Sc k lng

Phng php tchCc cu t c tch phn b gia pha tnh v pha ng

Mobile phase (Pha ng)Stationary phase (Pha tnh)1. Khi nim v k thut sc k lng

Qu trnh tch da vo tnh cht ha hc, vt l v ha l ca cc cht.Da trn 2 qu trnh:Hp phGii hp phXy ra lin tc gia 2 pha: Pha tnh: cht rn hoc lng Pha ng: cht lng (1 cht hoc hn hp nhiu cht)

Pha ng: ha tan v di chuyn cht phn tchPha tnh: gi cht phn tchSKL chia thnh 2 nhmSK lng p sut thng (sc k c in)SK lng p sut cao (SKL hiu nng cao: HPLC)(High Performance Liquid Chromatography)

Da vo bn cht ca qu trnh sc k, HPLC: - SK phn b - SK pha thng (normal phase chromatography - SK pha o (reversed phase chromatography) - Sk trao i ion (ion exchange chromatography) - SK ghp cp ion (ion pair chromatography)

Pha ng: LngPha tnh: Lng

Pha ng: lng Pha tnh: Rn

SK lng lng (LLC)(Liquid liquid chromatography)SK lng rn (LSC)(Liquid solid chromatography)- Da vo trng thi pha tnh

Khi ni vi u do (detector), HPLC cho php:nh tnh: da vo thi gian lunh lng: da vo chiu cao hoc din tch peak

2. Nguyn tc cu to ca h thng my HPLC

0: Ngun cung cp pha ng (mobile phase)- Bnh cha pha ng

123450

1: Bm cao p (h thng cung cp dung mi)Bm pha ng vo ct tchiu khin tc dng, p sut ca pha ng

2. Van bm mu (Injection valve):Bm mu PT vo ct tch theo nhng lng mu nht nh

Tim mu bng tayTim mu t ng (Auto sampler)

3: Ct tch (Column)Ct cha pha tnhYu t quyt nh qu trnh tch sc k Ct tch c kch c khc nhau Chiu di: 10 25cm ng knh: 2 5mm

4: u d (detector)Thit b pht hin cht phn tch (nh tnh v nh lng)C nhiu loi khc nhau ty mc ch phn tch: UV-VIS, Huynh Quang, o Dan, ien Hoa, Khoi Pho,

5. H thng ghi nhn v x l tn hiu:Thu thp v x l kt quRecorder, Computer + printer, software

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InjectorDetectorColumnMobile phaseH thng HPLC n gin

Detector UV-Vis: detector ph hp thu phn tXv nh cc cht c kh nng hp thu quang Hunh quang (Fluorescence detector): xc nh cc cht c kh nng pht hunh quang- Alflatoxin, Mycotoxin, Amino Acid, thuc tr su h Carbamate,. u d ch s khc x (Refractive Index Detector: RI)

u d dn (Conductivity detector): Xc nh cc ion v c, hu c u d khi ph (MS: mass spectrometry)Xc nh phn ln cc cht hu c

3. Cc qu trnh tch trong sc k lngQu trnh quan trng nht trong phng php sc kNhng cn bng ng xy ra gia pha tnh v pha ng trong ct sc kL s vn chuyn v phn b lin tc ca cht PT t u ct n cui ct

Cht phn tch lun phn b gia 2 pha, trong pha ng lun chy qua ct tch vi mt tc nht nh hoc gradientHiu qu ca qu trnh tch ph thuc rt nhiu vo tng tc gia cc cht trong pha tnh v pha ngMc ch chnh ca sc k l tch v nh tnh cc cht trong hn hp cht phc tp

Thi gian cht PT b pha tnh lu gi (thi gian lu) quyt nh bi: Bn cht ca pha tnh, cu trc v tnh cht ca cht PT Bn cht v thnh phn ca pha ng dng ra gii cht PT ra khi ct sc k (pha tnh)

Ghi li ton b qu trnh tch sc k ca hn hp cht PT sc k gm nhiu peak.c im ca peak PT:Cc peak c th tch ri nhau hon tonChp nhau mt phnChp nhau hon ton

Sc k phn nh qu trnh tch sc k trong ct tt hay khng tt.Tch tt: hn hp c bao nhiu cht c by nhiu peak ring bit khng chp nhauCht no b lu gi mnh s c ra gii ra sau cng, cht lu gi km s ra trc

