Download QUALITATIVE TESTS TO IDENTIFY FUNCTIONAL GROUPS ... and more Slides Biochemistry in PDF only on Docsity!
BZCYL-136 Physiology and Biochemistry: Laboratory
EXERCISE 7
QUALITATIVE TESTS TO
IDENTIFY FUNCTIONAL
GROUPS OF CARBOHYDRATES
(MONOSACCHARIDES) IN A
GIVEN SOLUTION: GLUCOSE
AND FRUCTOSE
Structure
7.1 Introduction
Objectives
7.2 Materials Required
7.3 Principles
7.4 Procedure
7.5 Results
7.6 Precautions
7.7 Terminal Questions
7.1 INTRODUCTION
In Exercises 1 to 6, you have performed Physiology Exercises. From this
Exercise onwards you will perform Biochemistry ones.
In the present Exercise, you will do a qualitative analysis of carbohydrates
especially monosaccharides (Glucose and Fructose). These tests are specific
to the functional groups present in that particular sugar molecule. As we all
know that carbohydrates are polyhydroxy aldehydes or ketones that occur in
nature as monosaccharides, disaccharides, oligosaccharides and
polysaccharides (Refer to Unit 9 of BZYCT-135). Due to the presence of free
hydroxyl group at anomeric carbon atom, they exhibit reducing property.
Hence, we call them reducing sugars. However, few carbohydrates do not
show reducing property for example: starch and glycogen. Various tests are
used for identification of carbohydrates. These tests are widely used in clinical
biochemistry for the diagnosis of diseases like diabetes and glycosuria. These
sugars undergo oxidation and react with phenylhydrazine to form osazones.
The specific shape of osazone crystals gives final confirmation. It is important
to know that in this Exercise we are focusing on qualitative analysis i.e., to
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides) in given Solutions: Glucose and Fructose
know which type of carbohydrate is present in the sample but not on the
quantitative analysis that means how much carbohydrate is present. Apart
from learning how to do various tests, weâll be discussing the principles and
preparation of reagents used for identification of glucose and fructose in a
given test sample.
For a better understanding of this Exercise, it is advised to go through the
physical and chemical properties of major biomolecules discussed in Unit 9 of
this course.
Objectives
After performing this exercise, you should be able to:
 enlist various tests used for identification of carbohydrates;
 explain the principle behind the specific test;
 distinguish between identification and confirmation tests; and
 identify specific monosaccharide in the test solution.
7.2 MATERIALS REQUIRED
i) Glassware: 10 mL boiling test tubes, pipettes (1-10 mL), dropper, glass
rod, microscope, glass slides and watch glass.
ii) Minor Equipment & other requirements: Water bath, light microscope,
test tube holder, spatula and blotting paper.
iii) Chemicals required: Conc. hydrochloric acid, Sodium hydroxide (10 N),
Conc. Sulphuric acid, Iodine solution, Phenylhydrazine hydrochloride,
Sodium acetate, Glacial acetic acid and distilled water.
Preparation of Reagents
Carbohydrate (sugar) solution [1% (w/v) in distilled water]: Prepare
individual sugar solutions of glucose and fructose respectively.
Molischâs Reagent [α-naphthol reagent (5% w/v in ethyl alcohol)]: Dissolve 5
grams of α-naphthol crystals in 100 mL ethyl alcohol.
Benedictâs Qualitative Reagent: Dissolve 17.3 grams of sodium citrate and
10 grams of sodium carbonate in 50 mL of distilled water and heat the solution
in a hot water bath, with constant stirring. Separately, prepare cupric sulphate
solution by dissolving 1.73 grams of this salt in 20 mL of distilled water. Mix
the cupric sulphate solution with sodium citrate-carbonate solution and make
up the volume to 100 mL.
Barfoedâs Reagent: Dissolve, 13.3 grams of cupric acetate in 80 mL of
distilled water. Add 2 mL of glacial acetic acid and make up the volume to 100
mL with distilled water.
Seliwanoffâs Reagent (0.05% (w/v): Dissolve 0.05 grams of resorcinol in 100
mL of 4 N hydrochloric acid.
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides) in given Solutions: Glucose and Fructose
Fig. 7.2: Reaction showing the principle working behind Benedictâs test.
Fig. 7.3: Schematic representation of colourâs developed during Benedictâs test and respective percentage of glucose.
The same principle is applicable for Barfoedâs test. However, in Barfoedâs test,
the reduction of cupric ions is under mild acidic condition and the test is more
rapid for monosaccharides than disaccharides.
(iii) Barfoedâs test: This test is useful for the identification of disaccharides
(Fig.7.4).
Fig. 7.4: Reaction showing the principle working behind Barfoedâs test.
(iv) Seliwanoffâs test: This test is useful for the identification of keto sugar.
