Chromatography & Separation Techniques Lecture (Fall 2024-2025) - Week 3 PDF

Chromatography& Separation Techniques Week 3 Assoc. Prof. Sameh Younis Associate Professor of Pharmaceutical Analytical Chemistry * Planar chromatography In which the stationary phase is supported on or in the pores of a flat support. The mobi...

Chromatography& Separation Techniques Week 3 Assoc. Prof. Sameh Younis Associate Professor of Pharmaceutical Analytical Chemistry * Planar chromatography In which the stationary phase is supported on or in the pores of a flat support. The mobile phase moves through the stationary phase by capillary action, sometimes assessed by gravity or an electrical potential. 2 Planar Chromatography - Types Thin layer chromatography (TLC) Sample: dried liquid samples mobile phase: liquid Stationary phase: A thin layer of alumina or silica gel supported by glass or plastic plate Paper Chromatography (PC) Sample: dried liquid samples mobile phase: liquid Stationary phase: water held to paper strip (cellulose fibers) (hydrated paper sheet) Electrophoresis: migration is carried out under the influence of an electrical field. 3 Principles of Planar Chromatography Capillary Action – the movement of liquid (mobile phase) within the spaces of a porous material without the assistance of, and in opposition to, external forces such as gravity. -due to the forces of adhesion, cohesion, and surface tension that can resist gravity. -The liquid is able to move up the planar stationary phase when adhesion power is greater than cohesive force. Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes dissolve into solvents that have similar properties. (Like dissolves like) This allows different solutes to be separated by different combinations of solvents. Separation of components depends on their solubility in the mobile phase and, by consequence, differential affinity to the mobile phase and the stationary phase. 4 N.B. Adsorbent: finely divided homogenous solid having uniform particle size and large surface area which is capable of attracting molecules to its surface. Resolution: the ability of any chromatographic process to separate pure compound. Rate of flow: distance travelled by solute / distance travelled by mobile phase (eluent) 5 THIN LAYER CHROMATOGRAPHY (TLC) 6 Introduction Thin layer chromatography (TLC) is carried out on a thin layer of chromatographically active particulate material (silica gel or alumina), which is dispersed on a surface of an inert support (flat glass or plastic plates). 7 In TLC, a liquid mobile phase moves along the stationary phase, carrying with it the component(s) of the sample applied. As the sample is carried by the solvent system, the components are separated by differential affinity between the mobile and the stationary phases. Although TLC is widely used for qualitative analysis, it does not in general provide quantitative information of high precision and accuracy. Recent changes in the practice of TLC have resulted in improved performance in terms of both separation and determination regarding quantitative analysis..(HPTLC)..(high performance thin liquid chromatography). 8 9 Basic concepts Basic Concepts 1. Preparation of the plate -For TLC, a variety of coating materials is available; silica gel is the most frequently used. -Slurry of the gel is uniformly spread over the plate by means of a commercially available spreader. -After air-drying overnight, or oven-drying at 80-90 C for about 30 minutes, the plate is ready for use. N.B. Ready to use thin layers (pre-coated plates) are commercially available. 10  Thin Layer Chromatography silica gel - silicon dioxide (SiO2)x (a common, inexpensive stationary phase) TLC plate O O O | | | −O−Si−O−Si−O−Si−O−H These exposed OH units | | | O O O give silica gel a | | | relatively polar surface. −O−Si−O−Si−O−Si−O−H | | | O O O bulk (SiO2)x surface 11 2. Sample application - Sample application is perhaps the most critical step in TLC, particularly for quantitative analysis. -Usually, the sample is applied as a spot 1 to 2 cm from the edge of the plate. - The origin line, to which the sample solution is applied, is usually located 2-2.5 cm from the bottom of the plate. - Manual application of sample solution is performed by touching a capillary tube containing the sample to the plate or by use of a hypodermic syringe. - For higher precision and accuracy of sample application, mechanical dispensers are preferably used (automatic applicators).  1. Sample Application 1. Sample Application (spotting) (spotting) TLC plate A. Draw “guide lines” lightly with pencil “finishing line”  1 cm. B. Dissolve solid sample in MeOH C. Use TLC capillary to transfer and spot dissolved sample “starting line” Sample A B C Ref. Ref. Ref.  1 cm. 13 3. Plate development 3. Plate development It is the process in which a sample is carried by a mobile- phase through the stationary-phase. It is usually developed by the ascending technique in which the plate is immersed in the developing solvent; care being taken to void direct contact between the sample and the solvent system. The tank or chamber is preferably lined with sheets of filter paper which dip into the solvent; this to ensure that the tank is saturated with the solvent vapour. Figure 14: Thin-layer chromatography setup. 14  The developing solvent travels up the plate, being drawn by capillary action between the fine particles.  As the developing solvent travels past the point of sample application, it dissolves the sample and carries it up the plate, with the sample distributing itself between the moving solvent and the stationary phase.  After the developer (solvent system) has reached about two thirds of the length of the plate, the plate is removed from the container and dried. The positions of the sample components are then located. 15 2. Development of TLC Plate {keep capped} A. Place spotted TLC plate TLC plate in developing chamber B. Developing solution is drawn up the plate by capillary action C. Remove TLC plate when solvent reaches top line NOTE: During this ~20 min. developing stage, compounds Developing solution } in the original spots are being (mobile phase) TLC Developing Chamber pulled through the silica gel. (just a glass jar with solvent in it!) 16 4. Locating sample components on the plate - The positions of separated sample components can be located by various methods. A ) Coloured substances can be seen directly when viewed against the stationary phase. B) The colourless substances can usually be detected by spraying the plate with an appropriate reagent, which produces coloured spot in the areas, which they occupy.  Visualization "semi-destructive" universal specific biological I2 reversible enzymatic detection complexation “aromatics” irreversible formation of coloured derivative destructive non destructive use of corrosive UV “aromatics+ conjugated systems” material as H2SO4 water spray for saponin 18 Examples of specific visualization: -Ninhydrin is used for amino acids -permanganate for oxidizable cpds. -2,4-dinitrophenylhydrazine is used for aldehydes and ketones.