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HPLC

(High Performance Liquid


Chromatography)
M. H. A
Outline

 Definition
 Principle
 Separation
 HPLC Applications
HPLC

 Kata kromatogrfi berasal dari bahasa Greek yaitu Chromato dan grafe yaitu
penulisan dengan warna (writing with colors).
 Prinsip: kromatografi merupakan cara pemisahan yang mendasarkan pada
partisi cuplikan antara fase bergerak (mobile phase) dan fase diam (stationary
phase).
Definition
 High-performance liquid
chromatography is a technique
in analytical chemistry used to separate,
identify, and quantify each component in a
mixture. It relies on pumps to pass a
pressurized liquid solvent containing the
sample mixture through a column filled
with a solid adsorbent material
Chromatography

 Ion Exchange muatan pada senyawa


 Gel Permeation column (GPC) ukuran partikel
 TLC/HPLC like disolvlike
 Ligan berdasarkan bentukan spesifik
Colomn HPLC

 Normal Phase (Fase normal)


 Fase diam polar dan fase gerak non polar
 Contoh Fase gerak: Chloroform, heksana dll.
 Reversed Phase (Fase terbalik)
 Fase diam non polar dan fase gerak polar
 Contoh fase gerak: air, metanol, asetonitril dll.
principle
Principle

Colomn HPLC/stationary phase


 Polar (“Normal” Phase):
 Silica, alumina

 Non-Polar (“Reversed Phase”)


 ODS Silica gel
 C18, C8
Principle
Solvent/The Mobile Phase
• Normal chromatography
Hexane ; dichloromethane; isopropanol; methanol

Increasing strength

• Reverse phase chromatography


water ; methanol; acetonitrile; tetrahydrofuran (THF)
Increasing strength
Comparing Chromatography to the Flow of a
River...

Light leaf
Water flow
Heavy stone

Base
Mobile Phase / Stationary Phase

• A site in which a moving


phase (mobile phase) and a
non-moving phase
Mobile (stationary phase) make
phase contact via an interface
Strong Weak that is set up.
• The affinity with the
mobile phase and
Stationary stationary phase varies with
phase the solute.  Separation
occurs due to differences in
the speed of motion.
Prinsip pemisahan
Separation
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents

High Performance Liquid Chromatograph


Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
Separations
Injector Chromatogram

Mixer mAU

Pumps

Start Injection time

Column

Detector

Solvents
The Chromatogram

to - elution time of unretained peak


tR- retention time - determines sample identity
tR

tR
mAU Area or height is proportional
to the quantity of analyte.

to

Injection time
Jenis Detektor HPLC
 UV-Vis Detector
 Photodiode array Detector (PDA = DAD)
 Refractive Index Detector (RID)
 Fluorescence Detector (FLD)
 Evaporative Light Scattering Detector (ELSD)
 Electrochemical Detector (ECD)
 Conductivity Detector
 Radiometric Detector
Hyphenated Systems
 LC-MS;
 LC-MS/MS; LC-NMR
PERHITUNGAN HPLC
Parameter
Area Theoritical Resolution Retention
HETP K'
Plate (Rs) Time (Rt)
67043 4944.345 50.563 427.488 3.646 4.322
267071 4742.723 52.712 426.925 3.198 4.332
465446 3450.100 72.462 433.095 2.892 4.341
MIN MIN
2000 1,8
THANK YOU

Have a Great Day!!

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