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  • 1. Purification of Substances Highlight!! Sneak preview of our first session included!!
  • 2. Have you wonder…….
    • How do chemists separate miscible mixtures (dissolve in one another)?
    • How is air separated into its various components?
    • How do you test if athletes have consumed banned drugs to enhance their performance?
    Miscible mixtures
  • 3.
    • Methods of Purification:
    • Chromatography
    Today’s lesson Objectives: Know their principles, procedures and application
  • 4. Chromatography
    • comes from a Greek word “ khroma ” meaning colour
    • 1 st used to separate colours, pigments and dyes
    • now can be used for colourless substances
  • 5.
    • Definition:
    • Paper chromatography is used to
    • separate and identify small amount of
    • the substances that are soluble in the
    • same solvent.
    Paper Chromatography Aim: To separate and identify mixtures of dyes.
  • 6. Examples: dyes , food colorings, forensics, Urine analysis etc. Paper Chromatography Solvent Solvent front Paper Lid Resolved spots
  • 7. Paper Chromatography – PROCEDURE 1
    • Prepare the chamber.
    • Prepare the stationary phase. Draw starting line with pencil . Why?
    • Spotting samples with fine capillary tubes . Ensure that the spots are small and concentrated . Why?
    • Developing the chromatogram. The chromatogram should be suspended in the chamber without the line touching the solvent . Why?
  • 8.
    • Points to note:
    • The starting line should be drawn with pencil rather than ink because the ink will also dissolve in the solvent together with the dyes and interfere with the results.
    • At the end of the experiment, the solvent line/front should be near the top of the paper because it is to ensure that the dyes are fully separated. (especially the faster moving ones.
    • The spots of solution and dyes on the starting line should be small and concentrated because it is to ensure that the dyes are clearly seperated in distinct lines/lanes.
    Paper Chromatography – PROCEDURE 1
  • 9. Paper Chromatography – PROCEDURE 1 At the Start At the End Chromatogram
  • 10.
    • The solvent will travel up the paper.
    • When the solvent reaches the top of the paper, take the paper out of the beaker and allow it to dry.
    • The end product is called a Chromatogram .
    Paper Chromatography – PROCEDURE 1
  • 11. Chromatography
    • Different types of chromatography
    • paper , column, gas-liquid, GC, HPLC etc.
    • Principles of chromatography:
    • Two phases involved – the stationary and mobile phase.
    • Dissolution of solutes in the mobile phase.
    • Adsorption of solutes in the stationary phase.
  • 12.
    • Dissolution of solutes in the solvent.
    • Different solutes have different solubilities in the same solvent.
    • The more soluble the component, the faster it will dissolve in the solvent.
    • The more soluble component will get carried along faster by the solvent and move ahead of the others.
    Paper chromatography
  • 13.
    • Adsorption of solutes by the paper
    • Components which are strongly adsorbed by the paper doesn’t move up.( ascending-type p.c. )
    • Components which are weakly adsorbed by the paper will redissolve in the solvent and move up
    • Rate of solutes moving up depends on:
    • how strongly it is adsorbed by the paper
    • how soluble it is in the solvent.
    Paper chromatography
  • 14. Paper Chromatography – PRINCIPLE 1
    • Based on differences in relative SOLUBILITIES of the mixtures in a particular solvent.
    • The more soluble the dye in the mobile solvent, the further the dye travels .
    • The less soluble the dye in the mobile solvent, the less the dye travels .
    Solubility in solvent
  • 15. Chromatography
    • The dyes move up at different rate because of the difference in
    • - solubility in the solvent
    • - adsorbance on the paper
  • 16. Paper Chromatography – PRINCIPLE 1
  • 17. Paper Chromatography – PRINCIPLE 2
    • Based on differences in ADSORBANCE of the component on the paper
    • The better the component is adsorbed to the paper, the less mobile the component.
    • The less the dye is adsorbed to the paper, the further the component travels.
    Mixed swarm of bees and hornets enter flower bed … Bees visit flowers; hornets don’t … Hornets leave the bed first …
  • 18. THREE KINDS OF CHROMATOGRAMS THAT CAN BE DEVELOPED
  • 19. Descending method is better because
    • the dyes will travel faster due to the force of gravity.
    • the separation will be greater.
    Two types of Paper Chromatography
  • 20. Paper Chromatography – Chromatogram
    • How to Read from a chromatogram?
    Orange Green Blue Red Black Unknown Known In the experiment, the solvent travels up the paper. The dyes on the pencil line dissolve in the solvent. The more soluble the dyes are in the solvent, the faster they move up the paper. So the dyes move up the paper at different speeds. Hence the dyes are separated. Colored Spot
  • 21.
    • Results
    • Identical dyes travel the same distance up the paper. The unknown dyes in the black ink can be identified by comparing them with known black dyes. The result of the experiment show that
    • Black ink is made up of 4 different dyes.
    • Three of the dyes are known. They are Red , Blue and Green dyes.
    • There is no Orange dye in the black ink.
    • Black ink consists 1 dye that is different from the four known dyes.
