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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 Keep test tubes in Staff to clear up any
breakage rack/waterbath to broken glass
prevent rolling of Cuts – seek first aid
bench
Copper sulphate Wear safety goggles Wash off with
solution - toxic 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 Volume of Final


mol dm-3 glucose deionised water concentration of
solution (cm-3) (cm-3) glucose solution
(mol dm-3)
10.0 0.0 0.08
7.5 2.5
5.0
2.5
0.0
Serial dilution
Making progressively more dilute solutions from the previous concentration
4% glucose solution =
We can produce a 2% solution 4g glucose in 100cm3
from a 4% solution : de-ionised water

5cm3 of 4% glucose solution


+ 5cm3 de-ionised water 5 cm3 original concentration
= 10cm3 of 2% glucose solution In a total of 10cm3
5/10 = 1/2 original
+5cm3 de-ionised
5cm3 water concentration

More More
concentrated dilute
solution solution
Making Glucose Concentrations.
Volume of Volume and
Notice that the table
de- conc. of Total Concentration is arranged with the
ionised glucose volume of glucose lowest glucose
water solution (cm3) solution (%) concentration at the
(cm3) (cm3)
top even though you
10 0 10 0.00 will start with the
greatest
5 10 0.25
concentration
5 10 0.50
5 10 1.00
5 10 2.00
0 10 of 10 4.00
4.00%
Textbook
pp112-114
Making Glucose Concentrations.
Volume of Volume and
de- conc. of Total Concentration
ionised glucose volume of glucose
water solution (cm3) solution (%) Control
(cm3) (cm3)
To show that....
10 0 10 0.00
5 10 0.25 You are given a
5 10 0.50 stock solution of 4%
glucose.
5 10 1.00 Complete this row
first, then think
5 10 2.00 about diluting the
solution you have
0 10 of 10 4.00 just made to get the
4.00% row above

Textbook
pp112-114
Volume of Volume and Concentration
conc. of Total volume of glucose
de-ionised glucose solution (%)
water (cm3)
solution
(cm3) (cm3)

10 0 10 0.00
5 5 of 0.5% 10 0.25
5 5 of 1% 10 0.50
5 5 of 2% 10 1.00
Why
5 5 of 4% 10 2.00 4.00, not
4?
0 10 of 4% 10 4.00

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 Dependent Dependent Mean


Variable Variable Variable Variable Trial result /
/units Trial 1/ units Trial 2/ units 3 / units units
Results table
Concentration of Final colour of Final cloudiness of
glucose solution (%) mixture tube (amount of
precipitate)

0.25

0.50

1.00

2.00

4.00
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
1cm3 9cm3 of de-ionised water
+
9cm3 water 1cm3 of 10% glucose solution

1cm3 of 10% glucose in 10 cm3 =


1/10 of original concentration
10% glucose = 1% glucose =0.1% glucose solution
solution 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 Colorimeter reading
reducing sugar (Absorbance)
solution (%)

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
Colorimeter reading
(Absorbance)

Textbook
pp113-114
Calibration curve 2
The unknown Mass of precipitate
solution gives a Calibration curve
value of 11g
18
Using the
calibration curve 16
this gives a
concentration of 14

2.75%
12

10
Mass of precipitate (g)
Linear (Mass of precipitate (g))
8

0
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Concentration of reducing sugar (%) 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!
A ….make an experiment more
1. A colorimeter reliable
B …..make an experiment more
2. Calibration curve valid
C …..gives quantitative objective
data
3. Repeats used to D ……make an experiment more
calculate a mean precise
E …..gives qualitative subjective
4. Controlling all the data
variables, and being
accurate F …….allows the concentration of
an unknown solution be found
5. A control, with no from a set of samples of known
concentration
glucose present,
G …..gives a colour standard to
compare against
Match up!
A ….make an experiment more
1. A colorimeter reliable
1C B …..make an experiment more
2. Calibration curve valid
2F C …..gives quantitative objective
data
3. Repeats used to D ……make an experiment more
calculate a mean 3A precise
4. Controlling all the E …..gives qualitative subjective
data
variables, and being
accurate
4B F …….allows the concentration of
an unknown solution be found
5. A control, with no from a set of samples of known
glucose present, 5G 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