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Lab - 1 Sugars Analysis

Lab sugar analysis

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SALMA BOUH
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26 views4 pages

Lab - 1 Sugars Analysis

Lab sugar analysis

Uploaded by

SALMA BOUH
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Download as DOCX, PDF, TXT or read online on Scribd
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Lab session 1: Mono-Di- and polysaccharides analysis

Objectives:
Carbohydrates are polyhydroxyaldehyde or ketone derivatives of
polyhydric alcohols with the general chemical formula Cn(H2O) n, and
these are largely distributed in animals and plants. Carbohydrates provide
energy through oxidation and supplies carbon for the synthesis of cell
components; they serve as a form of stored chemical energy.
Carbohydrates, along with lipids, proteins, nucleic acids, and other
compounds are known as biomolecules because they are closely
associated with living organisms (Table 1).

Table 1 : Classification of carbohydrates

The first attempts to separate sugar mixtures by thin-layer


chromatography (TLC) were made in 1961. Techniques concerning this
problem have continued to appear. Silica gel G, kieselguhr, gypsum,
cellulose powder as well as silica gel and/or kieselguhr impregnated with
boric acid, sodium tetraborate, bisulfite and acetatelhave been used on
chromatoplates for the separation of carbohydrates and many different
solvents were employed as developers. However thin-layer
chromatography of carbohydrates did not gain general acceptance in
scientific communities, however, it gives many insight about the
chromatographic behaviours and their chemistry. This lab session aims at
understanding the chemistry of carbohydrates via several assays as well
as it aims at analyzing the composition of unknown polysacchirides using
chemical hydrolysis and TLC.

Assay 1: Molisch’s Test (α-Naphthol Reaction)

Principle: Monosaccharide is treated with concentrated sulfuric acid


(H2SO4) or concentrated hydrochloric acid (HCl), -OH group of sugar are
removed in the form of water, and furfural is formed from pentose sugar,
and hydroxymethylfurfural is formed from hexose sugar. These products
react with sulfonated α-naphthol and give a purple (violet-red)-colored ring
at junction of two liquids.

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Pr. Jaouad ANISSI
Reagent Preparation: 5% solution of α-naphthol in alcohol.

Procedures: Add 2 drops of Molisch’s reagent to the 5 mL of sugar


solution in the test tube. Mix gently, and then add 2 mL H2SO4 (conc.) by
the side of the slanted test tube. The formation of violetred layer appears
at the junction. Acid goes down below the sugar solution as it is heavier.

Observations:

1. Purple (violet-red) color appears at the junction of two liquids.

2. A brown color due to charring must be ignored, and the test should be
repeated with a more dilute sugar solution.

3. Green color appears due to impurities or addition of excess Molisch’s


reagent.

Iodine Test

Principle: Starch forms adsorption complex with iodine and gives blue
color. The composition of red-brown or blue color is not defined.

Reagent: Iodine is dissolved in an aqueous solution of potassium iodide,


and 0.5 mL of iodine is diluted up to 5 mL using distilled water.

Procedure: Add 2 drops of iodine solution (0.05 N) to 2–3 mL of


polysaccharide solution (starch, dextrin, and glycogen).

Observation: Different colors appear for each of the polysaccharide


solution. In case of starch, dextrin, and glycogen, red-brown or deep blue
color appears, while blue color disappears on heating in case of starch and
color comeback on subsequent cooling. Color does not reappear in case of
dextrin and glycogen.

Questions:

Explain the reaction between iodine solution and polysaccharides

by giving the structures of related compounds.

Each polysaccharide tested gives different color results with the iodine
test. Explain the reason briefly.

Phenol/sulfuric acid assay

About 10 mg of samples dissolved in 100 µL of distilled water. 0.1 mL of


5% phenol added to the 100 µL, followed by 0.5ml of concentrated H2SO4.

Record the change in color.

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Pr. Jaouad ANISSI
Characterization of an unknown polysaccharides

You are provided with two different unknown polysaccharides along with
three standard polysaccharides; glycogen, carboxy methyl cellulose
(CMC), pectin. Also, a set of monosaccharides standards are also provided
to be used for TLC analysis.

Procedure:

For hydrolysis of the polysaccharides, weight 10 mg of each unknown and


put in a clean glass tube of 1 mL. Add 200 µL of H2O and adjust H2SO4 to
a final concentration of 2 M. adjust the volume to 500 µL. bring the
mixture to 120 °C and >960 mBar pressure for 1 h.

NB: The standards will be prepared by your instructor.

For TLC 1 % sugar solutions in 75% aqueous ethanol were employed. An


aliquot containing a suitable quantity of the pure sugars or their mixtures
was applied to the plates with a micro-pipette or a capillary tube. The
plates were developed by one of the following mobile phase:

(I) n-Butanol-acetone-water (4 : 5 : I, v/v),

(2) n-Butanol-pyridine-water (8 : 4 : 3, v/v).

TLC revelation of sugars:

1- Urea / hydrochloric acid

Spray with a solution of 5g urea in 20ml 2M hydrochloric acid, with 100ml


ethanol added and heat to 100°C.

Results: Ketoses and oligosaccharides containing ketoses turn blue.

2- Orcinol

Take 180 mg orcinol (e.g. Sigma), dissolve in 5 ml of water, add 75 ml


EtOH and cool on ice. Then add slowly under constant stirring 10 ml
H2SO4 conc. Keep the reagent in the dark (wrapped in foil) at 4 °C. When
you spray the TLC plate with this reagent, carbohydrates will be detected
as purple spots / bands upon heating in an oven).

3- Sulfuric acid and ethanol

10% sulfuric acid in methanol or ethanol.

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Pr. Jaouad ANISSI
Questions:

Present your analysis as follow:

1- Methods and Results

2- Discussion

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Pr. Jaouad ANISSI

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