In this article we will discuss about the tests for detection of carbohydrates in plants with the help of experiments.
Carbohydrates are the condensation products of polyhydroxyaldehydes or polyhydroxyketones and their derivatives. They contain carbon, hydrogen and oxygen, in which the ratio of hydrogen and oxygen is generally 2: 1.
In the living world there are a large number of carbohydrate materials, which may be conveniently classified as:
(a) Monosaccharides (contain only one unit of simple sugar, each unit contains 3-7 carbon atoms) such as glucose, fructose, galactose, etc.
(b) Oligosaccharides (contains 2-4 units of simple sugar i.e. di-, tri-, and tetra saccharides, respectively), such as sucrose, lactose, maltose, etc.
(c) Polysaccharides (contains many monosaccharide units) such as starch, glycogen, dextrin, etc.
Usually most of the carbohydrates are crystalline, water soluble, neutral compounds, but a number of them are acidic in nature such as pectic acid, gluconic acid, alginic acid etc. Due to the presence of free aldehyde and ketonic moiety, the monosaccharides are highly reactive showing a number of chemical and physical properties, while disaccharides and polysaccharides are lacking in such properties.
Requirements for Detecting Carbohydrates in Plants:
(a) Preparation of Sample Solution:
Carbohydrates are usually soluble in water, of course polysaccharides are soluble more readily in hot water. Thus aqueous solutions of purified samples are prepared for the qualitative detection of sugars present in the supplied samples. From plant tissue, aqueous extract (in cold water/in hot water) provides most of the sugars available in the specimen.
(b) Reagents:
1. α-naphthol solution (1 gm. in 100 ml of 70% alcohol)
2. Concentrated sulphuric acid
3. Fehling’s reagent: Equal vol. of Fehling’s A & B
(Fehling’s solution A: CuSO4. 5H2O — 35 gm. in 500 ml dist. water
Fehling’s solution B: NaOH — 50 gm. and Na-K-tartarate (Rochelle salt) — 173 gm. in 500 ml dist. water)
4. Benedict’s qualitative reagent: Dissolve sodium citrate 86.5 gms and sodium carbonate —50 gms in 350 ml dist. water and then dissolve copper sulphate 8.65 gm. in 50 ml dist. water. Finally mix together and dilute up to 500 ml
5. CuSO4.5H2O: 2.5 gm. in 100 ml dist. water
6. NaOH solution: 5 gm. in 100 ml dist. water
7. Barfoed’s reagent: Dissolve 13.3 gms of copper acetate in 200 ml of 1% acetic acid solution
8. Tannic acid: (20 gm. in 100 ml dist. water)
9. Seliwanoff’s reagent: 0.1 gm. of resorcinol in 100 ml of 50% (v/v) HC1
10. AgNO3 solution: 5 gm. in 100 ml dist. water
11. Phenyl hydrazine hydrochloride
12. Sodium acetate
13. Glacial acetic acid
14. Sodium carbonate (Na2CO3)
15. Concentrated HC1
16. Phloroglucinol solution: 0.5 gm. in 100 ml dist. water
17. Bial’s reagent: (5 gm. orcinol in 100 ml dist. water, Cone. HCl —30 ml and Ferric Chloride — trace)
18. Aniline acetate solution: 5 gm. in 100 ml dist. water
19. Ammonium molybdate solution: 5 gm. in 100 ml dist. water
20. Cobalt chloride solution: 5 gm. in 100 ml dist. water
21. Ammonium hydroxide (dilute 10%)
22. Iodine solution — 0.5 gm. I2 in 100 ml of 1% KI solution
Tests for Detection of Carbohydrates in Plants:
The following tests are performed to detect the nature of carbohydrate available in the supplied sample:
I. General Test:
Experiment:
Molisch’s test:
A few drops of sample solution is taken in a clean and dry test tube. Then a few drop of α-naphthol solution is added to it and shaken carefully. Finally conc. H2SO4 is poured into the test tube slowly.
Observation:
A violet ring or purple violet coloration is formed at the junction of the two liquids due to the formation of furfural compounds.
Inference:
This is a general test for all carbohydrates. Furfural compounds are easily formed from all carbohydrates, when treated with conc. H2SO4or HC1 and it can be given purple-violet coloured compounds in the presence of α-naphthol.
