Experiment No.

9
ANALYSIS OF RNA FROM YEAST

Experiment No. 9
GENERAL TESTS FOR CARBOHYDRATES AND HYDROLYSIS OF STARCH

Molisch’s Test

Molisch’s test is a general test for all carbohydrates. In this test, carbohydrates when reacted
with conc. H2SO4 get dehydrated to form furfural and its derivatives. When monosaccharide
are treated with conc H2SO4 or conc HCl, -OH group of sugar are removed in the form of water
and furfural is formed from pentose sugar and hydroxymethyl furfural is formed from hexose
sugar. These products reacts with sulphonated α- naphthol to give a purple (violet red) colored
complex.

Positive Molisch’s test: purple color complex

Negative Molisch’s test: no purple color

Anthrone Test

http://www.onlinebiologynotes.com/anthrone-test-objective-principle-reagents-procedure-and-result/
Anthrone test is also another general test for all carbohydrates. In this test also, carbohydrate gets
dehydrated when react with conc. H2SO4 to form furfural. This furfural reacts with anthrone to give
bluish green colored complex.

 positive test: all carbohydrate give test positive

Iodine Test

Principle of Iodine test for carbohydrate:

Starch when reacted with I2 forms absorbed compound that gives blue color. On heating or on addition
of alkali like NaOH or KIH, color disappears. This reaction is only physically association where I2 traps in
the coiled structure of polysaccharide. On heating or on addition of alkali; the coiled structure becomes
linear and the I2 molecules are released and the color disappears. The test will be answered by fructose,
sucrose and other keto containing carbohydrates.

 Positive iodine test: dark blue color (starch)

 Negative iodine test: glucose, fructose and sucrose

http://brilliantbiologystudent.weebly.com/iodine-test-for-starch.html

What are the expected observations and interpretation of the iodine test for starch?

Observation Interpretation
No change (Iodine remains brown) Starch is not present

A blue-black colour develops Starch is present

Are there any test samples which will not test positive for the presence of starch?

Recall that starch is a storage molecule found only in plants. Only plants and plant-based foods should
test positive for the presence of starch. Any animal product testing positive for starch may be
contaminated or mixed with plant product.

Note that the iodine test for starch cannot be performed on very dark solids or liquids which do not
permit the observation of a colour change. Results of such an experiment are inconclusive.

DISCUSSION: Questions & Answers

Describe the structure of starch and state which structural feature is key to the colour change in the
iodine test for starch.

Starch is a polysaccharide consisting of glucose units joined together by glycosidic bonds.The chains
formed during the condensation reaction are either linear or highly branched molecules.

.
Linear - both straight and helical - molecules of starch are referred to as Amylose.

Whereas branched molecules of starch are called Amylopectin.

Natural starches - from plants - consist of a mixture of amylose (10 - 25%) and amylopectin (75-90%).The
the structure of the helical amylose is key to the Iodine-starch reaction. A helix is a coil or a spring.

The tri-iodide and penta-iodide ions formed are linear and slip inside the helix of the amylose (form of
starch).

Describe the composition of the iodine/potassium tri-iodide reagent in the iodine test for starch.

Iodine on its own (small non-polar molecule) is insoluble in water. Therefore Potassium triiodide
solution - Iodine dissolved in potassium iodide solution - is used as a reagent in the test.

To be more specific, potassium iodide dissociates, and then the Iodide ion reacts reversibly with the
Iodine to yield the the triiodide ion. A further reaction between a triiodide ion and an iodine molecule
yields the pentaiodide ion.

Since molecular iodine is always present in solution, the bench iodine solution appears brown; the
iodide and triiodide pentaiodide ions are colourless.

Explain the principle or the basis of the colour change in the Iodine Test for Starch.

.
The starch-iodide complex is formed as charge - recall electrons are charged particles - is transferred
between the starch and iodide ions - tri-iodide or pentaiodide.

The transfer of charge between the starch and the iodide ion changes the spacing between the energy
levels/ orbitals.

