NGHIN CU C TH V CHT LNG SIU TI HN

Siu ti hn ( SFE ) l phng php chit s dng 1 dng dung mi c bit. Dung mi ny trng thi siu ti hn, c to ra mt nhit v p sut ln hn im ti hn . trng thi siu ti hn, dung mi ny: - Khng cn th lng ( do nhit cao ) - Nhng vn cha thnh th kh ( do p sut cao ) - C nht thp hn phase lng ( d xm nhp vo mu ) - C kh nng chuyn khi ln hn phase kh ( chit xut c cn kit hot cht ) Dung mi thng dng nht: Dioxytcarbon CO2 ( khng phn cc ) im siu ti hn 310C/73 atm nn d t , d duy tr. D p dng quy m cng nghip, khng chy n, an ton, thn thin vi mi trng. Lu cht siu ti hn c ng dng trong rng ri trong nhiu lnh vc khc nhau nh trong lnh vc mi trng, thc phm, trong cng nghip, trong y hc ng dng ca dung mi siu ti hn - Chit bng cht lng siu ti hn: (supercritical fluid extraction SFE) Cht lng siu ti hn c s dng trch ly cc hp cht

du d bay hi, cc hp cht thm v cc nguyn liu th ban u trong ngnh m phm (nh nc hoa) ng thi ko gy hi cng nh bin i mi ca nc hoa. Trong phng th nghim, CO2 siu ti hn c s dng nh mt dung mi trch ly. Chng hn nh xc nh tng hm lng hydrocacbon thu hi t mu rn, cht cn, bi tro v nhiu ci khc cng nh kim tra cc hydrocacbon vng thm PAHs (polycyclic aromatic hydrocarbon) trong cht rn v thi rn. Dng siu ti hn cng p dng trch ly hm lng hydrocacbon trong nc. - Ct phn on (supercritical fluid fractionation SFF): t hn hp lng. Ngy nay ngi ta ng dng ly trch hng liu t dch nhiu thnh phn c chng ct. Hoc dng tch lipid phn cc hay nhng polyme. - Sc k lng siu ti hn (Prerarative scale supercritical fluid chromatography- PSFC): dng tch phn on sau cng gm nhng cht c cu trc rt ging nhau. - c s dng nh mt polymer dng xp : CO2 siu ti hn c s dng trong polymer dng xp. CO2 siu ti hn c x l cng vi polymer trng thi n nh sau gim p sut v nhit th CO2 s nhanh chng gin ra v li cc l xp trong polymer to thnh polymer xp. Cc nh nghin cu vn ang tin hnh sn xut polymer xp vi kch thc l xp dng vi t bo i t CO2

- CO2 siu ti hn c s dng tng cng kh nng thu hi du trong giai on kt thc qu trnh thu hi. - Phn ng (supercritical fluid reactions SFR): lu cht siu ti hn c th xc tc cc phn ng tng hp, nht l phn ng hydrogen ha. - Lm kh v sch : CO2 siu ti hn cng c dng lm sch qun o thay cho PCE (perchloroethylene) hay l nc . Phng php mi ny c pht hin v thng mi ho bi tin s Joseph DeSimone,v cc chuyn gia v k s ho hc trng North Carolina State University v University of North Carolina, Chapel Hill.

Nghin cu chit tch mt s ch phm thin nhin c gi tr kinh t cao bng CO2 lng trng thi siu ti hn
1 i tng nghin cu 1.1L ch xanh, Camellia sinensis (L.) Kuntze, Theaceae Nguyn liu s dng trong nghin cu l ch thnh phm mua ca cng ty xut nhp khu H Ni. c im nguyn liu: Sn phm mua c l Ch xanh loi F, thnh phn ch yu t l 3 v 4, mnh ch c kch thc 0,5 1,5 mm. m nh hn 4,5 % theo quy nh i vi Ch xanh loi F ca cng ty

