PHUONG PHAP SAC KY BAN MONG 35 BAI 6: DINH TINH SACCHARINE BANG PHUONG PHAP sAc kv BAN MONG 1 NGUYEN TAC Chiét t&ch saccharine ti thyc phdm hote dé uéng bing ethy! acetate. Lam bay hoi dich chiét, hda tan phdn c&n con lai vao hén hgp dung mdi amoniac — nude — ethanol (1:1:2). Sau d6 chéim dung dich nay lén ban she ky lép ming, so sinh Ry va cutting a> huynh quang véi dung dich saccharine chun dui den tif ngoai 06 bude song ngéin 254nm. TH. DUNG CU 1- Micropipette c6 chia vach hofe cdc Sng mao quin 2- Binh dinh mute 10, 50, 250mé ‘8- Phu chiét 250m! 4- Ong nghi¢m c6 n&p 6- Binh cfu 250m 6 May 06 quay chin khong 1- Binh chay she ky 8- Tam kinh dé trang lép méng, kich thute 20 x 20em howe ding ban méng tréng sn Silicagel H 9- May sy t6e 10- Ben t ngoai c6 bude séng 264nm 11- B6p céch thily. THT. HOA CHAT 1- H,80, dung dich 1:1 2- NaOH dung dich 50% 3- NHg dung djch 25% 4- Eter ddu héa c6 diém sdi 40 + 70°C 5- Ethy! acetate 6- Ethanol 96% 7- Silicagel H . 8 Hn hgp amoniac - nude ~ ethanol theo t7 1g thé tich 1:1:2 thin hgp B) 9-H dung mdi chay sdc kj: n — buthanol ~ ethanol — amoniac ~ nutéc theo. {3 1g thé tich 40:4:1:9 (dumg méi A).36° CHUONG 4 TV- TIEN HANH Chudn bj dung dich saccharine chudn: Can chinh xée 10mg natri saccharinate cho vao binh dinh mtic 10m, dinh mite bing hin hyp B @ trén, lde déu (1m dung dich chugin chifa Img saccharine). Chudn bi mau thi: + Déi v6i dé udng 6 COs: dudi CO, bling céch dun mau trén bép céch thiy hode lie menh nhidu lan. ~ Déi vdi dé udng 06 edn: léy 50ml dé udng vAo mdt cdc thiy tinh, dun cdch thay trén bép dé dudi hét cén. Sau dé lam nguéi phdn con lai rdi dé vao binh dinh mife 50ml. Trang céc bing nuée ct, tap trung nu6c trang vao binh dinh mde réi dinh mic téi vach bing nude cét, Whe déu ~ Déi véi thye phém dang ran: nghién nhé méu, ly 5g mau da nghién nhé vao o8e thiy tinh, Thém 200m! nuée sbi vao cbc, tron déu va gitt trong 2 gid, thinh thodng khudy déu. Sau 46 loc hén hgp qua gidy loc vio binh dinh muc 260m. ‘Trang gidy loc nhiéu lan bing nuée céit, sau 46 dinh mute tdi vach bing nude cft va lic déu. Chiét tach saccharine: Axit h6a 50m! phan miu thit da due chudn bi é trén bing 10m! dung dich H,S0, 45%, sau dé cho vao phéu chiét, chiét bing 50m ether ddu héa. Lic manh, sau d6 dé yén cho phan lép. Thu Igi lép duéi c6 chita saccharine sang binh cdu. Chiét phan cdn lai trong binh chidt thi nh&t bing 50m/ dung dich ether dau héa nhv trén va cfing thu phdn Ip dusi vao binh chiét thé hai. Thém vao binh chiét thi hai 5d NaOH 50%, chiét hai lan bing ethyl acetate, mdi ldn 50m. Loc dung dich chiét ethyl acetate qua bong thiy tinh (di duge mita truée bing ethyl acetate) yao binh cdu cia may ¢3 quay chan khéng. Tign hanb cd quay cho dén khi cdn 2+ Sm thi chuyén sang Ong nghiém dung tich 10m. Tréng rita binh cdu bing ethyl acetate, thu nude tréng va éng nghiém. ‘Lam bay hdi dung méi trong éng nghi¢m trén bép cach thay, tét nhat 1a dudi ludng kh{ nite nhe. Sau 46 hda tan lugng e&n thu duge bang 2,5ml hin hgp B. Day kin Sng nghidm dé chay ede ky ban méng. Tién hanh chéim sdc kj: ‘Trude khi cham ste ky phai co sach I6p silicagen 6 canh déy va hai canh bén cia ban s&e ky dé tranh hién tugng mao dan. Ding bit chi va thude ké dénh déu vach xudt phat céch mép dutéi cia ban méng 2,5em. Dang micropipet cham 5 diémPHUONG PHAP SAC KY BAN MONG a7 dung dich mau thit va dung dich chudn, céc diém céch mép cia ban sc ky va eéeh nhau 2,5¢m. MBi ln chi chi'm khoang 1ul 46 duting kinh cfla méi vét chm khong qué 0,5em. Chaim xong mét chm phai lam kho bling may sy téc réi méi cham tiép cho d&n kkhi ché'm hét Iugng dung dich cén phai cham, Chim ede vét theo thi ty tit tréi sang phai nh sau: - 2,5yl dung djch saccharine chun ~ Syl dung dich saccharine chudn ~ Bul dung dich chiét cia mau thir = Bul dung dich chiét cia mau thit di duge pha long gap déi bing hin hgp dung dich B = 10u dung dich saccharine chuén, - Cham xong 4é bin méng kho trong khong khi. Tién hanh chay sde hg: Dang mét t8 gidy loc da duge tm ust bling dung mdi chay sie ky (dung méi A) phd xung quanh thanh binh phia trong, sau 46 dé dung moi A vao binh ti 45 cao Jem. Day kin nap binh va dé yén trong 30 phut dé c6 duge trang thai bio hda dung m6i trong binh. Dat ban mong shc ky da cham mau va duge lam khé vio binh, day nip Igi dé chi quyén trong binh ludn duge bao hia dung mai. Khi mie dung mi trén ban méng dat dé cao 10cm (sau khodng 1 gid), nhae bin méng ra, dénh dau chinh x4e mie dung mdi va lam bay hei dung méi cho tdi khi khéng nhin thay vach (kkhodng 10 phit). Quan sét bin s&c ky duéi anh dén tit ngoai ¢é bude séng 254nm va khoanh ving vét saccharine phat huynh quang (R; = 0,5). Danh gid otic hg dé: Do khodng cach di chuyén cia vét dung méi va eda vét saccharine, tinh todn R,, Déi chiéu so sinh she key dé cia mau thit v6i miu saccharine chufin vé gid tri Ry vi eudng d> huynh quang. Néu gidng nhau thi két luan: s&e ky dé do 1a eda hai chat gidng nhau.38 CHUONG 4 BAI 7: PHAN TICH AFLATOXIN i BANG PHUONG PHAP SAC KY BAN MONG T- NGuveN TAC Aflatoxin 14 nhém mycotoxin e6 die tinh cao do géy ung thu gan, duge sinh | bai m9t 86 loai n&im méc ma thudng ghp nhét 1a Aspergillus flavus. Day 1a mot nhém chat gdm khodng 20 Jogi cé nguén gée flavacumarin. Phin ti aflatoxin gim m6t géc cumarin, hai nhén furan va mt vng lacton. Cong thite chung (yy a) @) 07 ~O' ‘OCH, by (2) 07 *O ‘OCH, 2) AflatoxinB1 = R=H va ob ni d6i gia (1) va (2) AflatoxinB2 = R= va khong 66 n6i doi git (1) va (2) Aflatoxin M1 «-A=OH vA. ni di gida (1) va (2) AflatoxinM2 «A= OH va knOng c6 ndi d&i gida (1) va (2) | b) Afiatoxin G1 (06 61 d01 gid (1") va (2") Aflatoxin G2 khong 66 ni 601 gida (1') va (2") Céc aflatoxin trong ty nhién gém bén loai la B1, B2, G1, G2, Aflatoxin nhém B va nh6m G duge goi tén do tinh phat huynh quang duéi tia UV: B: blue; G: green. ‘Vé c&u tao héa hoc, ching khdc nhau ¢ ché nhém B chi cé m$t nhém chitc lacton trong khi nhém G e6 hai chic lacton. Thi ty 1,2 trong mdi nhém 1a do su di chuyén nhanh hay cham trén sic ky 46. Céc Jogi aflatoxin khée duge hinh thanh 1A do sy bién a6i héa sinh cia 4 aflatoxin ban d&u B1, B2, Gi, G2 bén trong cée sinh vat bj nhiém gdm ndm va dong vat. 1B phat hign va dinh ligng aflatoxin e6 hai nhém phuong php la phuong phép sinh hoc va phuong phép héa hoc, nhung phuong phép héa hoc cé tinh chon Ige han va e6 46 tin efy cao hon. Phuong phép héa hoc bao gdm cée bude co ban sau: - Léy mau = Chiét xudlt dBc 16HUONG PHAP dc KY BAN MONG 3g - Loai tap chat a die - Phan t4ch ede thanh phan bing siic ky - Dinh tinh va dinh lugng aflatoxin. ‘pé phn tich aflatoxin bang phuong phép sc ky ban méng sit dung dung méi 4é hda tan va trich ly aflatoxin tir nguyén ligu da duge nghién nhé. Ding Ot sie ky hap thy aflatoxin tt dich trich. Dung methanol gidi hp aflatoxin. Dinh tinh va dinh lugng aflatoxin trong dich trich bing phuong phép sic ky ban méng. Day 1& phuong phép duge sit dung nhiéu nhét trong cc phong thi nghiém dé dink lugng aflatoxin trong wong thye thye phdm, thie am gia sic... Phung phép nay ré han so vai HPLC va c6 46 nhay khé cao, cé thé phat hign tdi néng do Ippb. - DUNG CY HOA CHAT Dung cu thiét bj: - May soi huynh quang - May lke - May gia nhiét va théi khi nitrogen. - Binh trién kthai fic ky ban méng ~ Micropipette 20u! - Syringe nhya 20ml - Pipette loai 1ml, ml, 20m = Cot sc ky Ai lye mi&n dich = Cée minicoluran florisil ding 48 b4n dinh lugng ~ Ban méng sileagel o6 sén hoae tf ché tao - Gidy loe. Hée chat: - Methanol loai HPLC - Chloroform + Acetone - NaCl - Aflatoxin chudn 0,5yg/ml trong benzen-acetonitrile (98:2) ~ Dung méi trich ly aflatoxin 1 hén hop methanol-nuéc (55:45) - Hé dung moi tridn khai sc ky-chloroform-acetone (9:1)40 CHUONG 4 TH TEN HANH 1. Chiét xudit aflatoxin Dé chiét xufft aflatoxin e6 thé ding cée dung méi héu co nhu chloroform, dichloromethan, acetonitrile, aceton, methanol, Néu sii dung may khuly tron te do cao thi théi gian chiét xuft 1 « 8 phit, néu ding may Mc thi chiét trong 30 phut. Cé thé thém vao dung méi mt 86 chit dé lam ting kha nang chiét xudt aflatoxin nhu bé sung axit lam ddt céc lion két gitta toxin va protein, ding diatomit lam chit tra Ipc, bd sung mudi vo eg dé phd nbd, dung méi khong phAn eye dé tach ‘béo, nuge lam truong nd U6 bao. Thong dung nhdt 1A ding chloroform va nuée hote methanol : nuée. MAu phan tich duge nghién min va tron déu, Can 25g cho vao binh nén dung tich 500ml, thém 5g NaCl va 125ml hn hgp methanol : nude (55:45). Lie trén may Ie 30 phut dé trieh ly aflatoxin. Loc thu dich va ly 30md dem pha lodng véi 30m! nue edt. Lay 40m! tuong duong 4g miu, ding cho qua cot héip thy aflatoxin. 2. Logi tap chét Sy cé mat cila lipid va mét sé chat khac cé thé Iam dnh hudng dén két qua. nén can phdi loai sach tap cht khéi dich chiét truée khi c6 dae. C6 thé ding phéu chiét trich ly lang —léng dé logi tap chat do da 68 micotoxin ‘Khong phan eye nén ¢6 thé dang dung mdi loai lipid ma kchéng lam mat dc 6. Dang acetate chi hay hydroxide sit dé két tla gossipol, ding carbonate déng két tia chiorophyll, ding nitrate bac dé téch alkaloid... G8 thé loai tap chat bing sie ky edt hap phy vdi cde chat hap phy nh silicagel, aluminium oxide, polyamide florisil va sephadex, trong dé silicagel duce ding nhiéu nhét, Mot sé logi oft lam sin tign dung, gidm thi gian chudn bi va cho chat lugng én dinh. Séic k§ hép phu aflatoxin tit dich trich Dich chiét aflatoxin duge cho qua eft, aflatoxin hap phy trén cdt. Dang cdc dung m6i ria tap chat ra khdi cht sao cho khong héa tan aflatoxin. Cudi cng chon mot dung méi gidi hap aflatoxin ra khdi ot. Thu héi va ed die dung dich aflatoxin. Voi ky thudt lam sach aflatoxin bing cét silc ky 4i lve mién dich c6 thé loai ‘hoan toan tap chit ma khong gay that thodt aflatoxin. Thue hidn 6i edt ade hy di hee: Cdn syringe len gid a3, g&n cot s&e ky i lye tiép n6i vao syringe trung gian. Dat cOe hing dudi cot sde ky ai Itc. Dé dich chiét aflatoxin d& chudn bj 4 trén vao syringe va thao ndp d day cbt séc ky Ai luc.HUONG PHAP Sac KY BAN MONG a Dich chiét bat ddu chay qua cot sdc ky di lve va aflatoxin duge hélp phy lén et. Can duy tri te dé chay t6t nh&t khoang 2-2mbiphait. Néu chay cham cb thé ding bom tay bom nhe qua syringe. Khi két thie, ding bom diy hét