Lab # 2

Date: February 9th, 2023

Topic: Testing For Reducing and Non-reducing Sugars

Aim: To discover how reducing sugars can be detected using Benedict’s solution, how
this can be made semi-quantitative and how the test can be extended to register the
presence of non-reducing sugars as well.

Materials:
large beakers to act as a water bath (500 cm 3 or larger), six test tubes, test
tube rack, spatula, two syringes or graduated pipettes for measuring 2 cm 3, marker
pen, 20 cm 3 Benedict’s solution, distilled water, solid glucose, few cm 3 1% glucose
solution, solid sucrose, 0.1 mol dm -3 HCL, solid NaHCO 3 , test tube holder, rubber
bands.

Methods:

Testing for reducing sugars

The familiarization of colors obtained from the reaction with benedicts solution and reducing sugar, by
carrying out a test with glucose was first done.

1. A water bath of water was then heated in a large beaker to about 75 C. It was set up before the
preparation of the testing.
2. Then a small spatula load of glucose(reducing sugar) was dissolved in approximately 2cm3 water
and a roughly equal volume of benedict’s solution was also added. It was mixed thoroughly, then
the test tubes were placed in the water bath and were maintained there for four minutes. The test
tubes were gently agitated during heating for more consistent results.
3. The sequence of color change was carefully observed and recorded.

Making the test semi-quantitative

1. A serial dilution was set up starting with a 1% glucose solution. Six test tubes were labeled, 1-6,
and then 2cm3 of 1% glucose solution was placed in the first test tube and then 2cm3 distilled
water in the other five test tubes. Afterward, 2cm 3 of 1% glucose solution was added to test tube
#2 and mixed thoroughly before transferring 2cm3 to the next test tube until it reached test tube
#6. The content of test tube #6 was mixed and then 2cm3 was discarded. After that, 2cm 3 of
benedicts solution was added to each test tube and was again mixed thoroughly.
2. All the test tubes were placed in the boiling water bath and left there for a standard time(3
minutes)
3. All the test tubes were removed and arranged in order, while the color against the concentration
was recorded.
Test for non-reducing sugars
Although all monosaccharides are reducing, some disaccharides are not. When these
non-reducing sugars are hydrolyzed into their monosaccharide components, the
aldehyde or ketone groups are exposed and will produce a precipitate with Benedict’s
solution. Hydrolysis can be achieved using either mineral acids or enzymes.

1. A small spatula load of sucrose was dissolved in half a test tube of distilled water, and it was then
split equally between two test tubes labeled A and B. For test tube A, 1cm 3 of 0.1 mol dm -3
hydrochloric acid was added, while for test tube B the same volume of distilled water was added.
Both test tubes were heated in the boiling water bath for one minute.
2. Both test tubes were then removed and then cooled under a tap to room temperature. A small
amount of sodium hydrogen carbonate powder was added to test tube A. Additional small
quantities of the powder were added until there were no more effects of the result in
effervescence. This indicated that the acid was neutralized and the alkaline conditions necessary
for the Benidict’s test now prevail.
3. Equal amounts of Benidict’s solution were added to each tube that was then heated for a standard
time in the water bath. The test tubes were then removed and the final colors obtained were
compared.

Data Presentation:
IMAGES SHOWING COLOR CHANGE OF TRACES OF GLUCOSE FROM BENEDIC’S TEST

None . Low traces . Moderate High .

