Lecture outline
• • • • • Chromatographic techniques: 1- General principles. Chromatographic performance parameters. Physical basis of separation. Adsorption (liquid- solid).

• 2- Partition (liquid- liquid) • Gel filtration (steric exclusion). • Ion exchange chromatography.

• 3- Thin layer chromatography • High performance liquid chromatography. • Gas chromatography .

• Chromatography is the ability to separate molecules using partitioning characteristics of molecule to remain in a stationary phase versus a mobile phase. Once a molecule is separated from the mixture, it can be isolated , quantified through detector. • Partition coefficient KD • KD= Cs / Cm , • Cs is the concentration in stationary phase where Cm is the concentration in mobile phase

Terms used in chromatography • Chromatograph: chromatography. • Eluent: Fluid entering a column. Instrument employed for a • Stationary phase: Phase that stays in place inside the column. . either a liquid in LC or gas in GC. Can be a particular solid or gel-based packing (LC) or a highly viscous liquid coated on the inside of the column (GC). • Mobile phase: Solvent moving through the column.

• Chromatogram: Graph showing detector response as a function of a time.• Eluate: Fluid exiting the column. • Linear velocity: Distance passed by mobile phase per 1 min in the column (cm/min). ␣ Elution: The process of passing the mobile phase through • the column. . • Flow rate: How much mobile phase passed / minute (ml/min).

Chromatographic separation .

Chromatographic performance parameters .

Resolution .

Resolution case studies .

Plate height .

Plate height case studies .

The longer the solute band remains in the column. porosity. and the whole bed structure.Peaks broadening • Two factors promoting the broadening of analyte band and influenced by flow rate of the eluent through the column: • 1. depending on the particle shape. This is schematically shown below. the greater will be the extent of diffusion. • 2.Multiple pathways : The velocity of mobile phase in the column may vary significantly across the column diameter. .longitudinal diffusion: is the diffusion of individual analyte molecules in the mobile phase along the longitudinal direction of a column.

longitudinal diffusion .

Multiple pathways .

diffusion through packed column (is eliminated in GC) . • H~A+B/ū + Cū • We want H to be low = so all the parameters A.Van Deemter Equation • Van Deemter Equation tells us how the column and flow rate affect the plate height. and C should be as low as possible : • ū is average linear velocity (cm/min) • A multiple pathways.B.

faster is the interaction between analyte and stationary phase means smaller C. . • The smaller height plate.• B longitudinal diffusion (molecular diffusion) GC bigger molecule of gas used as a mobile phase. better separation . the narrower chromatographic band. the bigger B LC more viscous mobile phase => bigger B • C mass transfer – transfer of the analyte in and out of stationary phase.

B is reduced with smaller diameter packings. It is represented by practical problems such as sample and column degradation. • Diffusion along axis ↓ by ↑ flow rate is balanced by a back pressure of a column for LC. A is a problem with nonhomogenous particles as a packing. A reduces with a smaller homogenous packing and a smaller particle size • This is not a problem for GC. .• For packed columns. ↑ N with ↑ temp. • Related to transfer of solute between phases.


• Ion Exchange – attraction of ions of opposite charges. • Size Exclusion (gel filtration. for polar non-ionic compounds. • Affinity – specific interactions like a particular antibody to protein . gel permeation) – separates molecules by size. for ionic compounds anions or cations.based on the relative solubility of analyte in mobile and stationary phases. • Partition .Types of chromatography on the basis of interaction of the analyte with stationary phase • Adsorption – of solute on surface of stationary phase.


Adsorption (liquid.solid) .

• Other adsorbents are alumina and carbon. it has silanol (Si-OH) groups on its surface . . • This adsorption process occur at specific adsorption sites.• A classic form of chromatography. which are slightly acidic. is a typical adsorbent. have the ability to hold molecules at their surface. • It is based upon the principle that certain solid materials. non-ionic and hydrogen bonding type. that interact with functional group of analyte. called adsorbents. • It involves weak. • Silica.


Ion.exchange chromatography .

known as an ion exchanger. . • Cation exchanges posses negatively charged groups and these will attract positively charged cations and anion exchanges posses positively charged groups and these will attract negatively charged groups. nucleic acids and other charged molecules. and analyte. peptides. • Used to separate and purify proteins.• It relies on the attraction between oppositely charged stationary phase.


Partition chromatography .

• Elution may be gradually remove the staitionary phase. high capacity and broad selectivity. • Cheap . • Covalently attracted to the matrix. and distribution coefficents of the analytes using liquid stationary and mobile phases. . thereby altering the chromatographic condition.• Is based on separation of sample mixture according to their rtention factor (k) .

Size Exclusion chromatography .

relative molecular mass determinaton. solution concentration and desalting. .Size Exclusion chromatography • It based on separation of molecules on the basis of molecular size and shape. • Used in purification.