II. Test for Reducing Sugars:
All monosaccharides viz. trioses, pentose’s and hexoses and a few disaccharides, viz. maltose, have free aldehyde or ketone moiety for reactions. These are called reducing sugars.
Experiment:
1. Fehling’s Test:
In a clean and dry test tube 1 ml of Fehling’s solution A and 1 ml of Fehling’s solution B are taken, mixed thoroughly and then boiled for a minute. It is observed that the solution remains unchanged. Finally an equal volume of sample solution is added to it and boiled in a water-bath for 5-10 minutes.
Observation:
The solution initially turns yellow and then brick red. Precipitation of Cu2O takes place in the tube containing reaction mixture.
Inference:
Presence of reducing sugar in the supplied sample.
Cuprous hydroxide (CuOH) is formed due to the reaction of reducing sugar with Fehling’s reagent and then CuOH is reduced to cuprous oxide (Cu2O) on heating.
Reducing sugar + Fehling’s reagent → CuOH + acidic sugar
2. Benedict’ Test:
Experiment:
To a clean and dry test tube containing about 3 ml of sample solution, 2-3 ml of Benedict’s qualitative reagent is added and then boiled for 5 minutes.
Observation:
The solution turns green, yellow and finally Red, depending on the amount of reducing sugar present in the sample.
Inference:
Presence of reducing sugar in the supplied sample.
3. Barfoed’s Test:
Experiment:
About 1 ml of sample solution is added to about 3 ml of barfoed’s reagent is a test tube and then boiled for 1-2 minutes add finally cooled.
Observation:
Red precipitate of Cu2O is formed at the bottom of the test tube.
Inference:
Presence of reducing sugar in the supplied sample (Monosaccharides are more sensitive than reducing disaccharides).
Trommer’s Test:
Experiment:
About 1 ml of 2.5% CuSO4 solution and 2 ml of 5% NaOH solution are added to 3-5 ml of carbohydrate solution. The mixture is boiled in a water bath for 3-5 minutes.
Observation:
A blue precipitate of Cupric hydroxide is formed which dissolves to give a blue solution. A yellow or red precipatate of Cu2O is formed in presence of reducing sugar.
Inference:
Presence of reducing sugar in the supplied sample.
5. Moore’s Test:
Experiment:
To about 2 ml of sample solution, an equal volume of 5% NaOH solution is added and then boiled for 2-5 minutes in a water bath.
Observation:
The solution turns yellow and then reddish-brown due to the formation of a condensation product of the sugar.
Inference:
The sample contains reducing sugar, particularly glucose.
6. Precipitation Tests with Metallic Silver:
Experiment:
To about 5 ml of sample solution a drop of dilute NH4OH is added and then an excess of AgN03 solution is poured on the sample.
Observation:
A white precipitate is formed.
Inference:
Presence of reducing sugar in the supplied sample.
Experiment:
Then the mixture is kept in a boiling water bath for 5-10 minutes.
Observation:
A silver mirror is formed due to the precipitation of silver in presence of reducing sugar.
III. Tests for Non-reducing Sugars:
Except monosaccharides and a few disaccharides, all other carbohydrates are of non-reducing type. They do not respond to the tests for reducing sugar prior to their hydrolysis.
Hydrolysis of non-reducing sugars:
In a 100 ml capacity conical flask, about 20 ml of sugar solution is taken and 2.5 ml NH4SO4 is added. The mixture is boiled in a water-bath for 10-15 minutes, then cooled and neutralized by slow addition of Na2CO3 salt, till the effervescence continues. Finally, the mixture is filtered and tests for detection of sugars (reducing type derived from non-reducing type on acid hydrolysis) are made in the usual way as shown previously.
The complete hydrolysis is noticed by the positiveness of the reducing sugar tests. The neutralization of hydrolysed samples can also be done by addition of N/10 NaOH and N/10 H2SO4 solution using 1% phenolphthalein indicator.
The following tests are performed with hydrolysed samples:
Experiment:
1. Fehling’s Test
Observation:
Positive responses
Inference:
Presence of reducing sugar in the hydrolysed samples.
Experiment:
2. Benedict’s Test
Observation:
Positive reaction.
Inference:
Presence of reducing sugar in the hydrolysed samples.
