Presentation on CHROMATOGRAPHY TECHNIQUES
- 1. CHROMATOGRAPHIC TECHNIQUES By Y. Sai Kumar Assistant professor Dept. of Pharmacology
- 2. Chromatography is a laboratory method employed to separate a mixture into its individual components. The word "chromatography" is derived from the Greek terms "Chroma" (meaning color) and "grapho" (meaning to write), originally referencing its use in separating colored pigments. Today, chromatography is extensively applied in fields such as chemistry, biology, and environmental science for the analysis, purification, and isolation of different compounds. CHROMATOGHRAPHY Chromatography is based on the principle where molecules in mixture applied onto the surface or into the solid, and fluid stationary phase (stable phase) is separating from each other while moving with the aid of a mobile phase. Principle The factors effective on this separation process include molecular characteristics related to adsorption (liquid- solid), partition (liquid-solid), and affinity or differences among their molecular weights. Because of these differences, some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into mobile phase, and leave the system faster.
- 3. Based on the above approach three components form the basis of the chromatography technique. • Stationary phase: This phase is always composed of a “solid” phase or “a layer of a liquid adsorbed on the surface a solid support”. • Mobile phase: This phase is always composed of “liquid” or a “gaseous component.” • Separated molecules Fundamental concept: The type of interaction between stationary phase, mobile phase, and substances contained in the mixture is the basic component effective on separation of molecules from each other. Stationary phase in chromatography, is a solid phase or a liquid phase coated on the surface of a solid phase. E.g.: Silica Gel, Alumina, Cellulose, Polymer Resins, C18 (Octadecylsilane), Ion-Exchange Resins, Agarose Gel Mobile phase flowing over the stationary phase is a gaseous or liquid phase. E.g.: Water, Organic Solvents (e.g., Methanol, Acetone, Ethanol), Hexane, Acetonitrile, Toluene, Buffer Solutions, Gas (e.g., Helium, Nitrogen) If mobile phase is liquid it is termed as liquid chromatography (LC), and if it is gas then it is called gas chromatography (GC). Gas chromatography is applied for gases, and mixtures of volatile liquids, and solid material. Liquid chromatography is used especially for thermal unstable, and non-volatile samples. The purpose of applying chromatography which is used as a method of quantitative analysis apart from its separation, is to achieve a satisfactory separation within a suitable time interval. Various chromatography methods have been developed to that end, Some of them include
- 4. Types of chromatography • Column chromatography • Ion-exchange chromatography • Gel-permeation (molecular sieve) chromatography • Affinity chromatography • Paper chromatography • Thin-layer chromatography • Gas chromatography • Dye-ligand chromatography • Hydrophobic interaction chromatography • Pseudo affinity chromatography • High-pressure liquid chromatography (HPLC)
- 5. Column chromatography • When the mobile phase, along with the mixture to be separated, is introduced at the top of the column, the components of the mixture move at different speeds. • Those with weaker adsorption and affinity for the stationary phase travel more quickly, while those with stronger interactions with the stationary phase move more slowly. • As a result, the faster-moving components are eluted first, while the slower-moving components are eluted later. The adsorption of solute molecules to the column occurs in a reversible manner. The rate of the movement of the components is expressed as: (Retardation factor) Rf = the distance travelled by solute/ the distance travelled by the solvent • The fraction of the mobile phase that transports the sample components is known as eluent. The mixture of solute and solvent that exits the column is known as an eluate. • The eluate is made up of the mobile phase and analytes. A substance that separates and moves constituents of a mixture through the column of a chromatograph. • The eluent in liquid chromatography is a liquid solvent whereas in gas chromatography is a carrier gas. Purification of Organic Compounds, Pharmaceutical Industry, Biotechnology (Protein Purification), Environmental Science (Pollutant Separation), Food Industry (Essential Oils, Flavour Compounds), Forensic Science (Drug Testing, Toxicology), Petrochemical Industry (Hydrocarbon Separation)
- 6. Ion- exchange chromatography o Ion- exchange chromatography is based on electrostatic interactions between charged protein groups, and solid support material (matrix). o Matrix has an ion load opposite to that of the protein to be separated, and the affinity of the protein to the column is achieved with ionic ties. o 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. o While matrices bound with negatively charged groups are known as cation-exchange matrices, and adsorb positively charged proteins. Typical IC instrumentation includes: pump (provide a continuous constant flow), injector (range of 0.1 to 100 ml of volume with high reproducibility and under high pressure (up to the 4000 psi))., column (diameter from about 2mm to 5 cm and in length from 3 cm to 50 cm), suppressor (convert the ionic eluent to water as a means of enhancing the sensitivity), detector and recorder or data system (pre-programmed computing integrator).