Note: The reducing ability of carbohydrates is due to the presence of a free hydroxyl group at anomeric carbon atom (glucose). However, in certain sugars (sucrose) this hydroxyl group is involved in glycosidic bond formation and in some anomeric hydroxyl group of sugar blocked by either alkylation (α- or ÎČ- methyl glycosides). Hence, they do not respond to reducing tests. Interestingly, maltose and lactose being disaccharides possess a free anomeric hydroxyl group enabling it positive to reducing tests.
BZCYL-136 Physiology and Biochemistry: Laboratory
Principle
Keto sugars (eg. Fructose) when exposed to acid medium undergo
dehydration forming hydroxymethyl furfural more rapidly than aldohexoses.
This furfural condenses with resorcinol (m-dihydroxy benzene) to produce a
deep pink colour molecule (Fig. 7.5). This test is helpful to distinguish between
aldoses and ketoses.
Fig. 7.5: Reaction showing the principle working behind Seliwanoffâs test.
(v) Osazone test: This is the final and confirmatory test for qualitative
analysis of carbohydrates
Principle
Reducing sugars upon reacting with Phenylhydrazine produce osazones,
which are the characteristic derivatives of carbohydrates (Fig. 7.6). These
osazone derivatives have definite crystalline shape (Fig. 7.7). These crystals
made it easy to confirm the type of carbohydrate.
Fig. 7.6: Reaction showing the principle working behind formation of Osazone crystals.
7.4 PROCEDURE
We have explained the procedure under three phases. Phase-I focuses on
identification of the sample as mono or disaccharide. Phase-II is helpful in
identifying the sample as Aldose or Ketose sugar. Finally Phase-III is
confirmatory using osazone crystals. Learners need to note their observations
in the tabular format (Table 7.1) while performing this Exercise in practical
session.
Sugars lacking a free anomeric hydroxyl group (non-reducing sugars) do not respond to this test. Whereas glucose, fructose and mannose produce a similar type of osazone, i.e., glucosazone. An osazone crystal differs from the other with respect to time of crystallization, crystal shape and melting point.
BZCYL-136 Physiology and Biochemistry: Laboratory
- Benedictâs Test : Add few drops of sugar solution to 2 mL of Benedictâs reagent taken in a test tube, and place the tube in boiling water for 5 to 10 minutes.
Appearance of green/yellow/orange/ red color (the development of color depends on the concentration of sugar present in the solution)
Confirms the presence of reducing sugar
- Barfoedâs Test : To 2 mL of the reagent taken in a test tube, add few drops of the sugar solution and heat the contents in a boiling water bath.
Appearance of red precipitate at the bottom of the tube within 5 minutes
Presence of reducing monosaccharide
Note: If the time taken for the formation of the precipitate is more, it is suggestive of a reducing disaccharide.
- Seliwanoffâs test: Add few drops of sugar solution to 2 mL of the Seliwanoffâs reagent taken in a test tube, and place the tube in a boiling water bath for 5 minutes.
Appearance of pink or cherry red color
Presence of keto- sugar
- Osazone test: Take 3 mL of the sugar solution in a test tube, add 0.5 g of phenylhydrazine agent, add 0.1 g of sodium acetate and few drops of acetic acid. The contents are mixed well and placed in a boiling water bath for 15 minutes. Cool the solution to room temperature and observe the shape of the crystals formed, under a light microscope.
Microscopic examination of the osazones reveals the following shapes of crystals (Refer Fig. 7.7):
i) needle shaped crystals arranged singly or in groups (Feathery).
a) Crystals formed after 10 minutes
b) Crystals formed within 5- minutes
ii) Long fine needles.
iii) Sunflower shaped crystals.
i)
a) glucose is confirmed
b) fructose is confirmed
ii) Xylose is confirmed
iii) Maltose is confirmed
Exercise 7 Qualitative Tests to Identify Functional Groups of Carbohydrates (Monosaccharides) in given Solutions: Glucose and Fructose iv) Puff shaped crystals.
v) Broad glass piece shaped crystals formed within 30 minutes
vi) White colored irregular shaped crystals are formed after 45 minutes
iv) Lactose is confirmed
v) Presence of galactose
vi) Presence of Mannose
Fig. 7.7: Osazone Crystals.
7.5 RESULTS
The given test sample shows positive response to the following tests âŠâŠâŠ...,
âŠâŠâŠ.., and the osazone crystals observed are of âŠâŠâŠ.. shape and hence
it is confirmed that the given carbohydrate is âŠâŠâŠâŠâŠâŠ...
Note: It is a better practice to cross check the inference of all tests and
compare with the results obtained in osazone test before finalising your
results.
7.6 PRECAUTIONS
1. Ensure adequate distance from burner while boiling the reagents.
2. Always add reagents with the help of pipette or dropper.
3. Use separate pipettes or dropper to add reagents (avoid contamination).
4. Use clean and dry test tubes for individual test.
5. Avoid contact of microscope lens with osazone crystals.
