NurBio – Nursing Biochemistry

Laboratory Report

CARBOHYDRATES:
COMMON REACTIONS
Activity No. 5

RATIONALE

We study the reactions of various carbohydrates with reagents used to classify and identify these
compounds. One test for the presence of many simple carbohydrates is to use Benedict's reagent. It turns
from turquoise to yellow or orange when it reacts with reducing sugars. These are simple carbohydrates
with unbound aldehyde or ketone groups. Carbohydrates are classified according to the complexity of
their molecules. The simplest group, called monosaccharides, is the building blocks from which higher
sugars, (dissacharides) and more complex groups (polysaccharides) are formed. The ending ⎯ose is
generally used in naming carbohydrates and the term sugar refers to those possessing sweet taste.

The following are the objectives for this activity:

____________________________________________________________________________________________________
Natural Sciences Department, College of Science and Information Technology, Ateneo de Zamboanga University
Nursing Biochemistry
Laboratory Report

1. We learned about the common and specific reactions of carbohydrates.

2. We were able to do the Molisch test, fehling’s test, benedict’s test, barfoed test, picric acis test
and tollen’s test.
3. We were able to know the classification of unkown.

DATA INTERPRETATION

Table No. 1 Molisch Test

Test Solutions Added Reagent(s) Observation

The test tube is foggy on top but

clear on the bottom, with a
1% glucose subtle purple tint line in the
lower half.

In the test tube, there is a thicker

1% xylose magenta line. The upper part of
the substance is cloudy.
+ 1 drop Molisch reagent
The line is a thinner darker shade
+ 1 mL conc. sulfuric acid of violet with four layers. The
1% sucrose upper part of the substance is
less cloudy than the test tube that
has 1% xylose.

The upper part of the substance

1% starch is cloudy, it also has a thin violet
tint.

It is cloudy on top but clear on

Unknown sugar the bottom, and it has a fading
purple tint.
Interpretation: A chemical test called Molisch's test is performed to determine whether or not an analyte
contains carbohydrates. Hans Molisch, a Czech-Austrian botanist, is credited with discovering this test,
which bears his name. A few drops of concentrated H2SO4 (sulphuric acid) are then added to the mixture
after adding Molisch's reagent, which is an ethanol solution of -naphthol, to the analyte.

The presence of carbohydrates in the analyte is confirmed by the creation of a purple or a purplish-red
ring at the site of contact between the H2SO4 and the analyte + Molisch's reagent mixture.

Table No. 2 Reduction Test (Fehling’s Test)

____________________________________________________________________________________________________ 1
Nursing Biochemistry
Laboratory Report

Test Solutions Added Reagent(s) Observation

1% glucose It became color orange.

The substance has an orange

1% xylose
color that's a little faded on top.

The substance in the test tube

1% fructose became orange in color, with
some residue at the bottom.
+ 1 Fehling’s solution (A and B)
It is orange in color and more
1% lactose see-through on top, with the
Δ
residue still present.

The substance was clear and it

1% sucrose
has a bluish tint.

The substance is clear, but with a

1% starch
tint of blue

Unknown sugar It became color orange.

Interpretation: A chemical test called Fehling's test is used to distinguish between reducing and non-
reducing sugars. This test can also be used to distinguish between water-soluble carbohydrates and
carbohydrates belonging to the ketone functional group. A reddish-brown precipitate's appearance
denotes a successful outcome and the presence of reducing sugars. A negative result and a lack of
reducing sugars are indicated by the absence of the reddish precipitate or the presence of deep blue hue.
Using Fehling's test, which does not respond to any ketone sugars other than alpha-hydroxy-ketone, it is
possible to distinguish between the presence of aldehydes and ketones in carbohydrates. To identify the
presence of glucose in urine, medical institutions use Fehling's test. This aids in determining the patient's
diabetes status.

Table No. 3 Reduction Test (Benedict’s Test)

Test Solutions Added Reagent(s) Observation

1% glucose There is larger red precipitation.