dich chiét con Jai rong e6t. Gidi hdp phu, thu nhén aflatoxin tit cot sde k¥ éi le: D&t mot lo nhd (dung tich 5m) duéi o6t. Ding 2mi methanol cho qua syringe vao e6t sc ky 6i lye 6 gidi hap va thu nhén aflatoxin, Ding bom day hét methanol ra khéi eGt vao lo hing. 3- Phan tich dink tinh vat ban dinh lugng aflatoxin Iay Yl dich gidi hap cho qua minicolumn florisil dé ban dinh lugng. Minicolumn florisil 1a mOt logi vi c§t lim bing thug tinh 06 dung kinh 6 + 6mm va dai khodng 20em duge nbéi ede lép vat ligu o6 tinh hép phy gdm elumina, silicagel va florisil. Hai dau c6t 06 calcium sulfat 06 tinh gitt nude. Calcium sulphat ————w Atuorina ——— ae Sticage): ———e Flonsi Hinh 7.1 Cét hdp phy minicolumn florisil Khi cho dich chiét aflatoxin qua cdt ede chét h&p phy s@ giti aflatoxin lai va ta s& phat hign duge khi chiéu cbt duéi anh sfng dén UV thay mot bang phat mau huynh cuang xanh tim 6 phia trén lop florisil. So sénh véi mot minicolumn chuan ¢6 chia mOi luyng aflatoxin 4a biét (20 + 25ng), ta c6 thé dénh gié mau c6 chia aflatoxin nhiéu hay it hon so véi chudin, Phugng phap dinh tinh va ban dinh lugng aflatoxin bing se ky hap phy minicolumn nhanh va khong cain cdc thiét bi phie tap, tign Igi ngoai hign truving. Nhuye diém eda phuoag ph4p nay 1A khong phan bigt duge cde aflatoxin kh4e nhau va vige dinh lung chi mang tinh chat so sénh tuong déi.42 CHUONG 4 # Phan tich aflatoxin bing adic k§ ban méng Co thé tién hanh se ky bin méng mét chitu: duye thye hign véi pha dong trig Khai sde ky 1A mot dung méi hoge mot bn hop dung mii Ngoai ra, cing 06 thé thyc hin se ky ban méng hai chiéu bling céch chay hai in vubng géc, véi hai h0 dung méi khée nhau dé tang khé ning téch, dje biat IA khi cn phan tich ede miu ¢6 chita ham lugng doe t6 rit théip nhu AFM1 trong stfa va AFBI trong gan. hoe néu chét chiét e6 In nhidu tap chét nhur thie an gia sve hén hop hofe gu, Ige. Ban mang silicagel thudng o6 kich thuée 20 x 20em nhung 6 thé ekt thank ede ban ¢6 kich thuée nhé hon, 10 x 10cm hoke 7 x Tem cing déu cho két qua tot. Ste ky hai chigu véi ede bin méng e6 kich thude nhé tit kiam duge thiti gian va 06 thé thye hign trong m6t e6e thiy tinh thong thing. Ngoai ra cn eé thé chay nhiéu mu trong cing m9t he thong nén tiét kiém duge dung mai, Bang 7.1 Cac he dung méi ding phan tich aflatoxin bang sde hy bin méng Phan tich aflatoxin B1,62,G1,G2 Wie Benzen - methanol - CH,COGH 90:5:5 Ether - methanol - nude 96:3:1 CHCl - C:HCl:" n, amyl alcohol - HCOOH 80:15: 4:1 CHOI, -acetone - nude 88:12:18 | CHCl, - acetone - isopropanol nude 98:12: 4,5:4 CHI, - isopropanol 80:4 Phin tich aflatoxin M1 Ether « methane! - nuge 95:4:1 Ether - hexan - nude 85:18; 4:1 Viée phat hign va dinh lugng duge thye hign du¢i anh séng tia UV e6 do dai s6ng 865nm. Ding may do huyah quang (fluorometry) do outing d9 phat huynh quang, hay so sénh bing mat dg sang cila cde dém mdu va eda chutin, tit 46 suy ra néng ¢9 cia aflatoxin trong mau khdo sat. Dé thuc hién ky thuat sho ky hai chidu tién hanh chém mau & goc cia ban silicagel va Ichai trién hai ldn ydi hai ‘he dung moi. He dung méi dau tién 1a diethyl ether — methanol ~ nude (94:4,5:1,5) va hé dung moi thd bai la chloroform ~ aceton (9:1), Khdng dinh aflatoxin BE khang dinh aflatoxin, trénh gay nhdm ln véi mgt cht nao d6 06 thé c6 cing két qué TLC thi phai xéc dinh cu tric phan ti. Khi dé thudng ding phuong phép sdc kf ling ghép khéi phé nhung phuong phap nay ddi héi trang thiét bi d&tPHUONG PHAP SAC KY BAN MONG 43 tién, chi phi cao.va tén théi gion nén khé thye hign trong cée phdng thi nghiém thong thudng. Vi vay phuong ph4p phé bién hon la dong ky thu@i ndi chudn (internal standard), nghia 1a Ichi chm méu thi chém chéng lén miu mét lugng chat chudn xde din, sau d6 cho khai trién sée ky. Két qua, ede dém chuén va mu phai tring nhau. Mét phuong phép khéc 1A phun lén ban méng da khai trién mt thude thit ¢6 kha nang lam they déi mau huynh quang ciia cfc dém. Vi dy, khi phun axit sulfuric 25% lam déi mau céc 4m aflatoxin tit xanh chuyén sang vang. Néu chat Ichdo sat khéng phai IA aflatoxin, két qua x4y ra la 4m tinh. ‘Vide khang dinh aflatoxin cdn duge thye hién bang cach tao cde din xudt co kha nang lam thay déi tinh cht phat huynh quang cia phan ti aflatoxin. Trong key thudt may, ed chét chudn vA mdu Ichdo sét déu cho phan ting vi chat tao din xudt, Vi du, ding TPA (¢rifluoroacetic axit) sé chuyén héa aflatoxin B1 va aflatoxin G1 thanh aflatoxin Bla vA Gla. Sy giéng hét nhau vé din suit duge tao thank gitta chudn va mau 1a khang dinh che chdn ching 1a cling loai dde 16. Chudn bi m&u phan tich TLC Pha losing Imi dich edn lai véi Iml nuée edit va thém vao 2m! dichlomethane. Day chat nip va lc manh trong 10 gify, sau 46 dé yén 6 phat, hén hgp sé tach thanh hai lép. Dang pipette Imi hit bd I6p nuée phia trén. Thém khodng 0,5g Na,SO, khan vao lo, Ide nhe dé logi nuée du khéi dung dich chita aflatoxin. Ding pipette hit dung dich nay sang mt lp chia mu khée va thém 2m dichlomethane rita lai NaySO,. Dich rita cing duge hut sang lo chéa miu, trén 4éu. Dudi dung méi trong lp bang khi nitrogen & 65°C. Hoa tan cin bing 0,2mi hin hop benzen- acetone (98:2), Day 18 mu ding dé phan tich sdc ky ban méng. Phan tich sdic kj ‘Trén mot ban nhdm méng e6 tréng silicagel 10 x 10cm dung but chi ké vach xuit phét céch mép duéi 1,5cm va dutng dich céch mép trén 2em. Trén dutng xudt phét, dénh ddu 7 digm céch nhau lem. Digm ngoai cing e4ch mép 2em. Ding micropipette lan lugt cham Ién ban méng Ui tréi sang phdi dung dich chudn va mau nhu sau; ~ Cham ba diém mau lén lugt 1a 5, 10pd va 20pd = Diém thi tu ch&m 10p! mdu sau dé cham tiép 10u! chudn - C&e diém cdn Igi chém chudn 1Opl, 6! va 4ul Dé kh va d&t bdn méng vao binh trién khai sAc Ixy co sin 100m! hé dung mdi ade ky. Pay ndp. Dung dich trién Ichai bét dau chay Jén theo lye mao dAn. Cho t6i hic dung méi chay 4@n vgeh dich thi 1y ban mong ra. Dé kho44 cHUONG 4 Dat ban méng duéi may soi ti ngoai, 365nm. Che may, bat dén va doc két qui. Mau ngi chudn 4, chudn va mau phai cing nhm trén mét vat. Cac vt mau va chuan khée phai 06 cing Ry. Céch do va tinh Ry nhu ede bai trén. Tv TiNH KET QUA voi Ham lugng aflatoxin duge tinh theo céng thie: XxCxv Mxm i: M -lugng mau chim e6 dé sing tuong duomg vai chun, pl € - néng d9 chat chudin, 0,5ygimi X -lugng chudn cham o6 do sang tuong duong véi mau, wl V - téng thé tich dung dich mau aflatoxin ding phan tich se ky, 200u m--Ich6i lung nguyén ligu tuong dang v6i thé tich dich chiét. ding phan tich she ky, 2. Atppm)Chuong 5 PHAN TICH THUG PHAM BANG PHUONG PHAP sAc kY LONG HIEU NANG CAO Phuong php sie ky léng cao Ap (high pressure liquid chromatography) hay sac ky ling higu nang cao (high performance liquid chromatography) - HPLC, thuje loai s&e kf edt c6 pha dOng 1A chat Iéng. Khae voi edc loai sde ky léng khde, higu qua phan t{ch cao ciia HPLC dat duge la do vat ligu nhéi oft 6 kich thude hat rit bé (5+ 10um) va dé ding nbét cao lam tang dang ké sé dia ly thuyét, vige st dung bom cao ép duy tri dp suit cao & ddu eft nhim diéu chinh va én dinh van tée dang va thng téc qué trinh phan tich. Ngoai ra hé théng phan tich siic ky long cao dp c6 thé ed dung nhiéu logi dau dd khée nhau lam tang kha nang phat hign, tang hiéu qua phan tich cing 1A m6t uu viét rét lon cia phuong phap nay. Cet HPLC cé thé 1a cot sde ky phan bé, sée ky h&p phy, trao déi ion hay loc gel. Sy phan tach ede cfu tit dat duge nhd cde qué trinh phan bé, hap phy hoac trao 46; ion, phy thugc vao ban chat héa hoc cia pha tinh sit dung. HPLC 16 ra uu vidt hon so véi sie ky khi khi phfin tich ede hgp chat hiu co. Cée hgp cht nay khi phan tich 98 duge hoa tan vao dung mdi thich hgp va phan lén ede phén tich thye hién 4 nhiét 46 phng. Cée thanh phén thye phém phan 16n 14 ee hgp chat khong bay hoi va khdng bén nhiét c6 thé tién hah phén tich sée ky ma khong cin phan hiy hodc chuyén thanh céc din xudt dé bay hoi. Phan lén'phén tfch mau thye phdm dya trén co 80 sdc ky phan bO va thyi gian hodn tét trong vong 30 phiit. . ‘Trong sic ky phan bé, thong thudng ngudi ta sit dung pha tinh phén eye hon so véi pha dgng va goi lA se ky pha thufn (normal phase). Nhung trong kj thuat HPLC, nguge lai ngudi ta thudng ding pha tinh kém phén eye hon va goi dé 18 se kj pha do (inversed phase) (RP-HPLC). Khi 46 cdc dung mdi sit dung la dung m6i phén eye, thuding it doe hai hon, ré hon va khong gay @ nhiém moi trudng (thuting ding niée ¢6 bé sung methanol, ethanol, hoac acetonitrile). Cang ging nhu sie ky khi, thoi gian rifa gidi mot chat phy thude vio hé sé hip din cia né va dign tfch bé mat eda pha tinh. Chiéu dai ¢9t 1 mot yeu t quan ‘trong xe dinh higu qua phan tich. Chi efe hop chat cb hé sé hip d&n khéc nhau mdi cé thé phan ly trong HPLC. ‘Tinh thing dung cia HPLC, ngoai ly do vé kha ndng phén t{ch cho nhém déi tugng r&t da dang thi higu qué téch va tinh chon lgc eda phucng phép cing r&t cao.48 HUONG 5 He thong thiét bi Mot he thong HPLC bao gém binh cha pha dong, bom ting dp dé édy pha dong qua cét lam tang va én dinh van téc dong pha dong, bo phan nhap mau injector dua miu vao edt, edt se ky, déu dd thu nhan tin hiéu eiia cde cfu tit qua ©8t, duge ndi véi mot may ghi hay may tiah dé xit ly két qué va bé phéin thu nhan eée edu ti da phn ly qua cbt. Cét ding cho HPLC ngan, éng hep, ngn ch&t ede phdn ti pha tink tao diéu kign cho ede hgp chat ¢6 thé trao ddi nhanh chéng gia pha dong va pha tinh, Dé thu va ghi nhgn tin higu ra Ichi dau dd (detector), cde ehuong trinh may tinh duge ding dé kim sodt vige khéi dng va van hanh sc ky, tit dong héa viée tin hanh phan tich. Ky thugt bign déi lién tye thanh phén dung méi trong qué trinh chay sc ky boi 1A phuong phép nia gidi gradien hay phyong phap chung trinh héa dung mdi. Nhiéu khi nguti ta phéi phén tach se ky nhomg hén hgp phite tap gém nhiéu thanh phan ¢6 h@ s5 dung trong rat khdc nhau. CAe detector nhay cdm v6i a thay d6i thanh phan pha déng nhu la detector khuc xa ké vi sai, s2 khé hon trong viée sit dung ky thugt rua gidi gradient. Bé vige phan tfeh dinh Iugng duge chink xéc, phdi sit dung Jogi dung moi e6 49 sach cao chuyén dung cho s&e ky léng cao ép (HPLC grude). Detector phai 6 Kthodng d9ng hoc tuyén tinh réng (broad linear dynamic range) va cée chat dem do