High Performance Liquid Chromatography (HPLC) SMK Negeri 13 Bandung

CHROMATOGRAPHY Separation procedure of dissolved substance by a dynamic differential migration process in chromatographic syem Chromatographic system consists mobile phase and stationary phase‏ Usage Purification of chemical substance Qualitative and quantitative analysis of component in mixture According to the mobile phasei: -Liquid chromatography (mobile phase is liquid) - Gas chromatography (mobile phase is gas)‏

High Performance Liquid Chromatography (HPLC) : A method of liquid chromatography used to analyze organic compounds which are un volatile and thermolable (unstable on heat Basic principle of HPLC Separation of chemical compound from a mixture based on the physical properties difference

Based on the principle, HPLC divided in: Isocratic HPLC Used on analysis of chemical compound which are need no change of mobile phase composition, temperature ,pressure and flow during the injection and elution in column.. Gradient HPLC Used on chemical compound which are need change of mobile phase composition, temperature ,pressure and flow during the elution in column.

Based on the polarity of mobile phase and stationary phase, HPLC divided in: Normal phase chromatography The stationary phase is more polar than the mobile phase Reversed phase chromatography The mobile phase is more polar than the stationary phase

Chromatography Equipment 1. Eluent bottle 2. Pump 3. Injector 4. Column 5. Detector 6. PC and printer

Main component of HPLC Eluant bottle‏ Pump‏ A bottle for mobile phase contain pure eluant Pump‏ To pump injected sample or eluent. Much of pump have output pressure of 1.000–6.000 psi and produce flow up to 20 mL/ minute

Main component of HPLC Injector Column A place to inject sample in to column Column - Very important part of HPLC Made of plastic called cartridge, or made of stainless steel Consist of stationary phase (octyl, octa decyl, phenyl, nitrile, amine) chemicaly bounded with porous silica particle in the inner column.

Main component of HPLC Detector‏ Computer and printer UV-visible detector, fluorometer (especially for excitation and emission wavelength) and refraction detector Computer and printer Data processor sofware computer, moniter and printer to show chromatogram (graphic of sample absorbance to time)‏

High Performance Liquid Chromatography

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