Required practical 7: chromatography of leaf pigments - AQA A-Level Biology (Paper 3) Exam Questions

Practise 16 exam-style questions on Required practical 7: chromatography of leaf pigments for AQA A-Level Biology (Paper 3). Write your answer as you would in the exam and get it marked in seconds against the mark scheme, point by point, with the Study Helper on hand if you get stuck.

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Sample questions with model answers

Here are 4 of the questions in this topic, each with a model answer, an explanation of why it earns the marks and the mark scheme an examiner would use.

Question 1

2 marks

Leaf pigments can be separated by paper chromatography or by thin-layer chromatography (TLC).

Give two advantages of using TLC rather than paper chromatography to separate leaf pigments.

Model answer

1. TLC runs much faster than paper chromatography.
2. TLC gives smaller, sharper spots, so pigments with similar Rf values are separated more clearly.

Explanation

In TLC the stationary phase is a thin, even layer of fine particles (such as silica gel) on a plate, so the solvent rises quickly and the spots stay small and sharp. Because the plates are small, only small volumes of flammable solvent are needed, and the pigment spots can be scraped off and dissolved for measurement. Vague answers such as 'more accurate' do not gain credit.

Mark scheme

1 mark
TLC is faster / the solvent moves up the plate more quickly
Max 2. List rule: if more than two answers are given, each wrong or contradictory extra answer cancels one correct answer. Ignore 'more accurate', 'easier' and 'cheaper' unqualified.
1 mark
Better separation / smaller, sharper spots / pigments with similar Rf values are separated (more clearly)
Accept 'more distinct spots'.
1 mark
Smaller volumes of solvent (and extract) are needed, so less flammable / harmful vapour (safer)
Accept 'uses less solvent'.
1 mark
The spots can be scraped off to recover the pigments (for further tests, e.g. with a colorimeter)
1 mark
More sensitive / smaller amounts of pigment can be detected

Question 2

3 marks

A student made a chromatogram of a leaf extract. She used a ruler with millimetre divisions to measure the distance from the origin to the centre of a yellow-green spot, X, and from the origin to the solvent front.

Distance moved by spot X = 27 mm
Distance moved by solvent front = 90 mm

Each of these distances has an uncertainty of ±1 mm. In this solvent, the Rf value of chlorophyll b is 0.30 and the Rf value of another pigment, Y, is 0.31.

Use the uncertainties to calculate the largest and the smallest possible Rf values of spot X. Use your answers to explain whether the student can conclude that spot X is chlorophyll b.

Model answer

Largest Rf = (27 + 1) ÷ (90 - 1) = 28 ÷ 89 = 0.31
Smallest Rf = (27 - 1) ÷ (90 + 1) = 26 ÷ 91 = 0.29
The Rf value of spot X could be anywhere from 0.29 to 0.31. Both chlorophyll b (0.30) and pigment Y (0.31) fall within this range, so the student cannot conclude that spot X is chlorophyll b.

Explanation

The largest possible Rf uses the largest spot distance and the smallest solvent distance; the smallest Rf uses the reverse. If the range of possible values includes the Rf of more than one pigment, the measurement cannot tell them apart. Running pure pigments alongside the extract, using a second solvent, or letting the solvent run further (so the percentage uncertainty of each distance is smaller) would help.

Mark scheme

1 mark
Largest Rf = 28 ÷ 89 = 0.31 (accept 0.314, 0.315 or 0.3146); a correct value with no working gains this mark
Points 1 and 2 are calculations: a correct value with no working scores the mark for that value. Accept 0.3146. Accept a percentage-uncertainty method: (1 ÷ 27 + 1 ÷ 90) × 100 = 4.8%, so Rf = 0.30 ± 0.014, giving 0.314 and 0.286. Do not accept 0.30 (27 ÷ 90) as the largest value. Reject 0.32.
1 mark
Smallest Rf = 26 ÷ 91 = 0.29 (accept 0.286 or 0.2857)
Do not accept 0.30 or 0.28.
1 mark
No, because both 0.30 and 0.31 lie within this range, so spot X could be chlorophyll b or pigment Y
Allow ECF from incorrect largest and smallest values. Accept 'no, the uncertainty is too large to tell them apart'. Ignore 'yes, its Rf is 0.30' (does not use the range).

Question 3

4 marks

A student investigated whether storing picked spinach leaves in a refrigerator changes the amounts of photosynthetic pigments in the leaves. She picked a large number of leaves at the same time and put them in a refrigerator. After 0, 2, 4, 6 and 8 days, she made a chromatogram from an extract of some of the stored leaves. At the end of the 8 days, she laid the five chromatograms side by side on the bench and compared the size and colour intensity of the pigment spots by eye.

Give the dependent variable in her investigation.

Her teacher said that this method would not give a valid comparison of the amounts of pigment in the leaves after different storage times. Explain why, and suggest how she could improve her method.

Model answer

The dependent variable is the amount of each pigment, judged from the size and colour intensity of its spot.

Judging spots by eye is subjective and cannot detect small differences in the amount of pigment. The chromatograms were also of different ages when she compared them: the day 0 chromatogram was 8 days old, so its pigments may have faded in the light, while the day 8 chromatogram was fresh. In addition, the mass of leaf and the volume of extract used on each day may have been different.

She should use the same mass of leaf, the same volume of propanone and the same volume of extract each day, freeze each extract in the dark and run all five side by side on one plate at the end. She could then scrape off each pigment spot, dissolve it in a set volume of propanone and measure its absorbance with a colorimeter.