TABLE SHOWING THE COLOR CHANGE OF GLUCOSE(REDUCING SUGAR)

Test tube Solution First color Second color Third color Fourth color Final color
change change change change change

A Glucose + Light blue Green Green-yellow Dark yellow Brick red

Cu2+
IMAGE SHOWING RESULT OF COLOR CHANGE FOR THE SEMI QUANTITATIVELY METHOD
ON SUGARS

TABLE SHOWING THE TESTING RESULT FROM THE SEMI QUANTITATIVELY METHOD ON
SUGARS
Test tube Concentration (%) Initial color Final color

1 1 Light blue burnt orange

2 0.5 Light blue Dark brown

3 0.25 Light blue Light green

4 0.125 Light blue Light blue with minor

light green

5 0.0625 Light blue Light blue

6 0 Light blue Light blue

TABLE SHOWING RESULT FROM THE TESTING OF THE NONE-REDUCING SUGARS

Test tube Solution Initial color Final color

A Sucrose + H2O + HCl + Light blue Orange

NaHCO3 + Cu2+

B Sucrose + H2O + Cu2+ Light blue Light blue

Calculation of concentration (%) of glucose for semi-quantitative method

Tube 1: 1/1= 1.00
Tube 2: 1 / 2 = 0.50

Tube 3: 0.50/2 = 0.25

Tube 4: 0.25/2= 0.125

Tube 5: 0.125/2 = 0.0625

Tube 6
0 (water)
=0

Discussion: Carbohydrates are organic compounds that include things like sugars, starch, and cellulose.
They contain hydrogen and oxygen in the same ratio as water(2:1). More specifically, sugars are either a
monosaccharide or a disaccharide meaning it has either one or two sugar units. Reducing sugars also
known as reducing agent has either a ketone or aldehyde functional group and can effectively donate
electrons to other molecules through oxidation. While non-reducing sugars are the opposite meaning that
they do not contain a free aldehyde or ketone group but rather they are available in the formation of bonds
and also their inability to oxidize. Benedict's test cannot be done on none-reducing sugars because there
are no free ketone or aldehyde groups present therefore it has to first be hydrolyzed(break into two of its
reducing sugar counterparts for it to react with the benedicts solution).
To detect reducing sugars on the other hand a benedicts test is utilized, the basic principle behind it is that
when a reducing sugar is subjected to heat in the presence of an alkali, it gets converted to an enadiol
(powerful reducing agent) as such when the reducing sugar is present in the analyte the copper ions (Cu2+)
in the benedicts reagent are reduced to only Copper ions (Cu+) as a result the cuprous ions form Cooper(I)
oxide with the reaction mixture and precipitate a brick red colored compound which signifies a positive
benedicts test (reaction can be found below image#1). For the benedicts test, there are colors that reflect
the approximate % of reducing sugars. (image #2 below)

#1
#2

When there are no reducing sugars present the percentage is 0% glucose and this has a blue or light blue
color, then when its green there are traceable amounts present or approximately between 0.5-1g%, yellow
represents very low amounts of reducing sugar that ranges from 1-1.5 g%, a moderate amount of reducing
sugar is displayed by bright orange having 1.5-3 g%, and finally, the highest level of reducing sugars have
a brick red color which denotes that it has greater than 2% of glucose. As the color changed from brick
red which contained 1% glucose of approximately 0.056 Molarity then the molarity also changes since it
was diluted the molarity will also reduce by half until there is a very low molarity for the benedicts
solution to detect therefore no more color changes (light blue).

The semi-quantitative method(experiment #2) is a method that utilizes 1% glucose initially in the #1 test
tube, then 1% glucose in test tube #2 with 2cm3 water which would cut the concentration from 1% to
0.5% glucose, then 2cm3 of test tube #2 content is transferred to the remaining 3 test tube that all contain
2cm3 water, which makes it 5 test tube starting from 1% glucose concentration to approximately 0.0625%
glucose. For test tube #6, there was 0% glucose since it was diluted to the maximum however this and
other test tubes with glucose concentration less than 0.5% will still retain its light blue color since it's
closer to zero. This method only gives approximate estimates of the concentration because of the addition
of water and then the transfer of diluted content into the next test tube until it reached close to 0% of
glucose content. So essentially 1 is divided by two until it reaches close to zero however this may not be
the accurate concentration of the contents in the test tube but rather a close estimate. The contents in each
test tube after being diluted can be tested by benedict’s test to show different colors as was done in this
second experiment to determine the approximate concentrations of the diluted reducing sugar.

