2.1 Testing For Biological Molecules

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YOUR NOTES
2.1 Testing for Biological Molecules ⬇
CONTENTS
2.1.1 Biological Molecule Tests
2.1.2 The Benedict's Test
2.1.3 Testing for Non-Reducing Sugars
2.1.1 BIOLOGICAL MOLECULE TESTS

Testing for Key Biological Molecules
There are a number of tests that can be carried out quickly and easily in a lab to determine if
a sample contains one of the key biological molecules (carbohydrates, proteins and lipids)
The following tests are qualitative – they do not give a quantitative value as to how much of
each type of molecule may be present in a sample
The Benedict’s test for reducing sugars

Add Benedict’s reagent (which is blue as it contains copper (II) sulfate ions) to a sample
solution in a test tube
Heat the test tube in a water bath or beaker of water that has been brought to a boil for a
few minutes
If a reducing sugar is present, a coloured precipitate will form as copper (II) sulfate is reduced
to copper (I) oxide which is insoluble in water
A positive test result is, therefore, a colour change somewhere along a colour scale from blue
(no reducing sugar) to brown/brick-red (a high concentration of reducing sugar)
This test is semi-quantitative as the degree of the colour change can give an
indication of how much (the concentration of) reducing sugar present
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YOUR NOTES
The Benedict’s test for reducing sugars produces a colour change from blue towards red
if a reducing sugar is present
The iodine test for starch

To test for the presence of starch in a sample, add a few drops of orange/brown iodine in
potassium iodide solution to the sample
The iodine is in potassium iodide solution as iodine is insoluble in water
If starch is present, iodide ions in the solution interact with the centre of starch molecules,
producing a complex with a distinctive blue-black colour
This test is useful in experiments for showing that starch in a sample has been digested by
enzymes

YOUR NOTES
The emulsion test for lipids

Lipids are nonpolar molecules that do not dissolve in water but will dissolve in organic
solvents such as ethanol
Add ethanol to the sample to be tested, shake to mix and then add the mixture to a test tube
of water
If lipids are present, a milky emulsion will form (the solution appears ‘cloudy’); the more lipid
present, the more obvious the milky colour of the solution
If no lipid is present, the solution remains clear
The Emulsion test for lipids forms a milky colour

YOUR NOTES
The biuret test for proteins

A liquid solution of a sample is treated with sodium or potassium hydroxide to make the
solution alkaline
A few drops of copper (II) sulfate solution (which is blue) is added to the sample
Biuret ‘reagent’ contains an alkali and copper (II) sulfate
If a colour change is observed from blue to lilac/purple, then protein is present.

The colour change can be very subtle, it’s wise to hold the test tubes up against a
white tile when making observations)
If no colour change is observed, no protein is present

For this test to work, there must be at least two peptide bonds present in any protein
molecules, so if the sample contains amino acids or dipeptides, the result will be
negative

YOUR NOTES
2.1.2 THE BENEDICT'S TEST

The Benedict's Test for Reducing Sugars
Benedict’s solution can be used to carry out a semi-quantitative test on a reducing sugar
solution to determine the concentration of reducing sugar present in the sample
It is important that an excess of Benedict’s solution is used so that there is more than
enough copper (II) sulfate present to react with any sugar present
The intensity of any colour change seen relates to the concentration of reducing sugar
present in the sample
A positive test is indicated along a spectrum of colour from green (low concentration)
to brick-red (high concentration of reducing sugar present)
A semi-quantitative test can be carried out by setting up standard solutions with known
concentrations of a reducing sugar (such as glucose)
These solutions should be set up using a serial dilution of an existing stock solution
Each solution is then treated in the same way: add the same volume of Benedict’s solution to
each sample and heat in a water bath that has been boiled (ideally at the same temperature
each time) for a set time (5 minutes or so) to allow colour changes to occur
It is important to ensure that an excess of Benedict’s solution is used
Any colour change observed for each solution of a known concentration in that time can be
attributed to the concentration of reducing sugar present in that solution
The same procedure is carried out on a sample with an unknown concentration of reducing
sugar which is then compared to the stock solution colours to estimate the concentration of
reducing sugar present
Alterations
It is also possible to standardise this test but instead of waiting a fixed amount of time for a
range of colours to be observed, time how long it takes for the first colour change to occur
(blue to green)
The higher the concentration of reducing sugar in a sample, the less time it would
take for a colour change to be observed
To avoid issues with human interpretation of colour, a colourimeter could be used to measure
the absorbance or transmission of light through the sugar solutions of known concentration
to establish a range of values that an unknown sample can be compared against a calibration
curve

YOUR NOTES
Serial dilutions
Serial dilutions are created by taking a series of dilutions of a stock solution. The
concentration decreases by the same quantity between each test tube
They can either be ‘doubling dilutions’ (where the concentration is halved between
each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm-3)
Serial dilutions are completed to create a standard to compare unknown concentrations

against
The comparison can be:
Visual
Measured through a calibration/standard curve
Measured using a colourimeter
They can be used when:

Counting bacteria or yeast populations
Determining unknown glucose, starch, protein concentrations

