Chromatography

1st

Contents
1. 2. 3. 4. 5. 6. 7. Introduction Theoretical Considerations Thin Layer Chromatography (KLT) Paper Chromatography Column Chromatography Size Exclusion Chromatography (SEC) High Performance Liquid Chromatography (HPLC) 8. Gas Chromatography (GC) 9. Tandem Techniques 10.Chromatographic Analysis of Environmental and Food Toxicants

Assessment
Midle Test Final Test Lab Practice Quiz 25% 25% 25% 25%

Literatures
1. Chromatographic Methods : A. Braithwaite 2. Chromatography Theory: Jack Cases 3. Chromatographic Detectors Design, Function and Operation: Raymond P 4. Column Handbook for Size Exclusion Chromatography: ChiSan Wu 5. Analytical Gas Chromatography: Jennings 6. Basic Gas Chromatography: Harold M Mc Nair 7. Chromatography Handbook of HPLC: Elena Kantz 8. Other Related Literatures

Chromatography:

An Introduction

Kemampuan yang diharapkan:

i. Mampu menjelaskan pengertian kromatografi ii. Mengetahui sejarah perkembangan kromatografi iii. Mampu menjelaskan klasifikasi dari teknikteknik kromatografi

What Is ChromatographY??
a. Tswett: Chromatography is a method in which the components of a mixture are separated on an adsorbent column in a flowing system b. IUPAC: Chromatography is a physiscal method of separation in which the components to be separated are distributed between 2 phases, one of which is stationary while the other moves in a definite direction c. Simple definition: kromatografi merupakan suatu metode pemisahan komponen dari campurannya berdasarkan distribusi senyawa pada fasa diam dan fasa gerak.

Chromatography
Chromatogram - Detector signal vs. retention time or volume
Detector Signal 1 2

time or volume

Milestone Chromatography
Chromatography (from Greek :chromatos -- color , "graphein" -- to write) 1903 Tswett - plant pigments separated on chalk columns 1931 Lederer & Kuhn - LC of carotenoids 1938 TLC and ion exchange 1950 Reverse phase LC 1954 Martin & Synge (Nobel Prize) 1959 Gel permeation 1965 instrumental LC (Waters)

Tujuan Kromatografi
1. Analisis: Mengetahui komposisi kimia dari suatu sampel 2. Preparatif: pemisahan satu atau lebih senyawa dari campurannya untuk dianalisa lebih lanjut

Aplikasi Kromatografi
Real-life examples of uses for chromatography:
Pharmaceutical Company determine amount of
each chemical found in new product patients blood stream

Hospital detect blood or alcohol levels in a

Law Enforcement to compare a sample found at
a crime scene to samples from suspects pollutants in the water supply

Environmental Agency determine the level of

Manufacturing Plant to purify a chemical needed to make a product

Pemilihan Metode kromatografi

Jenis sampel yang dianalisis Sifat senyawa yang akan dipisahkan Tujuan Instrumen yang tersedia ( kolom. Detektor, dll)

Istilah dalam kromatografi

Term Solvent Definition

Mobile liquid phase with no affinity to the stationary phase (i.e. inert towards it) & no effect on solutes.
Any liquid with more affinity to the stationary phase than the solvent but less than solutes and just capable to move them through the column. Any liquid that passes out of the column. Any liquid that has lesser affinity to the stationary phase than solutes but is capable to move them out of the column. Fraction of eluent containing a required specific substance. (or retardation volume): Volume of mobile phase that passes out of the column, before elution of a specific substance.

Developer

Effluent

Eluent
Eluate Retention volume (VR)

Klasifikasi Kromatografi
1. 2. 3. 4. Berdasarkan prosedur pengembangan Berdasarkan jenis fasa gerak Berdasarkan pasangan fasa gerak-fasa diam Berdasarkan mekanisme pemisahan

Kromatografi berdasarkan metode pengembangan

Elusi Displacement Development Frontal Analysis

Elusi
Terjadi dalam kromatografi partisi Elusi aliran dari fasa gerak (eluting agent) secara terus menerus Syarat fasa gerak: tidak berinteraksi dengan fasa diam atau hanya sedikit diserap oleh fasa diam

Frontal analysis
Larutan campuran ditambahkan secara terus menerus sampai kolom jenuh Komponen yang paling lemah diserap akan keluar lebih dahulu dan didapatkan dalam keadaan murni Tidak bisa diperoleh pemisahan sempurna

Displacement development
Merupakan perpaduan antara elusi dan frontal analysis Sebagian kecil sampel dimasukkan di bagian atas kolom Komponen yang paling kuat serap diserap kolom akan ditambahkan secara terus menerus dari atas Senyawa ini merupakan displacer yang akan mengganti senyawa lain yang kurang diserap

Kromatografi berdasarkan Jenis Fasa Gerak

Kromatografi Fase gerak gas Fase gerak cair

Kromatografi Gas

Krom. Kolom

KLT

Krom. Kertas

KCKT

Kromatotron

KK. Terbuka

KK. Vakum

Mobile Phase
gas (GC) water (LC) organic solvent (LC) supercritical fluid (SCFC)

Kromatografi Berdasarkan Pasangan Fasa Gerak dan Fasa diam

MOBILE PHASE

LIQUID

FORMAT

Liquid-Liquid Chromatography (Partition)

Liquid-Solid Chromatography (Adsorption)

STATIONARY PHASE

Liquid

Solid

Normal Phase Mobile Phase Nonpolar Stationary phase Polar

Reverse Phase Mobile Phase Polar Stationary phase Nonpolar

Normal Phase

Reverse Phase

Gas Fasa diam

Solid

Liquid

GSC

GLC

Kromatografi Berdasarkan Mekanisme Pemisahan

Kromatografi Adsorbsi Kromatografi partisi Kromatohgrafi afinitas Kromatografi penukar ion Kromatografi molekular exclusion

Mode or type
Adsorption Chromatography Partition Chromatography

Stationary phase
Solid that attracts the solutes

Mobile phase
Liquid or gas

Mechanism
Solutes move at different rates according to the forces of attraction to the stationary phase. Solutes equilibrate between the 2 phases according to their partition coefficients Solute ions of charge opposite to the fixed ions are attracted to the resin by electrostatic forces & replace the mobile counterions.

Thin film of liquid Liquid or gas formed on the surface of a solid inert support Solid resin that carries fixed ions & mobile couterions of opposite charge attached by covalent bonds Liquid containing electrolytes

Ion Exchange Chromatography

Molecular Exclusion Chromatography

Porous gel with no Liquid attractive action on solute molecules

Molecules separate according to their size: 1.Smaller molecules enter the pores of the gel, and need a larger volume of eluent. 2.Larger molecules pass through the column at a faster rate. Special kind of solute molecules interact with those immobilized on

Affinity Chromatography

Solid on which specific molecules

Liquid or gas