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KROMATOGRAFI

Syarif Hamdani

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Kromatografi
 Adalah

teknik pemisahan fisik suatu
campuran zat-zat kimia yang berdasar pada
perbedaan migrasi dari masing-masing
komponen campuran yang terpisah pada
fase diam di bawah pengaruh pergerakan
fase gerak

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Sejarah kromatografi
 Pertama

kali diperkenalkan oleh W. Ramsey
pada tahun 1905
 Istilah kromatografi (artinya penulisan warna)
pertama kali diberikan oleh Mikhail
Semenovic Tswett pada tahun 1908
 KLT diperkenalkan oleh Izamailov dan
Shraiber pada tahun 1938

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com . memakai fase diam cair dan fase gerak cair Kromatografi dengan asas fitrasi. 4. 2. Kromatografi dengan asas adsorpsi. memakai fase diam padat yang mempunyai sifat fitrasi dan fase gerak cairan Kromatografi dengan asas suhu kritik. memakai CO2 dalam keadaan superkritik http://www. 3.Asas dan Dasar-dasar Kromatografi 1.catatankimia. memakai fase diam padat dan fase gerak cair atau gas Kromatografi dengan asas partisi.

com planar GFC .catatankimia.Klasifikasi kromatografi kromatografi gas GSC SFC GLC liquid colomn LSC TLC LLC PC BPC IEC EC GPC http://www.

catatankimia.com .Kromatografi Gas Macamnya :  Kromatografi gas padat : fase diam adalah butiran-butiran adsorben dan fase gerak adalah gas  Kromatografi gas cair : fase diam adalah cairan yang disalutkan pada permukaan tipis butiran padat dan fase gerak adalah gas http://www.

ECD. dll. IRD. FID. FPD.Kromatografi gas  Sampel berupa gas. Ar.  Analisis kualitatif dibandingkan terhadap BANK DATA http://www. N2 atau campuran He dan CH4  Detektor terdiri dari berbagai macam tergantung keperluan diantaranya : TCD. TID.catatankimia.  Gas pembawa : He.com . NPD. Untuk senyawa yang bukan gas dibuat turunan esternya.

Kromatografi Lapis Tipis Keuntungan :  Digunakan untuk tujuan analitik  Identifikasi komponen dapat dilakukan dengan pereaksi warna. radiasi UV  Dapat dilakukan elusi dengan mekanik (ascending) atau menurun (descending) atau dengan cara elusi 2 dimensi  Ketepatan penentuan kadar akan lebih baik karena komponen yang ditentukan merupakan noda yang tidak bergerak http://www. fluoresensi atau pemadaman fluoresensi.com .catatankimia.

com .Kromatografi Lapis Tipis  Identitas komponen dijabarkan dalam harga Rf (retardation factor) yang dalam penentuan kualitatif dibandingkan dengan standar  Untuk tujuan kuantitatif digunakan KLT preparatif (dikerok lalu senyawa diisolasi dalam pelarutnya) http://www.catatankimia.

com . karena selulosa (kertas) bersifat polar Banyak digunakan untuk pemisahan senyawa bahan alam Kekurangan : lebih lama karena panjang kertas bisa sampai 50 cm. fase mobil air dicampur pelarut organik Lebih banyak digunakan untuk pemisahan senyawa non polar.catatankimia.Kromatografi Kertas      Prinsip sama dengan KLT Fase diam adalah air yang didukung oleh pelat serat selulosa. http://www.

fase diam dan fase gerak sama dengan KLT http://www.catatankimia.Kromatografi Kolom  Digunakan untuk isolasi senyawa dari sample  Merupakan kelanjutan dari KLT  Prinsip pemisahan.com .

Kromatografi Cair Kinerja Tinggi  Dimaksudkan untuk mendapatkan pemisahan dan hasil analisa kuantitatif yang baik dengan waktu singkat  Pelarut pengembang harus dipilih dengan seksama  Kolom harus sesuai  Detektor harus memadai  Sample berupa larutan http://www.com .catatankimia.

com .HPLC Liquid Chromatography http://www.catatankimia.

The original development of HPLC used higher pressures than previously used ----High Pressure Liquid Chromatography However.com . over the years the preferred term has become: High Performance Liquid Chromatography http://www.catatankimia.

Advantages of HPLC  High resolution Speed Re-usable columns Great reproducibility Control of physical parameters  flow rate.com .catatankimia. polarity. and particle size.      http://www. Easy automation of instrument and data analysis. packing efficiency.

catatankimia.com .HPLC Chromatograph of Muscadine Grape Juice http://www.