1.2 Hoa bi, Citrus maxima (Burn.) Merrill, Rutaceae c im nguyn liu: Hoa bi dng bt u h n, cc cng hoa c gi nguyn vn, mu hoa t dp nt trnh mt tinh du, m trung bnh 78,4 %. 1.3 R Vetiver, Vetiveria zizanioides (L.) Nash., Poaceae c im nguyn liu: R Vetiver sau khi thu hoach x l bng cch phi trong bong rm mt. Chiu di ca r t 20-30cm, rz c mu nu hi ng trng. 1.4 Hoa Nhi, Jasminum sambac (L.) Ait., Oleaceae c im nguyn liu: Hoa bt u h n, cnh hoa c gi nguyn vn khng b dp nt. 2 Thit b v phng php nghin cu 2.1 Nghin cu k thut chit mu thc vt bng SCO2 a/ Thit b SFT-250

Hnh 2.1: nh chp thit b SFT-250

b/ Nguyn l hot ng

Chu trnh trng thi ca CO2 trong qu trnh chit Mu c s ch v nghin ri cho vo bnh chit. Bnh chit c lt Mng lc c phn nh v phn y trnh vic cc ht nh c th lt vo ng ng dn dung mi. Kh CO2 t bnh kh (45 - 55 bar) c dn qua ct than hot tnh v qua mng lc parafin loi b tp cht v tch nc. Sau , kh CO2 c a vo ha lng ti thit b lm lnh, nhit CO2 sau khi ngng t khong 0 C. Trc khi da vo bnh cha,
0

CO2 lng c th c trn them cc dung mi h tr khc nh etanol, methanol to thnh dung mi mi theo yu cu cng ngh. Kh CO2 lc ban u trong bnh cha trng thi I (hnh 2.3), thng l p sut trong khong 45-55bar, nhit 12-20 C. Khi c h nhit iu kin dng p t trng thi 1 sang trng thi 2 l trng thi CO2 th lng trong bnh cha CO2 lng. CO2 dng lng c th pha trn vi dung mi d dng. Quan trng hn l c th s dng bm cao p nn ln p sut cao v iu chnh lu lng vo thit b thun li. T bnh cha, CO2 lng c bm qua van iu chnh lu lng vo b phn trao i nhit iu chnh t trng v nht ph hp vi yu cu cng ngh. Dng CO2 lng t trng thi 3 c gi iu kin ng p v tng nhit dn dn chuyn CO2 lng sang trng thi siu ti hn 4. Trong sut qu trnh chit, nhit v p sut ca bnh chit lun lun c iu chnh gi n nh. Qu trnh chit nguyn liu thc vt bng CO2 siu ti hn c th thc hin lin tc hoc gin on ty theo yu cu cng ngh. Kt thc qu trnh tch chit, dch chit c dn vo bnh phn tch. Ti y qu trnh tch cht tan ra khi dung mi thnh nhng phn on ring c thc hin bng cch thay i cc thong s p
0

sut v nhit . t trng thi 4 tr v trng thi 5 l qu trnh gim p. Khi dch chit vo bnh tch (5-1) to cc phn on khc nhau th c th chn cc gi tr p sut P v nhit thch hp. c/ Ti u ha cc iu kin cng ngh chit bng CO2 theo quy hoch ha thc nghim Cc yu t nh hng n hiu sut v cht lng sn phm ca qu trnh chit xut: -Tnh trng mu: thi gian bo qun mu trc khi chit, kch thc mnh nguyn liu ca mu -Nng co-solvent trong CO2 - Thi gian ca qu trnh tin hnh chit xut - T l theo khi lng gia dung mi SCO2 v nguyn liu thc vt

3.

