Cac Phuong Phap Phan Tich Hoa Hoc Va Thuc Pham

7/22/2019 Cac Phuong Phap Phan Tich Hoa Hoc Va Thuc Pham

1/16

ng dng phng php phn tch cng c

Phng php phn tch cng cl phng php thuc bmn ca ngnh ha (Haphn tch) nghin cu vthnh 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,kthut, 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 hcc 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 sdichuyn 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, gingnhcc vn ng vin chy maratng. Mt cch l tng, mi thnh phn i quahthng 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 sphn bkhc 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 phtnh m chng di chuynqua, nhgiy, gelatin hay gel magnesium silicate. Ta phn loi phng php sck nhsau:

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)


2/16

+ 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 quhn cdo chng ta tm hiu v ng dngn trong thc t.

Nguyn tc hthng my HPLC:

O 1 2 3 4 5

O: Ngun cung cp pha ng (mobile phase)

- Bnh cha pha ng

1: Bm cao p (hthng 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


3/16

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

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

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


4/16

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


5/16

Hthng HPLC

DetectorUV-Vis: detector phhp thu phn tXc nh cc cht c khnng hp thquangHunh quang (Fluorescence detector): xcnh cc cht c khnng pht hunh quang

- Alflatoxin, Mycotoxin, Amino Acid, thuc trsuhCarbamate,.u o chskhc 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 svn chuyn v phn blin tc ca chtPT tu ct n cui ct- Cht phn tch lun phn bgia 2 pha,trong o pha ng lun chy qua ct tch vi mt tc nht nh hoc gradient- Hiu quca qua trinh tach phthuc rt


6/16

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 bpha 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 bqu trnh tch sc k ca hn hp cht PT sc k gm nhiupeak.- c im ca peak PT:Cc peak co thtch 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 blu gimnh sc ragii ra sau cng, cht lu gikem sratrc4. u im ca phng php sc k- C thphn 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- Thtich mu phn tch nh(1-100mL)

V d: XC NH AXIT HU CTL, VQUMNG CT BNGSC K LNG CAO P

TM TT

Axit hu ctrong l, vqumng ct kh c xc nh bng phng php

sc k lng cao p. L ti v vqumng ct kh c chit vi nc nhit 127 0C thi gian 30-60 pht di p sut 0,15 MPa. ng thi, vquba kh c chit bng dung mi (axeton v metanol) trong bchitsoxhlet 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 sdng etect210 nm. Axit hu cchyu trong l, vqumng ct kh c tm thy l axit (-) hyroxy xitric hm lng ln lt l


7/16

2,863 v 15,221 %. Phn axit cn li trong l, vqumng 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 tc khi lng nhv phc tp cchit tnhiu 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 schuynkhoa sc kho. l kh nng iu chnh qu trnh tng hp axit bo, shnhthnh lipit, s ngon ming, v gim cn. ng

phn ca (-)-HCA gp phn ln trong lnhvc dc hc nhtc nhn c vai tr quan trngtrong mc ch gim cn, bo vtim mch, hiuchnh trng thi bt bnh thng ca cc lipid,v khnng chu ng trong luyn tp ththao[3] [4] [5] [6]. Vquba 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 ddy rut, km tiu ho; Vim ming, bnh cn rng; Hora mu. Dng ngoi trbng, mn nht, su qung, eczema, dng mn nga, rt

cc vt n m vo tht; L ba c vchua thng c dng thi nhnu 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 chthc hin trn cc loi ba can . V vy, skhm 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 (hmng ct) - Clusiaceae.

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

2. Nguyn liu v phng php nghin cu

2.1. Nguyn liu

L, vquca cy mng ct (Garcinia oblongifolia Champ. ex Benth.) ti xHa Lin, huyn Ha Vang, TP. Nng v tthnh phHCh Minh.

COOHC

H

HO

COOHHO C

COOHCH

H

COOHC

H

COOHHO C

CH

H

C=OO

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


8/16

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, xl 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 khisdng (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, xl 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 khisd n chu n ch HPLC IR .

Nguyn liu nghin cu l, vqumng ct c chun btheo sphn2.2.1.

2.2. Phng php nghin cu

2.2.1. Snghin cu

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

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

L: Vqukh:

Phng php 1: Phng php2:


9/16

2.2.2. Phng php chit tch

Chng ninh trong ni p sut:L, vquba ti hoc kh c ct nhri cho vo cc thutinh 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:Vquba kh xoay nhcho vo bchit 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, vquBa ti cho vo cc thutinh c sy kh

v bit khi lng chnh xc. Cho cc thutinh c cha l, vquBa vo tsy

v sy 80o

C.Sau khi sy khong 3 gi, ta ly cc ra cho vo bnh ht m cho n khi ccthutinh 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 skhong 0.005g th dng qu trnhsy.

