1. Qualitative analytical tests for Carbohydrates

2. Objectives General information about Carbohydrates.
To differentiate between Carbohydrates .
Qualitative analysis of Carbohydrates .

3. Qualitative Analytical Tests
Is concerned with determining the identity of a substance.
Enables us to detect the presence of things which may be beyond the reach of our senses.

5. A functional groups: are the regions of a molecule that gives it particular properties. A single molecule can have more than one functional group as part of its structure. The active part in sugars is aldehyde or ketone group. The presence of aldehyde or ketone groups and hydroxyl groups causes that the sugars have typical reactions for aldehydes/ketones and alcohols.

6. Sugar nomenclature
For sugars with more than one chiral center, D or L refers to the asymmetric C farthest from the aldehyde or keto group.
Most naturally occurring sugars are D isomers.

7. a (OH below the ring).
b (OH above the ring).

8. Monosaccharides
Aldoses (e.g., glucose) have an aldehyde group at one end (C1)
Ketoses (e.g., fructose) have a keton group, usually at (C2).
The functional group is carbonyl group ( aldehyde or keto )

9. Polysaccharide Amylose is a glucose polymer with α(14) linkages.
Amylopectin is a glucose polymer with mainly α(14) linkages, but it also has branches formed by α(16) linkages.

10. Glycogen Glycogen has more α(16) branches.
The highly branched structure permits rapid glucose release from glycogen stores, e.g., in muscle during exercise.

11. Cellulose: a major constituent of plant cell walls, consists of long linear chains of glucose with b(1- 4) linkages.

12. Classification upon reducing end
Reducing Sugars
Oxidation: loss of electrons.
Reduction: gain of electrons.
A reducing sugar: is a carbohydrate possessing either a free aldehyde or free ketone functional group as part of its molecular structure.
Sugars that can be oxidized by mild oxidizing agents and the oxidizing agent is reduced in the reaction.
Can be oxidized to acid, reduces another compound.
All monosaccharides.
Maltose, Lactose.

13. All of the monosaccharides and most of the disaccharides can be oxidized.
Sugars exist in solution as an equilibrium mixture of open-chain and closed-ring (or cyclic) structures.
All monosaccharides have an open and closed form structure, but oligosaccharides have only closed structure.
When the cyclic structure opens, the aldehyde group is available for oxidation.

14. Opened and closed form of monosaccarides

15. Lactose is a reducing sugar, why?
One of the carbonyl groups are free
a carbonyl group is a functional group composed of a carbon atom double-bonded to an oxygen atom (C=O).

16. Reducing Sugars 2. Non Is not oxidized by mild oxidizing agents.
Sucrose
All polysaccharides

17. Sucrose is not a reducing sugar, why?
Sucrose is not a reducing sugar because it cannot revert to the open-chain form that would provide the aldehyde group needed to reduce the cupric ion. (the carbonyl groups are busy in the two side).

18. Aldose(glucose) carboxylate Ketose(fructose) hydroxyl carboxylate
Common oxidizing agents used to test for the presence of a reducing sugar are :
Benedict's solution.
Fehling's solution.
Tollen’s reagent.
picric acid solution.
An oxidizing agent (also called an oxidizer or oxidant) is referred to as: a chemical compound that readily transfers oxygen atoms or
A substance that gains electrons in a redox chemical reaction.(reduction)
Aldose(glucose) carboxylate
Ketose(fructose) hydroxyl carboxylate
oxidation
oxidation

20. Tollen’s test
Positive result (silver mirror)

21. 1. Benedict's Test Objective: Principle:
Detect the presence of reducing sugars (i.e. it distinguish between reducing and non-reducing sugars)
Principle:
Sodium citrate, sodium carbonate , CuSO4. 5H2O solution.
Cupric ions, which in an alkaline environment, oxidize the aldehyde group to a carboxylic acid. Cupric ions are reduced to cuprous oxide, which forms a red precipitate
RCHO + 2Cu2+ + 4OH RCOOH+Cu2O+2H2O
The color of the precipitate depending on the concentration of the reducing sugar.
Reducing sugars are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide.

