su (TTS)
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.
-
V d 2. Xc nh thuc tr su (TTS)
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
-
V d 2. Xc nh thuc tr su (TTS)
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.
-
V d 2. Xc nh thuc tr su (TTS)
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
-
V d 2. Xc nh thuc tr su (TTS)
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
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
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
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
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.
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
Vi Alloxan
CHO
NH2
+CH2
CO
OH
CH3
OH
N CH3
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
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
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
Tc cht l etylendiamin(en)
Vi Epinephrine
-
2.6.5.2. Tc cht l o Phenylen diamin (OPDA)
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.
-
2.6.5.3. Tc cht l phenol
Acid malic
-
2.6.5.3. Tc cht l phenol
Acid citric phn ng vi resorcinol
-
2.6.5.3. Tc cht l phenol
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
-
2.6.5.4. Phn ng to phc hunh quang
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+
-
2.6.5.4. Phn ng to phc hunh quang
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.
-
2.6.5.4. Phn ng to phc hunh quang
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
-
2.6.5.4. Phn ng to phc hunh quang
nh lng vitamin B1
Vitamin B1 (Thiamin) l hp cht khng hunh quang,
d tan trong nc, c cu trc ion.
-
2.6.5.4. Phn ng to phc hunh quang
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
-
2.6.5.4. Phn ng to phc hunh quang
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.
-
2.6.5.4. Phn ng to phc hunh quang
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.
-
2.6.5.4. Phn ng to phc hunh quang
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
-
2.6.5.4. Phn ng to phc hunh quang
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.
-
2.6.5.4. Phn ng to phc hunh quang
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 .
-
2.6.6. ng dng ph hunh quang trong ha
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.
-
2.6.6. ng dng ph hunh quang trong ha
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
-
Tn ch th Cng thc Khong pH i
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
Mt s ch th hunh quang trong chun acid - baz
-
Tn ch th Cng thc Khong pH i
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
Mt s ch th hunh quang trong chun acid - baz
-
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.
-
2.6.6.3. Ch th complexon
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Fluorescein complexon (Calcein hay Fluoresxon)
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Fluorescein complexon (Calcein hay Fluoresxon)
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Fluorescein complexon (Calcein hay Fluoresxon)
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Fluorescein complexon (Calcein hay Fluoresxon)
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Umbellicomplexon v xanthocomplexon
ch th xanh Calcein
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Umbellicomplexon v xanthocomplexon
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
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
Umbellicomplexon v xanthocomplexon
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
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+.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
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
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
8 oxyquinolin v 8 oxyquinolin sulfonic
N
OH N
OH
SO3H
8 oxyquinolin
8 oxyquinolin sulfonic
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
3 hydroxy 2 naphtoic acid
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.
-
2.6.6.3. Ch th complexon
Mt s ch th hunh quang kim loi thng dng
ng dng:
nh lng Al3+;
nh lng ng thi Al3+ v Fe3+ bng EDTA.
3 hydroxy 2 naphtoic acid
-
2.6.6.3. Ch th complexon
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
-
3.1. C s l thuyt ca phng php
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.
-
3.1. C s l thuyt ca phng php
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.
-
3.1. C s l thuyt ca phng php
ha pht quang
3.1.1. nh ngha v phn loi ha hc
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.
-
3.1. C s l thuyt ca phng php
ha pht quang
3.1.1. nh ngha v phn loi ha hc
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
-
3.1. C s l thuyt ca phng php
ha pht quang
3.1.1. nh ngha v phn loi ha hc
Pht quang in ho (PQH): l phn ng pht
sng do cc phn ng in ho xy ra ti in cc.
-
3.1. C s l thuyt ca phng php
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.
-
3.1.2. C ch ca qu trnh pht quang ha hc
C 3 c ch pht quang thng gp:
Phn ng (I) (II): pht quang trc tip.
Phn ng (III): pht quang gin tip.
-
3.1.2. C ch ca qu trnh pht quang ha hc
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.
-
3.1.2. C ch ca qu trnh pht quang ha hc
-CO2
-
3.1.2. C ch ca qu trnh pht quang ha hc
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.
-
3.1.2. C ch ca qu trnh pht quang ha hc
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:
-
3.1.3. Hiu sut ca qu trnh pht quang
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.
-
3.1.3. Hiu sut ca qu trnh pht quang
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.
-
3.1.3. Hiu sut ca qu trnh pht quang
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
-
3.1.3. Hiu sut ca qu trnh pht quang
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.
-
3.1.3. Hiu sut ca qu trnh pht quang
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.
-
3.1.3. Hiu sut ca qu trnh pht quang
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.
-
3.2. ng dng ha pht quang trong ha
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.
-
3.2. ng dng ha pht quang trong ha
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.
-
3.2. ng dng ha pht quang trong ha
phn tch
3.2.3. Thit b o trong ha pht quang
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.
-
3.2. ng dng ha pht quang trong ha
phn tch
3.2.3. Thit b o trong ha pht quang
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.
-
3.2. ng dng ha pht quang trong ha
phn tch
3.2.3. Thit b o trong ha pht quang
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
-
3.2.4. Xc nh hm lng cc cht bng phng php
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.
-
3.2.4. Xc nh hm lng cc cht bng phng php
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,
-
3.2.4. Xc nh hm lng cc cht bng phng php
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.
-
3.2.4. Xc nh hm lng cc cht bng phng php
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
-
3.2.4. Xc nh hm lng cc cht bng phng php
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.
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 1: Xc nh Cr(III) trong nc trn h thng tim
dng chy
H thng dng chy xc nh Cr(III) bng phn
ng gia Luminol vi H2O2
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 1: Xc nh Cr(III) trong nc trn h thng tim
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
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 1: Xc nh Cr(III) trong nc trn h thng tim
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
-
3.2.4. Xc nh hm lng cc cht bng phng php
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
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
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.
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
H thng tim dng chy xc nh paracetamol vi u d pht
quang ho hc
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
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)
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
Tn hiu ca 10 mu nn
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
Tn hiu theo nng Paracetamol chun
-
3.2.4. Xc nh hm lng cc cht bng phng php
ho pht quang
V d 2: Xc nh paracetamol bng phng php pht
quang ho hc trn h thng tim dng chy
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.
-
3.2.2.2. Ch th pht quang ho hc
Mt s ch th ha pht quang trong chun complexon
-
3.2.2.2. Ch th pht quang ho hc
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
-
3.2.2.2. Ch th pht quang ho hc
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.
-
3.2.2.2. Ch th pht quang ho hc
V d: Chun complexon Cu(II) dng Luminol lm
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.
-
3.2.2.2. Ch th pht quang ho hc
V d: Chun complexon Cu(II) dng Luminol lm
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)