Chromatographic Techniques PDF

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Chromatographic techniques  Learning outcomes  Express the basic principles of chromatography.  Categorize the types, basic components, and properties of: Paper Chromatography...

Biotechniques (BMS 34010A) Fall semester 2023 -2024 Dr. Tania Tahtouh [email protected] Chromatographic techniques  Learning outcomes  Express the basic principles of chromatography.  Categorize the types, basic components, and properties of: Paper Chromatography Column Chromatography HPLC Ion-exchange Chromatography Affinity Chromatography Gaz Chromatography  Protein Purification Steps  Given that the initial volume of the crude extract is relatively large a researcher typically applies a technique like ammonium sulfate precipitation to reduce the size of the sample and the number of proteins within it.  After ammonium sulfate precipitation, investigators typically apply column chromatography procedures to further purify the protein.  Chromatography  Chromatography is an important biophysical technique that enables the separation, identification, and purification of the components of a mixture. ▪ Chromatography is usually based on the principle of partition of solute between two phases. It usually consists of a Mobile Phase and a Stationary Phase.  3 components form the basis of the chromatography technique: ▪ Stationary (immobilized) phase: may be a solid, gel, liquid, or a solid/liquid mixture that is immobilized ▪ Mobile phase: may be liquid or gaseous, which is passed over or through the stationary phase after the mixture of analytes to be separated has been applied to the stationary phase. ▪ The mixture of analytes to be separated  Chromatography terms  Chromatograph is an equipment that enables a sophisticated separation  Eluent is a fluid entering the column/ solvent that carries the analyte.  Eluate is the mobile phase leaving the column.  Sample (analyte) is a substance analyzed in chromatography.  Solvent is any substance capable of solubilizing an other substance.  Retention time is the time required for a particular analyte to pass through the system (from the column inlet to the detector) under set conditions.  Factors influencing separation  The factors effective on this separation process include: Adsorption is the process by which ▪ Molecular characteristics related to adsorption (liquid-solid). ions, atoms or molecules adhere to ▪ Differences among the molecular weights. the surface of a solid material. ▪ Partition (liquid-solid). Partition coefficient (Kd) describes the way in which the analyte distributes between two immiscible phases. ▪ Affinity. Affinity is the degree to which a substance tends to combine with another.  Chromatogram  Chromatogram is a graphical presentation of detector response, concentration of analyte in the effluent*, or other quantity used as a measure of effluent concentration. The retention time or volume is when a solute exits the injector and passes through the column and the detector. Data represented by the chromatogram are used to help identify and quantify the solute(s).  Because eluting solutes are displayed graphically as a series of peaks, they are frequently referred to as chromatographic peaks. These peaks are described in terms of peak width, height, area. *Effluent is the stream flowing out of a chromatographic column  Classification  Based on shape of chromatographic beds: ▪ Planar chromatography ▪ Column chromatography  Based on the physical state of mobile and stationary phase: ▪ Gas chromatography ▪ Liquid chromatography  Based on mechanism of separation: ▪ Ion-exchange chromatography ▪ Affinity chromatography ▪ Adsorption chromatography  Paper chromatography  In paper chromatography support material consists of a layer of cellulose (thick filter paper) highly saturated with water. ▪ The solvent flows along the paper through the spots and, carries the substances from the spot ▪ Each of these substances will, if the solvent mixture has been well chosen, move at a different rate from the others. Used for identifying and separating colored mixtures like pigments.  Column chromatography  In column chromatography the stationary phase is packed into a glass or metal column.  The mixture of analytes is then applied and the mobile phase, commonly referred to as the eluent, is passed through the column either under gravity or by use of a pumping system or applied gas pressure.  The stationary phase is either coated onto discrete small particles (the matrix) and packed into the column or The general principle in applied as a thin film to the inside wall of the column. column chromatography  Column chromatography  As different proteins percolate through the column they are separated based on their physical properties. ▪ Net charge at a given pH (ion-exchange chromatography) ▪ Relative sizes (size-exclusion chromatography) ▪ Ligand binding specificity (affinity chromatography).  The effluent fraction containing the protein of interest can be identified based on an enzymatic or other type of assay.  HPLC  High-Performance Liquid Chromatography (HPLC) is basically a highly improved form of liquid chromatography.  Instead of a solvent (mobile phase) being allowed to drip through the column under gravity, it is forced through under high pressure. Advantage: High performance and high speed as compared with traditional column chromatography.  Ion-exchange chromatography  Ion-exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). 1. Light interactions: Proteins having a low density of net positive charge will tend to emerge first followed by those having a higher charge density. 2. Strong interactions: Proteins are separated from the column either by changing pH, concentration of ion salts or ionic strength of the buffer solution. ▪ Positively charged ion-exchange matrices are called anion- exchange matrices, and adsorb negatively charged proteins. ▪ Negatively charged ion-exchange matrices are called cation- exchange matrices, and adsorb positively charged proteins.  Affinity chromatography  It does not rely on differences in the physical properties of the proteins. Instead it exploits the unique property of extremely specific biological interactions to achieve separation and purification. ▪ It is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins. ▪ A ligand which can make a complex with specific protein binds the filling material of the column.  Affinity chromatography: steps  Choice of appropriate ligand  Immobilization of the ligand onto a support matrix  Binding of molecules of interest with the ligand  Removal of non specifically bound molecules  Elution of molecules of interest in purified form  Gas chromatography  Gas chromatography differs from other forms of chromatography in that the mobile phase is a gas and the components are separated as vapors.  It is thus used to separate and detect small molecular weight compounds in the gas phase. ▪ The sample is either a gas or a liquid that is vaporized in the injection port. ▪ The mobile phase for gas chromatography is a carrier gas, typically helium because of its low molecular weight and being chemically inert.  Gas chromatography  Gas chromatography is used in the analysis of: ▪ air-borne pollutants ▪ performance-enhancing drugs in athlete’s urine samples ▪ oil spills ▪ essential oils in perfume preparation Columns for Gas Chromatography References  Coskun O. Separation techniques: Chromatography. North Clin Istanb. 2016;3(2):156-160. Published 2016 Nov 11. doi:10.14744/nci.2016.32757