IDENTIFICATION OF THE UNKNOWN CARBOHYDRATE

I. Introduction

Carbohydrates are aldehyde or ketone derivatives of complex alcohols. The

aldehyde derivatives are spoken of as aldoses while ketone derivatives are spoken of
as ketoses (Hawk, Oser, and Summerson, 1916). Moreover, carbohydrates are
composed of simple sugars referred to as monosaccharides.

Glucose, also known as dextrose or grape sugar, is a monosaccharide, and is the

most important simple sugar in human metabolism. It is classified as a hexose due to its
six-carbon skeleton and as an aldose, because of the presence of an aldehyde group
on carbon 1. According to Michael McMilllin, the aldehyde group condenses with a
hydroxyl group in order to exists as a hemiacetal ring structure (Walker, Hall, and Hurst
1990, Chapter 141). In terms of chemical properties, glucose gives all typical reactions
including furfural formation, enolization, oxidation, and osazone formation.
Hydroxymethyfurfural is produced when glucose undergoes dehydration with the use of
concentrated sulfuric acid. Carbohydrates are further classified into reducing or non-
reducing sugars. Due to the free aldehyde group of glucose, it is a reducing sugar. In
alkaline medium, reducing sugars undergo tautomerization to yield their enediol forms,
which are strongly reducing in nature (Gupta, 2017). It also undergoes oxidation by
wherein certain metal ions become reduced. The reduction of the metal ion indicates
that the sugar is a reducing carbohydrate. These reducing sugars also have the
capability to form osazones, in case of glucose, it is termed as glucosazone.

Aside from these, glucose also plays an important role in the medical field. It is used
in monitoring the blood sugar level of people to avoid various diseases such as
diabetes, hypertension, heart failure and stroke. Moreover, glucose can also be used to
detect cancer. By using Positron Emission Tomography (PET), physicians are able to
detect cancerous tissue through the amount of radioactive glucose that are injected and
used up by the body (University of Bristol, n.d.).
 II. Materials and Methods

For test for reducing sugar, the group used Benedict’s Test, which contains cupric sulfate, sodium citrate, and sodium
carbonate giving off red precipitate as a positive result. This test was used as an indication if reducing sugar is
present in the solution. The unknown solution with Benedict’s reagent was subjected to boiling water bath for a few
minutes resulting a brick-red precipitate indicating a positive result. The students also followed Tollen’s test to classify
if it is aldehyde or ketone. Tollen’s reagent is consist of silver nitrate and ammonia. The test gave a silver mirror or
elemental silver on the inner surface of test tube which shows a positive result. This means the solution was either an
aldehyde or terminal α-hydroxy ketone. To further test the unknown solution, Ferricyanide Test which is composed of
potassium ferricyanide and sodium hydroxide was conducted. This test is test for reducing sugar and the visible
result shows a clear solution indicating reducing sugars or aldose is present in the solution. The unknown solution
was subjected to heat with Saturated Potassium Ferricyanide solution for ten minutes, the solution gave a clear
solution which was a positive result. The final test of the students for reducing sugar is Phenylhydrazine Test.
Phenylhydrazine test is composed of 2,4-dinitrophenylhydrazine, concentrated sulfuric acid and ethanol. It is a test
that shows a visible result of yellow to orange precipitation to reducing sugars, moreover it is also important to take
note the time of formation of osazone/crystals to identify the type of carbohydrate. The unknown solution became a
solution with yellow precipitate which further proves that the unknown solution was indeed a reducing sugar.
III. Results and Discussion
In determining the identity of the unknown, the group performed three kinds of tests:
general test, tests for aldoses and ketoses, and tests for reducing sugars.