phai doe lap kehdi bat eit mot eo chat giao thoa nao. Khoang déng hoc tuyén tinh ca mot chat la khodng do ma tin higu dap ting ciia detector ty 16 thuén vi ham lugng chét. Dé dat duge tinh link ding t6i da trong khi dinh lueng, khosng dong . hoe tuyén tinh phai ¢6 ba edip d9. Cie hé HPLC duge dinh chudn bling ofc dép ting peak trén dd thi, so sanh véi ede ndng’ do da bit cha mét chat chudn sii dung theo thi tue néi chudn ho&e ngoai chudn, Céc két qué dinh lugng ding trong pkuvng phap ngoai chudn dat duge n&u sit dung bé phan bom mau ty dng. Phuong ph4p ny cho phép d6i ching trye tiép cdc dap img peak trong phan tich sie ky riéng mdu thd va mu chudn. Néu.bom mau bang syringe, 6 4p sudt cao vige dinh lugng 8 khong chinh xée, Cée ket qué dink lugng t6t khi d6 dat due bing thi tye noi chun, mét lugng d& biét cda ch&t déi chimg khéng giao thoa goi 1a chat n@i chudn, duye bé sung vao mau thé va mdu déi chung, sau d6 so sénh ty 19 cdc dién tich pook (d4p tng peak) eda mau thit véi chit n6i chufn. , Vi nhing bign 4éi thong thuing ofa thidt bi, phy timg va ky thudt, yeu cdu hé thong phan tich phai sit dung duge lian tye. .PHAN TicH THUG PHAM BANG PHONG PHAP sdc KY LONG HiEU NANG CAC a Bai 8: PHAN TICH VITAMIN TAN TRONG NUGC BANG PHUONG PHAP SAC KY LONG HIEU NANG CAO Hé théng HPLC hién eé ctia phdng thi nghiém gém bom LO-10A8, dau do quang phé SPD-6AV va may ghi Chromatopac CR 6A. Sit dung oft ET 250/8/4 Nucleosil® 120-5 Cy, thude loai edt side ky ling pha éo cdn goila cot ODS hay RP-18 c6 nhém ankyl 18 Cis Haz 1a loai opt duge st dung rat phé bién do Gt c6 higu ning cao, thich hgp cho nhiéu adi tugng phan tich la cée chat tan trong dung mdi phan cyc va kém phan cc. Ct ¢6 49 chon loc cao v6i mBi hé dung mdi, cbt dat duge cén bing nhanh, ede peak can déi. Trén bé mat hat silicagel cdc nhém chite silenol (-SiOH) ¢6 hoat tinh cao duye cho tée dung véi cde nhém clo- hay alkoxysilkan tao thanh, Hat silicagel / 1 HnGre — GH,—SicH, 1 1 ‘CH,-Si- CH, 1 CHa 1- VAN HANH BOM J- Dit gid tri dp suét eve dai (P. MAX) Dat gid tr] dp sudt eye dai (P. MAX) nh&m bdo vé c@t va cdc hé thong khéc. ‘Theo khuyén cdo ciia cdc chuyén gia thiét bi HPLC, chi nén dgt P. MAX lén hon dp sudt oft 100kg//em?. Khi 4p suat vugt P. MAX bom sé ty dong t&t. "Tha tye dit P. MAX nhut sau: + Nh&n phim FUNC chuyén tin higu dén nhdy dén vi tri P. MAK ~ Nhép a6 ligu va nhén ENTER 2. Dat gidt han duit etka dp audtt (P. MIN) Mue dich 1 khOng cho khong kh{ vao hé théng khi 6 sy 06 hé trén be thong ya dé an toan khi do trong trutmg hop dung méi thodt ra ngoai, Tha tuc dat P.MIN tuong ty khi dat P.MAX. ‘Thong thutng dit P.MAX = 300-400; P.MIN = 2.48 CHUONG 5 Hinh 8.1 Cac phim diéu khién hoat dOng cia bom Bang 8.1 Gidi thich ede phim diéu khién hoat dong cia bom 86 Tén gol Chife nang 1 | Head hokier 86 gan du bum rda dudng dan, lam sach pitong 2_| Pump head Chuyén dng cia pitteng van chuy8n dung mot 3 | Drain pipe L6i ra cia dong ra pittorg 4 | Check vate IN| Van kid tra dudng alin vao 8. | Pump intet Au Ipc hat duge gin 8 du vao | Pump cutiet N6i ng dn vai injector 7_| tine fiser Gilt igi cin ban, ténh lam nget cot 8 | Drain vate Van giin vai cém ap, thay pha dong, loal kh 9 | Drain port Céng théo ndi vai ng d&n lu 10 | Check valve OUT | Van kidm tra dura a1 | Tray Ngan hifag dung méi n8ubj 0 a ti edu wa 3: Dat lan ligng pha déng (FLOW RATE) DE bio vé cot, khong nén dat lu veng wigt qué Imbiphit, trix tratmg hop can ria sach hé théng. ‘Thi tye dit: nhép sd ligu, nhén ENTER Khi cn thiét 06 thé dat chyong trinh Iu lugng. Vi dy: 06 thé lap mét chuong trinh luy lugng nhy sau:PHAN TicH THUG PHAM BANG PHUONG PHAP Shc KY LGne HEU NANG CaO 49 Luu fugng (muphuty (Tre gieriphut) = Ham Gia th 150 Flow 200 200 Flow 490. 300 stce Thal gian (phut) ‘Tha tuc tiga hanh nh sau: - han phim CE dé trd vé man hinh ban du (néu dang ¢ man hinh khéc) + Nha EDIT vi ENTER dé vao ché d6 soan théo chuung trinh - Nhap théi gian buée 1 (nh&n 1 6 va ENTER) khi dan chuyén sang FLOW thi nan ENTER rdi nhép gid trj luu lugng (nha 2 va ENTER) - Nhép thi gian buée 2 tuong ty + Nhap thoi gian bude 3 va nh&n FUNC chor. STOP réi nhgin ENTER - Nan CE dé tré v8 man hinh ban déu, Dé chay chwong trinh nh&n RUN. Chuong trinh s8 ty dong, dng’ thes che’ da dat, néu mudn dimg trong ki dang chay nha RUN, 8 On Dé x6a mot bude trong chugng trinh da soan, nhéx, BDIT wap shyang. Jam hign buée mun x6a bing eich. phn ENTER, v3 ghdn DEL 1 Thay Pn O08 ty Thay ahs déng.bdne.matnkrndon os ar Shysn. bi Ore pha doug ang in oe Mdywan wolauay~nguge chidu kim, ding. pd, 18 ids Ang sictropebugne IM i300 gecud! is AVRO oun 5 Goths ynsudy ob cat Nhdm PMBGE: bay, PUMR,. dé: nging: bom, dine vom my Ahh ion, st ‘Unliphiit dn 1,5mi/phut (OFS 350 you tie gadis wes egal sion NbSRJREIMP ho hom. chay.lé thes. .ahe, done; trope, e4c.nhAp. cba dai eo pha fy 16 BOAR I QUAY cic dow denis sin 26 at % anh molt seta Sageratig sid the degel ah!aage waBtea Paw aou eel Sa GMa ‘et sida Gnist BM ai OOS sate anita sidanwpolod) idy yi reliefs 5 id aid HidB al HOSS tHiny als AB bier He ed Sr dada owuge youn wil ena we gait, ease nah ee axe ae Gilqy yeuswp TAC gents b Av wim ofd isda 30 909 0 etn oer Lt yg ae wainie inal use od Bdyinel Bo ody ax ob ouUb Thay pha ding la dung diclydéms is by 784 adil gabds gouyb 19m Aerie grid 95D0ag.200mLnate eile, sach dung dich, Adm, trong he théngidQng.chdy va ashe déharnugebing whasingaeRdivsid aid dcddt mdyucs idx SE Ge 1G cosilie50 * cHUONG 5 SA, o6eara0 Hinh 8.2 Kiém tra hogt dong cla bom II- DETECTOR SPD-6AV Detector SPD-6AV cia hing Shimadzu Nhat Ban, thudc loai detector hap thu ti ngogi va kha kién (UV-VIS), do duge phd hp thu tir 195nm d&n 700nm do sit dung hai den gém dén Deutri (D) cho ving ti ngogi (UV) va dén Xenon tungsteng (W) cho vimg kha kién (VIS). Cé ba mite dép dmg nhanh (fast), trung binh (standard), chém (slow). ‘Tinh nang cia detector UV: DO dai s6ng 195 dén 350nm. Buée chuyén +2nm, sai 96 20,5nm. Bé rong phé 8nm. Ngudn sdng 1a den D2. Thé tich té bao Bu (Imm dutng kinh x 10mm chiéu dai khe séng), 4p suft 20kgflem?. Vat ligu tigp xae véi chat long IA SUS 316, thgeh anh, teflon. Nhiét d@ phan tich t6i uu trong khoang 10 dén 40°C, Thong thuding, ngudi ta sit dung mic dép ing (RESPOND) chudn (STD) c6 hing s6 thi gian cia dap (ing la 0,5 gidy. Tin higu ra & ngd AUX 8 mAt sai méy thuting ding & mic OAV/AU, néu thudng xuyén phan tich 6 d9 nhay cao thi sit dung mde 0,8V/AU. M@t chite ning khéc rét quan trong cia detector lA chinh dé hap thu va mite khong, sit dung nit ZERO. Hinh 8.3 trinh bay hé théng quang hoe cla thiét bj. Anh sdng bite xa (radiat héa) ti nguén sdng (den D2) di qua mdt cla thach anh, phdn xa qua guong M1 va ‘M2 va hoi ty vao khe adng, Qua che sdng, dnb sdng lai duye phan chiéu bai guong ‘MB va hi ty, sau d6 duge phan chia thanh cfc thinh phan quang phé béi mot tém chAn ty ghi (holographic grating) chita 1200 tia/mm. Anh séng bj phan chia béi tam ch&n, quay nguye thanh m6t chim tia séng chdi di rong dé chiéu lén cd hai té bao mAu va déi ching. Dai quang phé dnb sdng 6 chiéu dai bude séng tuong ty duge bic xp vao cd hai té bao sau khi nhiéu xa gay ra mot hudng 6 géc bén phai thanh mt dudng thang lién két véi cd hai té bao. - Sau khi di xuyén qua cde t& bao, anh sing chi€u vao che photodiode bing silicon D1 va D2 réi chuyén thanh tin higu ‘tuong ximg-véi cug d dnh sing.PHAN Tich THUG PHAM BANG PHUONG PHAP SAc KY LONG HIEU NANG GAO st Chidu dai buée séng cia 4nh séng bic xq vio 4 hai té bao (chiéu dai bude sing trong suét qué trinh phan tich) 1a do su chon géc cia tdm chén. Home position sensing disic Gung M, Té b4o quang h9e Stepping motor svang het DB, By (Cc photodiod bang sition Hinh 8.3 He théng quang hge cla detector UV TMJ CHROMATOPAC CR 6A May ghi chromatopac CR 6A gém céc bé phéin: ban phim dé cai dat chuong trinh vi cde phim didu khién, bg phan n6i véi detector thu nh§n tin higu, bo phan n6i cdm tng dé khdi dong déng thoi hé théng chay miu va hé th6ng ghi, b phan in c&m ing, xit ly di li@u theo c&c chuong trinh cai dat sn va in thanh she ky dé. Pint | S10P | sun]. 9 SoS 718 | 9 | oe aaave | int | S102 | SEM [ready TPR Close Power alals|o ee Sten Ren Sanat pase Request ae be tee Aral uj vi wl x ee eo Save ytztcho Lao ° ENTER wiatn | spe | driot [ainatea] TB. [stort] Aten | Speed mtn [Fowas| sew [ tami [window] 10 [Timor] eat Hinh 8.4 Ban phim cita chromatopac CR 6A82 cHUONG 5 I- Céch st dung ede phim Hau hét cdc phim déu cé hai hay ba chic nang. Chie nang uu tién duge ghi 4 phan trén eda phim, Muén sit dung chide nang ghi & phan dudi cia phim, nhan phim SHIFT DOWN trude va dé sit dung chife ‘nang thit ba (duige ghi bén canh phim) nhdn va gitt phim CTROL sau 46 nhén phim chite ning c&n. 2. Céch dat eae théng Nhén phim théng s@, gid tri muda dat va phim ENTER. Tat cd cdc théng sé déu e6 thé dat tai thai didm bat kp trong khi phén tich. ATTEN: Théng sé nay ¢6 11 gid tri ty 0 dén 10, gié trj cang nhé khoang biéu dign tron sie ky dé cing nbd. Tang gid tr} ATTEN len m@t don vi thi khong bidu dién trén sde ky dé tang 1én hai lan. ‘Thyc té phan tich cho thay véi ATTEN 5 thi c6 thé biéu din duye peak 6 ning 4 toi da 15ppm va nbu vay dit ATTEN 1 thi biéu diin duce peak 06 néng 43 0,94ppm. Néu néng 46 qué lén khi dat atten nhé, peak chi duve biéu dién mot phén nhung diéu nay khong anh hudng dén viée tinh toan dign tich hay chiéu cao ca peak. METHOD (phuong phép): Thong thutng ngudi ta st dung phuong phép 61. Trong phuong phép nay mdy ghi a8 cho két qué dign tich cde peak dd duge, ding théi dénh dau vi tri peak. Néu khéng mudn ddnh déu cdc peak thi sit dung method 41, khéng mu6n in dién tich peak thi dit phuong phdp 40 hay 60. SLOPE (d¢ nhay): Thong sé nay thé hign 49 nhay phat hién peak, SLOPE cang nhé d§ nhay cang cao (nghia IA phdt hién va ghi nhan duge cde peak nhé, néng dd nhé). Théng thuding slope nhé hon 200 thi tét. Cé thé dat slope hofe cho éat ty dong bing Iénh: SHIFT DOWN S.TEST ENTER Nhung chi dat ty dgng khi dutng nén aa én dinh. MIN.AREA (dién tich peak t0i thiéu): Chi nhiing peak e¢ dign tich 1én hon ‘hoge bang gid tri MIN.AREA méi duge ghi dién tich. Gia tri nay dat tay theo cdc peak quan tam, STOP.TIME (thoi gian nging ghi sc ky dé): Muén ty dong ngimg ghi ske