Chit xut Quercetin bng CO2 siu ti hn

Quercetin c nghin cu bi Caltagirone rng quercetin v apigenin c ch s tng trng, vic xm ln v tim nng di cn ca khi u c tnh. Quercetin c tim nng tr thnh mt liu php ha tr ung th tuyn tin lit nh bo co ca Xing (2001). Flavonols ny cng c xem l c chc nng chng oxy ha. C hai nhm cht chng oxy ha chnh: tng hp v t nhin. Mt trong cc xu hng quan trng nht trong ngnh cng nghip thc phm hin nay l nhu cu tm v v s dng cc cht chng oxy ho

t nhin. Chng an ton hn, t tc dng ph v t c tnh hn cc cht chng oxy ho tng hp. C hnh cha rt nhiu quercetin glucosides (quercetin-3,4'diglucoside v quercetin-4'-monoglucoside). Lp v ngoi cng ca c hnh chuyn sang mu nu v kh trong qu trnh gi i, v cc glycosides ca quercetin chuyn dng thnh quercetin t do. Herrmann (1976) cng cho rng trong c hnh kh cha ch yu l dng quercetin t do hn l dng glycoside. Theo Bilyk er al. (1984) th lp v kh pha ngoi c nhiu quercetin hn lp bn trong tt c tm ging hnh c nghin cu. Ging cao nht cha ti 34,15g quercetin/ kg v hnh kh. Cc ging khc cha t 1,14 16,53g quercetin/kg [11]. Chit xut flavonols t m hnh trong cc nghin cu trn c thc hin bng cch chit vi dung mi methanol. Nhng trong cng nghip vn pht sinh vi k thut ny l phi loi b dung mi hu c t cc sn phm cui cng, x l cht thi methanol, c tnh ca methanol cn ln trong sn phm,. Do , phng php chit quercetin mt cch nhanh chng, r tin v t c tnh l mt yu cu cp thit. Chit xut bng cht lng siu ti hn c nhiu li th hn cc phng php tch chit dung mi lng truyn thng nh: tnh chn lc c ci thin, t ng ha v thn thin vi mi trng.

Phng php ny c thc hin ti i hc bang Michigan ca M nm 2003, s dng CO2 lm cht lng siu ti hn. 3.1 Nguyn liu v cht chun: - Carbon dioxide cng nghip (CO2, BOC gases, Murray Hill, NJ) c s dng nh mt cht lng siu ti hn (SF). - Hnh v hnh vng c ly t cc trang tri Muck (i hc bang Michigan, East Lansing, MI). bo v nguyn liu trong giai on nghin cu hnh c lu tr ti 20oC. - Ethyl alcohol mua t Pharmco (Brookfield, CT). - Phosphoric acid 86,3% cung cp bi J.T. Baker (Phillisburg, NJ). - Quercetin chun c cung cp bi Sigma (St Louis, MO). 3.2 Trang thit b:

. S thit b chit quercetin bng Carbon Dioxide siu ti hn.

Ch thch:

1. Bnh cha CO2 2. My nn cao p 3. Bnh cha kh nn 4,7. My o p sut 5,10 iu khin p sut pha trc 8. Piston bm, 9. H thng cung cp nhit 11. Van micrometering 12. Ni thu mu 13. ng h o th tch kh TC: temperature control (iu khin nhit )

3.3 Qu trnh chit xut: Chun b mu: lp v c hnh c bc tch bng tay, loi b nhng phn h hng v ct thnh mnh nh khong 0,5 0,5 inch. Quy trnh chit xut: p sut v nhit c ci t 5700 psi v 40oC. Phi mt t 25 30 pht t c cc iu kin trn. Bnh chit c gia nhit 24h trc khi ly dch chit thng nht thi gian chit gia cc mu. Hnh ty thi nh v ethanol 5% (nng phn t so vi tng s mol CO2 s dng to p lc cho h thng) c thm vo bnh trc khi n c ng li. My bm c ci t 5 ml/pht. Khi cc iu kin mong mun t ti, cc van micrometering c m,

