K THUT TO DN XUT HU CT TRONG HPLC V NHNG NG DNG

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Ni Dung

I
II

Gii thiu khi qut v HPLC K thut to dn xut hu ct

ng dng ca to dn xut hu ct

III

I.KHI QUT V SC K LNG HIU NNG CAO (HPLC)

Ra i t nm 1967-1968. L mt phng php dng tch cc cht ra khi hn

hp trong pha ng l cht lng v pha tnh cha trong

ct l cht rn. Ngy cng pht trin v hin i ha cao nh s pht trin nhanh chng ca ngnh ch to my phn tch l cng c c lc trong phn tch cc loi thuc a thnh

phn cho php nh tnh v nh lng.

u im ln nht ca HPLC l c nhy cao, hiu sut tch cao, tn t thi gian cng nh tn t mu
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 Cu to ca h thng (HPLC) bao gm:

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 Cu to ca h thng (HPLC) bao gm:

+Bm cao p (pump)

+Van t ng bm mu (autosampler):
+Ct tch(HPLCcolumn) +Thit b pht hin cht phn tch (detector)

+Trang thit b hin th kt qu(Processing data & control)

II.TO DN XUT HU CT TRONG HPLC

1.Mc ch ca vic to dn xut trong HPLC Khc phc tnh trng mt s cht khng hp th nh sng UV/VIS hoc pht hunh quang. Gm 2 phng php: to dn xut tin ct (precolunm) v to dn xut hu ct (post-colunm).

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2.Phng php to dn xut hu ct (post-colunm derivitization)

Cht cn pht hin sau khi tch ra khi ct sc k, s c bm qua mt l phn ng t sau ct sc k.

Cht tch c s phn ng vi mt loi thuc th thch hp c bm vo to ra dn xut pht hunh quang hoc hp th nh sng UV/VIS.
Dn xut to ra c dn qua detector pht hin.

3.Cu to ca h thng to dn xut hu ct: H thng my HPLC B phn cha thuc th Bm t sau b phn cha thuc th L phn ng (hay b phn ng) Cc detector

4.Cc loi phn ng to dn xut Phn ng hu c: phn ng trao i, phn ng cng ha tr Phn ng in ha: phn ng oxi ha-kh 5.Cc loi thuc th to dn xut: gm 4 nhm

Thuc th khng hunh quang (ninhydrin, sunfonyl benzene): s dng trong ph UV/VIS
Thuc th tin hunh quang:

Thuc th hunh quang (ortho-phtalaldehyde (OPA) )

Thuc th oxi ha-kh

6.u v nhc im ca phng php to dn xut hu ct

u im

Nhc im

lp li cao Khng b nh hng bi cht trong mu ban u Hiu sut nh tnh v nh lng cao

Tiu th nhiu thuc th Chi ph cao Thuc th b gii hn

III.MT S NG DNG CA TO DN XUT HU CT

1.Xc nh c t Aflatoxin trong ng cc v thc n gia sc: Khi qut v Aflatoxin: L c t vi nm sn sinh bi mt loi nm mc (aspergillus ) C sn trong ng cc v thc n gia sc bo qun khng tt Gm 6 loi khc nhau (B1, B2, G1, G2, M1 v M3),B1:Cc c nh hng n tc tng trng v cc qu trnh s dng dinh dng ca gia sc. Tc nhn gy ung th

 Nguyn tc xc nh: Mu c chit v tch trn HPLC.

To dn xut pht hunh quang mnh vi dung dch iod.

Pht hin nh detector hunh quang

 iu kin tin hnh:

Qu trnh sc k Ct sc k thng Mu:tc dng:1ml/pht, t0=420C, V=10L Pha ng: methanol:acetonitril:nc (22:22:56) Qu trnh to dn xut Thuc th: 100mg iod + 2ml methanol+ 200ml nc. L phn ng: t0 =700C;1,4 ml Tc thuc th:0,4ml/pht

u im: gii hn nh lng:ng cc t:0,5-2g/kg, thc n gia sc :1 g/kg. Nhc im: chi ph cao

2.Xc nh d lng thuc tr su carbamat trong rau qu

Khi qut v Carbamat:

L dn xut ca axit carbamic (NH2COOH) .

L thuc tr su c ph tc dng hp hn thuc tr su gc ln hu c v Clo hu c

Dng iu tr mt s loi su nht nh trn cy trng

Gy c cp tnh kh cao, tc ng ln h thn kinh,tch ly nhanh.

 Nguyn tc xc nh
Mu c lm sch v tch trn HPLC . Thy phn trong mi trng kim sinh ra methyl amin

Methyl amin s c to dn xut vi o-phthaladehyde (OPA) v mercaptoethanol to ra dn xut hunh quang 1hydroxytylthio-2-metylisoindol .
Nhn bit bng detector hunh quang.

iu kin tin hnh:

Qu trnh sc k Qu trnh to dn xut

Ct sc k pha o: kch thc 20cm4,6mm5m Lng mu tim: 10L Pha ng: MeOH/nc theo gradient nng vi tc 1ml/pht

Nhit l phn ng:95-1020C VOPA:0,15ml/pht,nng:0,25mmol VNaOH:0,2ml/pht,nng :50mmol Nng Mercaptoethanol:125ppm

u im :phn tch c 10 loi carbamat khc nhau, gii hn nh tnh t 0,51-5ppb, gii hn nh lng:1,69-9,09ppb, hiu sut thu hi:73-95% Nhc im:Chi ph cao

3.Xc nh Acesulfame,saccharin v cyclamate trong mt s loi thc phm Khi qut v Acesulfame, saccharin v cyclamate L nhng cht to ngt nhn to

S dng rng ri trong nhiu loi thc phm v ung.

B gii hn v s lng s dng do nguy c gy ung th

Saccharin

Cyclamate

Nguyn tc xc nh:

Mu sau khi c chit tch trn HPLC

To dn xut pht hunh quang vi Hexandecyltrimethyl ammonium bromua v 1,6-Diphenyl-1,3,5-hexatrien . Xc nh bng detector hunh quang.

iu kin tin hnh:

Qu trnh sc k Ct pha o (C18, 2100mm) nhit :500C, tc dng: 0,2ml/pht Qu trnh to dn xut

Nnhit l phn ng:400C Tc thuc th 0,3ml/pht

Pha ng: KH2PO4 0,02M/ nc

(7:3)

u im: gii hn nh lng c th t c n 60ppm.

TI LIU THAM KHO

1.J. Chromatogr. A, 778 (1997) 103-110 A. Sabala et al. 2. Spector, A.A.; Yorek, M.A. Membrane lipid composition and cellular function. J. Lipid. Res. 1985, 26, 10151035. 3. Ghosh, S.; Strum, J.C.; Bell, R.M. Lipid biochemistry: functions of glycerolipids and sphingolipids in cellular signaling. Faseb. J. 1997, 11, 4550. 4. Shimizu, T. Lipid mediators in health and disease: enzymes and receptors as therapeutic targets for the regulation of immunity and inflammation. Annu. 1987 May 15;416(2):321-30. 5. Embree L, McErlane KM., J Chromatogr.1989 Nov 24;496(2):321-34. 6. Emara S., Biomed Chromatogr.1998 Jan-Feb;12(1):15-20. ..

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