Phan Tich Cong Cu

7/30/2019 Phan Tich Cong Cu

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ng dng phng php phn tch cng c

Phng php phn tch cng c l phng php thuc b mn ca ngnh ha (Haphn tch) nghin cu v thnh phn cu to v hm lng cc thnh phn canhng mu kho st. Ha phn tch thng c chia thnh Ha phn tch nhtnh v Ha phn tch nh lng nhng cng hay c chia thnh Ha phn tchv cv Ha phn tch hu c.

Cc phng php ca ha phn tch c thc chia thnh hai loi: nh tnh vnh lng. Ngoi ra cn c phn loi thnh cc phng php ha hc v ccphng php vt l.

Ha phn tch thc cht l ngnh phn tch ng vai tr quan trng trong khoa hc,k thut, trong nghin cu khoa hc; iu tra cbn pht trin tim nng, khai

thc ti nguyn khong sn; nh gi cht lng sn phm.Sc kl mt h cc kthut ha phn tch dng tch cc cht trong mt hnhp. N bao gm vic cho mu cha cht cn phn tch trong "pha ng", thngl dng chy ca dung mi, di chuyn qua "pha tnh." Pha tnh tr hon s dichuyn ca cc thnh phn trong mu. Khi cc thnh phn ny di chuyn qua hthng vi tc khc nhau, chng sc tch khi nhau theo thi gian, gingnh cc vn ng vin chy maratng. Mt cch l tng, mi thnh phn i quah thng trong mt khong thi gian ring bit, gi l "thi gian lu."

Trong kthut sc k, hn hp c chuyn chtrong cht lng hoc kh v ccthnh phn ca n c tch ra do s phn b khc nhau ca cc cht tan khichng chy qua pha tnh rn hay lng. Nhiu kthut khc nhau c dng phn tch hp cht phc tp da trn i tnh khc nhau ca cc cht trong mitrng ng kh hoc lng v i vi mi trng hp ph tnh m chng di chuynqua, nh giy, gelatin hay gel magnesium silicate. Ta phn loi phng php sck nh sau:

Sc k: - Sc k kh: + Sc k kh lng (GLC)

+ Sc k kh rn (GSC)

- Sc k lng: + Sc k lng-lng (LLC)

+ Sc k lng-rn (LSC)

+ Sc k trao i ion (IEC)


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+ Sc k loi tr (EC)

Trong cc phng php sc k th phng php sc k lng hiu nng cao hin nayl phng php hin i v hiu qu hn c do chng ta tm hiu v ng dngn trong thc t.

Nguyn tc h thng my HPLC:

O 1 2 3 4 5

O: Ngun cung cp pha ng (mobile phase)

- Bnh cha pha ng

1: Bm cao p (h thng cung cp dung

mi)- Bm pha ng vo ct tch- iu khin tc ng, p sut capha ng

2. Van bm mu (Injection valve):- Bm mu PT vao ct tach theo nhng lngmu nht nhTim mu bng tay


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Tiem mu tng3: Ct tach (Column)- Ct cha pha tnh- Yu t quyt nh qua trinh tach sc ky

- Ct tch c kch ckhc nhau- Chiu di: 10 25cm- ng kinh: 2 5mm4: u do (detector)- Thit b phat hin cht phn tch (nh tnh

v nh lng)- Co nhiu loi khac nhau tuy mc ch phn


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tch: UVVIS, Hunh quang....5. H thng ghi nhn v xl tn hiu:- Thu thp v xl kt qu- Recorder, Computer + printer,software


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H thng HPLC

Detector UV-Vis: detector ph hp thu phn tXc nh cc cht c kh nng hp th quang Hunh quang (Fluorescence detector): xcnh cc cht c kh nng pht hunh quang

- Alflatoxin, Mycotoxin, Amino Acid, thuc tr suh Carbamate,.u o ch s khc x (Refractive Index Detector: RI)u do dn (Conductivity detector):Xac nh cac ion v c, hu cu do khi ph (MS: mass spectrometry)Xac nh phn ln cc cht hu c3. Cac qu trnh tch trong sc k lng- Qua trnh quan trng nht trong phng php sc k- Nhng cn bng ng xy ra gia pha tnh v pha ng trong ct sc k- L s vn chuyn v phn b lin tc ca chtPT tu ct n cui ct- Cht phn tch lun phn b gia 2 pha,trong o pha ng lun chy qua ct tch vi mt tc nht nh hoc gradient- Hiu qu ca qua trinh tach ph thuc rt