ky 46 sau thai gian nao dé, dat gid tri thdi gian d6 cho thing s6 STOP.TM. Néu Khéng dat thdi gian ngiing ghi sdc ky dd, nh&n phim STOP dé nging. SPEED (téc 46 gidy): Toe a6 gidy (mm/phuit) thudng dimg 1A 5. Tuy nhién, imudin ti&t kigm gidy hohe dé peak cé dang sie nhon thi cé thé gidm SPEED. i3- Chuong trinh thet gian Chuong trinh nay cho phép thay d6i d$ nhay cia may ghi cding nhu ede thong {s6 khéc. Thi tue l4p chuong trinh théi gian nhu sau: (1) Nhén TIME.PRGENTER vao ché dQ soan thdo chuong trinh (2) Nhén SHIFT DOWN Y ENTER (3) Nh@p ede dong Ignh theo thif ty: thai gian bilt d4u d&t gid tri, tan thong 86 va gid trj mudn dat.PHAN Tich THUG PHAna BANG PHUONG PHAP sAc KY LONG HiEU WANG CAO 53 Vida: - Dat SLOPE = 100 ti hie 10.00 phat: 1 © ENTER SLOPE 10 0 ENTER - Dat ATTEN = 10 tir thoi didm 8,40 phut: 8.4 ENTER ATTEN 1 0 ENTER Néu dat nhidu thong $6 cing Ide, ngin cdch ode théng 5d bang dau “” Vi du: Dat SLOPE = 200 va ATTEN = 5 ké tir thai diém 5,00 phit: 5 ENTER SLOPE 2 00: ATTEN 5 ENTER (4) Két thie vige tao chuong trinh ding l@nh: END ENTER. (6) Kiém tra lai chuong trinh ding Ignh: LIST TIME.PRG Sau khi tao chudng trinh thdi gian, mi khi phan tich may ghi sé ty dong thay déi cée théng sé theo chung trinh da dat. Céch x6a mot bute trong chuong trinh da dat thye hign nhu sau: = Vao chuong trinh nh&n TIME.PRG ENTER -Nhén SHIFTDOWN N= ENTER - X6a mt bude nhéin SHIFT DOWN D, thei gian cia bude mun x6a va ENTER Vi du: X6a buée 6 thdi gian 10,00 phit nhén SHIFT DOWN D10 ENTER Néu x6a t&t cd cdc bude trong chuong trinh thdi gian thi 6 nghfa 1A x6a todn bo chuong trinh da dat. -Nhép lénh END ENTER May ghi c6 bd phn in cdm ting duge cai djt mot sé chung trinh xit ly dir ligu nbn tir detector. Ngoai ra cdn cb ede bo phan chuyén biét ndi véi cassette tape recorder digital 1/0 port and interface, b§ phfin ndi véi clin gat ella injector dm bao may ghi va hé théng hoat dong dang thai. IV- TIEN HANH PHAN TICH I- Chudin bi may Nude eft hay pha dong true khi bom vao ot phai dugc loi Ishi. Thudng logi Ihf bang siéu Am. . Nhung du loc vao binh chita pha dgng sach da loai Ichi. bé binh hing nude thai vac dubng dng théo. ‘Mé én 4p, nhén cong tac POWER cia bom, dé dign én dinh trong 15 phut. Kiém tra 4p suat cue dai va cue tigu d& twong ting chun? (4p sudl nim trong khong min 0, max 300kaflem"). Chir 4p suit én dinh. Xoay van théo 6 vi tri mé (DRAIN 180°), nhdn ndt ria “PURGE”, khi 46 bom sé vin hanh véi van tée khéang 9,9ml/phut.54 CHUONG 5 Quan sat nude ti Sng thao cho tdi khi cdc gigt chdy ra déu va khdng cd bot Khi (néu dung moi khong chay phai dang syring khdi dong pha dong nhu hinh 8.4). Hinh 8.4 Khéi déng pha d6ng Nhan “PURGE” hoac “PUMP” dé ding bom. at van tie dong. Xoay van théo 180° dé khéa chat. Nh&in PUMP 4é vn hanh bom. Kiém tra 4p sudt da én inh chia? Khi 4p suét én dinh thi mé hai module detector va may ghi. Pgi may ghi kiém tra xong, ligt ké ede thong s6 tron my ghi bing lénh: SHIFT DOWN LIST WIDTH ENTER va thay déi cdc thong sf néu edn. Dé hé théng hoat dong 15 phit rdi chinh d6 hép thu vé khOng bang céch nhan nat ZERO trén detector. Kiém tra dutng nén bing lénh: SHIFT DOWN PLOT ENTER ‘Trong qué trinh kiém tra néu duting nén bj léch thi vai phiit lai nhén ZERO trén detector mét lan. Dgi dugng nén thang lap lai lénh SHIFT DOWN PLOT ENTER dé chém dit kiém tra. Dén day hé théng da sin sang cho phan tich. Chit - Néu van th4o 6 vj tri md pha dong sé chay ra tif céng thdo. Gitt van théo déng suét thai gian tién hanh, ngoai trix lic van hanh van thdo. - Khi tang lu lugng, chi nén t&ng mdi lan ti 0,2 = 0,3mdiphiit va dgi eho 4p sudt 6n dinh mdi tang tiép va ctf tang dan nhu vay dén gid tri cai dat.PHAN TiGH THUC PHAM BANG PHUONG PHAP SAC KY LONG WiEU NANG CAO 55 - Néu pha dong cé chia mudi hay 1A dung dich dm thi phai rita pittong (plunge) dinh ky mdi 5 hay 10 phat trong khi bom dang hoat djng dé trénh su két tinh eda mudi lam hu dam, tét nhét dat déu Sng rita vao nude cdlt va cho rita lién tue. « Sau khi phan tich v6i pha dong chia mudi, phai ria ky hé théng bing nue eft. Truée khi phan tich mét mau mdi, can tin hanh rita he théng mét lan bang nuée cat vA mot ln bing pha déng. Dung pha dong chay khong tai qua oft dé rita sach edt, kiém tra dQ sach cia cpt cho tdi Ide nén 1A mOt dutng thdng. ‘Truge khi nap mu phai bom nuée cit rite injector, logi hét bot Iehi cd hai vi tri nap mau va tiém miu. 2. Chudin bj mau @ Chudin bj chét chudn: Ding axit ascorbic tinh khiét 1am chat chuéin Ikhi phan tich vitamin C ho%e ding cé¢ vitamin tinh khiét khéc lam chat chudn khi phan tich déng thi cdc vitamin. q Lugng mdu phan tich cho Ién eft khodng vai ppm. Pha loding chét chuén theo kiéu bée thang: can x mg chat chudn, dinh mic lén 100ml, ldc déu, dy 10mi pha loding tigp lén 100m!. Cit pha loang nbu vay cho tdi ndng 46 thich hop. b- Chudin bj mau thit: Nguyen ligu thye phdm cé chia cée vitamin edn phan tich. N6i chung, vige chudn bj cde mu thi déu phdi thyc hign cde céng dogn sau: nghién miu, trich ly bing dung mdi thich hgp cho ting déi tugng phan tich. Logi tap ch&t bing eée phuong phép két tia, thim tich, sde ky edt hay sée ky bin méng. Mau thif cang 06 ft tap chat thi két qué phan tich cang chinh xAc. Loc sach. Binh mie. Lay mét thé tich nhét dinh pha lodng tdi ndng dé thich hgp (vai ppm). Khir bot hf bing siéu am. Loai tap chat qua mang siéu loc 0,2 + 0,45yum true khi bom vao cot. Chudn bi mdu phan tich vitamin C (Can Sg nguyén ligu thye phém, nghién trong dung dich 4¢m pH 2,8, trich ly vitamin C, dinh muc bing nute c&t ti 100ml. Néu mau thye phém c6 nhiéu protein va cde tap chat keo thi bé sung tac nhan két tia trude khi dinh mic. Loc qua gidy loe thu dung dich mau M, chéa vitamin C. Lay chinh xe Imi dung dich M, dinh mie bAng nude off thinh 100ml, Dem loc dung dich miu qua mang siéu Toe 0,45 um 06 duige dung dich Ma, Mau cd nhiéu protein: Can 5g mau da nghién-nhé déng nhat. Thém 10m! TCA 10% va ti8p tuc nghién mau dé két tia protein va git méi tru’ng axit. Chuyén vao binh dink mic 100ml, ding nuée eft 4é trich ly vitamin C, thém nuée cat t6i vach. Lc déu. Dé ling va loc qua hai lan giy lee. ‘Trudng hop mdu 1A nude ép trai cAy, nude gidi khét c6 nbiéu axit thi chi edn My bmi, dinh mec bling nuidc oft t6i 100m! va loc bé tap chat.56 CHYONG 5 Dung dich mu phan tich HPLC cfn duge tigp tue pha loing t6i ning do vitamin C trong khoang 9 + Sppm va loe qua ming lige milipore 0,45pn. Trich ly déng thai cde vitamin tan trong nude Can 5 + 6g méu da nghi&n nhé vao binh tam gidc dung tich 250ml. Thém vao @5ml dung dich HCl 0,1M va dun céch thdy ¢ 100°C trong 30 phiit dé trich ly cde vitamin tan trong naée. Lam ngudi va chinh pH téi 4,5 bling dung dich axetat natri 2,5M, sau d6 thém 50mg amilase vi 500mg katadiastase vi mdt it nude va khudly. Dat ¢ 37°C dé phan tng thiy phan xAy ra qua dém. Chuyf2 toan bé khéi luong sang binh dinh mde 100ml va thém nue cat tdi vach, loc. Litu §: Nuée efit ding pha ché cic dung dich wrong thi nghi¢m va tat ed cde dung djch true khi bom vao cbt déu phai loc qua mang siéu loc 0,45 va pha bot khi bang sidu 4m trong vong 10+ 20 phit. ‘Dung dich miu edn sigu Am loai Ichéng khi truéc khi duge bom vao ot, trong qué trinh xif ly phi thao tée trong diéu kién tranh anh s4ng. . 3- Ché d6 van hanh 1. Dé phan tich vitamin C hode tach hin hgp thiamin va axit ascorbic dang pha dng 1A dung djch KH»PO, 0,06M, chinh pH 16i 2,8 bing HjPO. 85%. Cot ODS Crs. Bude séng 245nm. Lum lugng Iml/phut. Bé tang higu qué tach c6 thé ting hay gidm luu lugng. _ _ Cling 06 thé ding pha déng la hé Dém: methanol = 90:10; dénh siéu am trong § phit dé duéi khi trong pha dong. ih CAch pha dung djch dgm: hda tan 6,5¢ KH,PO, trong 1 lit nude, chinh pH dé 2,8 bing HsPO,. Loc qua mang Ipc 0,45pm. 2- Dé téch hdn hgp 4 vitamin thiamin, riboflavin, nicolinamide va anit ascorbic sit dung pha déng sau: “4°! Natriheptansulfonate: 1,08¢ usidlir a Kali chloride: 2,08 thaib Acetonitrile: Om! hie Methanol: 100mt Axit acetic: 10ml hunt Bhan nude. cdt t6i 1000ml. 11" C48 vitamin chudin duge pha trong dung dich axit acetic 1%. <9" $8 dung edt RP- Cys. Bude séng 260nm. ‘Lidl Lugne Iml/phut. Dé tang higu qua téch o6 thé t&ng hay gidm luu lugng. METHOD 41, ATTEN-5, MIN.AREA=2000, SPEED=3, STOP, TM=5pHAw TicH THUC PHAM BANG PHUONG PHAP SAC KY LON HEU WANG CAO a7 4 Cach lam Bom mau véo injector Nang edn injector ~/.0 vi tri “Inject”. Ding syringe chuyén dung 06 kim tig dau bing (khéng duge ding kim dau nhon) dé tiém mau vao injector cho téi khi du du, traoh dua vio bot khi. Trude khi bét déu phan tich thi nh&n ZERO trén TJotector va ding Iénh ZERO ENTER tréa may ghi dé dat g6e bat ahi Nhan START trén may ghi déng thdi gat dn injector xudng vi tri vao cot “Load”. Trung hap may ghi dupe n6i mbt 66 phan em dng voi injector the khi got cdn injector cho ru udo c6é, may ghi ciing ty kai dong ntn khéng edn nhdn START. May ghi bét dau lam vige. Cée peak duge ghi nhan cing thai gian hu Néw néng d6 qué cao, peals bi ti, phai pha loang hon. May ghi ty déng ding sau thoi gian da cai dat 6 STOP.TM. Khi cin ngtmg ghi cé thé nhan STOP trén may ghi. Ngiing ghi khi ki:sng can naak va dong nén 1a mot dutag thang. Két qua t6t Khi cd chun va miu déu cho ee peak e6 cling thdi gian lu, dién tich peak khOng qué nhé va khéng qué 1én Dé xde dinh ham lugng mot thanh phdn trong mau edn thue hign ede bude phan tfch sau: Bude 1: Tiem chun vio e6t xdc djnh peak oda chit chudn. Chay 6 lan dung dich chudn cing néng 46 dé kiém tra d9 lép Iai ofa két qua phan tich, thé hién bai dign tich peak. Xéc dinh a lech chudn tong 6i (relative standard deviation-RSD). vei: A, - dign tich peak mbi lén do A - gid tr} trang binh; x - sé ldn do. Buéc 2: Xay dung duting chun va chon khodng tuyén tinh dng 6, C.