Analysis of Carbohydrate
2.I. Qualitative Analysis
3.1. Molisch’s Test
Molisch's test (named after Austrian botanist Hans Molisch) is a sensitive chemical test for the presence of carbohydrates, based on the dehydration of the carbohydrate by sulfuric acid or hydrochloric acid to produce an aldehyde, which condenses with two molecules of phenol (usually α-naphthol, though other phenols (e.g. resorcinol, thymol) also give colored products), resulting in a red- or purple-colored compound.
4.1. Molisch’s Test
All carbohydrates – monosaccharides, disaccharides, and polysaccharides – should give a positive reaction, and nucleic acids and glycoproteins also give a positive reaction, as all these compounds are eventually hydrolyzed to monosaccharides by strong mineral acids.
Pentoses are then dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural.
Either of these aldehydes, if present, will condense with two molecules of naphthol to form a purple-colored product, as illustrated below by the example of glucose
5.1. Molisch’s Test
6.2. Seliwanoff’s Test
Seliwanoff’s test is a chemical test which distinguishes between aldose and ketose sugars. Ketoses are distinguished from aldoses via their ketone/aldehyde functionality.
If the sugar contains a ketone group, it is a ketose. If a sugar contains an aldehyde group, it is an aldose. When added to a solution containing ketoses, a red color is formed rapidly indicating a positive test. When added to a solution containing aldoses, a slower forming light pink is observed instead.
7.2. Seliwanoff’s Test
The reagents consist of resorcinol and concentrated hydrochloric acid (or H2SO4 & CH3COOH):
The acid hydrolysis of polysaccharide and oligosaccharide ketoses yields simpler sugars followed by furfural.
The dehydrated ketose then reacts with two equivalents of resorcinol in a series of condensation reactions to produce a molecule with a deep cherry red color.
Aldoses may react slightly to produce a faint pink color.
Fructose and sucrose are two common sugars which give a positive test. Sucrose gives a positive test as it is a disaccharide consisting of fructose and glucose.
8.2. Seliwanoff’s Test
9.3. Anthrone’s Test
Anthrone’s test is a tricyclic sweet-smelling ketone. It is utilized for a well-known cellulose measure and in the colorimetric determination of carbohydrates. The anthrones are utilized as a part of drug store as diuretic. They improve the movement of the colon and are in charge of less water reabsorption.
Starches are dried out with concentrated H2SO4 to frame “Furfural”, which gathers with anthrone to shape a green shading complex which can be measured by utilizing colorimetrically at 620 nm.
Anthrone reacts with dextrins, monosaccharide, disaccharides, polysaccharides, starch, gums and glycosides. If this happens, the yield of shading is where is to frame sugar to starch.
10.3. Anthrone’s Test
blue green-colored dye
11.4. Benedict’s Test
Benedict's reagent (often told as Benedict's Qualitative Solution or Benedict's Solution) is a chemical reagent named after an American chemist, Stanley Rossiter Benedict.
It is a complex mixture of sodium carbonate, sodium citrate and copper(II) sulfate pentahydrate. Benedict's reagent is a chemical reagent commonly used to detect the presence of reducing sugars, however other reducing substances also give a positive reaction. This includes all monosaccharides and many disaccharides, including lactose and maltose. Such tests that use this reagent are called the Benedict's tests.
12.4. Benedict’s Test
Generally, Benedict's test detects the presence of aldehydes, alpha-hydroxy-ketones, also by hemiacetal, including those that occur in certain ketoses. Thus, although the ketose fructose is not strictly a reducing sugar, it is an alpha-hydroxy-ketone, and gives a positive test because it is converted to the aldoses glucose and mannose by the base in the reagent.
A positive test with Benedict's reagent is shown by a colour change from clear blue to a brick-red precipitate.
13.4. Benedict’s Test
The principle of Benedict's test is that when reducing sugars are heated in the presence of an alkali they get converted to powerful reducing species known as enediols.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
14.4. Benedict’s Test
The color of the obtained precipitate gives an idea about the quantity of sugar present in the solution, hence the test is semi-quantitative. A greenish precipitate indicates about 1 g% concentration; yellow precipitate indicates 1.5 g% concentration; orange indicates 2.5 g% and red indicates 3,5 g% or higher concentration.
15.5. Barfoed’s Test
Barfoed's test is a chemical test used for detecting the presence of monosaccharides. It is based on the reduction of copper(II) acetate to copper(I) oxide (Cu2O), which forms a brick-red precipitate.
(Disaccharides may also react, but the reaction is much slower.) The aldehyde group of the monosaccharide which normally forms a cyclic hemiacetal is oxidized to the carboxylate.
