Testing for Reducing sugars
Benedict’s Test
All monosaccharides and some disaccharides are
reducing sugars
– (This means that they provide electrons that can carry
out a reduction)
Benedict’s Reagent detects the presence of a
reducing sugar
Lactose and maltose are directly detected by
Benedict's reagent, because each contains
glucose with a free reducing group
Sucrose is a non-reducing sugar
Benedict’s test
• Benedict’s reagent contains copper(II)sulphate CuSO4
• Heat a reducing sugar with Benedict’s reagent and an
insoluble precipitate is formed
• The copper is reduced by the sugar and insoluble
copper(I)oxide Cu2O produced
(OILRIG: Reduction is gain: Cu2+ gains an electron
and becomes Cu+)
• The clear blue solution changes colour depending on
the quantity of reducing sugar present
Textbook
pp112-114
Benedict test for reducing sugars –
risk assessment
Hazard
Precautions
Remedial measures
Glassware
breakage
Keep test tubes in
rack/waterbath to
prevent rolling of
bench
Staff to clear up any
broken glass
Cuts – seek first aid
Copper sulphate
solution - toxic
Wear safety goggles Wash off with
Carry dropper bottle
copious amounts
by glass
of water
Wash hands after
Ingested – seek first
practical
aid
Waterbath – hot
Care with hot water
Scalds – run under
cold water and
seek first aid
Learning Objectives
You will have been successful if by the end of the
lesson you can:
• Explain how to test for reducing sugars
• Demonstrate skilful and safe practical techniques using
suitable qualitative/quantitative method
– Follow instructions to practise the preparation of a serial dilution
• Make and record valid observations.
– Use the Benedict’s Test to carry out semi-quantitative method of
determining concentration of a reducing sugar and explain the
results.
Semi-quantitative
assessment of
reducing sugar
content
No reducing sugar
High
Produce a range of colour standards with known concentrations
of glucose and these can be used to compare the result of a test
carried out with an equal volume of an unknown solution e.g.
orange juice
Textbook
pp112-114
Concentration of sugar
One method for creating a range of dilutions
Volume of 0.08
mol dm-3 glucose
solution (cm-3)
Volume of
deionised water
(cm-3)
10.0
7.5
5.0
2.5
0.0
2.5
0.0
Final
concentration of
glucose solution
(mol dm-3)
0.08
Serial dilution
Making progressively more dilute solutions from the previous concentration
We can produce a 2% solution
from a 4% solution :
5cm3 of 4% glucose solution
+ 5cm3 de-ionised water
= 10cm3 of 2% glucose solution
5cm3
More
concentrated
solution
+5cm3 de-ionised
water
More
dilute
solution
4% glucose solution =
4g glucose in 100cm3
de-ionised water
5 cm3 original concentration
In a total of 10cm3
5/10 = 1/2 original
concentration
Making Glucose Concentrations.
Volume of
deionised
water
(cm3)
Volume and
conc. of
glucose
solution
(cm3)
10
5
5
5
5
0
0
10 of
4.00%
Total
volume
(cm3)
Concentration
of glucose
solution (%)
10
10
10
10
10
10
0.00
0.25
0.50
1.00
2.00
4.00
Notice that the table
is arranged with the
lowest glucose
concentration at the
top even though you
will start with the
greatest
concentration
Textbook
pp112-114
Making Glucose Concentrations.
Volume of
deionised
water
(cm3)
Volume and
conc. of
glucose
solution
(cm3)
10
5
5
5
5
0
0
10 of
4.00%
Total
volume
(cm3)
Concentration
of glucose
solution (%)
Control
To show that....
10
10
10
10
10
10
0.00
0.25
0.50
1.00
2.00
4.00
You are given a
stock solution of 4%
glucose.
Complete this row
first, then think
about diluting the
solution you have
just made to get the
row above
Textbook
pp112-114
Volume of
de-ionised
water
(cm3)
Volume and
conc. of
glucose
solution
(cm3)
Total volume
(cm3)
Concentration
of glucose
solution (%)
10
5
5
5
5
0
0
5 of 0.5%
5 of 1%
5 of 2%
5 of 4%
10 of 4%
10
10
10
10
10
10
0.00
0.25
0.50
1.00
2.00
4.00
Why
4.00, not
4?
Textbook
pp112-114
How can we display this data?
Is this data quantitative or qualitative?
What is the independent variable?
What is the dependent variable?
Where should these be positioned in a results table?
Independent Dependent
Variable
Variable
/units
Trial 1/ units
Dependent
Variable
Trial 2/ units
Dependent
Variable Trial
3 / units
Mean
result /
units
Results table
Concentration of
glucose solution (%)
0.25
0.50
1.00
2.00
4.00
Final colour of
mixture
Final cloudiness of
tube (amount of
precipitate)
Unknown solution
• Final colour of tube ………………………..
• Final cloudiness of tube ………………….
• Concentration of reducing sugar is
………………………………………………..
• Hmmm – we can’t be precise about the value – this is why this
method is semi-quantitative
• The answers here relate to the skilful
practice assessment of coursework
Benedict’s test
• Benedict’s reagent contains copper (II) sulphate
• Heat a reducing sugar with Benedict’s reagent and an
insoluble precipitate is formed
• The copper ions are reduced by the sugar and
insoluble copper (I) oxide produced
(OILRIG: Reduction is gain: Cu2+ gains an electron
and becomes Cu+)
• The solution is clear blue initially and changes colour
depending on the quantity of reducing sugar present
Learning Objectives
You will have been successful if by the end of the
lesson you can:
• Explain how to test for reducing sugars
• Demonstrate skilful and safe practical techniques using
suitable qualitative/quantitative method
– Follow instructions to practise the preparation of a serial dilution
• Make and record valid observations.
