Dai Cuong Ve HPLC

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Text of Dai Cuong Ve HPLC

AI CNG VE SAC KY LONG

SC K LNG CAO P (HPLC) L G?

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Performance L iquidC hromatography

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Priced L iquidC hromatography

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Pressure L iquidC hromatography

- BT U T NHNG NM 1970 NGI TA S DNG HPLC VO PHN TCH. - N GIA THP NIN 1970 TH K THUT CHO RA NHNG CT PHN TCH MI CHO HPLC V U D LIN TC (ONLINE-DETECTOR). - N NHNG NM 1980 HPLC C S DNG RNG RI TRONG K THUT PHN TCH.

SAC KY (CHROMATOGRAPHY) LA PHNG PHAP TACH DA TREN 2 QUA TRNH * HAP PHU * GIAI HAP PHU XAY RA LIEN TUC GIA 2 PHA: * PHA TNH : THNG LA RAN HOAC LONG * PHA ONG : CHAT LONG (MOT CHAT HOAC HON HP NHIEU CHAT)

KHI NOI VI CAC AU DO (DETECTOR) PHNG PHAP CHO PHEP NH TNH : DA VAO THI GIAN LU NH LNG : DA VAO CHIEU CAO HOAC DIEN TCH PEAK

INJECTORB MO ILE PHASE

CHROMATOGRAPHY

ES A HP Y RA NO T ATS I

DETECTOR

tM

tR1

tR2

tR3

tR4

Start

S O KHOI CUA MOT MAY SAC KY LONG N GIAN

HE ISOCRATIC : THANH PHAN DUNG MOI KHONG OI

S O KHOI CUA MOT MAY SAC KY LONG (tt)CAC BO PHAN C BAN BAO GOM :N CUNG CAP PHA ONG O (MOBILE PHASE) : LABNH LA NG PHA ONG HOAC O HOA DUNG MOI CHAY MAY. CHA MA

S O KHOI CUA MOT MAY SAC KY LONG (tt)CON GOI CO GO LAHETHONG CUNG LA HE THO CAP CA DUNG MOI : LC PUMP. BAO GM HE ISOCRATIC HE H PHA NG GRADIENT

S O KHOI CUA MOT MAY SAC KY LONG (tt)VAN BM MAU (INJECTION VALVE): GOM LOAI BM GO LOA TAY

VAHETHONG BM MAU VA HE THO TONG TO

LOCOT (COLUMN OVEN) : LO CO DUNG EIEU KHIEN DU E IE KHIE NHIET OCOT TACH NHIE O CO TA

S O KHOI CUA MOT MAY SAC KY LONG (tt)

CH : LANI XAY RA CAC QUA XA TRNH TCH CHAT. GOM COT PHAN GO CO TCH VACOT BAO VE VA CO BA VE

S O KHOI CUA MOT MAY SAC KY LONG (tt)AU DO (DETECTOR): CO NHIEU LOAI KHAC NHAU TUY THEO MUC CH PHAN TCH. DUNG E PHAT HIEN CHAT VA NH LNG : UV-VIS, HUYNH QUANG, O DAN, IEN HOA, KHOI PHO

S O KHOI CUA MOT MAY SAC KY LONG (tt)HE THONG GHI NHAN VA X LY TN HIEU (INTERFACE, SOFTWARE, PC) : DUNG E THU THAP VA TNH TOAN CAC KET QUA.

Injector

Column

Mobile phase

Detector

tM

Start

tR1

tR2

tR3

tR4

NGUYEN TAC CUA BM SAC KY LONG

-Gom 2 valve mot chieu :

+ Mot valve mot chieu vao, ch cho phep dung moi vao au bm. Khong cho dung moi ra. + Mot valve mot chieu ra, ch cho phep dung moi ra khoi bm, khong cho dung moi vao. + He thong bm piston chnh xac ieu khien bang mo t bc (step mo t)

HE THONG LOAI NHIEU CUA BM

DUAL PISTON

-Nhieu bm se lam tang nhieu nen do piston di chuyen se tao xung dong. - Giam nhieu bang hai cach : + Dung dch bm qua mot ong ng knh nho e giam xung. + Thay oi ket cau banh rang, s dung bm kep e piston di chuyen nhe va t nhieu.

