thong may HPLC 3. u dd Dau dé phai thoa man dieu kién: A=kC A: tin hiéu do ctia chat phéi C: nong dé ctia chat phan tick kz hiing s6 ctia diéu kién thee nghigm cia dau dé da chon; Tin higu do A cé thé: - Dé hap thu quang phan tr ctia chat phan tich; - Cuong d6 phat xa huynh quang cia chat phan tich; - Cuéng d6 phat xa vach phé nguyén tir; Loai do phé kh6i (cudng 46 ding khéi m/Z); ‘Cung d6 dong digén, dién thé do chat phan tich géy ra Dé din dign, d6 din nhiét, chiét suat, ... thong may HPLC B.1TDau di Dau do la mot bo phan thu nhan va phat hign cde chat hay hop chat phan tich dya theo mot tinh chat héa hoe hay héa ly’ hay tinh chat vat ly nao d6 cia chat phan tich. Yéu cau: - Khoang tuyén tinh (nong dé - tin higu) rong: - C646 nhay cao véi chat phan tich; Phai hoat dng 6n dinh va bén ving véi diéu kign nhat dinh ya d6 nhigu phai nho.thong may HPLC TNL Die dé Ubtray jolet /V ving UV -VIS Former ow cet samaie-nce ’ Reference site ‘ouput or data igieracent rg $8ct on Onicessno. ‘D, fama 90 3¢0rm) ‘enor (re ignt source s selected withthe angle ofthe micron) Figura 4. Disgramrrati illustrator of 2 WMO detector opteal eystom Be phan phat hign UV-VIS: = Sit dung duoc trong toaimg hop chay gradient, kh6ng pha buy: mau, khong bi anh hudng bai nhiét do. + Dap ding kém y6i cdc hop chat khéng hip thu UV. [¢ thong may HPLC 3.1 PR do 3.1.7.1 Dau do Ultraviolet / Visibie Diy lt dau dd sit dung phd bién nhdt trong HPLC dua hap thu bite xa UV- VIS cia chat phan tich, Ba cau hi dau dd hp thu UV-VIS: «. Pu do do & bude séng nhat dink Neuén sang la dén thuy ngan va dén Wonfarm ciing véi kinh loc dé chon mét bude séng niu 254 nm, 280 nm, 334 nm, 436 nn Han ché: Dau dé loai nay kém nhay nhung ré tién Doi voi céc chat phan tich khéng hap thu quang tai bude sOng nay thi khéng phat hign duge.é thong may HPLC 3 LTD Dau do Ultraviolet / Visible Lambert-Beer’s law:A=eC/ Concentration Nhuge diém: Loai iu dé nay khd kén chon hop chit hip thu UV rit kém, bude sing e& dinh Djnh lujt Lambert-beer 6 giéi han: Dung bigu dign mite hip thu A theo ning cua chit thuimg chi cé tinh tuyén tinh trong khong hep ve ning 6. 1.7.3 Dau dd huynh quang (Fluorescence detector) Te pjnh lugng dya trén co so: Iq=k.Cl Trong dé: k = 1,.0.E; 1,: Cuong do chim tia kich thich; 4: Higu suat huynh quang; E: Hé sé huynh quang cua phan tir chat phan tich.PHAP HPLC @ thong may HPLC BA. a hugnh quang (Fiiorescence detector) - Ding dé phiit hign cdc chat hitu co chira huynh quang ty nhién va cfc dan xuat c6 hujnh quang. Lad mét wong nhing detector ding wong LC cé.d6 whay.cao gdp “100-1000 lan so yai UV-VIS ya dé chon. loc cao. Fluorescence Detector ee ¢ thong may HPLC 3.1.7.2 Dau dd PDA — Photodiode Array - Vang lam viée rong va cé tinh 3 chiéu (d6 hap thu, bude song, thdi gian). - Dé nhay va d§ chon Igc cao. - Han ché su chen lan va tring song hap thu.i thong may HPLC 3 Dau do chi s6 khiic x@ (Refractive Index detector) n tc hoat dong: dua trén su khdc nhau gitra chi sé khac xa gitta pha dng va pha dong chita chit phan tich sau khi qué tinh tach qua cét sic ky. pnt eouree lamp Light coving ‘elernent Hinh: Bau do Khive xa }) nhay cia b6 phan phat hign khéc xa vi sai phu thudc vdo mhiéw yéu 16 nhc phiét a6, dp sudt va sw thay d6i thanh phan dung méi pha déng nén khong ding thi chay gradient Uegidung: Dé phan tich cdc loai duéng. Cac kiéu sic ky long higu ning cao Sic ky phan bé (bao gdm sic ky ghép cap ion) Sic ky hap phy Sac ky trao déi ion Sic ky loai cé Sic ky cdc déng pha quang hoc Sic ky di lucCac kiéu sic ky long higu nang cao 3.1 Sic ky hip phy (Adsorption Chromatography) éng trinh nghién ciru dau tién ky la qua trinh hap phy ciia chat phan tich lén bé mat chat ran - Pha tinh (a chat ran ma t cla né c6 kha nang hp phy; - Pha dong li chdt ling hogc khi. > Ag Ky » C) yr. 7 ® sinteractionsite; X, ¥ groups having different adsorption group energy inh: Corché hip ph a tinh 1a Silic oxyd (silica) va nhém oxyd (alumina): Silica trén bé mat cé nhém silanol & dang ty do. O dinh trong pH = 2-8 Alunina: ding tach céc base hitu co (anilin, pyridine..), On dinh trong - Pha tinh /@ chat rin ma bé mat ciia né c6 kha nang hap phy; Pha dong la chat léng hofic khi. Baar eae KO) OHO > * cinteractionsite; X, ¥ : groups having different adsorption group energy Hinh: Co ché hp phy a tinh 1a Silie oxyd (silica) va nhom oxyd (alumina): Silica trén bé mat c6 nhém silanol & dang ty do. O dinh trong pH = 2-8 Alunina: ding tach cic base hitu co (anilin, pyridine..), On dinh trongABsorption/partition ADsorption Hinh: Phan biét hdp thu va hdp phu Ving dung: Cac hop chat c6 khdi lugng phan tir duéi M<5000dvC, it fan trong nuéc hoac hén hgp hitu co nuée dung méi hitu co fac ky hdp phu cé thé manh trong vige tach déng phan vi tri 6 céc Bebop endt hieu co wong phap HPLC ¥longhiguningeao 3.3.2 Siic ky phiin bé (Liquid - Chromatography) ky, trong d6 chat phan tich duge tach riéng ra do sw can bang vé su phan bd solute disoiel é ia ji «lnliguid phase cua chat tan gitta pha tinh Lea chat long (chat long nay duge of solid support bao trén be mat mot chat ran si tro, khong tham gia vao qua trinh sac ky goi la chat mang) a pha déng co thé ling hoac hi. Hinh: Co ché phin bé-1 Se ky hap phy (Adsorption C hromatography) Pha déng: Trong sac ky hap phy pha dong anh huéng 16 rét dén hé phan bé cua chat phan tich. Dé ria gidi chat da hap phu, ding pl déng cé site ria gidi c,, ©, cdng lén, site nta gidi cing manh Bang: thong 6 lwe rita gidi cta ede dung mat Toren Toluene PO consid Pern Rctitad STUNDE eel 8 CURL Peroni t Methanol