CO2 tng ng 15 L kh nn (khong 4% tng s CO2 chim th tch bn trong bnh chit) i qua khay ly mu (nhanh nht c th duy tr ch tnh), sau cc van micrometering c ng li, thay cc chai thu tinh ng mu mi. Qu trnh ny ko di khong thi gian t 10-18 pht. Sau , mu tip theo c ly mt ln na vi vic m van micrometering. Mi mu chit 10 ln v chit vi 5 mu khc nhau. Cc thng s sc k: - Pha ng: dung dch acid phosphoric [0,5% (v/v)] - methanol (2:3 (v/v)) - Tc dng: 1ml/pht - Bc sng pht hin: 280 nm - Thi gian phn tch: 10-15 pht cho mi mu Quercetin t do c chit xut t v c hnh v vng, bng cch s dng CO2 siu ti hn nh mt dung mi v ethanol nh mt cht mang. 3.4 Kt qu Bng 2.1. Kt qu chit quercetin ca ging hnh vng v ging hnh . Mu s Thi Nng Tng gian chit (g/ (ph ml) lng querce T l g s g tin in/ Khi kg v % l (nng

Querce quercet lng Ethano Lt v hnh hnh

tin (g) /

t) Hnh 1 2 3 4 5 Trung 145 143 152 152 157 0,037 0,037 0,035 0,029 0,029 313 323 291 287 226

CO2 2,09 2,09 1,94 1,91 1,51 1,91 1,27 1,32 1,46 0,99 0,67 1,14 0,027 0,027 0,023 0,024 0,019 0,024 0,022 0,025 0,023 0,018 0,012 0,020 11,8 13,9 12,8 10,5 12,0 12,2 8,6 8,1 9,6 5,8 8,2 8,1

phn mol) 7,09 7,78 6,80 7,78 7,78 7,45 7,69 7,69 8,17 7,69 7,69 7,79

150 bnh Hnh vng 1 156 2 151 3 159 4 164 5 150 Trung 156 bnh

0,034 288 0,015 0,016 0,020 0,020 0,014 193 201 225 149 101

0,017 174

Quercetin t do c chit xut t v c hnh v vng, bng cch s dng CO2 siu ti hn nh mt dung mi v ethanol nh mt cht mang. Khi lng quercetin t do v th tch ethanol thu c trong mi ng thu t l thun vi nhau. Nghin cu ny cung cp mt phng php thay th cho phng php chit rn -lng thng thng chit quercetin. Tuy nhin, trong tng lai cn ch nhiu hn cc hp cht tng t khc c trong hnh c th nh hng n qu trnh chit xut v hiu sut chit.

4. Chit xut Quercetin bng nc siu ti hn Phng php ny c thc hin ti i hc Ewha womans Hn Quc nm 2010, s dng nc lm cht lng siu ti hn. 4.1 Nguyn liu v thit b: Chun b mu th: c hnh ty c ly t Uiseong, Gyeongsangbuk-do, Hn Quc, ch nhng c c bao ph bn ngoi lp v mu da cam mi c s dng. V c hnh ty c sy kh vi khng kh nng 60oC trong 10 gi v sau ct thnh ming (<10 mm) s dng mt my trn tc cao (Blender 7012S, Waring Co., Torrington, CT, USA). Tt c cc mu c lu tr trong t lnh duy tr 4oC. Cc mnh v hnh v diatomaceous earth (DE, to ct tri t) (ASE Prep DE, DIONEX Co., Sunnyvale, CA, USA) c pha trn v cho vo t bo chit thp khng g 34 ml (DIONEX Co.) c cha giy lc cellulose (30 mm, DIONEX Co.) nh hnh 2.5. Thit b: trnh by hnh 2.5.