HE THONG GRADIENTGRADIENT TRONG SAC KY LONG LA G ? -LA S THAY OI THANH PHAN PHA ONG TRONG QUA TRNH CHAY THEO NHNG NH HNG CU THE MUC CH : NHAM TANG CNG KHA NANG TACH CUA CAC CHAT TREN COT. NGUYEN NHAN : + NHIEU CHAT KHONG THE TACH C TREN COT TACH VI MOT THANH PHAN PHA ONG (DO TNG TAC CUA CAC CHAT CAN TACH VI PHA ONG VA PHA TNH GIONG NHAU) + NEU THAY OI THANH PHAN PHA ONG : TNG TAC GIA CHAT PHAN TCH VA PHA TNH, PHA ONG SE THAY OI. V VAY SE TACH C CAC CHAT.

HE THONG GRADIENT-BAO GOM HE GRADIENT AP SUAT THAP VA HE GRADIENT AP SUAT CAO -HE GRADIENT AP SUAT CAO (HIGH PRESSURE GRADIENT: HPGE) CO O CHNH XAC THANH PHAN PHA ONG CAO HN NHNG THIET B AT HN V TOI THIEU CAN 02 BM SAC KY LONG -HE GRADIENT AP SUAT THAP (LOW PRESSURE GRADIENT : LPGE) CO O CHNH XAC THANH PHAN PHA ONG THAP HN NHNG CH CAN MOT BM LC CO THE TRON VA THAY OI 04 DUNG MOI KHAC NHAU.

HE GRADIENT AP SUAT CAO THAP

HE GRADIENT AP SUAT

BO TIEM MAU-DUNG E A MAU VAO HE THONG -GOM VALVE 6 CONG -C CHE HOAT ONG NH SAU : + VALVE TIEM MAU CO VONG MAU (LOOP) CO THE TCH CO NH (THNG 20 uL)

LOOP

DUNG SYRING BM MAU VAO VONG CHA MAU V TR LOAD

BO TIEM MAU (tt)

V TR LOAD, THE TCH MAU CAN THIET SE GI TRONG LOOP, PHAN D SE THAI RA NGOAI. IEU NAY GIUP THE TCH MAU TIEM VAO MAY LUON GIONG NHAU

KHI CHUYEN VE V TR INJECT, MAU T LOOP SE C BM VAO HE THONG E VAO COT TACH

AU DO (DETECTOR)-QUAN TRONG NHAT LA AU DO UV VIS, (PDA-PHOTO DIODE ARRAY). -NGUYEN TAC GIONG NH MAY O UV-VIS THONG THNG -TUY NHIEN CELL O PHAI CO THE TCH NHO (DI 20 uL) E PEAK KHONG B DOAN CHAN VA PHAI CHU C AP SUAT CAO E KHONG B BE

AU DO HUYNH QUANG E PHAN TCH ALFLATOXIN, MYCOTOXIN, AMINO ACID, THUOC TR SAU HO CARBAMATE

Principle of Fluorescence detector(S1) Excited state (S )2

(S3)

excitation

emission

H (fluorescence)

Ground state S0 Mobile phase

AU DO CH SO KHUC XA (RI) DUNG E O CAC LOAI NG N, NG KEP

Khng s dng c ch chy gradient.

AU DO CH SO TN X (ELSD) DUNG E O CAC LOAI NG N, NG KEP

S dng c ch chy gradient.

MT S TNH CHT CA U D UV, HYNH QUANG, RI UV nhy chn lc ng Thng Fluorescence pg Cao RI g Khng xc nh Ln Khng

nh hng Nhit Chy Gradient

Nh c

Nh c

AU DO KHOI PHO (MS) E XAC NH PHAN LN CAC HP CHAT HU C

- Mt t cc (Single Quadrupole). - Ba t cc (Triple Quadrupole). - By ion (Ion trap). - Thi gian bay (Time of Flight-TOF).

KHI PH MT T CC

KHI PH MT T CC

KHI PH MT T CC

KHI PH BA T CC

KHI PH BA T CC

Q0

Q1

Q2

Q3

Precursor ion selection

Fragmentation

N2 CAD Gas

Multiple Reaction Monitoring (MRM)

NHY CA DETECTOR HPLC

CT HPLC

CHT NHI CT HPLC

CAC LOAI COT DUNG TRONG SAC KY LONG RAT NHIEU NHAN HIEU COT KHAC NHAU HIEN BAN TREN TH TRNG: C CHIA LAM NHIEU LOAI THEO MUC CH S DUNG, TRONG O CHU YEU LA : COT PHA AO RP C18 (ODS) COT PHA THNG CAC LOAI COT CHUYEN DUNG CHO TNG NHOM CHAT. COT C18 VA C8 C S DUNG CHO PHAN LN CAC HP CHAT