22. Procedure
Place 1mL of the following 1% carbohydrate solutions in separate, labeled test tubes: glucose, fructose, sucrose, lactose, maltose, and starch.
To each tube, add 1 ml of Benedict's reagent and heat the tubes in a boiling water bath for 5 minutes.
Remove the tubes from water bath. Note and record the results.
In the presence of a reducing sugar a precipitate which may be Red, Yellow will form.
-ve
+ve

23. 2. Barfoed's Test Objective: Principle:
Used to distinguish between mono- & di-saccharides
Principle:
Barfoed's reagent reacts with mono-saccharides to produce cuprous oxide at a faster rate than disaccharides:
RCHO  +  2Cu2+  +  2H2O   RCOOH  +  Cu2O  +  4H+
Reducing monosaccharides are oxidized by the copper ion in solution to form a carboxylic acid and a reddish precipitate of copper (I) oxide within three minutes. Reducing disaccharides undergo the same reaction, but do so at a slower rate.

24. Procedure A red precipitate will form if the test is positive.
Place 1 mL of the following 1% carbohydrate solutions in separate, labeled test tubes: glucose, fructose, sucrose, lactose, and maltose.
To each tube, add 1 ml of Barfoed's reagent, and heat in a boiling water bath for 10 minutes.
Remove the tubes from water bath. Note and record your observations.
A red precipitate will form if the test is positive.

25. 3. Bial's (orcinol) test for pentoses
Objective:
for the detection of pentoses(i.e. it distinguish between pentoses and hexoses).
Principle
Bial's reagent (0.1 % orcinol in concentrated HCl containing 0.1 % FeCl3.6H2O).
The action of concentrated acids causes the dehydration of sugars.
Bial’s test is used to distinguish between pentoses and hexoses. They react with Bial’s reagent and converted to furfural. Orcinol and furfural condense in the presence of ferric ion to form a colored product. Appearance of a blue green color or precipitate indicates the presence of pentoses and formation of muddy brown precipitate shows the presence of hexoses.

26. Procedure
Add about 1 ml of 1% xylose, glucose, fructose, maltose,, and arabinose solution to their respective labeled test tubes.
Add 1.5 ml of Bial's reagent to each tube and mix well.
Carefully heat each tube directly over the burner flame, until the mixture just begins to boil. Stop heating when the mixture begins to boil.
A blue-green color indicates a positive result. Prolonged heating of some hexoses yields hydroxymethyl furfural which also reacts with orcinol to give colored complexes.
-ve
-ve
+ve

27. Seliwanoff's (Resorcinol) Test
Objective:
used for detection of Ketoses
Principle
Seliwanoff’s test is used to distinguish between hexoses with a ketone group and hexoses that are aldehydes.
With ketoses, a deep red color is formed rapidly.
Aldoses give a light pink color that takes a longer time to develop.
Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as sucrose) form a cherry-red product.
Other sugars (e.g. aldose) may produce yellow to faint pink colors.

28. Seliwanoff's reagent (0.5 % resorcinol in 3N HCl).
When conc. HCl is added. ketoses undergo dehydration to yield furfural derivatives (5-hydroxymethylfurfural) more rapidly than aldoses.
5-hydroxymethylfurfural reacts with resorcinol present in the test reagent to produce a deep red color product within two minutes.
Aldohexoses reacts so more slowly to form the same product.

29. Procedure
Add about 3 ml of Seliwanoff's reagent to each labeled test tube.
Add 1 drop of the respective sugar solution to the appropriate test tubes, and mix well.
Place all the test tubes in the boiling water bath at the same time and heat for 3 min after the water begins to boil again. Record your observations.
A positive result is indicated by the formation of a red color with or without the separation of a brown-red precipitate.
-ve
+ve

30. 5. Picric Acid Test Objective: To detect reducing sugars. Principle
Picric acid (2,4,6-trinitrophenol) or TNP reacts with reducing sugars to give a red colored picramic acid C6H2.OH.NH2(NO2)2.

31. Procedure
Into a test tube add 1 ml of maltose solution, into the second tube, 1ml of sucrose solution.
Add into each tube 1 ml of a saturated solution of picric acid, and then add into each tube 0.5 ml of sodium hydroxide solution.
Heat both samples in a boiling water bath 5 min.
In the presence of reducing sugars, the solution stains red, a sodium salt of picric acid is formed.
5 MIN

32. The End