Hnh 2.5. S chit xut quercetin bng nc siu ti hn 4. 2Qu trnh chit xut: Chit bng nc siu ti hn: Vic chit xut c thc hin bng cch s dng mt dung mi nhanh (100 ASE, Cng ty DIONEX) vi nc Milli-Q (MRRO800, Cng ty TNHH Mirae ST, An Dng, Hn Quc) nh l dung mi duy nht. V hnh ty (1-2 g) v DE c trn t l hn hp gia 0,5:3,5 v 02:02. Qu trnh chit xut c thc hin nh hnh 2.5. Mu ny c np vo t bo chit thp khng g v in y dung mi trong khong 1 pht, tng p sut trong 5 pht. Sau khi p sut t 90 bar, t bo chit c cung cp nhit (tng trng cho

thi gian chit xut) trong khi p sut c duy tr 90-131 bar, nhit chit xut t 100-190oC. T bo chit ny c th c ra sch dung mi qua cc ng bm mu trong vng 30s. Vic ra t bo chit c thc hin khong 1-2 pht gia cc ln chit xut vi kh nit. Dch chit c thu vo mt l thy tinh (40-50 ml) bit khi lng. 4.3 Kt qu chit xut bng nc siu ti hn: Hiu qu chit ca SWE c nh gi bng cch so snh khi lng quercetin chit c (mg/g v c hnh) v % quercetin chit c ca t v c hnh qua cc ln chit. SWE c thc nhit l 165oC, thi gian 15 pht, t l hn hp v c hnh v DE l 1,5:2,5 cho kt qu nh bng sau: Bng 2.2. Hiu sut chit v phc hi ca SWE S ln Nng sut (mg/g v c phc hi (%)b a chit hnh) 1 16,29 0,75 92,40 2 1,28 0,11 7,26 3 0,06 0,02 0,34 4 Ndc 0 5 Nd 0 Tng cng 17,63 0,87 100 a Nng sut (mg/g v c hnh). Mi th nghim c thc hin 3 ln, d liu th hin di dng gi tr trung bnh lch

chun
b

phc hi (%) = nng sut/ tng lng quercetin chit c Khng pht hin Ngoi ra, thnh phn ha hc ca cc chit xut thu c t

bi SWE
c

SWE c so snh vi nguyn liu ban u (Bng 2.3) Bng 2.3. Thnh phn ha hc ca cc chit xut thu c t SWE v nguyn liu v c hnh ban u. Cht Carbohydrate (%) Protein (%) Lipids (%) Ash (%) Moisture (%) Quercetin (mg/g v c hnh) Quercetin-40-glucoside onion skin) Kempherol (mg/g v c hnh) Isorahmnetin (mg/g v c hnh) Rutin (mg/g v c hnh)
a

V hnh 47,63 0,87b

c Chit SWEa

xut ca

13,24 0,62

3,98 0,44 1,14 0,19 5,22 0,59 1,68 0,12 7,10 0,61 2,10 0,076 4,81 0,42 9,42 1,63 (mg/g 17,63 0,87 16,29 0,75 3,15 0,60

3,72 0,09 0,68 0,13 0,88 0,23 0,54 0,09 0,74 0,18 0,58 0,11
o

0,93 0,15 SWE c thc nhit l 165 C, thi gian 15 pht, t l hn hp

v c hnh v DE l 1,5:2,5

D liu th hin di dng gi tr trung bnh lch chun Kt qu trn cho thy cc thnh phn ha hc ca chit xut

SWE gim ng k so vi nguyn liu ban u. Tuy nhin, i vi flavonoid, chit xut bao gm mt lng ln quercetin (16,29 0,75 mg/g v c hnh), mt lng nh quercetin-4-glucoside (3,15 0,60 mg/g v c hnh), v c du vt cht flavonoid khc. Kt qu ny ch ra rng quercetin v quercetin-4-glucoside chim khong 99% tng lng flavonoid c trong chit xut v khong 92,40% tng lng quercetin so vi nguyn liu ban u. Cc kt qu trong nghin cu ny chng minh rng SWE l mt phng php rt hiu qu chit flavonol quercetin v do nc siu ti hn l gii php tuyt vi thay th dung mi hu c, gp phn nng cao hiu qu chit xut v thn thin vi mi trng.