MD100 Medical Biochemistry I Lab Exercise 2: Thin-Layer Chromatography PDF

MD100 Medical Biochemistry I Lab Exercise 2: Introduction to Thin- Layer Chromatography Fall Semester 2024  Objectives To be introduced to the general principles of Chromatography To understand the use of Thin Layer Chromatography [TLC] To separate and...

MD100 Medical Biochemistry I Lab Exercise 2: Introduction to Thin- Layer Chromatography Fall Semester 2024  Objectives To be introduced to the general principles of Chromatography To understand the use of Thin Layer Chromatography [TLC] To separate and identify amino acids in a mixture by TLC  Chromatography Chromatography is an analytical technique, available for the separation of closely related compounds in a mixture. There are different kinds of chromatography including paper, thin layer, column, size-exclusion, ion exchange, affinity and HPLC. The separation is affected by differences in the equilibrium distribution of the components between two phases, the stationary and the mobile phases. These differences in the equilibrium distribution are a result of nature and degree of interaction of the components with these two phases.  Principle of Chromatography ▪ Chromatography works on the principle that different 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. ▪ Some components of the mixture stay longer in the stationary phase, and they move slowly in the chromatography system, while others pass rapidly into the mobile phase, and leave the system faster. ▪ Once separation occurs, the individual components are visualized as spots at a different level of travel on the plate. Their nature or character are identified using suitable detection techniques.  Thin layer chromatography [TLC]: Thin layer chromatography (TLC) is a technique used to separate and identify compounds of interest. It provides qualitative information and with careful attention to details, it is possible to obtain quantitative data. A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support. TLC Plate  Stationary and Mobile Phase The silica gel acts as the stationary phase and the solvent mixture acts as the mobile phase (solvent or solvent mixture). In the ideal solvent system, the compounds of interest are soluble to different degrees. Separation results from the partition equilibrium of the components in the mixture.  Separation of Compounds Differential Partitioning: As the solvent moves up the plate, each compound in the sample mixture interacts differently with the stationary and mobile phases based on its polarity. Affinity for Stationary Phase: Compounds that are more polar interact strongly with the polar stationary phase (e.g., silica gel) and move more slowly. Affinity for Mobile Phase: Less polar compounds interact less with the stationary phase and more with the mobile phase, allowing them to travel farther up the plate.  Experimental Procedure 1cm 1cm 1cm 1. Draw a horizontal line with a pencil 2. Draw spots at the spotting line at the bottom, at the top and at the with equal distance between them edge of the TLC plate (1cm) Every spot represents a sample  Experimental Procedure 3. Using a capillary tube, a small spot of solution containing the sample is applied to a plate, about 1.0 cm from the bottom edge. Different samples can be placed in a row of spots the same distance from the bottom edge, each of which will move in its own adjacent lane from its own starting point. Use a pencil to mark starting point Dissolve sample in a solvent Use micropipette to transfer tiny drop of sample solution  Experimental Procedure 4. A small amount of an appropriate solvent is poured into a glass chamber to a depth of less than 1 centimeter. 5. The TLC plate is then placed in the chamber so that the spots of the sample do not touch the surface of the eluent in the chamber, and the lid is closed. The solvent moves up the plate by capillary action, meets the sample mixture and carries it up the plate 6. The plate should be removed from the chamber before the solvent front reaches the top of the stationary phase and dried. 7. The solvent front, the furthest extent of solvent up the plate, is marked.  Ninhydrin amino acid detection The plate is removed after an optimal development time and dried and the spots are detected using a suitable location reagent. 8. The TLC plate is sprayed by using Ninhydrin reagent. Ninhydrin reacts with the α-amino group of primary amino acids producing 'purple colure'. RF calculation RF Values