Kromatografi General

KROMATOGRAFI

Syarif Hamdani

http://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


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


Asas dan Dasar-dasar Kromatografi

1. Kromatografi dengan asas adsorpsi, memakai fase diam padat dan fase gerak cair atau gas

2. Kromatografi dengan asas partisi, memakai fase diam cair dan fase gerak cair

3. Kromatografi dengan asas fitrasi, memakai fase diam padat yang mempunyai sifat fitrasi dan fase gerak cairan

4. Kromatografi dengan asas suhu kritik, memakai CO2 dalam keadaan superkritik


Klasifikasi kromatografikromatografi

SFC liquid

GLC colomn planar

TLC

PC

LSC

LLC

BPC

IEC

EC

GPC

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


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


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


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)


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.


Kromatografi Kolom

Digunakan untuk isolasi senyawa dari sample

Merupakan kelanjutan dari KLT Prinsip pemisahan, fase diam dan fase gerak

sama dengan KLT


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


HPLC

Liquid Chromatography


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 Chromatography


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.


HPLC Chromatograph of Muscadine Grape Juice


SOLVENTS

Includes both liquid phaseand solid materials (Buffers) dissolved in the liquid.


•Solvent properties affecting detection•Solvent properties affecting separation•Solvent properties affecting flow

•Viscosity•Miscibility


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 Detection


BUFFERS

1)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.


Retention Time and pH in Reversed Phase

3 4 5 6 7 8 9pH

Rel

ativ

e R

eten

tio

n T

ime

pKa

partially charged

fully charged

not charged

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.

BasicCompound


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 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.


Solvent Selectivity TriangleRepresenting 3 “Polarity” factors 1) Each dot in the triangle

represent a different solvent2) Solvents can be groupedbased on their type of polarity3) Solvents and solvent mixturesare available for just about anyseparation you may desire.


Viscosity - resistance to flow

Difficult to force high viscosity solvents throughthe column.

Mixing solvents can drastically change viscosity


Viscosity of Water-Organic Solvent Mixtures


Viscosity vs. PressureThe higher the solvent viscosity, the harder it is for the

solvent to move through a column, and the more pressure inrequired to move the solvent at a specific velocity. The pressurerequired to move a solvent through a column can be estimated bythe following formula:

P = 250 L F / Dp2 Dc

2

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 Dc

2

EXAMPLE

column length = 15 cm, column diameter =.5 cm, particle diameter = 5 m, 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 = 0.54 250 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% methanol


SOLVENTS

Water 190 1.00 10.2 ---Methanol 205 0.55 5.1 YTetrahydrofuran 212 0.55 4.0 YPropanol 210 2.3 4.0 YAcetonitrile 190 0.38 5.8 YHexane 195 0.31 0.1 NEthyl Acetate 256 0.45 4.4 NChloroform 245 0.57 4.1 N

UVCutoff Viscosity Polarity

Misciblewith

Water?


– ToxicityToxicity

– FlammabilityFlammability– Reactivity Reactivity solvent should not react with samplesolvent should not react with sample

– Cost Cost – Disposal Disposal can be more than purchase costcan be more than purchase cost

Peripheral Properties


Geometry of HPLC Columns

DiameterLengthParticle Size

What is the effect on pressure?


P = 250 L F / Dp2 Dc

2

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)


Geometry of HPLC Columns

DiameterLengthParticle Size

What is the effect on Theoretical Plates?


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 Length

Remember 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!



Decreasing column diameter by half

halving the column diameter can also increase N slightly

For comparison purposes, let’s keep the mobile phasevelocity constant. Therefore, flow would be reduced 4Xand analysis won’t take any longer!

This reduces the amount of solvent used by 4X but alsoreduces the amount of sample that can be injected by 4X.



Decreasing particle size by half

However, halving the particle size can double N

Will increase pressure by 4X

Decreasing particle size and making the column half as long will keep N the same but cut sample time in half and solventuse in half.


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!


Kromatografi General

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