Created by Nick Buker 12/18/08

Created by Nick Buker 12/18/08
Chromatography Created by Nick Buker 12/18/08

Chromatography Merupakan teknik yg digunakan untuk memisahkan dan mengidentifikasi komponen dari suatu campuran . Molekul yg ada dalam campuran didistribusikan masing-masing diantara medium stasioner dan mobile. Molekul yg paling lama di fase gerak lebih cepat mengalir Created by Nick Buker 12/18/08

Metode pemisahan campuran molekul tergantung pada distribusinya diantara fase gerak dan fase diam Fase gerak (juga disebut sebagai pelarut ) bisa berupa cairan atau gas. Fase diam (juga disebut sebagai sorbent) dapat berupa padatan atau cairan, fase diam is held stationary oleh suatu padatan. Padatan yg mempertahankan fase diam cair merupakan matrik support. Molekul-molekul dalam kondisi tercampur yg dapat dipisahkan disebut solut. Created by Nick Buker 12/18/08

Apa itu Chromatography? Chromatography: metode pemisahan yg kuat yg biasanya terdiri dari fase gerak dan fase diam Kolom Fase gerak Elusi Fase diam Sampel Sampel dipisahkan pada kolom berdasar perbedaan dalam interaksi molekuler dari jenis molekul analit yg berbeda dengan fase gerak dan fase diam Created by Nick Buker 12/18/08

Bagaimana Chromatography Bekerja
Fase Diam/stasioner – Padatan tidak bergerak Fase Gerak – Larutan yg bergerak Created by Nick Buker 12/18/08

Chromatography dan sungai
Dua kelompok orang yg terbawa arus sungai : Kelompok kuat akan berpegangan di batu Kelompok lemah akan terbawa arus sungai lebih cepat Created by Nick Buker 12/18/08

Perenang dan molekul Kemampuan berpegangan ke batu akan mengontrol kecepatan perenang terbawa arus sungai Kemampuan berinteraksi ke padatan di fase stasioner akan mengontrol kecepatan molekul terbawa pelarut Created by Nick Buker 12/18/08

Chromatography Kolom Dilution & Peak broadening! Chromatogram

Chromatography: Pemisahan puncak

Chromatography: Resolusi puncak
Resolution jelek More separation Lass band spread

Chromatography Resolusi

RESOLUSI Created by Nick Buker 12/18/08

Retention Time Waktu yg diperlukan bagi sampel untuk bergerak dari bagian injeksi melewati kolom menuju detektor . Created by Nick Buker 12/18/08

Chromatography Retention Times
tM = retention time fase gerak (dead time) tR = retention time analit (solute) L = panjang kolom

Ekpresi hasil Rf= Jarak migrasi solute Jarak pergerakan solvent
Istilah "Rf" (relative flow) digunakan untuk mengekspresikan performance suatu solute dalam suatu solvent system /support medium. Istilah nilai Rf didefinisikan sbg jarak komponen mengalami migrasi dengan jarak migrasi pelarut. Nilai Rf konstan untuk komponen tertentu, sistem pelarut dan matrik tidak terlarut. Rf= Jarak migrasi solute Jarak pergerakan solvent Created by Nick Buker 12/18/08

Distribution Coefficient (Equilibrium Distribution ) Definition: Different affinity of these 2 components to stationary phase causes the separation. Concentration of component A in stationary phase Concentration of component A in mobile phase Created by Nick Buker 12/18/08

Pemisahan senyawa dg chromatography tergantung pada beberapa faktor : Partisi suatu solute diantara pergerakan fase pelarut dan fase cair stasioner. Solute bergerak sesuai aliran pelarut pada kecepatan yg ditentukan oleh kelarutan solute dalam fase bergerak. Jadi, komponen dg mobilitas tinggi lebih terikat ke fase organik bergerak daripada fase diam. Created by Nick Buker 12/18/08

