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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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Asas dan Dasar-dasar Kromatografi


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Kromatografi dengan asas adsorpsi, memakai fase diam padat dan fase gerak cair atau gas Kromatografi dengan asas partisi, memakai fase diam cair dan fase gerak cair Kromatografi dengan asas fitrasi, memakai fase diam padat yang mempunyai sifat fitrasi dan fase gerak cairan Kromatografi dengan asas suhu kritik, memakai CO2 dalam keadaan superkritik

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Klasifikasi kromatografi
kromatografi gas GSC GLC LSC LLC BPC IEC EC GPC
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SFC colomn

liquid planar TLC PC

GFC

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

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Kromatografi gas
  

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

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Kromatografi Lapis Tipis


Keuntungan :  Digunakan untuk tujuan analitik  Identifikasi komponen dapat dilakukan dengan pereaksi warna, fluoresensi atau pemadaman fluoresensi, 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
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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)

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Kromatografi Kertas
 

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

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Kromatografi Kolom
  

Digunakan untuk isolasi senyawa dari sample Merupakan kelanjutan dari KLT Prinsip pemisahan, fase diam dan fase gerak sama dengan KLT

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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

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HPLC
Liquid Chromatography

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The original development of HPLC used higher pressures than previously used ----High Pressure Liquid Chromatography However, over the years the preferred term has become: High Performance Liquid Chromatography

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Advantages of HPLC
    

High resolution Speed Re-usable columns Great reproducibility Control of physical parameters  flow rate, polarity, packing efficiency, and particle size. Easy automation of instrument and data analysis.

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HPLC Chromatograph of Muscadine Grape Juice

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SOLVENTS
Includes both liquid phase and solid materials (Buffers) dissolved in the liquid.

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Solvent properties affecting detection Solvent properties affecting separation Solvent properties affecting flow Viscosity Miscibility

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Solvent Properties Affecting Detection


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

1)Buffers are needed to control the pH differences caused by the sample matrix. 2)Buffers are used to control the ionization of compounds and therefore their retention by the column.

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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. Acids lose a proton and become ionized (negative charge) as pH increases. Bases on the other hand, gain a proton and acquire a positive charge as pH decreases.
not charged

partially charged

Basic Compound

Relative Retention Time

fully charged

pKa

6 pH

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SOLVENT SELECTIVITY
The less time a compound spends in the stationary phase, the faster it will move through the column (less retention time). If two compounds are added to the column, the ratio of their retention times is called the selectivity. The higher the selectivity, the better the separation. Selectivity can be increased by adjustment of the mobile and stationary phases.
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Solvent Selectivity Triangle Representing 3 Polarity factors

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.

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Viscosity - resistance to flow


Difficult to force high viscosity solvents through the column. Mixing solvents can drastically change viscosity

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Viscosity of Water-Organic Solvent Mixtures

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Viscosity vs. Pressure


The higher the solvent viscosity, the harder it is for the solvent to move through a column, and the more pressure in required to move the solvent at a specific velocity. The pressure required to move a solvent through a column can be estimated by the following formula:

P = 250 L L F / Dp Dc
Where P = pressure drop in psi. L = column length (cm) L= solvent viscosity (cP)
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F = flow rate (mL/min) Dp = particle diameter (Qm) Dc = column diameter (cm)

EXAMPLE
P = 250 L L F / Dp2 Dc2 column length = 15 cm, column diameter =.5 cm, particle diameter = 5 Qm, flowrate = 2.0 mL/min

For water n = 1.0 250 x 15 x 1.0 x 2 / 52 x .52 = 7125/6.25 = 1200 psi For methanol n = 0.54 250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psi For 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% methanol

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SOLVENTS

UV Cutoff

Miscible with Viscosity Polarity Water?

Water Methanol Tetrahydrofuran Propanol Acetonitrile Hexane Ethyl Acetate Chloroform

190 205 212 210 190 195 256 245

1.00 0.55 0.55 2.3 0.38 0.31 0.45 0.57

10.2 5.1 4.0 4.0 5.8 0.1 4.4 4.1

--Y Y Y Y N N N

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Peripheral Properties
Toxicity Flammability Reactivity solvent should not react with sample Cost Disposal can be more than purchase cost

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Geometry of HPLC Columns


Diameter Length Particle Size What is the effect on pressure?

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P = 250 L L F / Dp2 Dc2


Where P = pressure drop in psi. L = column length (cm) L = solvent viscosity (cP) F = flow rate (mL/min) Dp = particle diameter (Qm) Dc = column diameter (cm)

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Geometry of HPLC Columns


Diameter Length Particle Size What is the effect on Theoretical Plates?

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What is the effect of column geometry on Theoretical Plates?


Remember that separation is best on columns with high number of theoretical plates.

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!
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What is the effect of column geometry on Theoretical Plates? Decreasing column diameter by half
For comparison purposes, lets keep the mobile phase velocity constant. Therefore, flow would be reduced 4X and analysis wont 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.
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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.

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

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