(opm) | 2 4 é 8 ign tich poak, A: awa Phuong trinh duting héi quy G@ = fA) cda doan tuyén tinh o} dang C=kA baa & La Voi:58 CHUONG 5 Xée dinh h@ 86 tuong quan binh phuong: See, -GF RP =1-£1____ zx cy vi © 1a gid trj néng dé trung binh. Buée 3: Phan tich méu Bom mau vao eOt va c6 duge dién tich peak la Ay. Dya vao dutng chudn dé Yinh néng 49 vitamin C c6 trong dung dich bom Cy. Néu Ay nim ngoai khodng tuyén tinh eda dudng chudn thi efin phai pha lodng lai. ‘Tinh ham lugng vitamin C trong nguyén ligu theo céng thitc. Ta cing c6 thé so sénh két qué dinh lugng véi phuong phap thém chuén vao méu nhuf sau: Lan bom mdu thi hai, ly mét thé tich dung dich chugn va mot thé tich dung dich mAu tron lin theo tj 18 1:1. Phan tich hin hop mau thém chudn nay, ta c6 dign tich 8, tuong ting véi ndng d9 chun gidm mt nia va néng d) mdu cding gid mét nita. c=2 Coren Ti cde gid tri S va Co ta tinh duge Ci, 1A ndng 49 vi tamin C dia mu pha loang, Tinh ham lugng vitamin C trong mau bing céch nhén véi hé s0 pha loaing: Tinh néng 46 vitamin C trong dung dich m&u pha loaing: Cy opm) = Sasole 6, 8 Ham lugng vitamin C trong mau phan tich duge tinh bing céng thic sau: C wpm) = Cy Yi¥e, ™ trong dé: Cy - néng 46 (ppm) vitamin trong dung dich m&u phan tich C ~ ham lugng (mg %) vitamin C trong nguyén ligu phan tich Smo - dign tich pealk thu duge khi chay mdu thém chudn Sc - dign tich peak thu duge khi chay chudn Ce - néng 48 (ppm) dung dich vitamin chudn V) - thé tich dinh mie dung dich mau thu duge sau khi trich ly (100md) Vi - thé tich dung dich thu duge sau khi pha long ti néng 4 bom lén opt m - kh6i lugng (g) mu thye phim dinh lvgng vitamin C (5g),PHAN Tic THUG PHAN BANG PHUONG PHAP SAC KY LONG HIEU NANG CAO 39 V- CAC BIEU HIEN XAU VA CACH KHAc PHUC Khi chay sde ky thutng gap cde trubng hop sau: - Dinh peak bi ti, khong nhon: lugng mau vao cbt qué nhiéu, cn giém néng do bing céch pha loang. - Dinh peak bj ché: o6 nhiéu tap chat. Tap chat co thé do bt bj ban, phi rita Iai oft bhng ede hé dung méi ob 49 phan cye ting dan. Nhung kkhi phan tich cic mau ty nhién thi thu’ng gap trang hap tap chét ob trong mBu phan tich, Khi d6 phai loc lai hay xii ly Igi mdu dé Jogi tap. «Ap sudt ting khi khong ting tée 6 dong: e0t tach, dung An va cfc bo phn loc bj ban, cfn rifa loai tap chat, lam sach he thong. Dé nia nhanh tap chat khdi cbt, 06 thé quay nguge Adu odt khi bom dung méi. Cae dau loc c6 thé théo rdi thi ding dung méi ngém rita va diing siéu Am loni bot khi. - Duting nén khing én dinh: c6 khi trong dung méi ding si¢u 4m Joai khi. - Thoi gian Iwu thay déi: do thanh phan dung méi thay déi, hay nude ngumg 6 bd phan Joe, edn kiém tra thay rifa bang dung moi mdi. VI- BAO DUONG COT Trong hé théng HPLC, b6 phan gay sv 6 thutng xuyén nhat 1a cdt she ky, do cot 1a bd phan trvc tip chiu anh hudng cia mol tée dong: Ap sudt, h6a hoe, ln Jugng... Vige éuy tri bdo dung va hotn nguyén o6 tée dung loai tap chat, tang higu qua phan tich cia cot va kéo dai tudi tho cia edt. Quy trinh rita va hoan nguyén et pha ddo theo Phan vi¢n Kiém nghiém duge ligu TP Hé Chi Minh. Quy trinh dui diy 4p dung cho tt ed cfc Logi oot pha dao va oOt cyanva. Thé tich dung moi 16i thiéu dé rita opt 250/8/4 1a 6Orel, ebt 200/8/4 18 50m. ‘Luu lugng rita: Lmliphiit ‘Thai gian téi thiéu dé ria ebt: 60 +60 phiit. Quy trink rita cot ‘Negay sau kchi phin tich cdn ré e6t nhu sau: Bube 1: Rita bang nude eat qua loc, t6i thiéu 30mL Bute 2: Raa bing hé dang méi acetonitrile(nube (20:80 theo thé tich), ti thiéu 30mz Bube 3: Bao quan trong acetonitrile. Quy trinh hoan nguyen cot ‘Yeu cdu hodn nguyén edt tdi thiéu mot Jan mot tudn. Khi hodn nguyen ft thi rd nauge du c6t. Loai bom chuyén ding dé hodn nguyén bt 1A bom Hitachi 1.6000. Bude 1: Rita bing nue edt qua loc trong 30 phut, tuong duong 30m. Bute 2: Rita bang dung dich axcide acetic 1% qua. loc, 60m.60 CHUONG 5 Bude 3: Rta bing nude cat qua loc trong 60 phut, tuong duong 60m, Buéo 4: Rita bing hé dung méi methanol/nude (60:50), 60m. Bude 5: Rita bing methanol tinh khiét, 60ml, Buse 6. Rita bhng hé dung méi diclomethan/methanal (50:50), 60ml, Buée 7: Rita bing methanol tinh khiét, 6Omnl. Bute &: Néu tiép tue phan Uich bing hg pha dong kém phan eve hay khéng phn cye thi gift nguyén methanol trong cét. Néu tigp tye phan tich bang hé pha dong phan eye thi nap vao ebt hin hep acetonitrile/nuée (20:80). Néu cde bao quan C6t thi nap vao eét acetonitrile tinh khiét. Neoai ra, néu qué teinh phén tich kéo dai do tign hanh tren nhigu méu ma pha dong khéng chia muéi thi o6 thé bao quin e6t trong pha dong, cing 06 thé cho dung méi chay hoan Iuu qua dem 6 lay ligng nhd. BAI 9: PHAN TICH AXIT AMIN BANG PHUONG PHAP HPLC Thanh phan axit amin la m@t ehi tiéu quan trong eda thye phim. Trong phén lich thye phim, vige xée @inh chinh xde thanh phén axit amin cho phép dinh gid sid ti dink duong cia m9t nguén protein. Gid tri din duong cia protein va cde hve phaim giiu protein phu thude vao edn bing ede axit amin Khong thay thé (thiét yéu). Cae axit amin thiét yeu bao gbm methionine, lysine, threonine, valine, isoleucine, leucine, phenylalanine, tryptophan, arginine va. ii idine. Chat Iugng cia mot nguén protein duge so sénh tuong déi voi thanh phan, t¥ 1é cda cdc axit amin thiét yéu trong mét protein ly tuéng ¢6 gig tri sinh hoe cao, Hign nay trong sin xuit thye phém, phvong php lam gidu dinh dueng bing cfich bé sung axit amin khiém Khuyét, nhim dap ting can bing dinh duéng protein 1a mot phuong ph4p cdng nghé phé bién d nhiéu nude, Trong cong nghigp thy phdm thdi han tén trit clia mgt sé ching loai sin phdm cing phu thude vao thanh phan axit amin. Vi dy, két qua phan tich thanh phan axit amin ty do trong rugu vang va nude qué 1a mt thing s6 quan trong trong viée xde dinh tudi tho cia sain phdm. Viée dinh lugng lysine trong thye phém bing Phuong phap HPLC cho phép danh gig ham luong lysine sir dung duge, c6 trong thye Pham. Dé 1A do trong qué tinh ché bign va bdo quan sn phém, lysine 44 o6 thé bién 46i tao thnh ede din xudt Ikhdng hap thu duge. Phuong phép HPLC 06 un diém 1a e6 a) nhay phét hién cao, ¢6 thé dinh lugng chinh xe va nhanh chéng, e6 thé phan bigt mot cach rét higu qué e&e chat 6 célu tao héa hoc gan giéng nhau. Vi vay, n6 Ja Phuong phép thich hyp 6 x4c dinh dang lysine hap thu phan biét véi lysine khong hép thu va duge ding thay thé phuong phép phan tich bang quang phé true day.PHAN TicH THUC PHAN BANG PHUONG PHAP SAC KY LONG HiEU NANG CAO 61 Phuong phap HPLC cing cho phép xéc dinh cde axit amin dee tinh hay cic peptide sinh ra trong qué trinh ché bién thye phdm, chéng hen nhv lysinoalanine (LAL). Ngoai LAL, cde sin phdm phi dinh dv@ng khdc ¢6 thé hinh thanh trong qué trinh xit ly kiém déi véi protein 1a ornithinoalanine, lanthionine va B-amincalanine. Mét ting dung khde cia vige phan tfch axit amin bing HPLC la kiém nghigm (ham lugng) protein ciia thit dua vao chi sO 3-methylhistidine, vi day 1A m@t thanh phan tiéu biéu dic trung cho protein ea thit. Hay kiém nghiém histamin/histidine dé xdc di 1 d@ tuoi cda cA. ‘Trong nhtng nghién civ vé sy racemic héa axit amin, xdy ra trong khi protein bi xit ly kiém két hgp gia nbiét, thi HPLC eding la phvong phép phan tich hitu higu Ngoai ra, phén tich axit amin con nbim phat hién sy c6 mat ciia mot sO axit amin nhét dinh trong thye phéim nhu xde dinh ham Ivgng glutamic trong cae thye phdm c6 bé sung bot nggt monosodium glutamate; x4c dinh gelatine hay collagen trong the phém dya vao viée dinh lugng axit amin béc 2 1 hydroxyproline; xée dinh ham hyong taurine trong cdc sin phém sia danh cho tré em. Vé ky thuat phan tich axit amin thi phé bién nhat la ding cot sdc ky tra déi cation, vA phn lam hai phuong phap la phuong phap Li va phuong phap Na, dac bigt la phan tich thanh phdn axit amin ty do. Kidu Li thudng ding phan tich ede axit amin sinh hoc hay dé téch glutamin va axit glutamic. Ca ché gidi hip dao pha c6 thé tach phén Ién cfc loi axit amin nhung cing Ighéng thé tach duge tét cd cdc axit amin ty do. Do mot 88 axit amin ty do kém bén nén dé ting d6 nhay phét hién va kha ning tach ede axit amin thi hiéu qua hat 1a sit dyng phuong phép vi phn tién e6t co nghia 1A dung dich mau duye tao din mudt truGe khi dua vao edt phan tich. Sy két hgp gitia phuong phap tao din xudt vi phan tidn e6t va kiéu gidi hép déo pha cho phép phan tich nhanh chéng thanh phan axit amin 6 trong dich thay phan protein. Cac héa chat aé tao din xudt tién c6t nha OPA (ortho-phthelaldehyt), hay PITC (phenylisothiocyanate) duye ding phé bién. Ngoai ra, cdn sir dung phudng phép tao dan xudt vi phan tich hau c6t. Trong phuong phap nay, ede héa cht 48 tgo din xuft vi phan tich duge bé sung lién tye yao éung dich sau gidi hap. Phuong phap nay c6 wu diém néi bét 1a toan bo qué trinh vn hanh cé thé ty dong héa. Sy két hgp gita phvong phép vi phan hfju edt va phuong phap trao déi cetion cho phép phan tich mt gidi bién thién rong gém cfc axit amin ty do va san phém thiy phan ti protein. Cac thude thir phé bién trong phuong phép tao din xudt vi phan hau cot la OPA (48 phat hujnh quang) va ninhydrin, 2 oes -O pot ub td cal dais yaM 762 CHUONG 5 T- CHON CAC DIEU KIEN VAN HANH Mau phan tien Hirh the each Cot tity biéu Phat hien Acid amin tu do = Trao d6i cation ‘Shimpack AminoLi Shimpack AminoNa ‘Shimpack HRC-OOS CAPCELL PAK C005, Vipnan hau cot va phat huynh quarg vei OPA Trao a6 cation ‘Acid amie ir protein thoy phan Viphar én ot vva phat huyan quang v6i OPA, Khi phén tich thanh phdn axit amin trong céc thyc phd giau protein thi st dung ot sic ky pha ddo cing 1A mot phuong phép phan tfch rét phd bién. Hi¢n nay, dé tang higu qua vA gidm thdi gian phan tich thi ky that tao din xudt tién c§t (precolumn derivatization) dA duge ting dung. Khi 46, cdc hat silica lién két alkyl, kich thude 9 + Bum c6 kha ning phan tach mot hén hop din xuélt axit amin phic tap chi trong vong 10 phit. Két hop véi d} nhay eda detector (ddu a2) thi ky shugt nay cho phép phét hign axit amin 4 néng dO picomol va gi6i han phét hign cdn 6 thé thap hon na. Ske ky pha dio sit dung logi et hp phy ky nue octadecyleilane (Cys) duye ding thong dung nhét, Loi cot Cs cting duge ding nhung ft théng dyng hon V6 vige chon pha ding thi thye t& cho thay e6 thd sit dung nhigu hé dung mOi khde nhau, phn én ede hé dung moi nay gém e6: methanol, acetonitril va hiém hon IA ding tetrahydrofuran nh mgt thank phan hitu eg eda pha dong. Viée bé sung thanh phd hau co nh&m tang kha nang téch gt 86 cap axit amin, tuy nhién 6 lai c6 nguy co lam tAng dp sudt edt. Yeu cdu mgt hé thdng thiét bi HPLC 4é phan tich axit amin gém e6 cfc module sau: 1- Mot bom HPLC hay hai bom (néu chay gradient) 2. Mét bd kiém sodt gradient ty dong (automated. §radient controler) 3- M6t autosampler dé bom mau