GENERAL TEST
Test for Theoretical Results Actual Results Indication
Anthrone Test Presence of Blue- green Blue-green Positive;
Carbohydrate colored solution colored Carbohydrate is
s solution present
TEST FOR ALDOSES/KETOSES
Seliwanoff’s Test Ketoses Cherry red solution Yellow Aldose sugar is
solution present
Bial’s Test Pentoses Bluish green Clear solution Negative;
complex
Tollen’s Presence of Brick-red Yellow Negative;
Phloroglucinol Test Pentoses precipitate solution Sugar is a
hexose
TEST FOR REDUCING SUGARS
Benedict’s Test Presence of Brick-red Red Positive;
Reducing precipitate precipitate reducing sugar
sugar is present
Tollen’s Test Presence of Silver mirror Silver mirror Aldose is
aldoses present
Ferricyanide Test Aldoses and Clear colorless Clear Aldose/Ketose
Ketoses solution colorless is present
 solution
Phenylhydrazine Presence of Orange precipitate Orange Reducing sugar
test (Osazone reducing precipitate is present
Formation) sugars
Table 1. Visible results of the unknown

The table above presents the test used in

order to determine the identity of the
unknown. For the general test, the group
used the Anthrone test. The Anthrone test is
a general test for the presence of
Figure 1. Anthrone Test principle carbohydrates resulting in a blue-green
colored complex.

In figure 1, it shows that upon the addition of concentrated sulfuric acid, carbohydrates
undergo dehydration to form furfural which is shown as a bluish-green colored solution.
Comparing the theoretical and actual results, both gave the same color which indicates
that it is positive, and carbohydrate is present.

It was followed by the tests for aldoses and ketoses. In this part, the students
used the Seliwanoff’s test to distinguish whether the unknown sugar is a ketose or an
aldose sugar. This test gives off a cherry red colored complex for ketoses and a yellow
to faint pink color with aldoses. As a result, the solution turned out to be yellow which is
same with the theoretical results. This means that it is indeed positive, and an aldose
sugar is present.

The group then used to Bial’s test to determine if the unknown sample is a
pentose or hexose. A positive result of this test gives off a bluish green complex which
indicates the presence of pentoses. Hexoses, on the other hand, gives colors other than
blue green which makes it negative to this test. In the case of the unknown sample, it
gave a negative result shown by the clear colorless solution. Thus, the sample is a
hexose sugar.

To make sure that the sample is a hexose, the next test used was the Tollen’s
phloroglucinol test. This test is used to detect the presence of pentoses giving off brick-
red precipitate as its result. After adding the reagent to the sample, the solution turned
out to be yellow, thus indicating that it is negative, and the sugar is a hexose.

Knowing that the sample is a hexose sugar, the students then proceeded to the
test for reducing sugars. The first test used was the Benedict’s test. This test is used to
detect reducing sugars. The presence of the alkaline sodium carbonate converts the
sugar into a strong reducing agent called enediols. Upon heating the sugar mixed with
reagent, the copper (II) ions of the reagent are reduced to copper (I) ions and exists as
an insoluble cuprous oxide which is the brick-red precipitate at the bottom of the test
tube. In the case of the unknown, it resulted also to the formation of brick-red precipitate
which shows that the sample is a reducing sugar.

Another test used was the Tollen’s silver mirror test. This test is used for the
presence of aldoses by giving off a shining mirror on the walls of the test tube. The
formation of silver mirror is due to the reduction of Ag+ ions to elemental silver. In
relation to the sample, it also produced a silver mirror after the addition of the reagent.
This proves that the sample is indeed an aldose. This is also supported by the
Seliwanoff’s test wherein it gave a yellow color which is a result for aldoses.
The group made use of the Ferricyanide test, another test for reducing sugars, which
gives a positive result to both ketoses and aldoses. Ketoses in this test appears to have
a distinct color of orange while aldoses show a yellow color which later decolorizes
making the solution colorless. This is because the ferricyanide ions of the reagent under
alkaline condition becomes ferrocyanide ions which makes it colorless. Comparing
again the theoretical and actual results, it turns out that both are same having a clear
colorless solution. This shows that it is positive, and an aldose sugar is present.

The students also used to phenylhydrazine test to make sure that the unknown
sample is a reducing sugar. This test forms osazone which appears to be yellow or
orange as the sugar reacts with the reagent. Having all these tests shows that the
unknown sample is glucose.
IV. Conclusion and Recommendation

V. References

Gupta P. (2017). Qualitative Test for Carbohydrates. Retrieved from

https://www.ijccm.org/eReader/chapter/9789386056900/ch3
Walker, H., Hall, W. and Hurst, J., 1990. Clinical Methods. Boston: Butterworths.
Hawk, P., Oser, B., Summerson, W. (1916). Practical Physiological Chemistry. P.
Blakinston’s Son & Co. https://doi.org/10.1021/j150455a015