    Paper Chromatography – Chromatogram
  • 22. Which of the soft drinks contain a harmful dye? A) P and Q B) R and S C) Q and S D) P and R P Q R S X Y soft drinks dyes Interpreting Chromatograms
  • 23. What can be done if the substance is colourless ?
    • Use ultraviolet (UV) light to see the components. Many substances are coloured under UV light but colourless in white light.
    • Use a locating agent (eg ninhydrin solution). Ninhydrin solution forms a purple stain with amino acids (colourless).
    • The agent will react with the substances on the paper to form a coloured product .
    Chromatography
  • 24. chromatography What happen if there are more than 20 suspected substances in the sample? It would not be practical to put all the suspected substances on the chromatogram. We can make use of the R f values.
  • 25. Paper Chromatography
    • Interpreting data by calculating the R f values .
    It is easier and more sensible to - measure the R f value of each component and then identify them by looking at known R f in reference book. R f value = Distance travelled by the substance Distance travelled by the solvent
  • 26. Chromatography A B B A Solvent used : alcohol
  • 27.
    • is a technique used to
    • separate components in a mixture
    • identify the substances
    • determine if the sample is pure
    Chromatography
  • 28. Applications: Chromatography Detection of - drugs in athletes - pesticides sprayed on vegetables - illegal dyes in food
  • 29. Paper Chromatography – APPLICATION
    • Identify a compound by spotting known substances next to unknown substances on the same chromatogram.
    • Determine purity of a sample. An impure sample will often develop as two or more spots, while a pure sample gives only one spot.
    • To detect colorless liquids, a locating agent may be sprayed onto the chromatogram.
    • Locating agent : chemical that reacts with the substance to produce a visibly coloured product. Example: ninhydrin that reacts with colourless amino acids to produce a purple stain .
  • 30.                                                                             Drugs have provided the world of athletics with a fierce opponent ever since the emergence of systematic forms of doping in eastern Europe. But the most famous case in history is that involving a westerner - Canadian sprinter Ben Johnson. He won the 100 metres at the 1988 Seoul Olympics in a world record time but was later stripped of his gold medal and banned for two years after testing positive for an anabolic steroid. Since then he has made an unsuccessful attempt to get back into the Canadian team for the 2000 Olympics. There have also been some bizarre stunts such as a race with two horses and car for charity. The animals beat him although the car was stalled by wheelspin. Johnson has recently worked as personal fitness trainer for footballer Said Gaddafi, son of Libyan president Colonel Muamar Gadaffi. Meanwhile an incident in which a mugger took his wallet and outran him showed that the 38-year-old does not have the speed he once possessed. Drugs in world athletics BBC Sport website (Mon 31 July 2000) Tainted win: Johnson (second left) wins in Seoul A life ban from the track followed in March 1993 when he gave another positive test.
  • 31.  
  • 32. The Straits Times, 7 December 1988
  • 33.  
  • 34. Paper Chromatography – ADVANTAGES
    • Allows analysis of a small
    • amount of chemicals, e.g.. in
    • forensics or urine analysis.
    • Sensitive and highly reproducible .
    • Allows analysis of many samples at a time .
    • Relatively inexpensive .
  • 35.
    • A pure substance has
    • fixed melting point
    • fixed boiling point
    • one spot of dye on the chromatogram.
    What is a pure substance?
  • 36. Effect of Impurities in mixtures
    • The presence of mixture will
    • lower the melting point
    • raise the boiling point
    • result in more than one spot of dye on the chromatogram.
  • 37. TESTING THE PURITY OF SUBSTANCES
    • Melting Point
    • (Presence of impurities will lower the melting point)
    2. Boiling Point (Presence of impurities will raise the boiling point) 3. Chromatography (If impurities are present, there will be more than one spots on the chromatogram)
      • Impure substances or mixtures do not have fixed melting or boiling points.
  • 38. Chromatography
    • Questions
      • What is a chromatogram?
      • It is the paper that contains the results of chromatography.
      • What are the advantages of using chromatography?
      • 1. It only requires a small amount of sample is required.
      • 2. Gives quick result
      • 3. Can be used to test purity and identify of a substance.
  • 39.
    • separates mixtures for identification purposes
    • use a pencil to draw the starting line. (why?)
    Paper chromatography 1 pencil line
  • 40.
    • Add a small drop of a mixture to be separated on the line. (why can’t use a big drop?)
    Paper chromatography 2 black red blue green orange drops of ink on the pencil line
  • 41.
    • paper is dipped into a solvent and beaker is covered, making sure that the start line is above the solvent. (why?)
    • leave the paper to stand undisturbed in the solvent.
    • solvent travels up the paper.
    • inks dissolve in the solvent and move up the paper
    Paper chromatography 3 cover
  • 42.
    • dyes move up at different speeds are thus separated; identical dyes travel the same distance up the paper.
    • dyes are then identified.
    • each spot represents one component that is present.
    • paper with the separated components is called a chromatogram .
    Paper chromatography How many components does black ink has? 4 chromatogram of dyes in different coloured inks black red blue green orange