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Definition of Chromatography
Simplified Definition:

Chromatography separates the components of a mixture by their distinctive attraction to the mobile phase and the stationary phase.

Mobile phase passes through the stationary phase Mobile phase solubilizes the components Mobile phase carries the individual components a certain distance through the stationary phase, depending on their attraction to both of the phases

Separation mechanism (interaction between solute and stationary phase) in chromatography:

1. Adsorption
Using solid stationary phase and liquid or gas mobile phase. Solute was adsorbed in solid particle surface.

the relative polarities of solute and solid stationary phase determine the rate of movement of that solute through a column or across a surface

2. Partition
Stationary phase : liquid (sorbed solvent held on the surface, or within the grains or fibres of an inert solid supporting matrix)

3. Ion exchange
The stationary phase is a permeable polymeric solid containing fixed charged groups and mobile counter-ions which can exchange with the ions of a solute as the mobile phase carries them through the structure. Anion like SO3- (as cation exchange), or cation like N(CH3)3+ (as anion exchange) hold covalently in solid stationary phase , commonly: resin (polymer). Solute ion that have opposite charge will bonded in stationary phase by electrostatic force.

4. molecular exclusion
called gel filtration atau gel permeation chrom. This technique separate solute based on the size, the smaller solute will more retain.

5. Affinity
Involve specific interaction between one kind of solute molecule and the second molecule that bonded covalently (immobilized) by stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solute containing a mixture of proteins are passed by this molecule, only the specific protein is reacted to this antibody, binding it to the stationary phase. This protein is later extracted by changing the ionic strength or pH.

Klasifikasi metode kromatografi kolom

Klasifikasi Metode/ mekanisme Liquid Chromatography Liquid-liquid or (LC) partition mobile phase: liquid Liquid-bonded phase Liquid-solid or adsorption Ion exchange Size exclusion Gas chromatography (GC) Mobile phase: gas Gas-liquid Gas-bonded phase Gas-solid Supercritical fluid chromatography (SFC) Mobile phase: supercritical fluid Fs diam Liquid adsorbed on a solid Organic species bonded to a solid surface Solid Ion-exchange solid Liquid in intersticies of a polymeric solid Liquid adsorbed on a solid Organic species bonded to a solid surface Solid Organic species bonded to a solid surface Jenis kesetimbangan Partition between immiscible liquids Partition between liquid & bonded surface Adsorption Ion exchange Partition/ sieving Partition between gas & liquid Partition between gas & bonded surface Adsorption Partition between supercritical fluid & bonded surface


Konstanta distribusi
waktu retensi, tR tM ; tR Volum retensi Laju rata-rata solut dlm kolom Laju rata-rata fase gerak dalam kolom

Hubungan waktu retensi & konstanta distribusi

dengan kA: faktor retensi atau kapasitas faktor

Faktor retensi

Faktor selektivitas

efisiensi kolom
Teori lempeng (plate theory) N=L/H N: jumlah lempeng teoritik L: panjang kolom H: height equivalent of theoretical plates (HETP), tinggi ekivalen lempeng teoritik, tebal lempeng teoritik

Teori kinetik (kinetics theory)

N = L/H Tinggi plat dengan Banyaknya plat

Resolusi kolom (Rs) menunjukkan perhitungan secara kuantitatif kemampuan suatu kolom untuk memisahkan 2 analit

Pengaruh retensi & factor selektivitas terhadap resolusi Dengan asumsi