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nhiu vo tng tc gia cc cht trong pha tnh va pha ng- Mc ch chnh ca sc k l tch v nhtnh cc cht trong hn hp cht phc tpThigian cht PT b pha tnh lu gi (thigian lu) quyt nh bi:Bn cht ca pha tnh, cu truc va tinhcht ca cht PTBn cht va thanh phn ca pha ng dung ra gii cht PT ra khi ct sc k(pha tnh)Ghi li tan b qu trnh tch sc k ca hn hp cht PT sc k gm nhiupeak.- c im ca peak PT:Cc peak co th tch ri nhau han tanChp nhau mt phnChp nhau han tan

- Sc k phn nh qu trnh tch sc k trong ct tt hay khng tt.- Tch tt: hn hp c bao nhiu cht c by nhieu peak ring bit khng chpnhau- Cht no b lu gimnh sc ragii ra sau cng, cht lu gikem s ratrc4. u im ca phng php sc k- C th phn tch ng thi nhiu hp cht- Khng cn lm bay hi mu- phn gii cao nhqu trnh tch trn ct- nhy cao (ppm-ppb) nhu do- Th tich mu phn tch nh (1-100mL)

V d : XC NH AXIT HU C TL, V QU MNG CT BNGSC K LNG CAO P

TM TT

Axit hu ctrong l, v qu mng ct kh c xc nh bng phng php

sc k lng cao p. L ti v v qu mng ct kh c chit vi nc nhit 127 0C thi gian 30-60 pht di p sut 0,15 MPa. ng thi, vqu ba kh c chit bng dung mi (axeton v metanol) trong b chitsoxhlet nhit 75 0C trong thi gian 8 gi. Mu c bm vo my sck lng cao p cng vi axit photphoric 0,01 M v metanol vi tc dngl 0,7 ml/pht s dng etect210 nm. Axit hu cch yu trong l, v qumng ct kh c tm thy l axit (-) hyroxy xitric hm lng ln lt l


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2,863 v 15,221 %. Phn axit cn li trong l, v qu mng ct l lng nhaxit xitric. y l kt quu tin xc nh thnh phn cc axit hu c tmng ct (Garcinia oblongifolia Champ. ex Benth.).

1. Mu

Trong mt vi nm gn y, cc cu t c khi lng nh v phc tp cchit t nhiu loi mng ct (Garcinia Cowa, Garcinia cambogia, Garcinia indica,Garcinia antroViridis) trong c axit (-)-hyroxy xitric (HCA; 1,2-di hydroxypropan-1,2,3 tri cacboxylic axit; hnh 1), lacton ca (-) axit hydroxy citric (hnh 1))c tnh sinh hc l th gy ch i vi cc nh ho sinh cc bc s chuynkhoa sc kho. l kh nng iu chnh qu trnh tng hp axit bo, s hnhthnh lipit, s ngon ming, v gim cn. ng

phn ca (-)-HCA gp phn ln trong lnhvc dc hc nh tc nhn c vai tr quan trngtrong mc ch gim cn, bo v tim mch, hiuchnh trng thi bt bnh thng ca cc lipid,v kh nng chu ng trong luyn tp th thao[3] [4] [5] [6]. V qu ba c tnh sn da v hing, mt, hi c, c tc dng tiu vim, hnhit, lm sn da, hn vt thng; tr: Lot ddy, lot t trng; Vim d dy rut, km tiu ho; Vim ming, bnh cn rng; Hora mu. Dng ngoi tr bng, mn nht, su qung, eczema, dng mn nga, rt

cc vt n m vo tht; L ba c v chua thng c dng thi nh nu canhchua; Ht c o ht chua, n c, cng dng nu canh chua. Nha ba dng trbng [1]. Cc nghin cu chit tch ngun HCA ch thc hin trn cc loi ba can . V vy, s khm ph axit hu ctrong cy ba ti Vit Nam l ht sc cnthit, cy ba c tn khoa hc l Garcinia oblongifolia Champ. ex Benth., thuc hBa (h mng ct) - Clusiaceae.

Bi bo ny trnh by phng php chit tch, xc nh thnh phn axit hucchnh c trong l, v qu mng ct v cc thng s vt l khc.

2. Nguyn liu v phng php nghin cu

2.1. Nguyn liu

L, v qu ca cy mng ct (Garcinia oblongifolia Champ. ex Benth.) ti xHa Lin, huyn Ha Vang, TP. Nng v t thnh ph H Ch Minh.

