NANYANG JUNIOR COLLEGE H2 Biology

Practical 6: Membrane & Transport

(Chemicals on membrane permeability)

Name: ANSWERS Class: Date:

PRACTICAL 6: MEMBRANE & TRANSPORT

(INVESTIGATE THE EFFECTS OF CHEMICALS ON MEMBRANE PERMEABILITY IN
BEETROOT)

Beetroot cells contain a class of water-soluble pigments called betalains that give the tissue its
distinct dark purple-red colour. There are two types of betalains in beetroot, namely the red-
violet betacyanins and the yellow betaxanthins. The pigments are contained in the large central
vacuole.

Betalains cannot pass through the tonoplast or cell membrane of the beetroot cells as long as
these membranes remain intact. If, however, the integrity of the membranes becomes
compromised, betalains can leak out of the cells.

You are required to investigate the effect of different chemicals on membrane permeability to
betalains in beetroot cells.

You are provided with:

 Beetroot cylinders
 100 cm3 of distilled water, W,
 50% ethanol
 0.1M HCl
 0.1M NaOH
 colorimeter.

Beetroot juice will stain clothing (and temporarily, skin) but is not hazardous. It is
recommended that you wear gloves.

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

Additional notes for tutors:

Betalains are water-soluble nitrogenous pigments and are divided into two classes: red-violet
betacyanin and yellow betaxanthin, which provide beet roots with their typical colour. The
chemical structure of these pigments are derived from betalamic acid. It can be divided into two
structural groups according to the components bonded to the main structure. Betacyanins arise
when the group attached to betalamic acid is 3,4-dihydroxyphenylalanine, which may or may not
be glycosylated. While, betaxanthins take place if the same betalamic acid moiety is conjugated
with an amino acid or amine (Fig. 1).

Under alkaline conditions, most betacyanins (red-violet) change to betaxanthins (yellow).

Alkaline conditions cause aldimine bond hydrolysis while acidification induces recondensation of
betalamic acid with the amine group of the addition residue.

Tutors may also teach students about the control variables in this experiment:
 Size of beetroot
 Source/part of beetroot (Age of beetroot?)
 Volume of each chemical
 Time of incubation of beetroot
 Temperature at which the experiment was performed etc

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

Proceed as follows:

1 Cut 12 beetroot discs from the cylindrical beetroot strips on a white tile, each approximately
2mm thick.

2 Wash the discs with several changes of cold tap water until the water shows no sign of
colouration. Soak the discs in a fresh sample of cold water. Ignore any loss of stain which
occurs at this point.

3 Explain why it is necessary to wash the discs several times.

To rinse away the excess pigments (NOT dye!) that leaked due to mechanical stress/cellular
damage/cell breakage caused by cutting of the beetroot tissues;
So that the results obtained are more accurate / valid;
[2]

4 Label four test tubes, A, B, C, and D respectively. Place 3 beetroot discs into each of the
tubes.

5 Add 5cm3 of the respective solution into each of the 4 test tubes (indicated in the table).

Test Tube 5cm3 of solution

A Distilled water
B 50% ethanol
C 0.1M dilute hydrochloric acid
D 0.1M dilute sodium hydroxide

6 Leave the tubes aside in a test tube rack for 15 minutes. Shake the tubes periodically.

7 After 15 minutes, shake the test-tubes to evenly distribute the pigments in the solution.
Record the colour of the solutions observed in each test tube.

8 Label four cuvettes as A-D.

9 Decant solution from test tubes A-D into the respective cuvettes. Fill up to at least ¾ of the
cuvette.

10 Calibrate the colorimeter using cuvette A. Measure and record the transmission of blue light
through each of the cuvettes B-D. This gives a measure of the concentration of the betalain
pigment in the solution.

11 Repeat steps 1 – 9 to obtain a second set of readings to ensure reliability of results.

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

12 Tabulate your observations and data in a suitable format. [3]

Transmission of light / % OR
Type of solution Observations Percentage light transmission
Trial 1 Trial 2 Mean
distilled water Solution turned from colourless
to pale pink
50% ethanol Solution turned from colourless
to dark pink/red
0.1M HCl Solution turned from colourless
to dark pink/red
0.1M NaOH Solution turned from colourless
to yellow

Points to Note for Tables:

1. Each column in the table should be headed with the quantity and units (when
applicable).
2. The first column in the table should be independent variable, i.e. the variable that you
manipulated in the investigation.
3. The subsequent columns record the values for dependent variable which are the
measurements/observations you made.
4. Observations recorded should be phrased in this manner “solution turned from _____ to
_______”
5. Recorded values should be according to the precision of the colorimeter.
6. When there are two or more sets of readings, there should be an additional column to
process the mean data.