Cont.. Pengaruh Ion exchange : Banyak impuritas terion dalam support medium akan cenderung mengikat atau tertarik ion yg bermuatan berbalikan (solutes) dan oleh karena itu akan mengurangi mobilitas solut tersebut. Suhu : Juga merupakan faktor penting, Karena suhu dapat mempengaruhi kelarutan solut dalam suhu. Created by Nick Buker 12/18/08

Cont.. Berat molekul solute juga mempengaruhi kelarutan sehingga mempengaruhi performance chromatographic . Adsorpsi komponen (solute) ke support medium: Jika solut cenderung mengikat support medium, hal ini akan memperlambat mobilitasnya dalam solvent system. Created by Nick Buker 12/18/08

Cont.. Komposisi solvent: karena banyak komponen yg lebih larut dalam suatu pelarut daripada pelarut lainnya, campuran pelarut yg digunakan akan mempengaruhi pemisahan komponen. Created by Nick Buker 12/18/08

Structur molekuler dan Polaritas
Struktur Molekular mempengaruhi kecepatan Polaritas bertanggung jawab thd interaksi Polaritas Molekular dibangkitkan oleh area muatan Created by Nick Buker 12/18/08

PELARUT Pelarut Polar Water > Methanol > Acetonitrile > Ethanol > Oxydipropionitrile Pelarut Non-polar N-Decane > N-Hexane > N-Pentane > Cyclohexane Created by Nick Buker 12/18/08

Jenis chromatography Chromatography Partisi Chromatography Adsorbsi Gel filtrasi Ion exchange chromatography Created by Nick Buker 12/18/08

Jenis Chromatography Created by Nick Buker 12/18/08

Chromatography

Chromatography Partisi
Distribusi terlarut diantara 2 fase yg berbeda polaritasnya. Solute akan diditribusi dg sendirinya diantara 2 fase berdasarkan kelarutannya dalam masing-masing fase, hal ini disebut partisi. Created by Nick Buker 12/18/08

Contoh chromatography partisi
Dua jenis chromatography partisi yg paling umum adalah thin layer chromatography/TLC dan chromatography kertas. Fase stasioner kedua jenis tersebut merupakan cairan yang terikat ke suatu matrik. Pada chromatography kertas, fase stationary merupakan molekul air yang terikat ke matrik selulosa. Created by Nick Buker 12/18/08

Pada TLC, Fase diamnya berupa pelarut yg ditambahkan ke support untuk membentuk lapisan tipis sehingga pelarut dapat terikat ke matrik (support). Chromatography partisi utamanya digunakan untuk pemisahan molekul dengan berat molekul kecil . Created by Nick Buker 12/18/08

Chromatography kertas
Support sellulosa mengandung air terikat dalam jumlah banyak. Partitioning terjadi diantara air terikat yg berfungsi sbg fase diamnya dan pelarut merupakan fase geraknya. Created by Nick Buker 12/18/08

Deteksi spot Spots pada kertas kromatogram dapat dideteksi dengan 4 cara: Melalui warna alaminya Melalui fluorescencenya Melalui reaksi kimia yg terjadi setelah kertas dapat dispray dg berbagai reagent sbg contoh: selama paper chromatography amino acids, kromatogram dispray dg ninhydrin. Melalui radioaktifitas Created by Nick Buker 12/18/08

Identifikasi spot Spots biasanya diidentifikasi melalui perbandingan standart yg diketahui sbg nilai Rf . Created by Nick Buker 12/18/08

Thin layer chromatography
Dalam TLC, beberapa senyawa yg dapat dipisah dengan baik dan dibentuk menjadi lapisan seragam . Baik senyawa organik maupun anorganik dapat digunakan untuk membentuk lapisan seragam untuk TLC. Senyawa organik meliputi: cellulose, polyamide, polyethylene Anorganik : silica gel, aluminum oxide and magnesium silicate Created by Nick Buker 12/18/08

TLC Fase diamnya berupa pelarut yg digunakan untuk membentuk lapisan sorbent disebar seragam diatas permukaan plate plastik atau kaca Created by Nick Buker 12/18/08