ty dong 4- Mdt edt RP duge gia nhiet va én nhigt trong bo phan én nhiét 5- Dau dé (visible, UV hode fluorescenee) 6- May in slic ky dé va két qua 7. May tinh lun tra di lidu.PHAN Tich THUC PHAM GANG PHONG PHAP Shc KY LONG HEU WANG CAO 63 TI- PHAN TICH THANH PHAN AXIT AMIN BANG KY THUAT TAO DAN XUAT PITC I. Nguyén tde - Ding HCl age thiy phan cdc lién ket peptide gidi phéng axit amin ty do - 'Thdy phan hoan toan protein va peptide thanh axit amin - Tao din xuat tién oOt cla axit amin vi PITC - Phan tich thanh ph4n axit amin trén ot C18 sc ky pha déo. 2 Dung cy héa chét a- Héa chat Dung dich HCI 6M c6 bé sung phenol 1% (10pl phenol cho vo iml HCl) Binh khi nito - Phenylisothiooyanate (PITC), _- Triethylamine (TEA) - Ethanol logi ding cho HPLC; —- Nude cat hei lan Dung cu va thiét Bi hd tro - He théing sic ky lng cao 4p; - Dau dd UV, bude s6ng 254nm = Cot Pico Tag 3,9 x 150mm; + Bg trén dung moi - La didu nhigt; - May ghi. = pH kg; - May khuly tir - Giy loc; «Can phan tich 4 sf 16 - Binh dink mee; - Ong dong = Kim tiem may; - Bén siéu am ~ Méy Vortex; ~ H@ thong loc chan khong 4p suat thép. 3- Chudn bj mau Che dung ey dung miu 68 duye ria bling dung dich HCl 6M va rif lei bling nue efit, Thutng ding dng nghiém 25x 150mm bing théy tinh, e6 nép van lam b&ng teflon. Thiy phan protein, peptide Dét néng 1d é 105 + 112°C khodng 20 +90 phit trudc Ichi thiy phan. Thue phdm gidu protein: Can 0,1 mau bot cé chia khodng 20mg protein vio binh. Cho 10m dung dich HCl 6M chéta 1% phenol vao binh phan (ing, Tao chan khong trong binh phan ting va dat miu vao 1, Tién hanh thiy phan hoan totn protein va peptide thanh axit amin bing HC] 6M 6 110°C trong 24 gid trong diéu ki¢n khong cf O2. Sau thoi gian trén, ly binh phdn tng ra Ichdi 1 48 ngugi. Chuyén sang binh dinh mie, trdng binh phan ing bing nude eft hai Mn va dinh mac. Thite phim gidu chdt béo: Can 5g m&u phomat, edt nhd va trich ly chat béo, rita bling sidu Am v0i 100m! hé dung méi aceton: chloroform (3:1) ¢ nhigt 46 phong. Sau 46 Idy lugng mau tung duong 20mg protein dem thdy phan véi 165ml HCl 6M du6i ding No & 100°C trong 24 gid, bé sung a butyric axit ding lam chat ngi chudn.64 HUONG 5 Thue phém ngheo protein: Can lg m&u bot nga coc chia khedng 10mg nitrogen, oxy héa trong vong 16 gid ¢ O°C véi thuéc thi hin hop performic axit - hydrogen peroxide, chita 5mg phenol/ml. Lugng chit oxy héa du duge phan hily bing Na - pyrosulfite hoc HBr. Axit formic duge loai bang cé quay. MAu d& oxy h6a duge cho thiy phar véi 50m! HCl 6M 6 chita phenol, 4 110°C trong 28 gia, trong diéu kign c6 khi Np. Phuong phép xit ly mu nhu trén cho phép xée dinh véi do chinh xAc 95% céc axit amin: Asp, Glu, Gly, Ala, Leu, Phe, Lys, His, Tyr va Arg... ngoai trit tryptophan va tyrosin. Bo sai l¢ch gita thanh phan axit amin trong dich thay phan véi ham lugng cia ching trong protein ban ddu cé thé xay ra la do ede nguyén nhan sau: - Isoleucine va valine duge gidi phéng cham hon cdc axit amin khée - Threonin va serine bi phé hiy ~ Cysteine, cystine va methionine bj bién déi thanh cde sin ph&m khdc nhau phy thude vao mau ban ddu - Tyrosine c6 thé bj oxy héa béi sy ¢6 mat cia cht oxy héa va c6 thé bi bién 46i thanh cdc dan xudt chéa Chlorine, Vi dy, 3-Chlorotyrosine + Tryptophan bj phé hiy hoan toan cn asparagine va glutamine thi bj bién déi thanh céc axit tuwng ing 1a aspartic va glutamic. 4 Tao dén xudt tién cpt Phan dng tgo dain xudt: 8 i! NeC=S+H.N-CH, —e NH-C-NH-CH, R R Phenyithiocsbamylacsht) Vige to din xudt phai thye hién qua nhidu bute: BEA Ra LAUT fie, phan tgo duge din xudt hon toan thi phai logi het Ghd Vaowinaldy sgn dane dich mau duge lam bay hoi, 9 ho dudi chan khong,.Sau 46, hoa lai trong mot hon hgp dung di co a ee, née 10% | i Bim kha. C6 thé Lip lal val TAH death Saul Bok Qa REE Bul Ube tha tao i SURO Lh elim TEN Gdavece Bere AMAL Tiés' wep ay el otal ae Tha BY nial dite Gab ea ca) Seto eo hae es i PH a f Gad hoa che ‘eH thé dug? PHAR Haas hats aerate afd ab! prod Dé cia ‘enth, FestneeRCo ANIME « Borphitt. Made Hstratsn dis heh be! sti philn bap hai déu‘la chat 8 bay hai nén-Berdegu lost HE Nha, ‘Apaudt!tronp 80-440 phi) Maude tmailt + BULG, sow, aieytclads 96) SHE ban th 2000, trong. tA, Nhung:gay. la hoa saniigbmosrda dung diakodemtcphotnbah Soul, oH Zitfanetonitrila,, 96/6, chicos. the ‘Velde ide méengayd mbiet Se anbang hay 601170, 8b) A trong th laph,2 hors ifn dey ax6l grb anes oingud 9 yous Dd Sig MW yond D°0OL 8 Munch loudPHAN TCH THUC PHAM BiNG PHONG PHAP sdo KY LONG HIEU NANG C40 65 Quy trinh sly khé: - Héa chit: triethylamine, ethanol ding cho HPLC, nuge cat hai lan. - Dong dich dang sdy Ikhd lpi: hn hgp ethanol : nue edit hai In : TEA (2:2:1) - Lay 10 va 20,2 dung dich thay phan tif binh dinh mde cho vao hai dng nghiém dyng mu - Lily 10u hin hgp dung dich “sy kho Ini” cho vao e6e Sng dyng mau, xoay tron + D&t ng mu vao hat chan khong cho dén kh6. Chudin bi dung dich din xudt: Chuan bi hon hgp Ethanol : TEA : ouée efit : PITC = 7:1:1:1 Tao din xudt: - Rit 20u/ hén hgp dung dich tao din xudt méi pha 6 trén, cho vao méi dng mau da sdy Iho - Lae vai gidy - Dé yén 20 phat & nhiét 4d 20+ 25°C = Say khd. 5- Chudin bj dung dich axit amin chudn Ding pipette ty dong rat 3, 5 7, 10y2 dung dich axit amin chudn cho vao éng nghiém va lam dan xuét nhu di véi dung dich mau. Chi 9: - Séy kh miu tao din xudt dé loa bé nhang vét cudi cung cia PITC - Can chudn bj tgo din aut ¢ mdi lAn phén tich ~ O nhiét dd phong, cdc mfu da duge pha loang chi cho két qua én dinh trong vong 10 git, Con ede mau chifa pha loding thi e6 thé gitr duge trong 60 gi. Vi vay khong nén lum trix ede mAu da pha loaing, Nhing miu kh6 6 thé dé nhiéu tudn trong ti dong. 6- Phan tich axit amin trén may sdc k§ léng cao Gp Sau Kkhi tao duge din xudt, mau duge dinh mic 1000p) bing dung djch gm dung dich 48m phosphat SmM, pH = 7,4 va acetonitrile t7 1@ 96:5. Cui cling lay 20, dung dich dinh mie tiém vao HPLC. C6 thé chay sile ky theo hai ché d9 sau: = Tao din xust: PITC (precolumn) - Cot: Pico- tag column (water Cys) 8,9. 150 = Nhigt dg Gt CC): 95 + Pha dong: A) CHJCOONa 0,14 M chita 0,7mliL TEA pH = 6,7 B) Acetonitrile - nuéec theo ty 16 60:40 = Tée a6 dong (mi/phut): 1 va 1,5 Detection: UV 6 242m Théi gian chay: ‘Le'phat66 CHUONG 5 Hoae: - Tao din xuat: PITC (precolumn) - Cot: Nova- pak column (waters Cys) 3,9mm, 150 ~ Nhiét 49 edt CC): 38 ~ Pha dong: A) 940 ml CH,COONa 0,14M chita 0,05 % TRA (trietylacetic) pH = 6,4 tron v4i 60 ml acetonitriie B) Acetonitrile - nue theo ty 1 60:40 + Tée d9 ddng (mliphiit: 1 va 1,5 ~ Detection: UV 6 254nm + Thai gian chay: 12 phut Chudn bi dung dich: Hoa chat: - Acetate natri trihydrat; - Triethylamine ~‘Axit acetic; ~ Acetonitrile dang cho HPLC - Nude cfft hai ldn; - Na,HPO, ~ Axit phosphoric - Pha dong A: + Cn 19g acetat natri trihydrat + Thém vao 1 lit nude oft hai lén + Thém 0,5ml TEA + Chinh pH = 6,4 + Lge + Pha 940ml dung dich da loc v6i 60m acetonitrile, lde déu + Bénh logi bet khf trong bn siéu am. - Pha dong B: + Pha 600mi acetonitril vdi 400m nutic cAtt, Ide du + Dénh loai bgt kf trong ban siéu am. - Pha dung dich C: + Can 710mg NasHPO, + Thém 1 lit nuée et + Chinb pH = 7,4 bing dung dich axit phosphoric 10% + TrOn dung dich nay vdi acetonitrile dé dat duge dung dich 5% thé tich acetonitrile - Van hanh may: + Kiém tra hé thing méy + Dat nhiét do 1d 43° + Cho pha ding A va B chay qua hé thing bom + Cho hé thdng van hanh: cdi d&t buge song, céc thang sO méy ghi.PHAN TICH THUC PHAN BANG PHLONG PHAP Shc KY LONG HiEU NANG C40 67 - Lap chuong trinh chay gradient: [That atan cehay | Luts ang emvpray [6A *B ae Khdi dau 10 100 0 0 10,0 10 54 46 ° 10,5 10 0 100 0 115 10 ° 100 ° 12,0 15 0 100 0 12,5 15 100 ° ° 20,0 15 100 o ° 20,5 1.0 100 ° ) - Bom chét chun va miu vo may, Chay ste ky. 7 Xt lp hit qua Xem va so sinh she ky 6 cia chufn va miu: - Thong s6 thi gian iu - Dign tich peak - Chiéu cao ola peak trén sie ky 48 - NhAn dang peak va thet ty cdc axit amin trén sfc ky dé ~ Néng d@ chudn trong dung dich hn hgp chuén - Lp dung chudn tit cdc ndng 46 chudin. . Céng thie tinh két qué: Ham lugng mdi axit amin trong mAu phan tich duge | tinh bing s6 mg 6 trong 1g mAu bling ebng thie: Sycv Suv. A= My 1000 | Voi: A - néng 46 axit amin i, mg/g Sw - dign tich oda peak axit amin i trén se ky d8 cla mau | Sc - dign tich cia peak axit amin i trén sde ky 46 ciia chudn C - néng 6 axit amin chun é tinh theo dung chudin V - téng thé tich dinh mite mau M - ich6i lugng mau thyc phdm ding éé phan tich axit amin, g.Chitong 6 sAc KY Kui Ske ky khi cé pha dong la chét kh, chuyén ding dé phan tich cde hop chat a8 bay hoi. Mot s6 chat mac di c6 nhigt dé sbi cao nhung 06 thé tao duve eae din xudt 06 nhigt d¢ s0i thp edng thudng duge phan tich bing phusng phap sfc ky khi. Uu diém eda sie ky khi la van hanh don gian va khéng phai stt dung ede dung méi at tién, doe hai. Két qui phan tich sfc ky khf phy thude vao viée lia chon dang ¢Ot, chon ding nhigt 2 bom miu vao eft, nhiét do 1d, nhiét 6 8 detector va con phy thuge vao sy diéu chinh van tée déng khi mang vao cdt. Ché 6 nhiet do van hanh sic k¥ khi eé anh hudng rt én aén 49 phén gidi eda eft. Nhidt d6 qué cao lam cho cde chat e6 nhigt dd s6i thap bj ddy ngay ra Ichdi cot ma khong duge git lai béi pha tinh. Neuye Jai, nhigt 6 cot khong di cao sé gay nén ay hm git mu trong pha tinh va lam che thai gian phan tich kéo dai. Khi phan tich mat hin hop gém ahiéu thinh phéin 6 nhigt 40 66i Idhée nhau thi edn phi trién kthai theo ché dé gradient nhiét do. Trong link vyc phén tick thye phém, sd ky khi duge ung dung chi yéu dé Phan tich ede chat mdi ty nhién eda the phdm cing nhu cde chat mii sinh re trong qué trinh ché bién; phan tich cén, rugu, bia, trong truéng hop nay cdc she ky 46 mang tinh dic trung cho san phém giip phat hién hang gia; xéc dinh du lugng thude bao vé thye vat, ede chit hit og chia clo. Bang each tao ddn xuit co, nhigt d6 s0i thép hon, ede axit béo va axit amin cing duge phan tich bing | phuong phép sdc ky khf. |Ae KY Kui 6 BAI 10: PHAN TICH THANH PHAN AXIT BEO BANG PHUGNG PHAP SAC KY KHi (GAS CHROMATOGRAPHY) Dé phan tich thanh phin axit béo, thuting ding phuong php séc ky khi. Do céc dain xudt methyl ester cia axit béo thi c6 nhiét 46 héa hoi thp