chromatography or electrophoresis

• What is chromatography used for?

  To separate a mixture into different biological molecules
• What are the two main components of chromatography?

  The stationary phase and the mobile phase
• What materials can be used for the stationary phase in chromatography?

  A TLC plate or chromatography paper
• What is chromatography paper made of?

  Cellulose
• What is the role of the mobile phase in chromatography?

  It is the solvent that carries the biological molecules up the stationary phase
• How do different biological molecules behave during chromatography?

  They are adsorbed onto the surface of the TLC at different strengths
• What does a higher adsorption strength indicate about a molecule's movement on the TLC plate?

  The molecule will travel up the plate slower
• What is the definition of adsorption?

  It is where molecules bond to the surface of a substance
• What is a limitation of chromatography?

  Similar molecules may have similar Rf values, making them hard to distinguish
• What are the steps involved in the chlorophyll pigment chromatography method?

  1. Grind leaves with anhydrous sodium sulphate and propanone.
  2. Transfer to a test tube, add petroleum ether, and shake.
  3. Transfer liquid from the top layer.
  4. Draw a pencil line on the TLC plate.
  5. Use a capillary tube to place a dot of pigment on the line.
  6. Allow to dry and add more pigment for concentration.
  7. Add solvent mixture in a beaker.
  8. Place TLC plate in the beaker.
  9. Cover with a watch glass in a fume cupboard.

  10. Allow solvent to move up the plate and separate pigments.

  11. Mark solvent front and pigment locations.

  12. Leave to dry.

  13. Measure distances travelled by pigments and solvent.

  14. Calculate Rf values.

  15. Compare Rf values to identify pigments.
• What are the steps involved in the amino acid chromatography method?

  1. Draw a pencil line on chromatography paper.
  2. Place a concentrated spot of amino acid mixture.
  3. Add solvent (butan-1-ol, glacial ethanoic acid, water) to the beaker.
  4. Place chromatography paper in the beaker ensuring the pencil line is above the solvent line.
  5. Cover with a watch glass in a fume cupboard.
  6. Allow solvent to move up the paper and separate amino acids.
  7. Mark solvent front when it reaches near the top.
  8. Leave to dry.
  9. Spray with ninhydrin solution to visualize amino acids.

  10. Measure distances travelled by solvent and spots.

  11. Calculate Rf values.

  12. Compare experimental values to known values to identify amino acids.
• What are the steps involved in the electrophoresis method?

  1. Cut DNA into fragments with restriction enzymes.
  2. Pour agarose gel into a tray and let it solidify.
  3. Create wells at one end of the tray.
  4. Place gel tray in a gel box/tank.
  5. Position wells closest to the negative electrode.
  6. Add buffer solution to cover the gel.
  7. Use a micropipette to add loading dye and DNA sample to wells.
  8. Avoid piercing the bottom of the wells.
  9. Record which DNA sample goes into which well.

  10. Connect the gel box to the power source.

  11. Set power source to 100V.

  12. DNA fragments move towards the positive electrode.

  13. Smaller fragments move faster than larger ones.

  14. Run gel for 30 minutes or until dye is 2 cm from the end.

  15. Remove gel box and tip off excess buffer.

  16. Stain DNA fragments and rinse with water.

  17. Bands of DNA fragments become visible.

  18. Method can be used for RNA and proteins.

  19. Compare bands for similarities and differences.
• What is the purpose of using loading dye in electrophoresis?

  It helps the DNA fragment sink and makes them more visible
• Why do DNA fragments move towards the positive electrode during electrophoresis?

  Because DNA fragments are negatively charged
• How does the size of DNA fragments affect their movement through the gel?

  Smaller fragments move faster than larger fragments
• What should the power source be set to during electrophoresis?

  100V
• What happens to the bands of DNA fragments after staining?

  They become visible
• How can the bands of DNA in each sample be used after electrophoresis?

  They can be compared for similarities and differences
• Can electrophoresis be used for RNA and proteins?

  Yes, it can be used for RNA fragments and proteins
• What is done to proteins before they are run in electrophoresis?

  A chemical is added to make them all negatively charged
• What is the purpose of the buffer solution in electrophoresis?

  To cover the surface of the gel
• What is the significance of marking the solvent front in chromatography?

  It indicates how far the solvent has traveled
• Why is it important to allow the pigment dot to dry before adding more pigment in chromatography?

  To prevent the pigment from spreading out into a larger dot
• What should the pencil line be above in chromatography?

  The solvent line
• What is the purpose of using a watch glass during chromatography and electrophoresis?

  To stop the solvent from evaporating