– Use the Benedict’s Test to carry out semi-quantitative method of
determining concentration of a reducing sugar and explain the
results.
Learning Objectives
You will have been successful if by the end of the
lesson you can:
Evaluate data and practical skills by:
• Identifying and explaining the main limitations of the
data collection strategy.
• Suggesting and giving reasons for simple
improvements to the experiment;
• Commenting upon the reliability of the data collected;
and discussing the validity of the conclusions.
What are the limitations of this
method?
• Semi quantitative = subjective
• Difficult to describe the colour change and
cloudiness.
How to make the Benedict’s test
quantitative?
We need to know exactly how much of the Cu2+
has been reduced
1. Measure the cloudiness of the solution
(indicating the amount of precipitate) with a
colorimeter
2. Measure the ‘blueness’ of the remaining
copper sulphate solution using a colorimeter
3. Measure the mass of precipitate (will have to
be fully dry)
Another Serial Dilution
The test tubes contain a series
of solutions, each one more
dilute than before
9cm3 of de-ionised water
1cm3
+
9cm3 water
1cm3 of 10% glucose solution
1cm3 of 10% glucose in 10 cm3 =
1/10 of original concentration
10% glucose
solution
=0.1% glucose solution
= 1% glucose
solution
Benedict’s test results?
0.001%
0.01%
0.1%
1%
10%
• Look – more red precipitate, and the solution is
less blue
Using a colorimeter
Choose a filter with a
complementary colour to
your standard solution
The deepest colour should
absorb the most – and give
the highest absorbance
reading
Interactive
colourimeter
A graph can be plotted and used to determine the
concentration of glucose in an unknown solution
Textbook
pp113-114
Calibration curve
• Use solutions of known concentrations to
produce a set of values.
• Plot a calibration curve
• Find a value for the unknown
concentration
• Draw intercepts on the graph to determine
the concentration of the unknown solution
Textbook
pp113-114
sample results
Concentration of
reducing sugar
solution (%)
Colorimeter reading
(Absorbance)
4.00
0.05
2.00
0.25
1.00
0.65
0.50
0.96
0.25
1.15
Orange juice
0.12
Textbook
pp113-114
Textbook
pp113-114
Colorimeter reading
(Absorbance)
Calibration curve 2
Mass of precipitate
The unknown
solution gives a
value of 11g
Using the
calibration curve
this gives a
concentration of
2.75%
Calibration curve
18
16
14
12
10
Mass of precipitate (g)
Linear (Mass of precipitate (g))
8
6
4
2
0
0
0.5
1
1.5
2
2.5
3
Concentration of reducing sugar (%)
3.5
4
4.5
Textbook
pp113-114
Learning Objectives
You will have been successful if by the end of the
lesson you can:
Evaluate data and practical skills by:
• Identifying and explaining the main limitations of the
data collection strategy.
• Suggesting and giving reasons for simple
improvements to the experiment;
• Commenting upon the reliability of the data collected;
and discussing the validity of the conclusions.
Match up!
2. Calibration curve
A ….make an experiment more
reliable
B …..make an experiment more
valid
C …..gives quantitative objective
data
3. Repeats used to
calculate a mean
D ……make an experiment more
precise
1. A colorimeter
4. Controlling all the
variables, and being
accurate
5. A control, with no
glucose present,
E …..gives qualitative subjective
data
F …….allows the concentration of
an unknown solution be found
from a set of samples of known
concentration
G …..gives a colour standard to
compare against
Match up!
1. A colorimeter
2. Calibration curve
3. Repeats used to
calculate a mean
4. Controlling all the
variables, and being
accurate
5. A control, with no
glucose present,
1C
2F
3A
4B
5G
A ….make an experiment more
reliable
B …..make an experiment more
valid
C …..gives quantitative objective
data
D ……make an experiment more
precise
E …..gives qualitative subjective
data
F …….allows the concentration of
an unknown solution be found
from a set of samples of known
concentration
G …..gives a colour standard to
compare against
Answers to practical questions
1. stirred both / left to settle;
compared colours;
compared, amount of precipitate / opacity;
max 2
Answers to practical questions
2 not a precise match;
subject to colour judgement;
intermediate values;
colour of juice may impair;
difficulty in matching cloudiness by eye;
use of a background;
restirring to overcome settling;
take into account cloudiness or precipitate;
AVP;
max 6
Answers to practical questions
3
mark one method only
filter / settle;
colorimeter;
ref transmission / absorbance;
use more intermediates of colour comparisons;
filter;
dry;
take mass;
max 3
Answers to practical questions
4
five concentrations used gives a (wide) range;
different results obtained for each % of glucose;
ref to anomalous results;
range of standards encompasses that of juices;
standardised (boiling) time;
long enough for reduction of, Benedict's / Cu;
volume of Benedict’s to test solution gives excess Cu ions / AW;
timing of stirring consistent;
standardised volumes;
measure accurately with syringes;
clean syringes;
clean dry rod;
reason for clean syringes or rod / non-contamination;
using cloudiness and colour enables two ways to make a comparison;
results did not have to be collected while test tubes were in water bath;
results did not have to be taken at same time;
therefore results more likely to be accurate;
AVP;
max 9
Answers to practical questions
5 other reducing sugars may be present;
1