Correct heading with units;

Correct precision to one decimal point for percentage transmission;
Correct values and observations and mean data;
Reject only mentioning ‘test tube’ without mentioning the solution in the heading
Reject ‘average’, it can refer to mean / median / mode.
1dp is due to precision and not because it’s mean.
‘/’ unit NOT ‘( )’ unit

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

13 Explain how the different chemicals affect membrane permeability of beetroot cells to
betalain pigments.

1. (Betalains are contained within the vacuole of the plant cell.) The different chemicals
increase the permeability of the cell surface membrane and tonoplast, resulting in gaps
in the membranes, causing the red pigments to leak out of the cell;
2. Ethanol: It dissolves the phospholipids as the polar –OH group in ethanol can form
interactions with polar / hydrophilic phosphate head of the phospholipid;
3. HCl and NaOH (affects concentration of H+ ions, therefore pH of solution): A change in
pH affects charged and polar R groups and disrupts ionic bonds and hydrogen bonds of
membrane proteins;
Extra point: Denaturation of membrane proteins, thereby resulting in change in its
specific 3D structure/conformation. More gaps appear in the membrane increase
membrane permeability of beetroot cells to betalain pigments

Reject ethanol being non-polar

Reject no optimum pH as we are not looking at enzymes
Reject excess H+/ OH- interacting with phosphate groups of phospholipids and disrupting H
bonds between phospholipids
[3]
14 Identify two sources of experimental errors that could affect the accuracy of the results.

1. There is time lag in adding the solutions into all the test-tubes at the same time / in
decanting the solutions from all the test-tubes at the same time. Therefore, the pigments
could continue leaking for those test-tubes where the discs were soaked for a longer period
of time before decanting;
2. The temperature at which the experiment is conducted was not kept constant which can
affect the rate of diffusion;
3. There is inconsistent shaking across the test-tubes which can affect the rate of diffusion;
4. The discs may not be cut to exactly 2mm in thickness, therefore leading to differing amounts
of pigment present in the beetroot discs at the start of the experiment;

® Even though measures were taken to ensure that excess red pigment from the beetroots
were rinsed and washed away when they were cut, not all of the excess red pigment might
have been removed; (This issue has been addressed by the fact that there’s control tube A)

® Betalain pigments were more concentrated in some parts of the beetroot/unequal

distribution of betalain pigments in the beetroot sample; (Some sections of the beetroot is
darker than the others. It is impossible to ensure that all of the beet discs have the same
concentration of pigments. But this issue has been addressed by the use of 3 beetroot discs
per test tube)
Reject fingerprints, different mass, different volume of solution in cuvette as this will not
affect % light transmission, discs from different parts of beetroot so different pigment conc

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

(same cylinder and same source/ part of beetroot is used to produce the discs), the
pigments not evenly distributed (quite a few thought it’s like suspension)
[2]

15 Describe two improvements to this investigation which would increase the confidence in
your results. [2]

Accept use of mould cutter, time stagger / increase the duration of time which discs sit in
solution to decrease percentage error caused by time lag, to reduce time lag, consistent
shaking, use of water bath to control temp at which expt is conducted.
Reject repeat the expt, because already in the steps.
Reject do expt on individual tubes separately to reduce time lag, because if do individually
the whole expt will take v long.

16 A fifth test tube, E, was added to the experiment set-up, containing 5cm3 of 0.1M lead (II)
nitrate solution. (Lead nitrate is poisonous). Predict and explain what you will observe.

Predict: The surrounding solution will turn red/pink as the betalain pigments leak from the
beetroot cells;
Explain: Lead is a heavy metal that is positively-charged and forms strong bonds with the
negatively-charged carboxyl R groups of membrane proteins, disrupting the ionic bonds,
causing the proteins to denature / polar regions of phospholipid is susceptible to damage by
lead;
As a result, more gaps appear in the membrane, allowing the pigments to escape;
[3]

Reject mention of ‘dark color’ without indicating the color.

