Introduction Chromatography is a technique for separating mixtures into their components in order to analyze, identify, purify, and/ or quantify the mixture or components. Chromatography was invented about ninety years ago by Mikhail Tsvet, a Russian scientist studying plant pigments.Why Chromatography? It is used to separate low-molecular-mass compounds based on their distribution between stationary phase and mobile phase. Due to its low cost and availability of various protocols for the separation of compounds, paper chromatography is considered a powerful analytical tool. So it is taught and used extensively to separate liquids soluble in a solvent, but having differert acisorption abilities on an adsorbent. Scientists Use Chromatography Analyze — examine a mixture, its components, and their relations to one another * Identify — determine the identity of a mixture or components based on known components * Purify — separate components in order to isolate 1 one of interest for further study © Quantify — determine the amount of mixture and/or the components present in the sampleTypes of Chromatography * Liquid Chromatography - separates liquid samples with a liquid solvent (mobile phase) and a column composed of solid beads (stationary phase) * Gas Chromatography - separates vaporized samples with a carrier gas (mobile phase) and a column composed of a liquid or of solid beads (stationary phase)* Paper Chromatography —Paper chromatography is an analytical method used to separate coloured chemicals or substances. It separates dried liquid samples with a liquid solvent (mobile phase) and a paper strip (stationary phase). “hromatography *Thin-Layer Chromatography - separates dried liquid samples with a liquid solvent (mobile phase) and a glass plate covered with a thin layer of alumina or silica gel R, values Saivert rantMaterials Required 6 beakers or jars 6 covers or lids Distilled H20 Isopropanol Graduated cylinder © 6 strips of filter paper * Different colors of Sharpie pens * Pencil ¢ Ruler * Scissors Tape ePrinciple of Paper Chromatography Partition Chromatograph Partition Chromatography: Partition chromatography swewe — 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. Exch solu srt at ance etna hase ard me neAdsorption Chromatography: 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: 1. Ascending Paper Chromatography — The techniques goes with its name as the solvent moves in an upward direction. 2. Descending Paper Chromatography - The movement of the flow of solvent due to gravitational pull and capillary action is downwards, hence the name descending paper chromatography. 3. Ascending — Descending Paper Chromatography — In this version of paper chromatography, movement of solvent occurs in two directions after a particular point. Initially, the solvent travels upwards on the paper which is folded over a rod and after crossing the rod it continues with its travel in the downward direction.4, Radial or Circular Paper Chromatography — The sample is deposited at the center of the circular filter paper. Once the spot is dried, the filter pi is tied horizontally on a Petri dish which contains the ss 5, Two-Dimensional Paper Chromatography — Substances which have the same rr values car be resolved with the help of two-dimensional paper chromatography. Events of Paper Chromatography ¢ Capillary Action — the movement of liquid within the spaces of a porous material due to the forces of adhesion, cohesion, and surface tension. The liquid is able to move up the filter paper because its attraction to itself is stronger than the force of gravity. ¢ 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 both their solubility in the mobile phase and their differential affinity to the mobile phase and the stationary phase.PROCEDURE 1. Preparing the Isopropanol Solutions: Prepare 15 ml of the following isopropanol solutions in appropriately labeled beakers: - 0%, 5%, 10%, 20%, 50%, and 100% 2. Preparing the Chromatography Strips: ° Cut 6 strips of filter paper * Draw a line 1 cm above the bottom edge of the strip with the pencil * Label each strip with its corresponding solution * Place a spot from each pen on your starting oe 73. Developing the Chromatograms: * Place the strips in the beakers. * Make sure the solution does not come above your start line. * Keep the beakers covered. * Let strips develop until the ascending solution front is about 2 cm from the top of the strip. * Remove the strips and let them dry.Solvent | front ee | Blue +9 Purple | rf | Red J Origin An overview «i: _ Purpose: To introduce students to the principles and terminology of chromatography and demonstrate separation of the dyes in Sharpie Pens with paper chromatography. Time Required: Prep. time: 10 minutes Experiment time: 45 minutes What Is Retention Factor (Ri)? The amount that each component of a mixture travels can be 4, Quantified using retention factors 1 | Particular material is the ratio of the distance @bove the origin to the distance the solvent front moved above _the origin, (Rf). The retention factor of a the spot movedObservations The Dyes separate out slowly at different positions. * The above are chromatogram condition at different time interval.Applications of Chromat, graphy Ograph ‘a * [Pharmaceutical Compan determine amount of each chemical found in new product. ® [Hospital detect blood or alcoho! levels in a patient’s blood stream. 2-20 2? ee We’ Foren saenad to compare a sample found at a i 4 crime scene to samples from “suspects. */Environmental Agen determine the level of pollutants in the water supply.DOD PA Dg im L Food Industry Pos = = Ez 5 ae The technique of chromatography plays a crucial role in the food industry for the determination of the shelf life of several food substances through helping in the analysis of the point wherein the food tends to spoil. Also, the presence of chemical additives in the food items is determined by using this technique. The food sample’s nutritional value is also determined with the help of chromatography techniques.OP te High Performance | quid Chromatography (ELC) AM PC for Data Acquisition f} Waste HPLC Column, Injector Detector When it comes to Molecular Biology, the studies of metabolomics and proteomics often tend to involve the usage of several hyphenated techniques of chromatography, out of which the most notable is EC- LC-MS. The nucleic acid research is known for making extensive usage of these chromatographic techniques. A special kind of chromatography technique is called HPLC which is widely used in protein separation applications, This kind of chromatography technique is Used in the purification of enzymes, insulin purification, and plasma fractionation.§/ ; 8] Cenelusion &| chromatography is not just a Method to 5 &! components of a mixture, but eparate S| inour lives and lives of other cit; technique to S€parate plant d §| the chemical content of medici §| pollutants in the wa iter of agricultural field and many more. It is also a part of the developing Sciences like S$ biotechnology and microbiology, From just beinga laboratory technique, it has become an integral § technique of many fields as mentioned above. So, it is {essential that we have knowledge of this technique because you never know when and where would we need it.