SOLVENTS Includes both liquid phase and solid materials (Buffers) dissolved in the liquid.catatankimia. http://www.com .

catatankimia.•Solvent properties affecting detection •Solvent properties affecting separation •Solvent properties affecting flow •Viscosity •Miscibility http://www.com .

catatankimia.com .Solvent Properties Affecting Detection UV Cutoff -Solvent may interfere with detection For peptide analysis UV = 215 nm. Refractive Index of Solvent vs Sample for Refractive Index detection (Carbohydrates) Volatility needed for HPLC Mass Spectrometry (trifluoroacetic acid is a typical volatile buffer) http://www. Solvents that absorb UV at this wavelength would not be good candidates for the mobile phase.

catatankimia.com . 2)Buffers are used to control the ionization of compounds and therefore their retention by the column.BUFFERS 1)Buffers are needed to control the pH differences caused by the sample matrix. http://www.

gain a proton and acquire a positive charge as pH decreases. not charged Basic Compound Relative Retention Time partially charged fully charged 3 http://www.catatankimia. Bases on the other hand.Retention Time and pH in Reversed Phase When an acid or a base is ionized it becomes much less hydrophobic and will elute much earlier.com 4 pKa 5 6 pH 7 8 9 . Acids lose a proton and become ionized (negative charge) as pH increases.

the better the separation.catatankimia. the ratio of their retention times is called the selectivity. If two compounds are added to the column. the faster it will move through the column (less retention time). http://www.SOLVENT SELECTIVITY The less time a compound spends in the stationary phase.com . The higher the selectivity. Selectivity can be increased by adjustment of the mobile and stationary phases.

com 1) Each dot in the triangle represent a different solvent 2) Solvents can be grouped based on their type of polarity 3) Solvents and solvent mixtures are available for just about any separation you may desire.Solvent Selectivity Triangle Representing 3 “Polarity” factors http://www. .catatankimia.

catatankimia.resistance to flow Difficult to force high viscosity solvents through the column.com . Mixing solvents can drastically change viscosity http://www.Viscosity .

Viscosity of Water-Organic Solvent Mixtures http://www.com .catatankimia.

Pressure The higher the solvent viscosity.com 2 F = flow rate (mL/min) Dp = particle diameter (m) Dc = column diameter (cm) .catatankimia. and the more pressure in required to move the solvent at a specific velocity. L = column length (cm) = solvent viscosity (cP) http://www.Viscosity vs. the harder it is for the solvent to move through a column. The pressure required to move a solvent through a column can be estimated by the following formula: 2 P = 250 L  F / Dp Dc Where P = pressure drop in psi.

com .0 250 x 15 x 1.52 = 7125/6.0 x 2 / 52 x .9 x 2 / 52 x .25 = 1200 psi For methanol n = 0.5 cm.54 250 x 15 x .52 = 2025/6.25 = 2280 psi 40% methanol http://www.EXAMPLE P = 250 L  F / Dp2 Dc2 column length = 15 cm.catatankimia. particle diameter = 5 m.52 = 7125/6. flowrate = 2.0 mL/min For water n = 1.25 = 648 psi For 60% water n = 1. column diameter =.54 x 2 / 52 x .9 250 x 15 x 1.

1 4.31 0.8 0.3 0.00 0.38 0.1 --Y Y Y Y N N N .4 4.55 0.2 5.SOLVENTS Water Methanol Tetrahydrofuran Propanol Acetonitrile Hexane Ethyl Acetate Chloroform http://www.45 0.0 5.55 2.0 4.com UV Cutoff 190 205 212 210 190 195 256 245 Miscible with Viscosity Polarity Water? 1.1 4.catatankimia.57 10.

Peripheral Properties – Toxicity – Flammability – Reactivity solvent should not react with sample – Cost – Disposal can be more than purchase cost http://www.com .catatankimia.

com .catatankimia.Geometry of HPLC Columns Diameter Length Particle Size What is the effect on pressure? http://www.

com F = flow rate (mL/min) Dp = particle diameter (m) Dc = column diameter (cm) . L = column length (cm) = solvent viscosity (cP) http://www.catatankimia.P = 250 L  F / Dp2 Dc2 Where P = pressure drop in psi.

Geometry of HPLC Columns Diameter Length Particle Size What is the effect on Theoretical Plates? http://www.catatankimia.com .

catatankimia.What is the effect of column geometry on Theoretical Plates? Remember that separation is best on columns with high number of theoretical plates.com . N=L/H where N is number of plates. H is plate height and L is Column Length Therefore. doubling the column length will double N but this will double analysis time and pressure! http://www.

Therefore. let’s keep the mobile phase velocity constant.What is the effect of column geometry on Theoretical Plates? Decreasing column diameter by half For comparison purposes. http://www. flow would be reduced 4X and analysis won’t take any longer! halving the column diameter can also increase N slightly This reduces the amount of solvent used by 4X but also reduces the amount of sample that can be injected by 4X.catatankimia.com .

What is the effect of column geometry on Theoretical Plates? Decreasing particle size by half Will increase pressure by 4X However. halving the particle size can double N Decreasing particle size and making the column half as long will keep N the same but cut sample time in half and solvent use in half.com . http://www.catatankimia.

and can plug easier. and columns CLEAN! http://www. mobile phases.catatankimia. smaller samples.but require higher pressures.com . …. The problem with plugging should not be underestimated and care should be exercised in keeping the sample.In general small diameter columns with small particles are best for rapid separation.

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