+ 1 mL Benedict’s reagent
1% xylose There is less red precipitate and
it has faded slightly.

____________________________________________________________________________________________________ 2
Nursing Biochemistry
Laboratory Report

There is less red precipitate and

1% fructose
it has faded more.

There is very little red

1% lactose
precipitate, but it is still evident.

1% sucrose There is no precipitation.

Δ

Unknown sugar There is larger red precipitation.

Interpretation: A chemical test called Benedict's test can be performed to determine if an analyte
contains reducing sugars. This test can thus be used to identify simple carbohydrates that include a free
ketone or aldehyde functional group. The test is based on a compound combination of sodium citrate,
sodium carbonate, and the pentahydrate of copper(II) sulfate known as Benedict's reagent, also referred to
as Benedict's solution. A brick-red precipitate that forms as a result of Benedict's reagent's interactions
with reducing sugars is a sign that the test is successful.

It should be mentioned that the Benedict's test may be utilized to determine whether there is glucose
present in a urine sample. Since glucose is an aldose whose open-chain forms an aldehyde group and this
test identifies any aldehydes and -hydroxy ketones, the presence of glucose in the analyte results in a
positive test result. However, the presence of homogentisic acid, ascorbic acid, and other reducing agents
in urine can also result in a favorable response. As a result, a positive Benedict's test result does not
automatically indicate that the test participant has diabetes.

Table No. 4 Reduction Test (Barfoed’s Test)

Test Solutions Added Reagent(s) Observation

1% glucose A red precipitation has formed.

+ 1 mL Barfoed’s reagent
1% lactose There isn't any precipitation.
Δ
A red precipitation has formed.
Unknown sugar

Interpretation: A chemical test known as the Barfoed's test is used to identify the presence of
monosaccharides and can identify reducing monosaccharides when disaccharides are present.
Disaccharides might be utilized in this process, although it would proceed extremely slowly. Reducing
monosaccharides in the sample are found by looking for red precipitate. If the color develops within the

____________________________________________________________________________________________________ 3
Nursing Biochemistry
Laboratory Report

first few minutes, reducing monosaccharides are present in the sample. However, the sample is of
decreasing disaccharides if the color doesn't show during the first three minutes.

Table No. 5 Reduction Test (Picric Acid Test)

Test Solutions Added Reagent(s) Observation

1% glucose It's mahogany in color.

It's darker now, and there are

1% xylose
stains.
+ 10 drops saturated picric acid
1% fructose solution Mahogany and stains remain.
+ 4 drops 10% Na2CO3
Δ
1% lactose The color is golden orange.

It became color yellow and

1% sucrose
similar to the original.

Unknown sugar It's mahogany in color.

Interpretation: The presence of reducing sugars may be detected chemically using the picric acid test for
carbs. In order to get Picramic Acid, which is red in color, reducing sugars must first react with picric
acid, which is a poisonous yellow crystalline solid also known chemically as 2,4,6-trinitrophenol (TNP).
All monosaccharides and disaccharides that have an aldehyde or ketone group that might be free have
reducing characteristics. Sugars that are being reduced have a free aldehyde or ketone group that has a
reducing property. When in an alkaline solution, several organic acids are reduced. The solution is either
basic or alkaline by adding sodium carbonate (Na2CO3). As a result, picric acid (yellow solution) is
reduced to picramic acid by reducing sugars (mahogany red solution).

