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AS Biology CIE Your notes

2.1 Testing for Biological Molecules

Contents
Biological Molecule Tests
T he Benedict's Test
Testing for Non-Reducing Sugars

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Biological Molecule Tests

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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
The Benedict's Test Diagram

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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
enz ymes
Iodine Test Diagram

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Iodine solution can be used to test for the presence of starch

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
Emulsion Test Diagram

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The Emulsion test for lipids forms a milky colour

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
Biuret Test Diagram

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Biuret solution can be used to test for the presence of proteins in a sample of food

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The Benedict's Test

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The Benedict's Test for Reducing Sugars
Method
Add Benedict's reagent (which is blue as it contains copper (II) sulfate ions) to a sample solution
in a test tube
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
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

Semi-quantitative Benedict's test

A semi-quantitative test can be carried out by setting up standard solutions with known
concentrations of 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
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

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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

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Making serial dilutions

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Colourimeter
A colourimeter is an instrument that beams a specific wavelength (colour) of light through a Your notes
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 (e.g. 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
Colorimeter Diagram

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Your notes

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A colourimeter is used to obtain quantitative data that can be plotted to create a calibration curve to be
used to find unknown concentrations

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Testing for Non-Reducing Sugars

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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
If Benedict's test has been carried out on a solution and it shows that no reducing sugars are
present then a modified version of the test can be carried out to test for the presence of non-
reducing sugars

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 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 (orange-red precipitate), a non-
reducing sugar is present

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

Reducing & Non-reducing Sugars Table

Reducing Sugars Non-Reducing Sugars

Sucrose (the only one you need to know)

Galactose

Glucose

Fructose

Maltose

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