hon rét nhiéu 50 vidi axit béo ty do nén hdn hgp axit béo duge tao thanh din xudt methyl ester truée khi dua vao phan lich. Cung 06 thé chuyén axit béo thanh ethylester nhung nhiét 49 héa hoi sé cao hon, Dé chuyén axit béo thanh methyl ester (fatty axit methyl ester, FAME) 06 thé ding ed ic téc kiém hodc axit, tuy nhién edn chi ¥ 1a khi dang BF; lAm xtc tae trong phuong phép axit sé lam bién déi edu tric khong gian ciia én két dai thanh ede dang lién hgp kiéu CLA, vi vay lam sai léch két gud Ichi phén tich CLA (axit linolee néi doi lién hgp - C = C-C =C-), 1 DUNG CY, THIET B] - Phéu chiét 100mdé, binh edu 250m! 6 hai c3, Ong nghiém, pipette ty dong, syringe tiém mau 10 mierolit. - May ly tam. - Hé théng thiét bj phan tich sée ky khi. T- HOA CHAT 1- Hexan, 2- Dung dich metanolat natri: Can 7,5mg phenolphtalein vi 1,15g Na; hoa tan vio 30m! metanol va 20m! at hign dung dinh metanolat natri mau dé cé ning do Lmol/t. benzen, 3 3- Dung dich metanolic HCl: Cho vao bin hai cé 30m! H,80, dam dic. Lip phéu chit 06 chita 15m! HCI 4am dac vo mét cé binh qua nut cao su. Pau kia eda binh duge din 60m! metanol yao dung dich, Ter ti nhd HCI yao F280, dam dae s8 gidi phong ra khi HCI duge ‘hép thy vRo metanol. Dung dich metanolic thu duge s& 6 néng 46 khodng 13%. TH PHUONG PHAP CHUAN By METHYL ESTER I: Nguyén tj ‘Triglycerid duge chuyén ester héa véi methanol trong méi trung CH,ONa. Axit béo ty do con Iai sau phan ting duge methyl ester hoa bing méi rung axit, Siin phiém methylester cua axit béo duge trich ly ra khdi hin hop bing hexan.70 GHUONG 6 Phan ting chuyén este héa cia glycerid ¢6 thé duge xtc tée bling axit hay kiém (1), trong kbi phan ting ester héa chi c6 thé duge xic tae bing axit (2]: CH,~O~CO— RF, R,coocH, GH, — OH H', OCHS CH —O—CO—R, + 3CH,OH ===" A,COOCH, + CH —OH (ty 1 CH, —O = CO— R, R,COOCH, CH,—OH w RCOOH + CH,OH === RCOOCH, + HOH re) 2. Phuong phap tao dén xudt methyl ester cila céc axit béo Can chink xc khoang 20 + 30mg lipid, cho vao trong 6g nghiém. Sau dé cho vio 1ml hexan va 0,15mi dung dich metanolat natri, lc that kj trong khodng 1 phit r6i dé yén 8 nhigt 46 thudng 30 phat. Tis theo, cho vao Im! metanolie HCl, Ide tht kj ri 48 cho phan img xdy ra trong 46 phit, Sau a6 ly tam Ong aghiém 3000 vong/phuit. Dung dich trong éng nghigm sé bj téch lm 3 pha. Ding pipet ty dong hit Idy pha trén cing 06 chia methyl ester cia axit béo dem phan t{ch trén méy séc ky khi. Dén xu&t methyl ester duge béo qudn trong ampule trong didu kign chéng oxy’ hoa: duge lam khan, ding khi nitrogen du6i khong kh, han kin ampule va bie quan lenh cho tdi khi phn tich. TV- PHAN TICH THANH PHAN METHYL ESTER TREN MAY SAC KY KHI 1. Gidi thidu vé he thing thiét bi 1+ binh chifa khi mang; 2-b6 phan di6u chinh 4p suat; 3- van; 4- b§ phan loc; 5-10; 6- d€u bom mau; 7- cot mao quan; 8- ddu dd; 9- bd phan xii ly da ligu Hinh 10.1 So dé he théng adc ky i Pha déng: Khi mang (carrier gas) phai la loai khi tro vé héa hoe 6 nhiét db phan tich véi bat ect mét thanh phdn nao cla mdu va phai c6 dé nhét thifp. CAtSic KY Kit n Joai khi mang hay duge et dung 14 No, He, Ar, Hy, Van tée dong khi thutng trong khoang 20+ 100mJ/min (do & dau ra cia cot). Ap sudt du vao cia dong khi thutng trong khodng 10-50psi. BO phén tiém mdu (injection port): LA mot khodng khéng gian rit nhd duge gia nbiét, mét diu duge day kin bling mét septum bing cao su, du kia duge ndi véi oft phan tich. Mau tiém vao, truée khi vao dén oft phii dam bdo duye héa khi hoan ‘toan va phi di vao cbt duéi dang m$t dai méng. Vi vay, hit 4} 6 bd phan tiem mAu thuing phi lén hon nbiét 4} bay hoi cia thanh phén khé bay hoi nhét trong mau ft nhat 14 10°C, ahung phai nhd hon nhigt 4 lam phan hiy mdu. Lugng mu bom vao cot c6 thé dao dong trong khodng 1+ 10p/ 461 voi m&u léng; 0,5 + L0ml ddi véi mdu khi, Khi sit dung cot mao qudn, chi cin mt lugng mau rat ahd, doi khi Khang thé do duge mét efch chinh x4c. Pé diéu chinh luu luong Ichi vao oft, sit dung cbt chia dang (splitter). Smvmin 100mlmin Dong vao edt = 105- 100-3 = 2mvmin Ty la chia dong = 50:1 Hinh 10.2 B3 phan chia dong Cot sde ky khE (GC column): Sit dung cbt mao quan 1a mot Ong silic nung chay boc ngoai bing mét phim poliimid chju nhiét dén 370 + 400°C, hode:bing mot lép nhém chju nhiét dén 480°C. Pha tink thudng duge trang tran ‘ba mat trong cia éng nh@ mt phan ting héa hoc. Cdt sde ky khf o6 nhidu logi. Tinh chat phan eve eda pha tinh s& quyét djnh thit ty phan tich cia cdc axit béo. Kich 8 oft: 7 = Dai th6ng thuting: 12; 25; 80; 50; 60; 100m - Dudng kinh trong: 0,1; 0,18; 0,22; 0,25; 0,32; 0,58mm ~ Bé day phim: 0,12; 0,2; 0,25; 0,4; 1;57] cHUONG 6 Hinh 10.3 C6t mao quan ding cho sdc ky kht V6 boc polyinide }— 6ng sie nung chay Pha finh lién két hoa hoe tran be mat trong cite ct EEE Déu do: Trong she ky khi, mot dau dd ly tudng edn 06 ede die diém se + Phi ¢6 kha nang phat hién chat can phan tich ngay Khi n6 ra khéi edt ~ Di nhay dé 06 thé phat hign ed khi chat phan tich c6 ham Iuong rat nhd - Phat hign phai dap ung véi sy thay adi néng dé dé 06 thé ghi duge hét cde mii va ede mai khéng bi bién dang, do dép ting khong phy thude tée a> dong - Dép ting tuyén tinh trén mot khodng néng do rong - Dap ting gidng nhau cho moi loai héa chit - On dinh va chinh x40, dung nén {t nhigu, khong trOi - Nhigt d6 sit dung len dén 400°C - Khéng bay mau + Dé sit dung. Str dyng dau dd ion hoa bang ngon lita, FID (lame ionization detector). Day 1a mot ddu dd 06 dé nhay cao, dé tuyén tinh tong 46} rong, Ikha nhay néu chat hou ¢9 chifa nhiéu CH. Nguyén ly hoat dong cia ddu do nay la sé dét chay nhitng chit ra khéi cot phan tich bing ngon Ita ¢6 khi hydro dang chay. Khi mét chat hitu ed bj dét chay dui tae dung ngon lita tao ra béi sy dét chéy hydro, 6 sy tao thanh cde géc CHn*, OH*, 0," 6 bién ngon lita, Cée ge ty do phan tng sinh ra CHO": C*+ OH* —* CHO + &| she ky Kut 3 Tin higu ghi nan dya trén ion (CHO") sinh ra, Ion sinh ra tao dong dién, duge khuéch dai va bign déi thanh mii sée ky. Ong 96p ——— 89 phén daah ita Khong kb ‘Bau ra cha o6t Hinh 10.4 Déu do ion hoa ngon lita (FID) ding trong sic hy kht Bé phan xi lj dit ligu: Nhan tin higu ty dau do, xti ly tin higu, chuyén thanh dang dé thi duge danh dau bing thai gian lu 2. Tién hanh phan tich mau Trinh tu tién hanh: - Khéi dng may; - Cho chay pha dong - Cai dat chuong trinh chay séc ky; - Bom mau - Chay sde ky phan tich ru; - Thu nhan va wit ty dif liu - Béo cao trinh bay két qué phén tich. Thue hién: Diéu kién phan tich : - Sit dyng et mao quan DB-1 (ID = 0,26n72; 2 = 30m), 1a logi oft khéng phan eye - Pha tinh: 100% dimethylpolysitoxane - Pha dong: khi nito - Detector: FIDm” CHUONG 6 Cai dat chung trinh chay se kp: - Nhiét dé injector: 250°C - Nhigt 49 detector: 250°C - Lita lugng Hy: 40ml/min ~ Limi lugng dong khi mang No: 400ml/min ~ Nhiét d9 cbt: 180°C trong 2 phit, ting 2,5°C/phut t6i 205°C, tang 3,5°C/phut tdi 280°C, dimg 2 phuit, tang 5°C/phuit t6i 250°C. - Ap suét dau vao cia khf mang: 14psi - Split ratio: 125, Holic ding ot DB-6 (0,26mm; 30m) 1A loaj edt phan cye y8u. Pha tinh c6 95% 1a dimethylpolysilosane, 5% diphenylpolysilosane véi chuong trinh chay phan tich: + Naiét d6 injector: 270°C - Nhiét do detector: 270°C ~ Ché dg bom mau eé chia dong, split ratio: 1/60 + Luu lugng Hy: 40ml/min - Luu lugng dong khi mang Nz: 400ml/min ~ Nhiét 49 oft: 180°C trong 2 phit, tding 2,5°C/phiit t6i 205°C, tang 3,5°C/phit t6i 230°C, dimg 2 phut, tang 5°C/phut t6i 275°C, - Ap sudt dau vao cia khi mang: L4psi. Bom mau: Ding syringe véi kim tiém ddu nhon, bom trye tifip Kholing 2 microlit din xuét metylester olia cde axit béo vao cot ske ky khf qua bo phén tiém miu, C6 thé thay di chuong trinh nhiét d) nhim gidm thdi gian phn t{ch khi c6 cde khodng tring khéng ¢6 miii. Trong qué trinh phan t{ch phai gitt van téc truyén thang ciia khi mang khi qua et 14 khéng éi dé mai séc ky nhon va d8i xing. ‘Théi gian phan tich mdu 1a do ché do nhiét 4 di dat, thong thutng khong 20 phat, DE xdc dinh vj tri cde axit béo trén sé ky dé dung phuong phép ni chudn 1A bs sung mdt chat chudn c6 néng dd biét true ngay trong mAu true khi xit ly. Thudng ding axit ¢6 cacbon 1é (015:0) hope axit. khong 06 trong mu. Bé xéc dink trinh ty c6 thé ding hai chun é hai d4u. Néag do chat bé sung phdi gan néng do axit béo trong mau. Hay ding phuung phép ngoai chufn, tie 1a ding chat chudn xdy dung dudng chudin. Khi d6 phdi 6 da, chét chudn, thye hign phdi cing diéu kign, lugng mau bom vao phai e6 dink, dung chufin phai tuyén tinh. Hé 86 tuong quan eda dung chuén R? phi gén bing 1.SKC KY Kat A 8. Xi lf hot qua Xem va so sinh s&e ky dé cba chudn va mu. Dya vao thi gian Iu cba céc peak chudn dé xAc dinh vi tri eda cdc axit béo va thi ty cilia cde exit béo trén she key dd. So sénh dign tich peak va chiéu cao peek eta cdc axit béo trén she ky dé cia méu va cdc néng d@ chudn trong dung dich hén hop chudin. Lap dutng chudn tif cdc néng 46 chudn. Céng thie tinh két qué: Ham lugng mii axit béo trong méu lipid ph4n tich duge tinh bing ty 1é % theo cong thie: SyC aye? véi: By - néng d6 axit béo i, % Sy - dién tich eda peak axit béo i trén she ky dé cia mau Se - dian tich eda peak axit béo é trén s&c ky dé cia chudn C - néng d@ axit béo chudn i tinh theo dung chudn ES, - téng dién tich eda cdc peak axit béo trén sdc ky dé cia mau. B,PHAN III PHU LUCPhu luc 1 COT SAC KY DUNG TRONG HPLC | T- PHAN LOAI COT Chéit nhéi cbt Lil - octadecyl silane lién két héa hoc vdi cfc vi hat 6 18, bing silic hay | ceramic, duting kirih tis 3+ 10pm, 12 - octadecyl silane lién két héa hoc véi hat silica gel c6 bé mat c6 15 xde dinh, lién két thanh Iép vé rén dudng kinh 30+ 50um. | ~ 18 - hat silic 06 15, dudng kinh 3+ 10um. ' 14 - silica gel bé mat ¢6 18 xdc dinh, lién két thanh Iép vé rin dudng kinh j 30 + 50pm. LS - oxyt nhém bé mt o6 13 xde dinh, lién két thanh Iép vé rén dung kink 80+ 50m. i L6 - cht nh6i la nhua trao déi cation manh bing polyme. , 4: Phén logi dya vito bén chélt oGt ligu eda chét mang : Cée chat mang rén ding lam vat ligu nhdi cét st dung trong HPLC duge phfin lam ba ahém nbu sau: Logi silica gel: 6 4$ bén co hoc cao, dé xay nghién thanh dang hat rat min, higu qua cao. Loai polyme: ¢6 a6 bén trong mot khodng pH rong. Logi hydroxyapatit: ¢6 