RCHO + 2Cu2+ + 2H2O → RCOOH + Cu2O↓ + 4H+
16.6. Fehlings’s Test
In this test the presence of aldehydes but not ketones is detected by reduction of the deep blue solution of copper(II) to a red precipitate of insoluble copper oxide (Cu2O). The test is commonly used for reducing sugars but is known to be NOT specific for aldehydes. For example, fructose gives a positive test with Fehling's solution as does acetoin.
Two solutions are required:Fehling's "A" uses 7 g CuSO4.5H2O dissolved in distilled water containing 2 drops of dilute sulfuric acid.Fehling's "B" uses 35 g of potassium tartrate and 12 g of NaOH in 100 ml of distilled water.
17.7. Osazone’s Test
The famous German chemist Emil Fischer developed and used the reaction to identify sugars whose stereochemistry differed by only one chiral carbon. Glucosazone and fructosazone are identical. Osazones formation test involves the reaction of a reducing sugar (free carbonyl group) with excess of phenylhydrazine when kept at boiling temperature.
All reducing sugars form osazones. Therefore, sucrose, for example, does not form osazone crystals because it is a non reducing sugar as it has no free carbonyl group. The reaction involves formation of a pair of phenylhydrazone functionalities, concomitant with the oxidation of the hydroxymethylgroup in alpha carbon (carbon atom adjacent to the carbonyl center).
18.7. Osazone’s Test
The reaction can be used to identify monosaccharides. It involves two reactions. Firstly glucose with phenylhydrazine gives glucosephenylhydrazone by elimination of a water molecule from the functional group. The next step involves reaction of one equivalent of glucosephenylhydrazone with two equivalents of phenylhydrazine (excess).
First phenylhydrazine is involved in oxidizing the alpha carbon to a carbonyl group, and the second phenylhydrazine involves in removal of one water molecule with the new-formed carbonyl group of that oxidized carbon and forming the similar carbon nitrogen bond. The alpha carbon is attacked here because its more reactive than the others.
19.7. Osazone’s Test
Osazones are highly coloured and crystalline compounds and can be easily detected. Each sugar has a characteristic crystal form of osazones.
Maltose forms petal-shaped/sun flower-shaped crystals
Lactose forms powder puff-shaped crystals
Galactose forms rhombic-plate shaped crystals
Glucose, fructose and mannose form broomstick or needle-shaped crystals.
20.7. Osazone’s Test
21.8. Tollens’s Test
Tollens' reagent is a chemical reagent used to determine the presence of an aldehyde, aromatic aldehyde and alpha-hydroxy ketone functional groups.
The reagent consists of a solution of silver nitrate and ammonia. It was named after its discoverer, the German chemist Bernhard Tollens.
A positive test with Tollens' reagent is indicated by the precipitation of elemental silver, often producing a characteristic "silver mirror" on the inner surface of the reaction vessel.
22.8. Tollens’s Test
This reagent is not commercially available due to its short shelf life, so it must be freshly prepared in the laboratory. One common preparation involves two steps. First a few drops of dilute sodium hydroxide are added to some aqueous silver nitrate. The OH−ions convert the silver aquo complex form into silver oxide, Ag2O, which precipitate from the solution as a brown solid:
2 AgNO3 + 2 NaOH → Ag2O (s) + 2 NaNO3 + H2O
In the next step, sufficient aqueous ammonia is added to dissolve the brown silver(I) oxide. The resulting solution contains the [Ag(NH3)2]+ complexes in the mixture, which is the main component of Tollens' reagent. Sodium hydroxide is reformed:
Ag2O (s) + 4 NH3 + 2 NaNO3 + H2O → 2 [Ag(NH3)2]NO3 + 2 NaOH
23.8. Tollens’s Test
24.9. Iodine Test
The iodine test is used to test for the presence of starch. When treated with KI solution-iodine dissolved in an aqueous solution of potassium iodide-the triiodide anion (I3−) complexes with starch, producing an intense blue/purple colour. To put it simply, when the iodine solution comes into contact with starch, it turns dark blue/purple. Otherwise, it will remain brown in color.
25.9. Iodine Test
26.9. Iodine Test
Amylose, a linier chain component of starch, gives a deep blue color.
Amylopectin, a branched chain component of starch, gives a purple color.
Glycogen, gives a reddish brown color.
Dextrins, Amylo, Eryhthro and Achrodextrins, form as intermediates during hydrolysis of starch gives violet, red, and no color with iodine
Left to right : Iodine solution, starch solution, starch solution with iodine