TLC Fase gerak berupa cairan Aliran sampel terjadi pada thin layer pada solid support. Substansi yg kurang tertarik ke padatan atau lebih larut dalam cairan bergerak lebih cepat Created by Nick Buker 12/18/08

TLC Fase diam > Silica Gel Fase gerak > pelarut Created by Nick Buker 12/18/08

TLC Created by Nick Buker 12/18/08

Kelebihan TLC dibanding paper chromatography
Resolving power lebih besar karena adanya difusi spot yg lebih rendah. Kecepatan pemisahan lebih tinggi Pilihan bahan sbg sorbents lebih banyak Created by Nick Buker 12/18/08

LIQUID COLUMN CHROMATOGRAPHY Campuran sampel melewati suatu kolom yang dilapisi dengan partikel padat yang dapat atau tidak dapat di coating dengan cairan lainnya . Dengan pelarut yg sesuai, kondisi pengemasan, beberapa komponen dalam sampel akan bergerak menuju kolom lebih lambat daripada lainnya yang menghasilkan pemisahan sesuai dengan keinginan. Created by Nick Buker 12/18/08

Schematic Diagram of Liquid Chromatography
Created by Nick Buker 12/18/08

Diagram of Simple Liquid Column Chromatography Created by Nick Buker 12/18/08

EMPAT DASAR LIQUID CHROMATOGRAPHY 4 dasar model liquid chromatography dinamakan sesuai dengan mekanisme yg digunakan : 1. Liquid/Solid Chromatography ( chromatography adsorpsi) A. Normal Phase LSC B. Reverse Phase LSC 2. Liquid/Liquid Chromatography ( chromatography partisi) A. Normal Phase LLC B. Reverse Phase LLC 3. Ion Exchange Chromatography 4. Gel Permeation Chromatography ( chromatography exclusi) Created by Nick Buker 12/18/08

LIQUID SOLID CHROMATOGRAPHY Mekanisme pemisahan dalam LSC didasarkan pada kompetisi komponen dari campuran sampel bagi sisi aktif pada absorbent seperti Silica Gel. Created by Nick Buker 12/18/08

LIQUID SOLID CHROMATOGRAPHY Created by Nick Buker 12/18/08

VITAMINS LARUT AIR Created by Nick Buker 12/18/08

LIQUID-LIQUID CHROMATOGRAPHY Permukaan padatan diam dilapisi dengan pelarut kedua (fase diam) yg immiscible dalam pelarut (fase gerak). Partisi sampel diantara 2 fase menunda atau mempertahankan beberapa komponen daripada komponen lainnya untuk mempengaruhi pemisahan. Created by Nick Buker 12/18/08

Pembuat Gradient Created by Nick Buker 12/18/08

Bahan Reversed phase Created by Nick Buker 12/18/08

Prinsip RP Created by Nick Buker 12/18/08

ION-EXCHANGE CHROMATOGRAPHY
Pemisahan Ion-exchange Chromatography didasarkan pada kompetisi komponen ionik yg berbeda dari suatu sampel bagi sisi aktif di ion-exchange resin (column-packing). Created by Nick Buker 12/18/08

MEKANISME ION-EXCHANGE CHROMATOGRAPHY DARI ASAM AMINO

Chromatography Asam Amino Created by Nick Buker 12/18/08

Ion exchange Created by Nick Buker 12/18/08

CM and DEAE Created by Nick Buker 12/18/08

Jenis ion exchange Created by Nick Buker 12/18/08

Stepwise elution for ion exchange

GEL-PERMEATION CHROMATOGRAPHY
Gel-Permeation Chromatography merupakan suatu mekanisme sorting molekul-molekul berdasarkan ukuran molekul dalam larutan . Molekul kecil dapat melewati/ permeate lebih more pores , oleh karena itu, dipertahankan lebih lama daripada molekul besar. Created by Nick Buker 12/18/08

