KROMATOGRAFI

KROMATOGRAFISyarif Hamdanihttp://www.catatankimia.com

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 kromatografiPertama kali diperkenalkan oleh W. Ramsey pada tahun 1905Istilah kromatografi (artinya penulisan warna) pertama kali diberikan oleh Mikhail Semenovic Tswett pada tahun 1908KLT diperkenalkan oleh Izamailov dan Shraiber pada tahun 1938http://www.catatankimia.com

Asas dan Dasar-dasar KromatografiKromatografi dengan asas adsorpsi, memakai fase diam padat dan fase gerak cair atau gasKromatografi dengan asas partisi, memakai fase diam cair dan fase gerak cairKromatografi dengan asas fitrasi, memakai fase diam padat yang mempunyai sifat fitrasi dan fase gerak cairanKromatografi dengan asas suhu kritik, memakai CO2 dalam keadaan superkritikhttp://www.catatankimia.com

Klasifikasi kromatografihttp://www.catatankimia.com

Kromatografi GasMacamnya :Kromatografi gas padat : fase diam adalah butiran-butiran adsorben dan fase gerak adalah gasKromatografi gas cair : fase diam adalah cairan yang disalutkan pada permukaan tipis butiran padat dan fase gerak adalah gashttp://www.catatankimia.com

Kromatografi gasSampel berupa gas. Untuk senyawa yang bukan gas dibuat turunan esternya.Gas pembawa : He, Ar, N2 atau campuran He dan CH4Detektor terdiri dari berbagai macam tergantung keperluan diantaranya : TCD, FID,ECD, NPD, FPD, IRD, TID, dll.Analisis kualitatif dibandingkan terhadap BANK DATA http://www.catatankimia.com

Kromatografi Lapis TipisKeuntungan :Digunakan untuk tujuan analitikIdentifikasi komponen dapat dilakukan dengan pereaksi warna, fluoresensi atau pemadaman fluoresensi, radiasi UVDapat dilakukan elusi dengan mekanik (ascending) atau menurun (descending) atau dengan cara elusi 2 dimensiKetepatan penentuan kadar akan lebih baik karena komponen yang ditentukan merupakan noda yang tidak bergerakhttp://www.catatankimia.com

Kromatografi Lapis TipisIdentitas komponen dijabarkan dalam harga Rf (retardation factor) yang dalam penentuan kualitatif dibandingkan dengan standarUntuk tujuan kuantitatif digunakan KLT preparatif (dikerok lalu senyawa diisolasi dalam pelarutnya)


Kromatografi KertasPrinsip sama dengan KLTFase diam adalah air yang didukung oleh pelat serat selulosa, fase mobil air dicampur pelarut organikLebih banyak digunakan untuk pemisahan senyawa non polar, karena selulosa (kertas) bersifat polarBanyak digunakan untuk pemisahan senyawa bahan alamKekurangan : lebih lama karena panjang kertas bisa sampai 50 cm.http://www.catatankimia.com

Kromatografi KolomDigunakan untuk isolasi senyawa dari sampleMerupakan kelanjutan dari KLTPrinsip pemisahan, fase diam dan fase gerak sama dengan KLT


Kromatografi Cair Kinerja TinggiDimaksudkan untuk mendapatkan pemisahan dan hasil analisa kuantitatif yang baik dengan waktu singkatPelarut pengembang harus dipilih dengan seksama Kolom harus sesuai Detektor harus memadaiSample berupa larutan


HPLCLiquid Chromatographyhttp://www.catatankimia.com

The original development of HPLC used higher pressuresthan previously used ----High Pressure Liquid Chromatography

However, over the years the preferred term has become:

High Performance Liquid Chromatographyhttp://www.catatankimia.com

Advantages of HPLCHigh resolutionSpeedRe-usable columnsGreat reproducibilityControl of physical parameters flow rate, polarity, packing efficiency, and particle size.Easy automation of instrument and data analysis.


