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
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. Instrument employed for a

Stationary phase: Phase that stays in place inside the column. 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, either a liquid in LC or gas in GC. Eluent: Fluid entering a column.

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

Chromatographic separation

Chromatographic performance parameters

Resolution

Resolution case studies

Plate height

Plate height case studies

Peaks broadening
Two factors promoting the broadening of analyte band and influenced by flow rate of the eluent through the column: 1- longitudinal diffusion: is the diffusion of individual analyte molecules in the mobile phase along the longitudinal direction of a column. The longer the solute band remains in the column, 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, depending on the particle shape, porosity, and the whole bed structure. This is schematically shown below.

longitudinal diffusion

Multiple pathways

Van Deemter Equation

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

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

 For packed columns, 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. Diffusion along axis by flow rate is balanced by a back pressure of a column for LC. B is reduced with smaller diameter packings. Related to transfer of solute between phases. N with temp. It is represented by practical problems such as sample and column degradation.

Types of chromatography on the basis of interaction of the analyte with stationary phase

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

Adsorption (liquid- solid)

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

Ion- exchange chromatography

 It relies on the attraction between oppositely charged stationary phase, known as an ion exchanger, and analyte. Used to separate and purify proteins, peptides, nucleic acids and other charged molecules. 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.

Partition chromatography

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

Size Exclusion chromatography

Size Exclusion chromatography

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