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 [Link] 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: [Link] molecules enter the pores of the gel, and need a larger volume of eluent. [Link] 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