IV. Detection of Types of Monosaccharides:
(A) Tests for Pentose Sugars
1. Bial’s Orcinol Test:
Experiment:
A little Bial agent is heated to boiling and then a few drops of sugar solution are added.
Observation:
The solution turns green or purple in colour.
Inference:
Presence of pentose sugar in the supplied sample.
2. Aniline/Acetate Test
Experiment:
A filter paper strip soaked with aniline acetate solution is placed over the vapour of boiling pentose sugar for 1-2 mins.
Observation:
The paper turns white to Pink
Inference:
Presence of pentose sugar in the supplied sample.
(B) Tests for Hexose Sugars:
1. Seliwanoffs Test:
Experiment:
To about 1 ml of sample solution, 3 ml of Seliwanoffs reagent is added and then boiled in a water bath for 1-2 mins.
Observation:
A red colouration is obtained
Inference:
Presence of Ketohexose (fructose) in the supplied sample.
2. Ammonium Molybdate Test:
Experiment:
To about 2 ml of sample solution, 2-3 ml of ammonium molybdate solution is added and then the mixture is heated to boiling in a water bath for a while.
Observation:
The solution turns bluish green.
Inference:
Presence of Ketohexose (fructose) in the supplied sample.
3. Tollen’s Phloroglucinol Test:
Experiment:
10 ml of conc. HCI and 8 ml of 0.5% phloroglucinol solution is mixed together. Then about 4.5 ml of this mixture is added to 1-2 ml of sample carbohydrate solution and then boiled in a water bath.
Observation:
The solution turns yellow to red
Inference:
Presence of galactose in the sample
4. Cobalt Chloride Test:
Experiment:
To about 3 ml of sample solution, 1-2 ml of cobalt chloride solution is added and then the mixture is heated to boiling. Finally, after cooling, a few drops of NaOH solution are added.
Observation:
The solution turns greenish blue, or The solution turns purplish violet, or the upper part of the solution greenish blue and lower part purplish.
Inference:
Presence of glucose in the sample or Presence of Fructose or Presence of a mixture of glucose and fructose (as in hydrolysed samples of sucrose)
(C) Tests for Non-reducing Disaccharides like Cane-sugar (Sucrose):
Experiment:
1. Fehling’s test without hydrolysis of the sample.
2. Hydrolysis followed by tests for reducing sugars.
3. Cobalt chloride test: (after hydrolysis of the sample).
Observation:
No reaction.
Positive reaction.
Both greenish blue and purplish- violet ppt. is formed
Inference:
Absence of reducing sugar.
Presence of reducing sugar on hydrolysis of the sample. Presence of a mixture of glucose and fructose in the sample.
(D) Tests for Non-reducing Polysaccharides 1. Iodine Test
Experiment:
1. Iodine Test:
To about 2-3 ml of sample solution a few drops of dilute iodine solution are added.
Observation:
The solution turns blue immediately due to the formation of starch-iodine complex, or the solution turns reddish-brown. The colour may disappear on boiling the solution and reappear again on cooling. The colour may disappear on addition of a few drops of 10% NaOH, but reappear on neutralizing with acids.
Inference:
glycogen-type polysaccharose’s in the sample.
Presence of polysaccharide (e.g. starch or glycogen) in the sample.
2. Tannic Acid Test:
Experiment:
In a clean and dry test tube about 4-5 ml of sample solution is taken. Then a pinch of salt mixture (phenyl hydrazine hydrochloride and sodium acetate in the ratio of 2 : 3) is added, followed by addition of a few drops of glacial acetic acid. Then the mixture is shaken thoroughly and placed in a boiling water bath for about 20-30 mins. Finally the mixture is cooled.
Observation:
Yellowish, osazone crystals of definite shape separate out as the tube cools.
The osazone crystals are examined under microscope :
(a) Long needle-shaped crystals arranged in sheaves (Glucosazone)
(b) Elongated strips and plates (Galactosazone)
(c) Stellate clusters of broad- bladed crystals (Maltosazone)
(d) Close tufts of short fine crystals (Lactcsazone)
Inference:
Reducing sugars mostly produced osazone crystals.
Presence of glucose, fructose and mannose in the sample.
Presence of galactose in the sample.
Presence of maltose in the sample. Presence of lactose in the sample.
Glucose + Phenyl hydrazine hydrochloride → Glucose-phenyl → Glucosazone hydrazine complex (Yellowish crystals)