YOUR NOTES
Making serial dilutions

YOUR NOTES
Colourimeter
A colourimeter is an instrument that beams a specific wavelength (colour) of light through a
sample and measures how much of this light is absorbed (arbitrary units)
They provide a quantitative measurement
They contain different wavelengths or colour filters (depends on the model of colourimeter),
so that a suitable colour can be shone through the sample and will not get absorbed. This
colour will be the contrasting colour (eg. a red sample should have green light shone
through)
Remember that a sample will look red as that wavelength of light is being reflected
but the other wavelengths will be absorbed
Colourimeters must be calibrated before taking measurements

This is completed by placing a blank into the colourimeter and taking a reference, it
should read 0 (that is, no light is being absorbed)
This step should be repeated periodically whilst taking measurements to ensure that
the absorbance is still 0
The results can then be used to plot a calibration or standard curve

Absorbance against the known concentrations can be used
Unknown concentrations can then be determined from this graph

YOUR NOTES
A colourimeter is used to obtain quantitative data that can be plotted to create a

calibration curve to be used to find unknown concentrations

YOUR NOTES
2.1.3 TESTING FOR NON-REDUCING SUGARS

Testing for Non-Reducing Sugars
Sugars can be classified as reducing or non-reducing; this classification is dependent on their

ability to donate electrons (a reducing sugar that is able to donate electrons is itself oxidised)
OILRIG in Chemistry
Benedict’s solution is a blue solution that contains copper (II) sulfate ions (CuSO4 ); in the
presence of a reducing sugar copper (I) oxide forms
Copper (I) oxide is not soluble in water, so it forms a precipitate
The more reducing sugar present in a sample, the greater the colour change observed from
blue to brick-red
Copper (I) oxide forms a brick-red precipitate, but a range of colours are seen during a
Benedict’s test depending on how much reducing sugar is present and how much
copper is reduced
Reducing & non-reducing sugars table
To test for non-reducing sugars:

Add dilute hydrochloric acid to the sample and heat in a water bath that has been brought to
the boil
Neutralise the solution with sodium hydrogencarbonate

Use a suitable indicator (such as red litmus paper) to identify when the solution has
been neutralised, and then add a little more sodium hydrogencarbonate as the
conditions need to be slightly alkaline for the Benedict’s test to work
Then carry out the Benedict’s test as normal; add Benedict’s reagent to the sample and heat
in a water bath that has been boiled – if a colour change occurs, a reducing sugar is present

YOUR NOTES
Explanation
The addition of acid will hydrolyse any glycosidic bonds present in any carbohydrate
molecules
The resulting monosaccharides left will have an aldehyde or ketone functional group that can
donate electrons to copper (II) sulfate (reducing the copper), allowing a precipitate to form
Exam Question: Easy

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Exam Question: Medium
Exam Question: Hard

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CIE AS Biology (9700) exams from 2022 Revision Notes savemyexams.co.

2.1.2 The Benedict's Test

2.1.3 Testing for Non-Reducing Sugars

2.1.1 BIOLOGICAL MOLECULE TESTS

The Benedict’s test for reducing sugars

CIE AS Biology (9700) exams from 2022

The iodine test for starch

CIE AS Biology (9700) exams from 2022

The emulsion test for lipids

If no lipid is present, the solution remains clear

The Emulsion test for lipids forms a milky colour

CIE AS Biology (9700) exams from 2022

The biuret test for proteins

If a colour change is observed from blue to lilac/purple, then protein is present.

If no colour change is observed, no protein is present

CIE AS Biology (9700) exams from 2022

2.1.2 THE BENEDICT'S TEST

CIE AS Biology (9700) exams from 2022

each test tube) or a desired range (e.g. 0, 2, 4, 6, 8, 10 mmol dm-3)

Serial dilutions are completed to create a standard to compare unknown concentrations

Measured through a calibration/standard curve

Measured using a colourimeter

They can be used when:

Determining unknown glucose, starch, protein concentrations

CIE AS Biology (9700) exams from 2022

Making serial dilutions

CIE AS Biology (9700) exams from 2022

They provide a quantitative measurement

Colourimeters must be calibrated before taking measurements

The results can then be used to plot a calibration or standard curve

Unknown concentrations can then be determined from this graph

CIE AS Biology (9700) exams from 2022

A colourimeter is used to obtain quantitative data that can be plotted to create a

CIE AS Biology (9700) exams from 2022

2.1.3 TESTING FOR NON-REDUCING SUGARS

Sugars can be classiﬁed as reducing or non-reducing; this classiﬁcation is dependent on their

Copper (I) oxide is not soluble in water, so it forms a precipitate

Reducing & non-reducing sugars table

To test for non-reducing sugars:

Neutralise the solution with sodium hydrogencarbonate

CIE AS Biology (9700) exams from 2022

Exam Question: Easy

CIE AS Biology (9700) exams from 2022

Exam Question: Medium

Exam Question: Hard

CIE AS Biology (9700) exams from 2022

> CHECK YOUR ANSWERS AT SAVEMYEXAMS.CO.UK

CIE AS Biology (9700) exams from 2022

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