COOHC

H

HO

COOHHO C

COOHCH

H

COOHC

H

COOHHO C

CH

H

C=OO

Hnh 1: Cu trc ca (-) axit hydroxy citricv lacton ca (-) axit hydroxy citric


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lp li01 ln

10 g mu (ct nh) + 100 mL nc

Ni p sut 0,15 MPa, 30 pht

Lc bng vi muslin

Trn ln dch 02 ln chit + 4g thanhot tnh: ngm trong nc m 30'

Lc bng giy lc, than hot tnh

c ra 02 ln vi 15 mL nc vlc li nc ra

Trn ln dch lc v ra, c t n20mL, x l vi 100mL etanol

15 pht k t ta h t pectin.

Ly tm 10 pht, tch phn dch niv kt ta ring. Phn kt ta ra 02

ln vi 20mL etanol v ly tm

Trn cc dch ni, c c n thtch 50 mL v lu gi40C n khis dng (chun , chy HPLC, IR).

25 g mu+axeton hoc etanol (150 mL)

Chit Soxhlet 75 0C trong 8 gi

Lc b ng gi y lc v c c

Etanol v axeton chit ha tan trong20mL nc + 4g than hot tnh

Ngm trong nc m 30 pht v lcbng giy lc, than hot tnh c ra

02 ln vi 10 mL nc.

Dch chit gp chung thnh 50 mL

lp li01 ln

10 g mu + 150 mL nc

Ni p sut 0,15 MPa, 60 pht

Lc bng vi muslin

Trn ln dch 02 ln chit + 4g thanhot tnh: ngm trong nc m 30'

Lc bng giy lc, than hot tnh

c ra 02 ln vi 15 mL nc vlc li nc ra

Trn ln dch lc v ra, c t n50mL, x l vi 100mL etanol

15 pht k t ta h t pectin.

Ly tm 10 pht, tch phn dch niv kt ta ring. Phn kt ta ra 02

ln vi 20mL etanol v ly tm

Trn cc dch ni, c c n thtch 50 mL v lu gi40C n khis d n chu n ch HPLC IR .

Nguyn liu nghin cu l, v qu mng ct c chun b theo sphn2.2.1.

2.2. Phng php nghin cu

2.2.1. S nghin cu

L ba: loi b l gi, l su, ra sch, hong kh tin hnh o m v thchin chit tch axit theo s sau.

Qu ba: loi b qu h, dp, qu cha chn, ra sch, hong kh, tch bphn rut qu, ct nh tin hnh xc nh m v sy kh nhit 800C, xoaynh lm nguyn liu chit tch axit theo 02 phng php s sau.

L: V qu kh:

Phng php 1: Phng php2:


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2.2.2. Phng php chit tch

Chng ninh trong ni p sut: L, v qu ba ti hoc kh c ct nhri cho vo cc thu tinh 500 ml, chng ninh trong ni p sut p sut 0,15 MPa,nhit 127 oC trong thi gian 30-60 pht v lp li 02 ln chit tch ht lngaxit hu cc trong mu vi dung mi l nc.

Chit soxhlet: V qu ba kh xoay nh cho vo b chit soxhlet tin hnhchit nhit 75 oC, trong vng 8 givi dung mi l axeton hoc methanol.

2.2.3. Phng php trng lngCn khong 10g l, v qu Ba ti cho vo cc thu tinh c sy kh

v bit khi lng chnh xc. Cho cc thu tinh c cha l, v qu Ba vo t sy

v sy 80o

C.Sau khi sy khong 3 gi, ta ly cc ra cho vo bnh ht m cho n khi ccthu tinh ngui hn th tin hnh cn tnh khi lng trn cn phn tch. Sau , ckhong 30 pht ta li tin hnh qu trnh trn mt ln cho n khi khi lng giahai ln cn lin tip l khng i hay c sai s khong 0.005g th dng qu trnhsy.

Da vo cc kt qu thu c, ta tnh c khi lng l, v qu Ba trcv sau khi sy. T, ta tnh c m l da vo cng thc sau:

Trong : H: m (%); m0: khi lng l hoc v qu ti trc khi sy(g); m1: khi lng l hoc v qu sau khi sy (g).

2.2.4. Phng php chun [2]Xc nh hm lng axit tng s trong l, v qu ba theo tiu chun TCVN

4589-88.