Gel filtration Created by Nick Buker 12/18/08

350 min on 60 cm 75 micron ID 3.5 beads 100 nl/min

SPPVGPAL TIC MS MS/MS Created by Nick Buker 12/18/08

LC-MS display of differently expressed proteins
Differently expressed peptide M/Z Time Time Created by Nick Buker 12/18/08

Gas Liquid Chromatography
Fase geraknya berupa gas yang tdk reaktif (seperti Nitrogen) yg mengalir melalui suatu tabung Dan fase diamnya berupa liquid tidak volatil held on particles of a solid support. Prinsip Partisi molekul diantara gas (fase gerak) dan cairan (fase diam ). Created by Nick Buker 12/18/08

Fase Diam yg paling umum
1. Pemisahan campuran komponen polar Carbowax 20M (polyethylene glycol) 2. Pemisahan campuran komponen non polar OV101 or SE-30 (polymer of methylsilicone) Methylester dari asam lemak DEGS (diethylene glycol succinate) Created by Nick Buker 12/18/08

Pada animasi dibawah ini molekul yg berwarna merah lebih mudah larut dalam cairan (atau kurang volatil) daripada molekul yg berwarna hijau. Created by Nick Buker 12/18/08

In practice the Column is contained in a thermostatic oven. (Why ?) About 1μL of liquid is injected into one end of the column. As each component reaches the other end it is detected and registered on a chart recorder. The Retention Time is characteristic of a particular substance. (for the same column, temperature, gas flow etc.) The area under each peak indicates the relative quantities. Created by Nick Buker 12/18/08

Injection port Recorder Oven Detector Column Nitrogen cylinder Created by Nick Buker 12/18/08

Chromatogram of petrol
Suggest identities of some of the unlabelled peaks. Created by Nick Buker 12/18/08

Gas Chromatography Filters/Traps Data system H RESET Regulators Syringe/Sampler Air Hydrogen Gas Carrier Inlets gas system inlet column detector data system Detectors Column Created by Nick Buker 12/18/08

Skema Diagram Gas Chromatography Created by Nick Buker 12/18/08

DETEKTOR Flame Ionization Detector (Nanogram - ng) High temperature of hydrogen flame (H2 +O2 + N2) ionizes compounds eluted from column into flame. The ions collected on collector or electrode and were recorded on recorder due to electric current. Created by Nick Buker 12/18/08

Skema Diagram Flame Ionization Detector Exhaust Chimney Igniter Hydrogen Inlet Column Effluent Polarizing Electrode Collector Electrode Created by Nick Buker 12/18/08

Skema Diagram Flame Ionization Detector
Collector Detector electronics  volts Flame Chassis ground Jet Signal output Column Created by Nick Buker 12/18/08

Thermal Conductivity Detector Mengukur perubahan konduktifitas thermal berkaitan dengan sampel (mg). Sampel dapat direcovery. Created by Nick Buker 12/18/08

Detektor Konduktifitas Thermal
Principal: The thermal balance of a heated filament Electrical power is converted to heat in a resistant filament and the temperature will climb until heat power loss form the filament equals the electrical power input. The filament may loose heat by radiation to a cooler surface and by conduction to the molecules coming into contact with it. Created by Nick Buker 12/18/08

Dasar Konduktifitas Thermal
The TCD is a nondestructive, concentration sensing detector. A heated filament is cooled by the flow of carrier gas When the carrier gas is contaminated by sample , the cooling effect of the gas changes. The difference in cooling is used to generate the detector signal. Flow Flow Created by Nick Buker 12/18/08

When a compound elutes, the thermal conductivity of the gaseous mixture of carrier gas and compound gas is lowered, and the filament in the sample column becomes hotter than the other control column. Its resistance increased, and this imbalance between control and sample filament resistances is measured by a simple gadget and a signal is recorded Created by Nick Buker 12/18/08

