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  1. 1. What is chromatography? Chromatography (from Greek word for chromos for colour) is the collective term for a family of laboratory techniques for the separation of mixtures into their individual compounds. The basic principle of chromatography is that different compounds will stick to a solid surface or dissolve in a film of liquid to different degrees. Chro mato g raphy is use d e xte nsive ly in fo re nsics, fro m analyzing bo dy fluids fo r the pre se nce o f illicit drug s, to fibe r analysis, blo o d analysis fro m a crime sce ne , and at airpo rts to de te ct re sidue fro m e xplo sive s. It ca identifyhow m n anycom ponents in a m ixture. It is quick, sim and CHEAP ple, !
  2. 2. What is chromatography? We can use chromatography to separate the components of inks and dyes, such as those found in pens, markers, clothing, and even candy shells. Chromatography can also be used to separate the colored pigments in plants or used to determine the chemical composition of many substances. We will be using thin layer chromatography or TLC
  3. 3. What do we do?• A TLC plate is a sheet of paper, glass, metal or plastic which is coated with a thin layer of a solid adsorbent.• A mobile phase is selected based on substances possible identity and placed in a tank• A pencil line is drawn near the bottom of the plate above the solvent line. An equal small amount of the mixture is spotted near the bottom of the plate• The TLC plate is carefully placed inside the chamber and observed as the solvent moves up• The chromatogram is removed BEFORE the solvent runs out the top and dried to calculate Rf
  4. 4. • Why Does it Work! As the solvent moves past the spot , each component of the mixture has a different affinity to the adsorbent and mobile phase• As the solvent moves up the medium, the differences in affinities will separate the components in the mixture• The more affinity to the solvent the faster the component moves• A UV lamp is utilized to be sure all components are identified
  5. 5. Illustration of Chromatography Stationary Phase Separation Mobile Phase Mixture Components Components Affinity to Stationary Phase Affinity to Mobile Phase Blue ---------------- Insoluble in Mobile Phase Black   Red   Yellow          
  6. 6. Developing the Chromatograms
  7. 7. Identification of Components of a Mixture on Chromatograms• Components of a mixture may be identified by matching Rf values to known compounds. (Best to run the sample and knowns on the same plate at the same time. ) A close match suggests that the compounds are the same. This is not fool-proof • The position of spots on a chromatogram are described by giving the Rf of the spot. Distance Sample Travels Rf = Distance Solvent front Travels
  8. 8. Separation of components of a Red Maple Leaf
  9. 9. Thin-Layer Chromatography Uses paper, thin plastic or glass trays to identify the composition of pigments, chemicals, and other unknown substances. It is very important not to disturb the chamber during the developing process and slosh the solvent, the spots should be the same size, the TLC plate should remain flat and not fold over or curve and the solvent mark should be indicated upon removal from the chamber
  10. 10. Mixtures & Compounds Mixture – Two or more substances that are mixed together, but not chemically combined. Examples of mixtures ... Air – mixture of gases Bowl of cereal – mixture of cereal and milk Soda pop – mixture of soda syrup, water, and CO 2 gas Fog –water suspended in air Kool-Aid – mixture of water, sugar, and flavor crystals Compounds – Two or more elements that are chemically combined. Examples of compounds ... Salt –Sodium and chlorine combined chemically Water –Hydrogen and oxygen combined chemically Carbon Dioxide – Carbon and oxygen combined chemically
  11. 11. Solutions Solutions are mixtures in which one substance is dissolved in another. Solutions have two parts: solute and solvent The solute is the substance that is dissolved. The solvent is the substance that does the dissolving Identify the solute and solvent in each solution ... Solution Solute Solvent Lemonade Soda pop Ocean water Solubility - A measure of how much of a given substance will dissolve in a liquid. A substance that does not dissolve in water is called insoluble. A substance that does dissolve in water is called soluble.
  12. 12. Paper Chromatography Lab• Obtain the supplies you’ll need. Tape – Label with marker – 1 large beaker (or plastic cup) Pencil – 1 small beaker (or plastic cup) filled with water Filter – 4 pieces of filter paper Paper – 4 black markers for testing – 4 small pieces of masking tape – Pencil (to attach to the top of the filter paper) Ink – Permanent marker Mark – Timer• Write the pen number on a piece of masking tape with a permanent marker and place it at the top of the strip.• Choose one of the testing markers and draw a thick line near the bottom of the filter paper - about ¼ inch from the bottom.• Pour a small amount of water into the large cup and then hang the paper strip in the cup. Make sure the ink line does not touch the water – only the bottom of the filter paper.• Allow the water to move up the paper for 5 minutes and then remove the strip from the water. Hang it on the side of the table to dry.
  13. 13. Other Examples of Chromatography Liquid Chromatography Used to identify unknown plant pigments & other compounds. Gas Chromatography Used to determine the chemical composition of unknown substances, such as the different compounds in gasoline shown by each separate peak in the graph below.