Nh NaOH 0,1N t buret xung, cho n khi dch th c mu hng nht bn

vng.

Tnh kt qu: axit ton phn theo phn trm (X1) tnh bng cng thc:

100.10

m

mmH

=

P

100.

V

Vcd.n.KX1 =


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trong : n: s ml NaOH 0,1N s dng chun V ml dch th; Vcd:th tch mu c c; P: trng lng mu th, tnh bng gam; K: h s ca loiaxit, K = 0,006904.

2.2.5. Ph

ng php xc

nh c

u trc ho hc b

ng quang ph

(IR)Cc phn t lun dao ng khng ngng. Tn s dao ng ca cc phn t

ph thuc vo hng s lc ca lin kt v khi lng ca chng, do cc nhmchc khc nhau s c tn s hp th khc nhau nm trong vng t 5000 200 cm-1.Mi nhm nguyn t (nhm chc) xc nh c tn s xc nh v khng i trongbt k hp cht no c cha nhm nguyn t. V vy, khi phn tch trn quangph hng ngoi ta c th xc nh c cc nhm nguyn t m cht em phntch c c.

2.2.6. Phng php sc k lng cao p (HPLC) [3] [4]Cht chun l (-)-Calcium threo-hydroxycitrate tribasic hydrate, cung cpbi hng Sigma-Aldrich, cng thc C12H10Ca3O16.xH2O.

Chun b HCA t do: Calcium threo-hydroxycitrate tribasic hydrate (50mg) cho vo cc 50-mL cha 5.0 mL nc, v x l vi 500 mg Dowex 50 [H+].Hn hp c khuy trong thi gian 10 pht s dng khy t. Tch ly phn dungdch, v nha c ra vi nc c pH trung tnh. Nc ra v dung dch dctrn ln v lm thnh 25 mL, khuy trn, v lc s dng giy lc. Chun b 05

dung dch chun HCA c nng thay i t 10 ppm n 320 ppm.Chun b dung dch chun axit xitric cho HPLC: Dung dch chun axit

xitric c chun b ring bit c nng t 2 n 30 ppm s dng nc ct 3 lnct.

HPLC phn tch: H thng sc k lng cao p c s dng nghin cugm my sc k lng cao p hng Knauer trang b vi bm loi low pressure hng

Knauer, v lp ct sc k Knauer C18: 250 mm x 4,6 ID x 5m. B tng hp dung

mi: quaternary LP Gradient, hng Knauer. Qu trnh d c thc hin bngetectKnauer UV: khong bc sng 190-740 nm. Pha ng gm (A) metanolMeOH v (B) l axit photphoric 0,01 M vi tc dng 1,5 ml/m. Qu trnh tchcc pic tt khi cht A trong B thay i t 10-30% trong thi gian 0-25 pht, 90% Atrong B trong thi gian 30 pht, sau 5 pht cn bng vi 90% A. Cht chun v

mu c lc qua Millipore lc 0,45 m v tim vo HPLC.


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Xy dng ng chun: Phng php xy dng ng chun c thchin bng cch da vo kt qu phn tch mt chui cc mu HCA chun. Nmmu dung dch chun cha 10-320 ppm HCA t do c tim vo HPLC, thao tcra gii c thc hin nh phn tho lun trn, v kt qu thu c cc din

tch pic. ng cong HCA c v da trn biu din s ph thuc gia nng ca HCA v din tch pic (trung bnh ca 03 ln chy). Tng t, ng chun caaxit xitric c tim vo HPLC, c thc hin nh cch tho lun trn, v kt quthu c cc din tch pic.

Khong nng mu chun cn xy dng ng chun c xc nhda vo nng thc ca HCA c trong mu. Khong nng ny tnh t gi trgii hn di (LLOQ) n gi tr gii hn trn (ULOQ).

Xc nh gi tr gii hn: Gi tr gii hn (LOQ) c nh ngha nhnng thp nht ca HCA m c th xc nh vi tin cy v chnh xc


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3. Kt qu v tho lun

3.1. Kt qu xc nh m trong l v qu mng ctKt qu xc nh m trong l, v qu ba c trnh by trong bng 1 sau

y:

Bng 1. Kt qu xc nh m trong l, v qu Mng ct

Mu Sttmu

Khi lng ltrc khi sy

(g)

Khi lng lsau khi sy (g)

Khi lngnc trong l

(g)

m

(%)

1 9.726 2.789 6.937 71.32

2 9.96 2.898 7.062 70.9Lba

3 10.172 3.022 4.17 70.49

1 10.6012 1.6767 8.9245 84.18

2 9.7521 1.547 8.2051 84.14

Vquba

3 10.5966 1.6458 8.9508 84.47

T kt qu trn cho thy m trong l Ba ti khong 70,70 0,62 %, m trung bnh trong l l 70,70 %. m trong l Mng ct cao, chim khong70% khi lng l. m trong v qu mng ct khong 84,31 0,16 %, mtrung bnh trong v qu l 84,31 %. m trong v qu mng ct l rt cao, chimkhong 84 % khi lng v qu.