Explanation

A valid comparison needs the storage time of the leaves to be the only thing that differs between the samples. Here the chromatograms were different ages when compared (pigments fade in the light), the amounts of leaf and extract were not controlled, and the comparison was by eye, which is subjective. Storing the extracts in the dark and running them together, controlling the mass of leaf and the volumes, and measuring each pigment quantitatively would make the comparison valid. An improvement should remove a specific problem, not just 'repeat it'.

Mark scheme

1 mark
Dependent variable: the amount / concentration of (each) pigment (in the leaves) OR the size and colour intensity of the pigment spots
Max 4: point 1, then max 3 from points 2 to 9, with max 2 from points 2 to 5 (why the comparison is not valid) and max 2 from points 6 to 9 (improvements). For point 1, do not accept 'Rf value' or 'type of pigment'; ignore 'time' unqualified.
1 mark
Judging the size or colour intensity of spots by eye is subjective / not quantitative / cannot detect small differences in the amount of pigment
Accept 'different people would judge the spots differently'. Ignore 'human error' unqualified.
1 mark
The chromatograms were of different ages when they were compared (the day 0 chromatogram was 8 days old), so the pigments on the older ones may have faded / broken down (e.g. in the light) after the chromatogram was made
Accept 'the spots fade over time, so earlier chromatograms would look paler'.
1 mark
The mass of leaf, the volume of solvent or the volume of extract put on the origin may have differed from day to day ('some of the stored leaves'), so the amount of pigment in a spot does not depend only on the storage time
Any one of these uncontrolled quantities gains the mark.
1 mark
The conditions of each run may have differed (e.g. temperature, solvent composition, evaporation of solvent, how far the solvent ran), changing the size or spread of the spots
Ignore 'not repeated' for this point (see point 9).
1 mark
Use the same mass / area of leaf, the same volume of propanone and the same volume of extract on the origin each time
Accept any one of these controls for the mark.
1 mark
Store each extract (or leaf sample) frozen / cold in the dark and run all the chromatograms at the same time / side by side on one plate
Accept 'compare the chromatograms immediately after each is made, against the same standard' only if a standard is described.
1 mark
Measure each pigment quantitatively, e.g. scrape off each spot, dissolve it in a set volume of solvent and measure its absorbance with a colorimeter
Accept 'photograph the plates under the same lighting and measure the intensity of each spot with image software'.
1 mark
Make several chromatograms from different samples of leaves on each day and compare the means
Ignore 'repeat it' unqualified.

Question 4

6 marks

Golden privet is a variety of privet with yellow-green leaves. Green privet has dark green leaves. A student wanted to find out whether the leaves of golden privet contain less chlorophyll a and less chlorophyll b than the leaves of green privet.

She had: leaves of both varieties, a cork borer, sand, a pestle and mortar, propanone, TLC plates, a beaker with a lid, a chromatography solvent, capillary tubes, a scalpel, a colorimeter with cuvettes, and the Rf values of chlorophyll a and chlorophyll b in this solvent.

Describe how she could use this apparatus to compare the amounts of chlorophyll a and chlorophyll b in the leaves of the two varieties.

Model answer

Use the cork borer to cut 10 discs from leaves of each variety, avoiding the veins. Grind each set of discs with a little sand and 2 cm³ of propanone, and filter. Draw a pencil origin line on a TLC plate and put the same number of drops of each extract on the origin, drying between drops to keep the spots small. Stand the plate in solvent below the origin in the covered beaker, remove it before the solvent reaches the top and mark the solvent front. Calculate the Rf value of each spot and use the given Rf values to identify chlorophyll a and chlorophyll b. Scrape each chlorophyll spot off with the scalpel and dissolve it in the same volume of propanone. Set the colorimeter to zero with propanone and measure the absorbance of each solution with a red filter: the higher the absorbance, the more pigment. Repeat with leaves from several plants of each variety and compare the mean absorbances.

Explanation

Chromatography separates the pigments, but on its own it shows only whether a pigment is present. To compare amounts, each identified spot can be scraped off, dissolved in a fixed volume of solvent and measured with a colorimeter, because absorbance increases with the concentration of pigment. A valid comparison needs the same area or mass of leaf, the same volume of solvent and of extract, and repeats from several plants. Chlorophylls absorb red and blue light most strongly, so a red or blue filter gives the most sensitive readings.

Mark scheme

1 mark
Cut the same number of discs / the same area of leaf with the cork borer from each variety (avoiding the veins)
Max 6. Accept the same mass of leaf. Accept steps in any sensible order. Comparing only the size or colour of the spots does not gain points 7 to 9.
1 mark
Grind (each sample) with sand and the same volume of propanone
Accept 'filter or decant the extract'.
1 mark
Put the same volume / number of drops of each extract on an origin drawn in pencil on a TLC plate (as a small, concentrated spot)
Accept 'on separate plates' or 'side by side on one plate'.
1 mark
Stand the plate in the solvent with the solvent level below the origin, in the covered beaker
Ignore 'cover the beaker' on its own.
1 mark
Remove the plate before the solvent reaches the top OR mark the solvent front (in pencil) as soon as the plate is removed
1 mark
Calculate Rf values (distance moved by spot ÷ distance moved by solvent front) to identify the chlorophyll a and chlorophyll b spots
Accept 'compare with the Rf values given'.
1 mark
Scrape off each chlorophyll spot (with the scalpel) and dissolve it in the same volume of propanone
Accept 'elute'.
1 mark
Measure the absorbance of each solution in the colorimeter (with a red or blue filter), set to zero with propanone
Accept 'set the colorimeter with a blank of propanone'.
1 mark
Higher absorbance means more pigment, so compare the absorbances for each pigment in the two varieties OR use a calibration curve to find the mass of each pigment
1 mark
Repeat with leaves from several plants of each variety and calculate means
Accept 'use a statistical test to compare the means'.

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