CHromatograhpy

Question 1

What is chromatography used for>

Answer 1

• identifying, separating and measuring quantitites of susbtances extracted from biological material

Question 2

How is chromatography done?

Answer 2

• drops of soln are placed at the start line at the base of a piece of chromatography paper or a layer of fne-grtained material, such as silica gel, on a solid support
  
  • latter is TLC
• placed in a solven, which moves upwards

Question 3

What does the degree of movement depend on?>

Answer 3

• depending of their solubility in the solvent and their attraction to the solid phase

Question 4

What is done when the solvent has reached 10mm from the end? What if the substances are not observable?

Answer 4

• paper or plate is removed and a line is drawn to indicatte the solvent from
• pigments, such as those in chloroplasts, are visible in chromatograms
• some substances are not visible with the naked eye, and so the chromatograms must be sprayed with a special chemical or observed under UV light

Question 5

What are the safety precautions for observing chloroplast pigments?

Answer 5

• wear gloves throughout the procedure and goggles

Question 6

How is the photosynthetic pigment sample prepartd?

Answer 6

• hold 2 slides to scrape out the contents of several leaves
• transfer scraping to a watch glass
• add 10 drops of propanone (acetone) and pulverise with a glass rodd
• use a pipette to transfer as much as possible of the dark green liquid to a second watchglass
• use a hair dryer to evaporate all the propanone and water
• add 2-3 drops of porpanone and use a fie paint brush to mix with the extract

Question 7

How is the TLC plate prepared?q

Answer 7

• draw a pencil line 10mm from one end
  
  • put a thin pencil spot on the middle of the line
• use a fine capillary tube to transfer the extract to the pencil spot
  
  • wait until the extract is dry before repeating with another drop
  • continue doing this to make a dark green spot no wider than 2mm

Question 8

Once the TLC plate is prepared, what is done?

Answer 8

• place TLC plate in a split bung
• pour the running solvent into a specimen tube just below 10mm
  
  • running solvent = peteroleum ether + propanone (2:1)
• put the bung into the specimen tube immediately
  
  • keep out of direct sunlight
• watch the solvent from move up the |TLC plate and the seperation within the extract
  
  • take the bung out when the sovent front is about 10mm from the end
• mark solvent front
• take photo

Question 9

What is recorded?

Answer 9

• record colour of each pigment and the distance travelled

Question 10

How can a control be made?

Answer 10

• run pure substances alongisde th extract
• such as beta-carotene, and lutein,
  
  • avialable in health food shops

Question 11

What is Rf?

Answer 11

• retention factor
• ratio between the distance travelled by a susbstance and the distance t ravelled by the solvent fron
• distnace travelled by substance / distnace travbelled by solvent front

Question 12

What are Rf values used for>

Answer 12

• iudentify a substance
• it is indpendent of the length of the chromatogram or distance travelled by solvent front