The aldehyde or ketone groups that are exposed when non-reducing sugars are hydrolyzed into
monosaccharide components will precipitate with benedict's solution. Mineral acids or enzymes can be
used for hydrolysis. In this experiment, HCl was used, and when it was heated with the disaccharide
sucrose, the sucrose hydrolyzes and transforms into its two reducing sugar counterparts(Test tube A).
While in test tube B there was only sucrose and water therefore it remained light blue since the sucrose
didn't break down into its reducing sugar counterparts because of the lack of either mineral acid/enzymes.
Test tube A, however, changes color from blue to orange as it separates into its two counterparts(reducing
sugars). After the hydrolysis of sucrose the reducing sugars, fructose, and glucose % would be reduced to
50% each and then if they were to be separated using Chromatography then they can be compared
semi-quantitatively until they are no more color change visible after being diluted.

It is unsafe however to assume the state of concentration for reducing sugar other than the glucose
because a semi-quantitive method is used however this is only an approximation and not the actual
concentration as well as not all disaccharides are reducing sugar and therefore it cannot be tested for
concentration.
Condensation is the reaction in which simple sugars combine to form larger ones. eg. glucose and fructose
combine to form sucrose. While hydrolysis is the breakage of glycosidic bonds converting a sugar into
two. eg. Sucrose into glucose and fructose

Source of error: A source of error could be if more water was added in each of the semi-quantitative test
tubes resulting in less color change because of it being more diluted.
Another source of error could be if the none-reducing sugars never hydrolyzed correctly resulting in no
color change.
Limitation: A limitation could be if the glucose concentration was not 100% accurate.

Conclusions:
A reducing sugar is determined using benedict's solution which shows the amount of sugar present while
none reducing sugars are first hydrolyzed (broken into two) into their monosaccharide components, the
aldehyde or ketone groups are then exposed and will produce a precipitate with Benedict’s
Solution. As such, particular colors such as light blue, green, yellow, orange-red, and brick red are used to
make an approximate estimate of the % of glucose present.
Reference:

Libretexts. (2022, September 21). 26.1: Monosaccharides. Chemistry LibreTexts. Retrieved

February 12, 2023, from
https://chem.libretexts.org/Bookshelves/Introductory_Chemistry/Introductory_Chemistry_(CK-12)/
26%3A_Biochemistry/26.01%3A_Monosaccharides#:~:text=A%20monosaccharide%20is%20a%2
0carbohydrate,monosaccharides%20are%20glucose%20and%20fructose

Admin. (2022, November 14). Benedict's test - reagent preparation, principle, procedure, reaction.
BYJUS. Retrieved February 12, 2023, from
https://byjus.com/chemistry/benedicts-test/#:~:text=Benedict's%20test%20is%20a%20chemical,be
%20identified%20with%20this%20test.

Reducing sugar - definition and examples - biology online dictionary. Biology Articles, Tutorials
& Dictionary Online. (2022, June 16). Retrieved February 12, 2023, from
https://www.biologyonline.com/dictionary/reducing-sugar

Benedict´s Test for Reducing Sugars ( A&I)

Discuss reducing/non-reducing sugars (3 marks)

-include chemical reaction (redox) between reducing sugar ad Benedict´s solution [1]
-explain why non-reducing sugars do not react with Benedict´s [1]
-describe how Benedict´s test can be used semi-quantitatively [1]
Analysis of Results (7 marks)
-accurately calculate concentrations (%) of 6 glucose solutions [1]
-describe what is meant by condensation & hydrolysis with respect to sugars and starch [1]
-discuss the effect of hydrolysis of sucrose on the Benedict´s test [2]
-discuss the effect of hydrolysis of starch on the Benedict´s test [2]
-compare semi-quantitatively the amount of reducing sugar released after hydrolysis of both sucrose and
starch [1]
Accurate Sources of error/limitations/assumptions
-any two [2]
-less than two [1]