3.2. Kt qu xc nh cu trc bng phhng ngoi (IR)

Kt qu chp ph IR cho thy xut hin pic c bc sng t 3300 3600cm-1, l ph dao ng ca nhm OH c lin kt hiro, v pic c bc sng t1600 1800 cm-1, l ph ca nhm C=O trong nhm COOH (hnh 2, 3).


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0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

A

bsorbance

5001000150020002500300035004000

Wavenumbers (cm-1)

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

0.22

0.24

Absorbance

5001000150020002500300035004000

Wavenumbers (cm-1)

Hnh 2: PhIR mu v qu ba chittrong nc

Hnh 3: PhIR mu l ba chit trongnc

T nhng ph hng ngoi trn, c th kt lun trong mu chit t nc cal v v qu mng ct c s tn ti ca axit hu cc nhm OH.

3.3. Kt qu xc nh axit hu ctrong l, v qu mng ctBng 2: So snh axit hu ctrong l, v qu mng ct bng HPLC v phng

php chun

Axit hu cxcnh bng HPLC

(g/100 g)

Mu Dung mi

chit

HCA Axit xitric Chun axit-baz

(g/100g)L ba Nc 2,863 0 3,554

V qu bakh

Nc 15,221 0,406 17,468

V qu bakh

Axeton 12,999 0,239 13,711

V qu bakh

Metanol 9,508 0,078 10,492

S mu n=3, kt qu tnh trung bnh

Thnh phn axit hu cxc nh bng phng php sc k lng cao p vphng php chun c th hin trong bng 2. Tng axit xc nh bng


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phng php chun axit-baz c kt qu ln hn tng axit xc nh bngHPLC. T bng trn cho thy, chit bng dung mi nc cho lng axit l lnnht, tip n l axeton v metanol. Gi tr thu c ch yu ca phng phpHPLC c tnh n ch l HCA, bi v gi tr thu c t din tch pic ca HCA

ln nht. Axit ch yu c tm thy trong l, v qu ba bng HPLC l HCA,c th hin trn sc k hnh 5, 6, 7, 8. Pic th yu c xc nh l pic caaxit xitric. Trn sc k, HCA cho pic n trong tt c cc mu chit. Ring ivi mu l ba chit trong nc khng xut hin pic ca axit xitric. Xc nh picHCA c da vo pic ca axit HCA chun xut hin thi gian lu l 4,939pht, tng t pic ca axit xitric xc nh nhpic axit chun xut hin thi gianlu l 5,623 pht (hnh 4). Thi gian lu ca HCA v axit xitric c tm thytrong tt c cc mu ln lt l 4,992 0,065, 5,609 0,010 pht.

ng chun c xc nh bng cch thay i nng ca 05 mu chun.ng chun c xy dng vi nng t 10 n 320 ppm, phng trnh ngchun: C = 1,37A-6,88 vi A l din tch pic ca HCA, C l nng HCA, h stng quan R2=0,999698.

Hnh 4. Sc k mu axit HCA v

axit xitric chun

Hnh 5. Sc k mu v qu mng ct

kh chit trong metanol


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Hnh 6: Sc k mu v qu mng

ct kh chit trong nc

Hnh 7: Sc k mu v qu mng

ct kh chit trong axeton

4. Kt lun

Bng phng php sc k lng cao p (HPLC) xc nh c hm lngaxit hu ctrong l, v qu ba. Kt qu cho thy axit (-) hyroxy xitric l thnhphn axit ch yu c trong l, v qu ba. Hm lng HCA tm thy trong l, vqu ba kh l kh cao, c th HCA trong l, v qu ba kh ln lc l 2,863 v15,221 %.

Hnh 8: Sc k mu l mng ct chit


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T kt qu ny c th tip tc nghin cu su sn xut thc phm cha trbo ph t cy ba Vit nam.

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