This change results in the starch-iodide complex absorbing light at a different wavelength - than any
other species aforementioned - resulting in an intense purple colour; Biologists call this colour blue-
black.

https://www.chemistryviews.org/details/education/10128441/Why_Does_Iodine_Turn_Starch_Blue.ht
ml

Starch is a carbohydrate found in plants. It consists of two different types of polysaccharides that are
made up of glucose units which are connected in two different ways. One is the linear amylose and the
other is the branched amylopectin (pictured below).
Amylose is the compound that is responsible for the blue color. Its chain forms a helix shape, and iodine
can be bound inside this helix (pictured below).

Charge-Transfer Complexes

The colors are caused by so-called charge transfer (CT) complexes. Molecular iodine (I2) is not easily
soluble in water, which is why potassium iodide is added. Together, they form polyiodide ions of the
type In–, for example, I3–, I5–, or I7–. The negatively charged iodide in these compounds acts as charge
donor, the neutral iodine as a charge acceptor. Electrons in such charge-transfer complexes are easy to
excite to a higher energy level by light. The light is absorbed in the process and its complementary color
is observed by the human eye.

In the case of the aqueous solution of polyiodides, the absorptions of the different species lead to an
overall brownish color. Once amylose is added, it forms another CT complex, Here, the amylose acts as a
charge donor and the polyiodide as an acceptor. This complex absorbs light of a different wavelength
than polyiodide, and the color turns dark blue.

Polyiodide Chains
The exact structure of the polyiodides inside the amyloid helix is not clear. The amylose-iodine complex
is amorphous (i.e., it does not form ordered crystals), which has made it difficult to determine its
structure. It has been proposed that the species inside the helix are repeated I3– or I5– units.

However, Ram Seshadri, Fred Wudl, and colleagues, University of California, Santa Barbara, USA, have
found evidence that infinite polyiodide chains Inx– are contained in the amylose-iodine complex [1]. The
team investigated a related system, a pyrroloperylene–iodine complex, to study its properties as an
organic electronic conductor. The material is crystalline, and therefore, the team was able to determine
its structure using X-ray crystallography. They found nearly linear polyiodide chains in-between stacks of
pyrroloperylene. It turned out that the material containing these chains absorbs light at very similar
wavelengths to the amylose-iodine complex, which supports the hypothesis that similar polymeric
chains form in the iodine test for starch.

Reference

 [1] Infinite Polyiodide Chains in the Pyrroloperylene-Iodine Complex: Insights into the Starch-
Iodine and Perylene-Iodine Complexes,
Sheri Madhu, Hayden A. Evans, Vicky V. T. Doan-Nguyen, John G. Labram, Guang Wu, Michael L.
Chabinyc, Ram Seshadri, Fred Wudl,
Angew. Chem. Int. Ed. 2016, 55, 8032–8035.
DOI: 10.1002/anie.201601585

Polysaccharides, such as amylase or amylopectin, are polymers of glucose molecules. Starch

can form an intense, brilliant, dark blue or violet colored complex with iodine. The straight chain
component of starch (or amylose) gives a blue color, while the branched component (or
amylopectin) yields a purple color. In the presence of iodine, amylose forms helixes, where the
iodine molecules assemble as long polyiodide chains. The helix– forming branches of
amylopectin are much shorter than those of amylose. Therefore, the polyiodide chains are also
much shorter in the amylopectin–iodine complex than in the amylose–iodine complex. The
result is a different color (purple). When starch is hydrolyzed and broken down to small
carbohydrate units, the iodine will not give a dark blue (or purple) color. The iodine test is used
in this experiment to indicate the completion of the hydrolysis.
https://msu.edu/course/lbs/145/luckie/Lab1.html

Iodine (iodine-potassium iodide, I2KI) staining distinguishes starch (a polysaccharide) from

monosaccharides, disaccharides, and other polysaccharides. The basis for this test is that starch is a
coiled polymer of glucose. Iodine interacts with these coiled molecules and becomes bluish black. Other
non-coiled carbohydrates do not react with iodine. Therefore, a bluish black color is a positive test for
starch, and a yellow-ish brown color (i.e., no color change) is a negative test for starch. Glycogen, the
common polysaccharide in animals, has a slight difference in structure and produces only an
intermediate color reaction. Test each of the known sugars for the presence of starch.

Acid Hydrolysis of Starch