Konduktifitas Thermal Relative
Compound Relative Thermal Conductivity Carbon Tetrachloride 0.05 Benzene 0.11 Hexane 0.12 Argon Methanol 0.13 Nitrogen 0.17 Helium 1.00 Hydrogen 1.28 Created by Nick Buker 12/18/08

Responds to all compounds Adequate sensitivity for many compounds Good linear range of signal Simple construction Signal quite stable provided carrier gas glow rate, block temperature, and filament power are controlled Nondestructive detection Created by Nick Buker 12/18/08

Detektor Electron Capture
For pesticide analysis (picogram). Accept electrons of carrier gas. Created by Nick Buker 12/18/08

ECD detects ions in the exiting from the gas chromatographic column by the anode electrode. 3H or 63Ni which emits  particles. Ionization : N2 (Nitrogen carrier gas) +  (e) = N2+ + 2e These N2+ establish a “base line” X (F, Cl and Br) containing sample +  (e)  X- Ion recombination : X- + N2+ = X + N2 The “base line” will decrease and this decrease constitutes the signal. Insecticides, pesticides, vinyl chloride, and fluorocarbons Created by Nick Buker 12/18/08

Gas Chromatography Application

ANALYSIS ASAM LEMAK SEMI- QUANTITATIVE

IDENTIFIKASI KOMPNEN YG TDK DIKETAHUI TENTATIVE

Retention Times Created by Nick Buker 12/18/08

KELEBIHAN GLC 1. Pemisahan sangat baik 2. Waktu (analysis singkat ) 3. Membutuhkan sampel sedikit - ml 4. Sistem deteksi bagus 5. Analisis Quantitativ Created by Nick Buker 12/18/08

KEKURANGAN GAS CHROMATOGRAPHY
Bahan harus dibuat volatil pada suhu 250C tanpa terjadi dekomposisi . Created by Nick Buker 12/18/08

Gas Chromatogram of Methyl Esters of Fatty Acids

The Effects of OH groups of Carbohydrates
HO CH 2 1 3 4 5 6 OH O HO CH 2 1 3 4 5 6 Created by Nick Buker 12/18/08

Derivation of Glucose with Trimethylchlorosilane
OH O HO CH 2 1 3 4 5 6 Si CH 3 5Cl + Glucose Trimethylchlorosilane 6 CH O-Si(CH3)3 2 O 5 + 5HCl 4 1 O-Si(CH3)3 (CH3)3-Si-O O-Si(CH3)3 3 2 O-Si(CH3)3 Created by Nick Buker 12/18/08

Pengaruh Derivatisasi
1. Butuh waktu 2. Reaksi samping 3. Kehilangan sampel Created by Nick Buker 12/18/08

Detektor Thermal Conductivity
The detector contains two filaments: one exposed only to carrier gas, while the other is exposed to the carrier gas for sample analysis. When the gas for the sample analysis is only carrier gas , the two filaments can be balanced. Instead of a direct measurement of filament temperature, the filament resistant, which is a function of temperature, is measured. Created by Nick Buker 12/18/08

The ability of a colliding molecule to carry off heat depending on its thermal conductivity. Hydrogen and helium have high thermal conductivity and therefore will be more efficient at “cooling” a heated filament than other gases will Created by Nick Buker 12/18/08

The TCD will respond to any substance different from the carrier gas as long as its concentration is sufficiently high enough. Created by Nick Buker 12/18/08

Electron capture compound, X (highly electonegative element), tends to capture free electrons and increase the amount to ion recombination X (F, Cl and Br) + e  X- Ion recombination : X- + N2+ = X + N2 The current will decrease and this decrease constitutes the signal. Halogens, lead, phosphorous, nitro groups, silicone and polynuclear aromatics. Insecticides, pesticides, vinyl chloride, and fluorocarbons Created by Nick Buker 12/18/08

Protein’s properties used in purification

Salting in and out Created by Nick Buker 12/18/08

Solubility in ammonium sulfate

Created by Nick Buker 12/18/08

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