4. u im ca phng php sc kC th phn tch ng thi nhiu hp chtKhng cn lm bay hi mu- phn gii cao nh qu trnh tch trn ct - nhy cao (ppm-ppb) nh u d- Th tch mu phn tch nh (1-100L)

Hn hp cht tch khi nhau th no ? Pha tnhFlow

Ti sao li c s khc nhau?MnhDo lc tng tc khc nhauYu

Cc cn bng trong ct HPLCMu PT i qua ct: tn ti ng thi 3 thnh phn:SP khng chuyn ngCht PT va chuyn ng t u ct n cui ct, ng thi phn b li gia SP v MP:B SP hp ph, ri ra gii bi MPMP: dung mi ra gii v chuyn ngTng tc vi cc cht, li ko v mang cht PT ra khi ct

3 tng tc chnh:S tng tc v cn bng ca cht PT vi SPS tng tc v cn bng ca cht PT vi MPS tng tc ca SP v MP

PTSPMPFaFbFc

Ftot = Fa + Fb + FcCht no c lc tng tc ln nht s b gi li lu trn ctCht no c F nh b ra gii u tinFtot khc nhau nhiu th qu trnh tch tt

Qu trnh tch din ra trong ct sc kcolumnVt liu nhi ct 3- 5m

columnmixed sampleQu trnh tchMobile phase

5. Sc k lng pha thng v pha o

Ct nhi trong sc k pha thng- Ct silica trung tnh:

B mt c cha nhm phn cc (a nc): -OHXc nh cc cht khng phn cc hoc t phn cc

SiSi

Ct silica trn nn mch carbon:-Si-CH2CH2CH2CN-Si-CH2CH2CH2NH2-Si-CH2CH2CH2OCH(OH)-CH2(OH)+ Ct cyano: a mc ch+ Ct amino: phn tch ng+ Ct diol: protein

Dung mi ch yu: khng phn cc+ Hydrocarbon: hexan, pentan, octan+ Aromatic hydrocarbon: benzen, toluen, xylen,+ Chloroform CHCl3+ Methylene chloride CH2Cl2Dung mi ph: phn cc hoc hi phn cc+ Ethanol, methanol,.

Lin kt hydrogen v thi gian luSiOHSiOHMnhYuRt yuPhn cc1221Rt

Lin kt hydrogen Mu phn tch c:COOH: nhm carboxylOH : nhm hydroxylNH2 : nhm amino Mu phn tch c nhm tert-butyl hoc cc nhm khng phn cc ln LK hydrogen s yu

Lin kt hydrogen mnh

Ct nhi trong sc k pha oCt: khng phn ccPha ng: phn ccCt silica alkyl ha cc nhm OH trn b mt silica trung tnh:Ct C18 (ODS)Ct C8 (octyl)Ct C4 (butyl)Ct phenylB mt khng phn cc hay t phn cc(k nc)

Xc nh cht phn cc, khng phn cc v t phn cc

Thi gian lu v lin kt k ncC18 (ODS) MnhYu1212

Dung mi trong HPLC pha oDung mi phn cc:Methanol (CH3OH: MEOH), acetonitrile (CH3CN: ACN)Dung dch m Ti u ha t l dd m/ dung mi rt quan trng qu trnh tch tt

So snh pha thng v pha o

ng dng ca HPLCCh yu xc nh cc hp cht hu c kh bay hi trong nhiu i tng khc nhau:+ Amino acid+ Acid hu c+ Thuc tr su+ .

Sc k ion (Ion Chromatography) Ion ExchangeN+RRRSampleSO3-Sample+++++++++++++Lc ion

Phn tch cc hp cht ionC 2 loi ct:+ Ct trao i cationStrong cation exchange: (R-SO3-)Weak cation exchange: (R-COO-)+ Ct trao i anion:Strong anion exchange: (R4N+)Weak anion exchange:diethyl aminoethyl)

Pha ng: thng l dung dch m trong dung mi ncAnion:Carbonat/bicarbonate (Na2CO3/NaHCO3)Potassium hydroxide (KOH)Cation: Nitric acidTartaric acidTartaric acid/dipicolinic acid Tartaric acid/citric acid

n tpKhi nim v phn tch nh lngTnh ton v x l s liu phn tchPhng php phn tch dng cPP ph hp th phn tPP ph nguyn tPP in haPP sc k lng4. Cc bi thc tp, tnh ton s liu t s o thc nghim

Inject syringe