Table No. 5 Reduction Test (Tollen’s Test)

Test Solutions Added Reagent(s) Observation

+ 3 drops Tollen’s reagent

Mudded in hue, yet shinny and
1% glucose
metallic in appearance.
Δ
Interpretation: Chemical analysis known as the Tollens' test is used to distinguish between reducing and
non-reducing carbohydrates. Based on the results of this test, it is also known as the silver mirror test. A

____________________________________________________________________________________________________ 4
Nursing Biochemistry
Laboratory Report

positive result suggests that the provided sample includes reducing sugars or aldoses when a dark grey
precipitate or silver mirror forms on the bottom and sides of the test tube. A negative result shows that
there are no reducing sugars, aldoses, or -hydroxy ketoses in the test sample. This precipitate's absence
demonstrates this. Tollen's test is frequently used in chemical laboratories to discriminate between
aldehydes and ketones during qualitative organic analysis. Additionally, reducing sugars and non-
reducing sugars are distinguished using this test.

Classification of Unknown: the classification of the unknown is starch.

ANSWERS TO QUESTIONS

1. Why do all sugars give a positive result for Molisch’s test?

ANSWER: The Molisch test yields a positive result for all carbohydrates (monosaccharides,
disaccharides, and polysaccharides). Based on the formation of an aldehyde from the dehydration of the
carbohydrate by sulfuric acid, which then condenses with two molecules of -naphthol to produce a purple
ring at the interface.

2. What compounds other than -naphthol can be used as a reagent for the test?

ANSWER: Other than napthol, concentrated hydrochloric acid can also be used. The results will still be
the same.

3. Why is it important to use freshly prepared Fehling’s reagent?

ANSWER: The Fehling's solution decomposes over time and is no longer useful for the tests, so they are
prepared as needed and not in advance!

4. How are the conditions for Barfoed’s test different from those for Benedict’s test?

ANSWER: The only differences between Barfoed's reagent and Benedict's reagent are that the pH is
lower (about 4.5) and the heating period is only two minutes. Barfoed's test would establish whether the
sample is a monosaccharide or disaccharide, and Benedict's test would indicate whether it is a reducing
sugar.

5. Account for the black precipitate in Nylander’s test.

ANSWER: Under alkaline conditions, glucose or fructose converts bismuth oxynitrate to bismuth. A
black precipitate of metallic bismuth is created when the Nylander's reagent, which is composed of

____________________________________________________________________________________________________ 5
Nursing Biochemistry
Laboratory Report

potassium sodium tartrate, potassium hydroxide, and bismuth nitrate, is introduced to a solution
containing reducing sugars.

CONCLUSION
The study of the reactions of various carbohydrates with reagents used to classify and identify these
compounds. Carbohydrates are classified according to the complexity of their molecules. The simplest
group, called monosaccharides, is the building blocks from which higher sugars, (dissacharides) are
formed.

Benedict's test can be used to identify simple carbohydrates that include a free ketone or aldehyde
functional group. A brick-red precipitate that forms as a result of Benedict's reagent's interactions with
reducing sugars is a sign that the test is successful. Sugars that are being reduced have a free aldehyde or
ketone group that has a reducing property. Negative result shows that there are no reducing sugars,
aldoses, or -hydroxy ketoses in the test sample.

Barfoed's test would establish whether the sample is a monosaccharide or disaccharide, and Benedict's
test would indicate whether it is a reducing sugar. A black precipitate of metallic bismuth is created when
the Nylander's reagent, which is composed of potassium sodium tartrate, potassium hydroxide, and
bismuth nitrate, is introduced to a solution containing reducing sugars.

REFERENCES
Biosciencenotes. (n.d). Picric acid test for the detection of reducing sugars. Retrieved from
https://www.biosciencenotes.com/picric-acid-test-for-the-detection-of-reducing-sugars/

Byjus. (n.d.). Benedict’s test. Retrieved from https://byjus.com/chemistry/benedicts-test/

Byjus. (2022). Fehlings test. Retrieved from https://microbenotes.com/fehlings-test/

Byjus. (n.d.). Molischs test. Retrieved from https://byjus.com/chemistry/molischs-test/

Micro benotes. (2022). Barfoeds test. Retrieved from https://microbenotes.com/barfoeds-test/

Micro benotes. (2022). Tollen’s test. Retrieved from https://microbenotes.com/tollens-test/

____________________________________________________________________________________________________ 6