Da vo cc kt quthu c, ta tnh c khi lng l, vquBa trcv sau khi sy. T, ta tnh c m l da vo cng thc sau:

Trong : H: m (%); m0: khi lng l hoc vquti trc khi sy(g); m1: khi lng l hoc vqusau khi sy (g).

2.2.4. Phng php chun [2]Xc nh hm lng axit tng strong l, vquba theo tiu chun TCVN

4589-88.

NhNaOH 0,1N tburet xung, cho n khi dch thc 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 =


10/16

trong : n: sml NaOH 0,1N sdng chun V ml dch th; Vcd:thtch mu c c; P: trng lng mu th, tnh bng gam; K: hsca loiaxit, K = 0,006904.

2.2.5. Ph

ng php xc

nh c

u trc ho hc b

ng quang ph

(IR)Cc phn tlun dao ng khng ngng. Tn sdao ng ca cc phn t

phthuc vo hng slc ca lin kt v khi lng ca chng, do cc nhmchc khc nhau sc tn shp thkhc nhau nm trong vng t5000 200 cm-1.Mi nhm nguyn t(nhm chc) xc nh c tn sxc nh v khng i trongbt khp cht no c cha nhm nguyn t. V vy, khi phn tch trn quangphhng ngoi ta c thxc nh c cc nhm nguyn tm 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 xl vi 500 mg Dowex 50 [H+].Hn hp c khuy trong thi gian 10 pht sdng 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 t10 ppm n 320 ppm.Chun bdung dch chun axit xitric cho HPLC:Dung dch chun axit

xitric c chun bring bit c nng t2 n 30 ppm sdng nc ct 3 lnct.

HPLC phn tch:Hthng sc k lng cao p c sdng nghin cugm my sc k lng cao p hng Knauer trang bvi bm loi low pressure hng

Knauer, v lp ct sc k Knauer C18: 250 mm x 4,6 ID x 5m. Btng 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 t10-30% 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.


11/16

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

tch pic. ng cong HCA c vda trn biu din sphthuc 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 nhcch 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 tgi trgii hn di (LLOQ) n gi trgii hn trn (ULOQ).

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


12/16

3. Kt quv tho lun

3.1. Kt quxc nh m trong l vqumng ctKt quxc nh m trong l, vquba c trnh by trong bng 1 sau

y:

Bng 1. Kt quxc nh m trong l, vquMng 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

Tkt qutrn 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 vqumng ct khong 84,31 0,16 %, mtrung bnh trong vqul 84,31 %. m trong vqumng ct l rt cao, chimkhong 84 % khi lng vqu.

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

Kt quchp ph IR cho thy xut hin pic c bc sng t3300 3600cm-1, l phdao ng ca nhm OH c lin kt hiro, v pic c bc sng t1600 1800 cm-1, l phca nhm C=O trong nhm COOH (hnh 2, 3).


13/16

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 vquba chittrong nc

Hnh 3: PhIR mu l ba chit trongnc

Tnhng phhng ngoi trn, c thkt lun trong mu chit tnc cal v vqumng ct c stn ti ca axit hu cc nhm OH.

3.3. Kt quxc nh axit hu ctrong l, vqumng ctBng 2: So snh axit hu ctrong l, vqumng 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

Vqubakh

Nc 15,221 0,406 17,468

Vqubakh

Axeton 12,999 0,239 13,711

Vqubakh

Metanol 9,508 0,078 10,492

Smu n=3, kt qutnh 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


14/16

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 chl HCA, bi v gi trthu c tdin tch pic ca HCA

ln nht. Axit ch yu c tm thy trong l, vquba bng HPLC l HCA,c thhin trn sc k hnh 5, 6, 7, 8. Pic thyu c xc nh l pic caaxit xitric. Trn sc k , HCA cho pic n trong tt ccc 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 tpic 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 ccc 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 t10 n 320 ppm, phng trnh ngchun: C = 1,37A-6,88 vi A l din tch pic ca HCA, C l nng HCA, hstng quan R2=0,999698.

Hnh 4. Sc k mu axit HCA v

axit xitric chun

Hnh 5. Sc k mu vqumng ct

kh chit trong metanol


15/16

Hnh 6: Sc k mu vqumng

ct kh chit trong nc

Hnh 7: Sc k mu vqumng

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, vquba. Kt qucho thy axit (-) hyroxy xitric l thnhphn axit chyu c trong l, vquba. Hm lng HCA tm thy trong l, vquba kh l kh cao, cthHCA trong l, vquba kh ln lc l 2,863 v15,221 %.

Hnh 8: Sc k mu l mng ct chit


16/16

Tkt quny c thtip tc nghin cu su sn xut thc phm cha trbo ph tcy ba Vit nam.

Documents

Cac Phuong Phap Phan Tich Hoa Hoc Va Thuc Pham