HPLC Chromatograph of Muscadine Grape Juicehttp://www.catatankimia.com

SOLVENTSIncludes both liquid phaseand solid materials (Buffers) dissolved in the liquid.http://www.catatankimia.com

Solvent properties affecting detectionSolvent properties affecting separationSolvent properties affecting flowViscosityMiscibilityhttp://www.catatankimia.com

UV Cutoff -Solvent may interfere with detection

For peptide analysis UV = 215 nm. Solvents thatabsorb UV at this wavelength would not be goodcandidates for the mobile phase.

Refractive Index of Solvent vs Sample forRefractive Index detection (Carbohydrates)

Volatility needed for HPLC Mass Spectrometry(trifluoroacetic acid is a typical volatile buffer)Solvent Properties Affecting Detectionhttp://www.catatankimia.com

BUFFERS1)Buffers are needed to control the pH differences caused bythe sample matrix.

2)Buffers are used to control the ionization of compoundsand therefore their retention by the column.http://www.catatankimia.com

Retention Time and pH in Reversed Phase3456789pHRelative Retention TimepKapartially chargedfully chargednot chargedWhen 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.BasicCompoundhttp://www.catatankimia.com

SOLVENT SELECTIVITYThe 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 theirretention 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.http://www.catatankimia.com

Solvent Selectivity TriangleRepresenting 3 Polarity factors1) Each dot in the trianglerepresent a different solvent2) Solvents can be groupedbased on their type of polarity3) Solvents and solvent mixturesare available for just about anyseparation you may desire.http://www.catatankimia.com

Viscosity - resistance to flowDifficult to force high viscosity solvents throughthe column.

Mixing solvents can drastically change viscosityhttp://www.catatankimia.com

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

Viscosity vs. Pressurehttp://www.catatankimia.com

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 ( F / Dp2 Dc2

Where P = pressure drop in psi.

F = flow rate (mL/min)

L = column length (cm)

Dp = particle diameter (m)

(= solvent viscosity (cP)

Dc = column diameter (cm)

P = 250 L F / Dp2 Dc2EXAMPLEcolumn length = 15 cm, column diameter =.5 cm, particle diameter = 5 mm, 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 psiFor methanol n = 0.54250 x 15 x .54 x 2 / 52 x .52 = 2025/6.25 = 648 psiFor 60% water n = 1.9 250 x 15 x 1.9 x 2 / 52 x .52 = 7125/6.25 = 2280 psi 40% methanolhttp://www.catatankimia.com

ToxicityFlammabilityReactivity solvent should not react with sampleCost Disposal can be more than purchase cost

Peripheral Propertieshttp://www.catatankimia.com

Geometry of HPLC Columns

DiameterLengthParticle SizeWhat is the effect on pressure?http://www.catatankimia.com

P = 250 L F / Dp2 Dc2Where P = pressure drop in psi.F = flow rate (mL/min)L = column length (cm)Dp = particle diameter (mm) = solvent viscosity (cP)Dc = column diameter (cm)http://www.catatankimia.com

Geometry of HPLC Columns

DiameterLengthParticle SizeWhat is the effect on Theoretical Plates?http://www.catatankimia.com

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

What is the effect of column geometryon Theoretical Plates?Decreasing column diameter by halfhalving the column diameter can also increase N slightlyFor comparison purposes, lets keep the mobile phasevelocity constant. Therefore, flow would be reduced 4Xand analysis wont take any longer!This reduces the amount of solvent used by 4X but alsoreduces the amount of sample that can be injected by 4X.http://www.catatankimia.com

What is the effect of column geometryon Theoretical Plates?Decreasing particle size by halfHowever, halving the particle size can double NWill increase pressure by 4XDecreasing particle size and making the column half as long will keep N the same but cut sample time in half and solventuse in half.http://www.catatankimia.com

In general small diameter columns withsmall particles are best for rapid separation,

.but require higher pressures, smaller samples, and can plug easier.

The problem with plugging should not be underestimatedand care should be exercised in keeping the sample, mobilephases, and columns CLEAN!http://www.catatankimia.com

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KROMATOGRAFI