PAG 6: Chromatography + Electrophoresis Flashcards
What is chromatography used for in biology? (1)
Analytical method used to separate a mixture into different biological molecules.
What are the two main components of chromatography? (2)
Stationary phase - either a TLC plate or chromatography paper
Mobile phase - the solvent that carries the biological molecules
What is the role of the stationary phase in chromatography? (2)
The stationary phase adsorbs biological molecules
Slowing their movement depending on their interaction with the surface
What is the role of the mobile phase in chromatography? (1)
The mobile phase moves biological molecules up the stationary phase, separating them based on solubility
What does the term ‘adsorption’ mean in chromatography? (1)
Adsorption is when molecules bond to the surface of a substance (e.g., the TLC plate)
Why do different molecules travel at different speeds in chromatography? (2)
Molecules that adsorb more strongly to the stationary phase move more slowly
Whereas molecules that interact more with the mobile phase travel faster.
What are the limitations of chromatography? (1)
Molecules with similar properties may have similar Rf values, making it difficult to distinguish between them
Describe the method for separating chlorophyll pigments using chromatography. (12)
- Grind up leaves using a pestle and mortar with anhydrous sodium sulfate and propanone
- Transfer the liquid to a test tube and add petroleum ether; shake to separate layers
- Extract pigment solution from the top layer and add anhydrous sodium sulfate
- Draw a pencil line 2cm from the bottom of a TLC plate
- Use a capillary tube to place a concentrated dot of pigment on the pencil line
- Allow the dot to dry and add more pigment to concentrate it
- Add a small amount of solvent to a beaker so the pencil line is above the solvent
- Place the TLC plate in the beaker and cover with a watch glass
- Allow solvent to move up the plate, separating the pigments
- Mark the solvent front and pigment locations once the solvent has nearly reached the top
- Measure the distance travelled by each pigment and the solvent front
- Calculate the Rf value
How is an Rf value calculated in chromatography? (1)
Distance travelled by the solute ÷ Distance travelled by the solvent
How can chromatographic Rf values be used to identify pigments? (1)
Rf values can be compared with reference values for known pigments
Describe the method for separating amino acids using chromatography. (11)
- Draw a pencil line near the bottom of a piece of chromatography paper
- Spot the amino acid mixture onto the paper
- Add a small amount of solvent (butan-1-ol, glacial ethanoic acid, and water) to the beaker
- Place the chromatography paper in the solvent, ensuring the pencil line is above the solvent level
- Cover the beaker with a watch glass to prevent solvent evaporation
- Allow the solvent to move up the paper, separating the amino acids
- Remove the paper when the solvent nears the top and mark the solvent front with a pencil
- Let the paper dry completely
- Spray the amino acids with ninhydrin solution to make them visible as purple spots
- Measure the distance moved by the solvent and the amino acids
- Calculate the Rf values for each amino acid and compare to known values to identify amino acids
Why is a pencil line used in chromatography instead of a pen? (1)
Pencil is used because it is insoluble in the solvent and will not interfere with the results
Why should the chromatography paper be placed in the solvent with the pencil line above the solvent level? (1)
To prevent the amino acids from dissolving directly into the solvent before separation can occur.
What is the purpose of covering the beaker with a watch glass during chromatography? (1)
To prevent the solvent from evaporating, ensuring consistent movement up the paper
Why are amino acids sprayed with ninhydrin solution after chromatography? (1)
Amino acids are not coloured, so ninhydrin reacts with them to make them appear purple for visibility
Explain why a student measured to the centre of each pigment spot when measuring the distance travelled by each pigment. (1)
It standardises readings and allows comparisons to be made.
Explain why a substance may not move up the chromatography paper at all. (1)
Some substances may be insoluble in the solvent
Suggest the advantage of a plant containing several different photosynthetic pigments. (2)
Different pigments absorb different wavelengths of light
So having a range of pigments maximises the wavelengths of light that can be absorbed and so maximises the rate of photosynthesis.
What is the purpose of electrophoresis? (1)
Used to separate DNA fragments based on size by applying an electric current through a gel
Describe the process of preparing a gel for electrophoresis. (3)
- Pour agarose gel into a tray and leave it to solidify
- Create a row of wells at one end of the gel tray
- Place the gel tray into a gel box with the wells closest to the negative electrode
Why must the wells be placed near the negative electrode? (1)
DNA fragments are negatively charged and will move towards the positive electrode
What is the purpose of adding buffer solution to the gel box? (1)
The buffer solution covers the gel and helps conduct electricity for the movement of DNA fragments
Describe how to load DNA samples into the gel. (3)
- Using a micropipette, mix DNA samples with an equal volume of loading dye
- Carefully add the mixture into the bottom of the wells without piercing them
- Repeat for each DNA sample using a clean micropipette
What is the purpose of the loading dye? (2)
It helps DNA fragments sink to the bottom of the well
It makes the fragments more visible when running the gel