tinh chon Ige duy nh&t cho ede polyme sinh hee, vi dy nhu protein. C&c loai e¢t HPLC cia Shimadzu duge phan loai theo ba nhém trén nhy sau: Loai silica gel: - Nhém shim - pack (ODS, Cs, TMS, CN, phenyl, NH,, SIL), nhém CAPCELL - PACK (Cis, Cs). - Nh6m zorbax (ODS, C;, TMS, CN, NH, SIL...) - Nhém STR (ODS). Logi polyme: - Polystyrene : Nhém shim - pack (Amin, ISC, ISA, SCR, IC, ION, GPC) - Polyaerylate : Nhém shim - pack (PA, IC) - Polyvinyl alcohol : Nhém asahipak (GS, BS, ODP). Loai hydroxyapatit: Nhém shim - pack (HAC).80 PHY LUE T Phén logi theo phuong thite gidi hdp Loai sitica get: - Gidi hap pha thudn SIL (shim - pack, zorbax);__ Phospholipit NH, (shim - pack, zorbax); -Sacarit, tocopherol GN (Shim - pack, Zorbax), __ Hormon steroit. - Gidi hap pha dio ‘ODC(Cx») (shim - pack, zorbax, CAPCELL PAK, STR) Cae chat phan tir lugng nha Ca(shim - pack, zorbax, CAPCELL PAK) (fe chat phan tit lugng shd CN (shim - pack, zorbax) Ce cht phan tit lugng nhd TMS (shim - pack, zorbax) Cac cht phan tit lugng nhd Phenyl (shim - pack) Cée chat phan tit lugng nhd - Trao déi ion Shim - pack WAX (trao déi anion baze yéu) Nucleotit, protein Shim - pack WCX (trao ddi cation axit yu) Protein Zorbax SAX (trao déi anion bazo yéu) Nucleotit Zorbax SCX (trac déi cation axit yéu) Catecholamin = Loe gel Shim - pack diol Protein Zorbax GF Protein - Sie ky tuong tae ky nude Shim - pack HPC Protein, axit nucleic Logi polyme: + Gidi h&p pha dao Asahipak ODP - 50 (polyvinyl alcohol} Peptit, protein, cto chét phan tc lugng nhé. + Trao déi lién két Shim - pack amino, ISC (polystyrene) Axit amin, guanidin Shim - pack ISA (polystyrene) Sacarit Shim - pack IC (polyacrylate, polystyrene) Cae ion vO eo Shim - pack PA (polyacrylate) Protein, axit nucleic Asahipak ES (polyvinyl alcohol) Protein + Ton Shim - pack SCR-101H, 102H (polystyrene sulfonat) Axit héu eo Lge gel Shim - pack ION (polystyrene sulfonat) —_—_—Polysacarit, Asahipak GS (polyvinyl aleohol) Cée hyp chét coo phan ti tan trong nudecOr sac KY DUNG TRONG HPLC 8t - GPC Shim - pack GPC (polystyrene) Ce dai polyme téng hgp, eno su Lowi hydroxyapatit: Shim- pack HAC Protein, axit nucleic 2. Phan logi theo kich thuée e6t ~ Cot ding trong HPLC tay theo kich thude duge chia hai loai la c6t 48 phan tich va edt ding dé tach thu nhgn ché phdm ~ Logi ot phan tich lai duge phan logi sau hon thaah ede nhém = Cot dang thu ché phdm 20mm ® x 26em Ban tach phan 80mm ® x 25em Ban téch phan 50mm @ x 25em Tach phan ~ Ct ding cho phan tich + Cot chudn 4.0mm © x 16 hode 25cm; 4.6mm w x 16 hoe bom 60mm @ x 15cm 8.0mm © x 80cm + Séc ky long nhanh (FLC) . 46mm © x Bem +Nonghep * 2,5mm © « 16cm + Nong siéu hep (microcolumn) 10mm & x 25 hoge 50cm COt kich thuée chudn Loi edt o6 kich thute dung kinh 4 +8mvh va chidu dai 16 + 30cm, duge sit dung rong ri nhét va thich hop trong mét Ikhodng bién thién rong. Cét siéu nep (MBC micro bore columns) Gy. MBS cia Shimadzu chi cé logi dutng kinh trong rong 1,0mm. Tieu thy pha d§ng chi t6n 1/20 so v6i khi si dung edt c6 dudng kinh trong réng 4,6mm vi van téc chy chi tténg dudng 1/20. Céc logi kim bom mdu, té bao chdy sudt va ede Sng néi loai nhé déu 6 thé sit dung thay thé, - COt ndng hep (SBC small bore columns) C$t SBS cia Shimadzu 1a loai dutng kinh trong 2,5mm. ‘Tiéu thy pha dong bling 1/3 so véi logi opt 6 dung kink trong 46mm. Mie di loai oft nay 6 thé thich hgp véi mt hé thing thiét bi HPLC thing thutng, tét hon 1a nén si dung cho loai ¢6 don vi nang lve nhd.82 PHY LUCY - Cot ding cho stic ky long nhanh (FLC) Cot FLC cia Shimadzu Ja loai edt ¢6 du’ng kinh 3ym va chi dai Sem. Ching cing thich hgp vi hé théng HPLC théng thudng va phan tich rat nhanh. 8- Cée logi e6t ODS Cot ODS duge nhéi béi vat ligu nhéi c6 nhém octadecyl lién két héa hoe trén bé mat va duge sit dung rong rai nhat trong HPLC. Cot ODS e6 thé c6 cde tinh chit phan tach khée nhau tidy thude vao kiéu end- capping va ede dae tinh cia silicagel duge sit dung lam cht mang ran. Ching 06 thé Ihde nhau 6 mach nhénh. Nhimng khée bigs va cfc tinh chat phan ly duge sit dung dé thanh cing trong nhiing phan tich kh. Shimadzu ¢6 nhigu iogi cbt OSD khac nhau dép ung vide ing yng cho mét linh vye rong. ok, CH, j < 2 LagSin Star Liots dogo, a Ha 4 dog a a) >) ° 4 Hinh 1 Pha tinh trong cde logi e6t C18 (ODS) " 6-0-6-6,,H,, (pro-8 Chait mang rdn silica gel vd pha tinh en-capped Shim - pack CLC-ODS, shim - pack FLC-ODS, shim - pack MBC-ODS, shim - pack SBC-ODS, shim - pack PREP-ODS, -ODS(E), -ODS(L), -ODS(K), -STS ODS-H Chét mang silicugel rdn boc polyme va pha tinh en-capped CAPCELL PAK Cis Chat mang-rdn silica gel va pha tink non en-capped Zorbax ODS . Chat mang rdn bang polyme Asahipak ODP - Phuong phap dé géin cae nhém Cys - Chon et ODS Bang duéi day 14 huéng din vin t&t.dé chon cht ODS06T SAC KY odNG TRONG HPLC 83 Tén ogt Bie didm Gidi ben pH pha dong ‘Shim-pack HAC-ODS | Day ob boc mang nhim gidm thigu Anh hudng cla | 2-76 sivene. ede din xudt silanol va khit chiBu ota céc chat kiém Zorbax ODS. Strdung Anh hung cia ofc hap chit gée silanol 2-78 CAPCELL PAK Cy ‘Chat mang silicagel rin boc polyme silicon !am tang 2-10 4 én 66t trong pha dbng kiém tinh Co thé sit dyng cho pha dng cd pH tai 13. 2-13 ‘Tinh chon Ige 1A khée bi6t so vdi tinh chen lec ofa | tse sticaget 1H- HUGNG DAN CHON CAc DIEU KIEN VAN HANH 9 phan ove Khong tan trong nude Tan tong nus LEO Khong phan eye Phan ly ion PhAn cyc, khéng phan ly ion Phan ti lugng 10° SK trao di ion Gol fttration Hinh 2 Su iva chon hidu sde ky long tren co sd phan ti lugng ve.-tinh chat phan eye, hoa tan, ion hay phi ion cia ede cfu tit cin phan tich (Theo “Principles of instrumental analysis” Douglas A. Skooy)a PHU LUG 1 1. Axit amin Axit amin la hgp chat lu@ng eye chia nhém cacboxyl va nhém amin cé cong thie chung: = NH,-RCH-COOH Cée axit amin duge phan thanh ede nhém axit, trung tinh va bazo tay theo sf luyng cdc nhém chic. Ching cing duye phan thanh nh6m mach thang hay mach vong tiy thude vao ban chat g6e R. Riéu stic kp léng (LC mode) Phé bién nbat 1A ding sc ky trao déi cation, va phan lam hai phuong php la phusng php Li va phuong phép Na. Kiéu Li thutng ding phan tich ofc axit amin sinh hoc hay dé tach glutamin va axit glutamic. Hinh thie gidi hap ddo pha ¢6 thé t4ch phan Ién céc logi axit amin nhung cing khéng thé tach duge tt ed cic axit amin bf do, Dé tach cfc axit amin thi higu qué nhat la vi phn tich ede din xuét tién o6t. Phuong phép phat hign Phyong php hdp thu ti ngoai duye ding dé hap phy cde nhom cacboxyl & bude song 200 +210nm khong thuting ding, mac di né cé thé phat hién mét s0 loai axit amin. Phuong phép vi phan tién ct va hau edt thutng ding a6 phét hign axit amin. Phuong php vi phén tién eft: Dung dich mu duyc tao dan xuit truée Ichi dua vao cit phan tich. Sy k6t hop gita phuong phép tao din xuft vi phan tién cpt va kiéu giai hap dio pha cho phép phan tich nhanh chéng cée axit amin 06 do thiy phan protein. Céc thutc thi dé tgo dan xudt tién oft nhu OPA (ortho-phthalaldehyt) sn o6 trén thi truéng duge ding khé phé bién. Phuong phap tao dan xuat dé vi phan tich hau cdt. Cae hoa chat dé tao din xudt vi phan tich duge bé sung lién tue vao dung dich sau gidi hép. Phuong php nay 6 wu diém néi bat Ja toan bd qué trinh van hanh c6 thé ty dong héa. Sy két hop gida phvong phép vi phan hau cot va phuong phép trao déi cation cho phép phan tich mét gidi bién thién rong gém cdc axit amin ty do va sin phdm thiy phan wy protein. Cée thuée thi phé bign trong phuong phép tao din xudt vi phan hau cét Ja OPA (4é phét hujnh quang) va ninhydrin.OT Sic KY DONG Thone HPLC 85, Chon ede diéu kign vén hanh ‘Mau phan tich Hinh thie tach Céttiau bidu Each) — Vi phan hau cot Aadamin w co L{ Trae déi cation ‘Shumpack aminot va pratnayne auang vai Om Trao aéi cation ‘Shimpack HAC.ODS. Vi phan tién et CAPCELL PAK hat 61,098. quang véi OPA 2. Peptit vé. protein Peptit va protein cfu tgo tix céc axit amin lién két voi nhau bang lién két Deptit. Ce tinh chat ky nude va ion khée nhau ty thude vao thanh phan va's6 lung cia axit amin, Kiéu sde hy long (LC mode) Peptit va protein thudng duge phan tich bing mot trong sé céc phuong phép sau: - Loc gek: ‘Tach bang kich thude phan tir + Trao déi ion: Tach biing dién tich phan ti - Pha dao: ‘Tach bling tinh ky nude cia phan tit - Hydrophobic: Tach bang phan tng phitc tap - Hydroxyapatit: Tach bing phan ting phitc tap. Phuong phép phat hign: Phat hign bing phuong ph4p quang phé tit ngoai. Phin Jén trudng hop sit dung bude sing 280am. Phat hién ¢ 210nm thudng ding Adi véi cde protein c6 nhidu axit amin mach thang va phat hign protein, peptit e6 néng do thap. Logi e6: Loe gel: Shimpack Diol, Asahipak GS, Zorbax GF ‘Trao d6i cation: Skimpack WCX-1, Shimpack PA-CM, Shimpack PA-SP, Asahipak ES-502C Treo déi anion: Shimpack WCX-2, Shimpack PA-DEAE, Shimpack PA-QA, Asahipak ES.502N Pha dao: Shimpack HRC-ODS, Asahipak ODP-50, CAPCELL PAK Cig Hydrophobic: Shimpack HPC Hydroxyapatit: Shimpack HAC86 PHY LUC 1 3- Axit nucleic Kiéu sde kg téng (LC mode) Chia ba nhém phén tich gdm ede bazo nit, nucleozit va nucleotit. Céc bazo nite va aucleozit duge phin tich theo kiéu trao adi cation hode gidi hap pha dao. ‘Nueleotit phan Ién duge phan tich bing trao 46i anion, Kiéu chiét xuat pha dao cing duge sit dung bang efch chon pha dong thich hop. Khi phan tfch cde méu e6 phén tit lugng Iéa nhu cde oligonueleotit, ADN ho&e ARN, tiy diéu kign sé Iva chon trong ede phuong phap loc gel, hydrophobic hay trao di anion. Phuong phap phat hign Axit nucleic va cae hgp chat tuong ty thudng hép thu manh 6 ving tit ngoai, vi véy thudng ding phuong phép quang phé tit ngoai 250+ 260nm dé phat hign. Logi e6t Cae baze nits vd nucleozit ‘Trao di cation: Shimpack WCX-1, Pha déo: Shimpack HRC-ODS, Asahipak ODP-50, Asahipak GS. Nueleotit Trao déi anion; Shimpack WAX-1 Pha dao: Shimpack HRC-ODS, Asahipak GS. ADN vit ARN Lge gel: Shimpack Diol, Asahipak GS ‘Trao déi anion: Shimpack WAX-2, Shimpack PA-DEAE, Shimpack PA-QA, Asabipak ES-502N Hydrophobic: Shimpack HPC Hydroxyapatit: Shimpack HAC 4. Dung Monotacarit Pentoza: Xyloza, arabinoza... Hexoza: Glucoza, fructoza, galactoza... Disacarit: “ Sacaroza, maltoza... Oligosacarit: —_Dextrin... Polysacarit: Tinh bt, xenluloza, pectin...