- 7. Gel- permeation (molecular sieve) chromatography The basic principle of this method is to use dextran containing materials to separate macromolecules based on their differences in molecular sizes. This procedure is basically used to determine molecular weights of proteins, and to decrease salt concentrations of protein solutions. In a gel- permeation column stationary phase consists of inert molecules with small pores. The solution containing molecules of different dimensions are passed continuously with a constant flow rate through the column. Molecules larger than pores can not permeate into gel particles, and they are retained between particles within a restricted area. Larger molecules pass through spaces between porous particles, and move rapidly through inside the column. Molecules smaller than the pores are diffused into pores, and as molecules get smaller, they leave the column with proportionally longer retention times. Sephadeks G type is the most frequently used column material. Besides, dextran, agarose, polyacrylamide are also used as column materials.
- 8. This chromatography technique is used for the purification of enzymes, hormones, antibodies, nucleic acids, and specific proteins. A ligand which can make a complex with specific protein (dextran, polyacrylamide, cellulose etc.,) binds the filling material of the column. The specific protein which makes a complex with the ligand is attached to the solid support (matrix), and retained in the column, while free proteins leave the column. Then the bound protein leaves the column by means of changing its ionic strength through alteration of pH or addition of a salt solution. Affinity chromatography Gas chromatography • In this method stationary phase is a column which is placed in the device, and contains liquid stationary phase which is adsorbed onto the surface of an inert solid. • Gas chromatography is a “gas-liquid” chromatography. Its carrier phase consists of gases as He or N2 Mobile phase which is an inert gas is passed through a column under high pressure. • The sample to be analyzed is vaporized, and enters into a gaseous mobile phase. • The components contained in the sample are dispersed between mobile phase, and stationary phase on the solid support. Gas chromatography is a simple, multifaceted, highly sensitive, and rapidly applied technique for the extremely excellent separation of very minute molecules. It is used in the separation of very little amounts of analytes.
- 9. Thin Layer Chromatography can be defined as a method of separation or identification of a mixture of components into individual components by using finely divided adsorbent solid / (liquid) spread over a plate and liquid as a mobile phase. Thin-layer chromatography is performed on a sheet of glass, plastic, or aluminium foil, which is coated with a thin layer of adsorbent material, usually silica gel, aluminium oxide (alumina), or cellulose. This layer of adsorbent is known as the stationary phase. After the sample has been applied on the plate, a solvent or solvent mixture (known as the mobile phase) is drawn up the plate via capillary action. Because different analytes ascend the TLC plate at different rates, separation is achieved. It is thus based on the principle of adsorption chromatography or partition chromatography or combination of both, depending on adsorbent, its treatment and nature of solvents employed. The components with more affinity towards stationary phase travels slower. Components with less affinity towards stationary phase travels faster. Once separation occurs, the individual components are visualized as spots at a respective level of travel on the plate. Their nature or character is identified by means of suitable detection techniques. Thin Layer Chromatography
- 10. Components of Thin Layer Chromatography (TLC) 1.TLC plates, preferably ready made with a stationary phase: These are stable and chemically inert plates, where a thin layer of stationary phase is applied on its whole surface layer. The stationary phase on the plates is of uniform thickness and is in a fine particle size. 2.TLC chamber- This is used for the development of TLC plate. The chamber maintains a uniform environment inside for proper development of spots & prevents the evaporation of solvents, and keeps the process dust free. 3.Mobile phase- This comprises of a solvent or solvent mixture The mobile phase used should be particulate-free and of the highest purity for proper development of TLC spots. The solvents recommended are chemically inert with the sample, a stationary phase. 4.A filter paper- This is moistened in the mobile phase, to be placed inside the chamber. This helps develop a uniform rise in a mobile phase over the length of the stationary phase. The stationary phase is applied onto the plate uniformly and then allowed to dry and stabilize. These days, however, ready-made plates are more commonly used. 1.With a pencil, a thin mark is made at the bottom of the plate to apply the sample spots. 2.Then, samples solutions are applied on the spots marked on the line in equal distances. 3.The mobile phase is poured into the TLC chamber to a leveled few centimeters above the chamber bottom. 4.A moistened filter paper in mobile phase is placed on the inner wall of the chamber to maintain equal humidity (and also thereby avoids edge effect). 5.Now, the plate prepared with sample spotting is placed in TLC chamber so that the side of the plate with the sample line is facing the mobile phase. Then the chamber is closed with a lid. Procedure:
- 11. 6.The plate is then immersed, such that the sample spots are well above the level of mobile phase (but not immersed in the solvent) for development. Sufficient time is given for the development of spots. 7. The plates are then removed and allowed to dry. 8. The sample spots are then seen in a suitable UV light chamber, or any other methods as recommended for the given sample. Some common techniques for visualizing the results of a TLC plate include • UV light, KMnO4 stain (organic molecules) • Iodine Staining: is very useful in detecting carbohydrates since it turns black on contact with Iodine • Ninhydrin Reagent: often used to detect amino acids and proteins Retention Factor (Rf ) Value The behaviour of a compound on a TLC is usually described in terms of its relative mobility or Rf value. Rf or Retention factor is a unique value for each compound under the same conditions. The Rf for a compound is a constant from one experiment to the next only if the chromatography conditions below are also constant: solvent system, adsorbent, thickness of the adsorbent, amount of material spotted, temperature Since these factors are difficult to keep constant from experiment to experiment, relative Rf values are generally considered. Relative Rf” means that the values are reported relative to a standard, calculated using the following equation:
- 12. 1. In monitoring the progress of reactions 2. Identify compounds present in a given mixture 3. Determine the purity of a substance. 4. Analyzing ceramides and fatty acids Applications of Thin Layer Chromatography (TLC) Detection of pesticides or insecticides in food and water Analyzing the dye composition of fibers in forensics Assaying the radiochemical purity of radiopharmaceuticals Identification of medicinal plants and their constituents Chromatography technique that uses paper sheets or strips as the adsorbent being the stationary phase through which a solution is made to pass is called paper chromatography. Paper Chromatography The principle involved can be partition chromatography or adsorption chromatography. Partition chromatography because the substances are partitioned or distributed between liquid phases. The two phases are water held in pores of the filter paper and the other phase is a mobile phase which passes through the paper. When the mobile phase moves, the separation of the mixture takes place. The compounds in the mixture separate themselves based on the differences in their affinity towards stationary and mobile phase solvents under the capillary action of pores in the paper. Adsorption chromatography between solid and liquid phases, wherein the solid surface of the paper is the stationary phase and the liquid phase is the mobile phase. Types of paper chromatography: Ascending Paper Chromatography Descending Paper Chromatography Ascending – Descending Paper Chromatography Radial or Circular Paper Chromatography Two Dimensional Paper Chromatography
- 13. High-pressure liquid chromatography (HPLC) Using this chromatography technique it is possible to perform structural, and functional analysis, and purification of many molecules within a short time. This technique yields perfect results in the separation, and identification of amino acids, carbohydrates, lipids, nucleic acids, proteins, steroids, and other biologically active molecules. In HPLC, mobile phase passes through columns under 10–400 atmospheric pressure, and with a high (0.1–5 cm//sec) flow rate. In this technique, use of small particles, and application of high pressure on the rate of solvent flow increases separation power, of HPLC and the analysis is completed within a short time. Essential components of a HPLC device are solvent depot, high- pressure pump, commercially prepared column, detector, and recorder. Duration of separation is controlled with the aid of a computerized system, and material is accrued. To study the process of fermentation and ripening. To check the purity of pharmaceuticals. To inspect cosmetics. To detect the adulterants. To detect the contaminants in drinks and foods. To examine the reaction mixtures in biochemical laboratories. To determine dopes and drugs in humans and animals.
- 14. Application areas of chromatography in medicine Affinity chromatography's (i.e.. ion-exchange chromatography) are more effective in the separation of macromolecules as nucleic acids, and proteins. Paper chromatography is used in the separation of proteins, and in studies related to protein synthesis; Gas-liquid chromatography is utilized in the separation of alcohol, ester, lipid, and amino groups, and observation of enzymatic interactions. Molecular-sieve chromatography is employed especially for the determination of molecular weights of proteins. Agarose-gel chromatography is used for the purification of RNA, DNA particles, and viruses. Chromatographic technique is also used in the separation of vitamins, and proteins. Pseudo affinity chromatography Some compounds as anthraquinone dyes, and azodyes can be used as ligands because of their affinity especially for dehydrogenases, kinases, transferases, and reductases. The mostly known type of this kind of chromatography is immobilized metal affinity chromatography (IMAC).
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