 Laboratory technique for the Separation of mixtures  Chroma -"color" and graphein - "to write”.  Colour bands - separation of individual compounds  Measured or analysed.

 Analytical Determine Chemical composition of a sample  Preparative Used to purify sufficient quantities of a substance

 Chromatograph - equipment that enables a sophisticated separation EX. Gas chromatography or Liquid chromatography  Eluent - Fluid entering column/ solvent that carries the analyte.  Eluate - Mobile phase leaving the column.

 Retention time -Time takes for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.  Sample (Anylate)- Substance analyzed in chromatography.  Solvent - Any substance capable of solubilizing another substance.

 Techniques by Chromatographic bed shape › Column chromatography › Planar chromatography  Paper chromatography  Thin layer chromatography  Techniques by Physical state of mobile phase › Gas chromatography › Liquid chromatography  Affinity chromatography › Supercritical fluid chromatography

Mobile PhaseStationary Phase SolventBonded Phase Separation is based on the analyte’s relative solubility between two liquid phases Partitioning

Normal Phase. - Polar stationary phase and non-polar solvent. Reverse Phase. - Non-polar stationary phase and a polar solvent.

1) Solvent reservoirs, 2) Solvent degasser, 3) Gradient valve, 4) Mixing vessel for delivery of the mobile phase, 5) High-pressure pump, 6) Switching valve in "inject position" Switching valve in "load position", 7) Sample injection loop, 8) Pre-column (guard column),guard column 9) Analytical column, 10) Detector (i.e. IR, UV), 11) Data acquisition, 12) Waste or fraction collector.

Ion Exchange Gel Filtration Affinity Charge Size Conformation

Partition chromatography uses a retained solvent, on the surface or within the grains or fibres of an "inert" solid supporting matrix as with paper chromatography; or takes advantage of some additional coulombic and/or hydrogen donor interaction with the solid support. Molecules equilibrate (partition) between a liquid stationary phase and the eluent. Known as Hydrophilic Interaction Chromatography (HILIC) in HPLC, this method separates analytes based on polar differences. HILIC most often uses a bonded polar stationary phase and a non-polar, water miscible, mobile phase. Partition HPLC has been used historically on unbonded silica or alumina supports. Each works effectively for separating analytes by relative polar differences, however, HILIC has the advantage of separating acidic, basic and neutral solutes in a single chromatogram.paper chromatographycoulombichydrogen donorstationary phasemiscibleacidicbasic

Ion-exchange chromatography is a process that allows the separation of ions and polar molecules based on their charge. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality controlionspolar moleculesproteinsnucleotidesamino acids Ion exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. This type of chromatography is further subdivided into cation exchange chromatography and anion exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.analytecoulombic

A charged solid phase or matrix Liquid phase contains molecules of different charges Solutions (eluant) of different charges to influence interactions between liquid and solid phases

 1. A cation exchanger: › Matrix negative charge › Exchanges cations  2. An anion exchanger: › Matrix positive charge › Exchanges anions Solid matrix exchangers :-

Size exclusion chromatography (SEC), also known as gel permeation chromatography or gel filtration chromatography, separates particles on the basis of size. It is generally a low resolution chromatography and thus it is often reserved for the final, "polishing" step of purification. It is also useful for determining the tertiary structure and quaternary structure of purified proteins. SEC is used primarily for the analysis of large molecules such as proteins or polymers. SEC works by trapping these smaller molecules in the pores of a particle. The larger molecules simply pass by the pores as they are too large to enter the pores. Larger molecules therefore flow through the column quicker than smaller molecules, that is, the smaller the molecule, the longer the retention time.tertiary structurequaternary structure

 Gel permeation(GPC), gel filtration(GFC) chromatography  Technique applicable to separation of high-molecular weight species  Rapid determination of the molecular weight or molecular-weight distribution of larger polymers or natural products  Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase

 Specific pore sizes-average residence time in the pores depends on the effective size of the analyte molecules › larger molecules › smaller molecules › intermediate size molecules

This is the most selective type of chromatography employed. It utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. For example, the immobilized molecule may be an antibody to some specific protein. When solutes 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.

Affinity Chromatography Surface bound with Epoxy, aldehyde or aryl ester groups Metal Interaction Chromatography Surface bound with Iminodiacetic acid + Ni 2+ /Zn 2+ /Co 2+ Affinity Chromatography

Binding Capacity (mg/ml) medium 12mg of histag proteins (MW= 27kDa) Depends on Molecular weight Degree of substitution /ml medium ~15  mol Ni 2+ Backpressure ~43psi Change the guard column filter

Pump Injector Column Detector Gradient Controller

Restek ® ULTRA C-18 and CN Columns (250mm x 4.6mm, 5µ), Mobile Phase: (1:1 Methanol:Water), 1.5 mL/min.

A Supelcosil LC-PAH Columns B Conditions: A: 150mm x 4.6mm, 5µ. Flow Rate: 1.5 mL/min Conditions: B: 50mm x 4.6mm, 3µ. Flow Rate: 3.0 mL/min