Reject idea of poisoning the cells
Reject the idea of lead (II) nitrate being an inhibitor of enzyme / carrier proteins transporting
betalain

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

17 The same apparatus was set up at different temperatures using distilled water to investigate
the effects of temperature on membrane permeability in beetroot cells. At each temperature,
the beetroot discs were soaked for 5 minutes. The results of the investigation are shown in
Table 1.1.
Table 1.1

percentage light transmission

temperature / 0C
trial 1 trial 2 mean
30.0 93.9 95.0 94.5
40.0 80.1 77.0 78.6
50.0 62.4 63.1 62.8
60.0 26.3 29.9 28.1
70.0 13.5 12.6 13.1

Correct calculation of means and rounded to 1dp; due to precision

[1]

18 Calculate the mean percentage of light transmission at each temperature and plot the mean
data on the grid below. Draw a line of best fit. [4]

Points to Note on Plotting Graph:

1. Title of graph: Graph of y-axis/units against x-axis/units.
2. The x-axis should be the independent variable (the variable you are manipulating).
The y-axis should be the dependent variable (the variable that you are measuring).
3. Label axis with quantity/units.
4. Use data symbols either x or . If plotting multiple data sets, write a key / legend.
5. Draw a line of best fit. These lines should pass through or near as many data points as
possible. They can either be straight lined, or a smooth curve. Look for the pattern to
decide which is most appropriate.
6. Use appropriate scale to ensure that data is graphed as large as possible in the grid
provided.

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

Award 1 mark for each of the following: Q15

(i) Use of sensible scale. Awkward scales S Appropriate Scale:
(e.g. 3:10) are not allowed. Scales must
be chosen so that the points occupy at
least half the grid in both the x and y
directions;
If the scale is awkward the points do not need
checking.
(ii) all points plotted accurately to within half a P Correct plot points. Make sure data
small square on the grid; points are clearly plotted for all points
using x or 
(iii) correct choice of axes complete with Ax x-axis: temperature / 0C
labels and correct units;
y- axis: mean percentage
transmission no need to put %
(iv) points joined by straight lines of best fit F Straight lines of best fit /
and key/labels to distinguish the different Data symbol allowed  and  only
sets of data;
No extrapolation beyond data points

Lines of best fit should not be extrapolated

beyond extreme plotted points.

Things to take note:

Use appropriate scale to ensure that data is graphed as large as possible in the grid provided.
Scale may or may not start at 0. Do indicate the values that x and y axes start with. The scale
on each axis goes up in equal intervals. Each axis should be scaled using multiples of 1, 2, 5 or
10 for each 20 mm square on the grid. This makes it easy for you to plot and extract data.

Recall how to draw a line of best fit:

- These lines pass through or near as many data points as possible, & even number
of points on both sides. They can either be straight lined, or a smooth curve. Look
for the pattern to decide which is most appropriate. It is appropriate to use a line or
curve of best fit when we can safely assume the intermediate points would lie along the
line i.e. you can see a clear trend in which you have confidence.
- For best-fit line, you should ensure that approximately the same number of points
(don’t need to be exact), but the exact same perpendicular distance from the line, lie
above and below it (± half a square). There is no need for your line to go through
either the first or the last point – these points are no more ‘special’ than any of the
others, so should not get special treatment.

Do not extend the line beyond the plotted points (extrapolate).

Erase properly, and use 2B pencil to draw to show a clear graph.

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 NANYANG JUNIOR COLLEGE H2 Biology
Practical 6: Membrane & Transport
(Chemicals on membrane permeability)

19 Account for the results above.

(Account means “describe” and “explain”)

1 Describe the trend with figures (1m): Generally, as temperature increases, the mean
percentage light transmission decreases. For instance, when temperature increases from
30.00C to 70.00C, the mean percentage light transmission decreases from 94.5% to
13.1%; (values should be quoted from best fit line drawn)
2 Betalain pigments are contained within the vacuole of beetroot cells, surrounded by a
membrane called tonoplast. When the membrane breaks / gaps appear in membrane, the
red betalains leak out into the surrounding solution.
3 At high temperatures, molecules have higher kinetic energy, thus they vibrate faster and
both the beetroot cell membrane and tonoplast become more fluid. Phospholipids move
far apart frequently, creating gaps in the membranes, increasing their permeability.
4 Ionic and hydrogen bonds within the membrane proteins hold them in specific 3D
conformation/structure which can be broken due to the high temperature. When these
membrane proteins denture, gaps appear in the membranes, allowing the pigments to
escape.
[4]

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