PAG - chromatography

Question 1

what is chromatography

Answer 1

a technique that can be used to separate a mixture into its individual components

Question 2

what mixtures can be separated by chromatography

Answer 2

carbohydrates
proteins
vitamins
nucleic acids

Question 3

what are the two phases in chromatography

Answer 3

mobile phase
stationary phase

Question 4

outline the principals and process of paper/thin-layer chromatography

Answer 4

• use capillary tube to spot solution onto pencil ‘start line’ (origin) 1cm above bottom of paper
• place chromatography paper in solvent (origin should be above solvent level)
• allow solvent to run until it almost touches other end of the paper. Molecules in mixture move different distances based on relative solubility in solvent/attraction to paper

Question 5

what are Rf values? how can they be calculaed

Answer 5

ratios that allow comparison of how far molecules have moved in chromatograms
- Rf value = distance between origin and centre of pigment spot / distance between origin and solvent front

Question 6

what is the term TLC

Answer 6

thin layer chromatography

Question 7

why should you wear gloves/use forceps when holding the TLC plate

Answer 7

to prevent oils from your finger affecting how far the mixture travels

Question 8

why do you draw the origin line in pencil and not pen

Answer 8

pencil is insoluble
pen ink will dissolve in the solvent and contaminate

Question 9

why do you ensure solvent is below the height of origin line

Answer 9

so that the solvent does not mix with the mixtures
mixtures will not be able to separate properly

Question 10

why do you suspend the TLC plate vertically without touching the sides of the tank

Answer 10

the TLC is horizontal to measure Rf

Question 11

why do you place a lid on the tank

Answer 11

to prevent evaporation - measure accurate Rf value

Question 12

why do you stop the chromatogram before solvent reaches the top of the TLC plate and mark the solvent front with a pencil

Answer 12

measure solvent front - calculate Rf value so pigment can be identifiable
see the end point

Question 13

Rf value calaculation

Answer 13

distance moved by the pigment / distance moved by the solvent

Question 14

example question:
a group of students wanted to use a TLC plate to identify four amino acids
to produce the chromatogram the students:
- drew the pencil line 1 cm from the bottom of the TLC plate and put the solvent into the beaker to a height of approx 0.9 cm
- held the chromatography plate firmly in the middle with their hands and lowered it into the beaker
- left the apparatus to stand

suggest four ways you would refine the method used by the students

Answer 14

• use ninhydrin to see / visualise amino acids
• repeat and find mean Rf value
• place lid over beaker to prevent evaporation
• use a stand to hold the TLC plate to keep plate at a constant height
• reduce the height of the solvent to prevent it mixing with the amino acid mixtures
• wear gloves to prevent your hands contaminating the results
• space the mixtures evenly across the origin to prevent the mixtures from mixing together

Question 15

what is ninhydrin solution used for

Answer 15

a chemical which reacts with amino acids producing an easily visible blue-violet colour

Question 16

what is the stationary phase

Answer 16

chromatography paper or silica attached to plastic - TLC plate

Question 17

what is the mobile phase

Answer 17

the mixture is dissolved in solvent

Question 18

describe how to carry out practical chromatography

Answer 18

step 1 - sample preparation
* proteins extracted from cells are hydrolysed using protease enzymes or hydrochloric acid to produce a mixture of amino acids
step 2 - producing chromatogram
* wearing gloves / using forceps / only toughing the edges of the TLC plate draw origin line in pencil 1 cm from the end
* add one drop of mixture to center of origin line using a capillary tube, allow to dry and then repeat many times, to ensure that the spot is concentrated
* if there are multiple mixtures, make sure that each drop is in even spacing
* add ninhydrin to the drops of amino acid mixtures to make them visible
* place TLC plate / chromatography paper vertially into a TLC chamber / tank containing 0.5 cm depth of solvent so that it does not touch the side of the sube and that the solvent does not touch the amino acid mixture and mix / contaminate it
* just before solvent reaches the top of the TLC plate, remove it from the tube and quickly mark the solvent front in pencil
step 3 - interpreting chromatogram
* calaculate Rf value of each amino acid / pigment spot
* use published Rf values of identify amino acid / pigment