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Iodine Test for Starch Explanation

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
Benedict Test for Reducing Sugar Explanation

What is Benedict's Test for Reducing Sugars?

Benedict's Test for non-reducing Sugars is a test which determines the presence of non-reducing
sugars in a test solution.

The principal reagent in Benedict's Test for Reducing Sugars is Benedict's Solution which contains

 copper(II) sulphate
 sodium carbonate
 sodium citrate

What are reducing sugars?

Sugars are classified as reducing or non-reducing based on their ability to act as a
reducing agent during the Benedict's Test. A reducing agent donates electrons during a
redox reaction and is itself oxidized.

The aldehyde functional group is the reducing agent in reducing sugars. Reducing
sugars have either an aldehyde functional group or have a ketone group - in an open
chain form - which can be converted into an aldehyde.
Reducing sugars are simple sugars and include all monosaccharides and most
disaccarides. Some examples of monosaccharides are glucose, fructose and
galactose.Examples of reducing disaccharides are lactose and maltose.

Note that the disaccharide sucrose is not a reducing sugar. In fact, sucrose is the most
common non-reducing sugar.
Is the Benedict's Test for reducing sugars qualitative or
quantitative?
The test may be qualitative, or it may be quantitative.

The qualitative test produces a colour change from blue to green to yellow to orange to brick
red. The qualitative test is also regarded as semi-quantitative as the colour obtained correlates to
the concentration of reducing sugars in the solution ( see observations below). This allows for a
rough estimation of the amount of reducing sugar present. The qualitative test is discussed here.

The quantitative test involves the use of potassium thicyanate and the production of copper
thiocyanate as white or pale green precipitate. This precipitate can then be titrated.

PROCEDURE

What is the procedure for the Benedict's Test for reducing

sugars?
A liquid food sample does not need prior preparation except dilution if viscous or concentrated.

For a solid sample prepare a test solution by crushing the food and adding a moderate amount of
distilled water. Decant the suspension to remove large particles. Use the decanted liquid as the test
solution.

 Add 2 cm3 of the sample solution to a test tube.

 Add an equal volume of Benedict's solution to the test tube and swirl or vortex the mixture.
 Leave the test tube in a boiling water bath for about 5 minutes, or until the colour of the
mixture does not change.
 Observe the colour changes during that time as well as the final colour.
 To prepare a control, repeat the steps above using 2 cm3 of distilled water instead of sample
solution

What are the expected observations for the Benedict's Test for
reducing sugars?

Observations Interpretations

No Colour Change (Blue) No reducing sugars present

Green Trace amounts of reducing sugars present

Yellow Low amounts of reducing sugars present

Orange Moderate amounts of reducing sugars present

Brick-red Large amounts of non-reducing sugars present

DISCUSSION
What is the principle of the Benedict's Test for reducing
sugars?
Reducing Sugars have an aldehyde functional group which can reduce soluble copper (II) ions - in copper
(II) sulphate - to insoluble copper (I) ions - in copper (i)oxide. The copper (I) oxide is seen as a precipitate.

State the role of copper sulphate in Benedict's Solution.

Reduced Species. The blue copper(II) ions from copper(II) sulphate are reduced to red copper(I) ions by
the aldehyde groups in the reducing sugars. This accounts for the colour changes observed.

The red copper(I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for
the precipitate formed.

As the concentration of reducing sugar increases, the nearer the final colour is to brick-red and the

State the role of sodium carbonate in Benedict's Solution.

Alkalinization. Sodium carbonate provides the alkaline conditions which are required for the redox
reaction above.

State the role of sodium citrate in Benedict's Solution.

Stabilization. Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to
copper(I) ions during storage.

Are there alternative tests to Benedict's Test for

Reducing Sugars?
The Fehling's Test for non-reducing sugar is an alternative to the Benedict's Test. However it is less popular
as it less sensitive and requires that the reagents - Fehling's solutions A and B - be kept separate until the
experiment is carried out.

Biuret Test for Protein Explanation

What is the Biuret Test for Proteins?

The Biuret Test is often used to determine the presence of peptide bonds in protein. As a student you will
most likely will be testing for the presence of protein in foods.

The Biuret test for proteins may also be extended to quantitatively

measure the concentration of total proteinusing spectrometric methods.

The Biuret reagent is the sole reagent in the Biuret Test for proteins. The
Biuret reagent contains

 Hydrated Copper sulphate

 Potassium hydroxide solution
 Potassium sodium tartrate

Are the alternative reagents for the Biuret Test for Proteins?
As an alternative to the Biuret Reagent, either of the following solutions may be used to yield
comparable results.
  Sodium Hydroxide and Copper Sulphate Solutions
 Fehling’s Solutions A and B

PROCEDURE
Three procedures are outlined below using different reagents.
For each of the procedures, a liquid sample must be prepared from solid foods as follows.

PREPARATION OF LIQUID SAMPLE

Crush the solid food, add a little de-ionized water and decant the liquid. This liquid should be used
for the food test. The quantity of food crushed and water used depends on the number of tests to be
conducted.

What is the procedure for the Biuret Test for proteins using
Biuret Reagent?
Add 2 cm3 of the liquid food sample* to a clean, dry test tube

 Add 2 cm3 of Biuret Reagent.

 Repeat steps the steps above with de-ionized water to prepare a negative control and with
albumin (egg white) to prepare a positive control.
 Shake well and allow the mixture to stand for 5 minutes
 Observe any color change.

What is the procedure for the Biuret Test for protein using
sodium hydroxide and copper sulphate solutions?
 Add 1 cm3 of sodium hydroxide solution (40% or bench solution) and 1% copper (II)
sulphate solution dropwise (one drop at a time) to the food sample

 Repeat the steps above with de-ionized water to prepare a negative control and with albumin
(egg white) to prepare a positive control.
 Shake well and allow the mixture to stand for 5 minutes
 Observe any color change.

Who or what is Biuret?

The biuret reagent is not named after z famous scientis but after a substance
called biuret (H2NC(O)NHC(O)NH). Biuret is the result of the condensation of 2
molecules of urea. The reagent is so named because the peptide bonds in biuret give a
positive result for the test.

What is the composition of the Biuret reagent?

Biuret Reagent contains

 Hydrated Copper sulphate. This provides the Cu (II) ions which form the chelate complex.
Cu (II) ions give the reagent its characteristic blue color.
 Potassium hydroxide solution does not participate in the reaction but provides the alkaline
medium.
  Potassium sodium tartrate (KNaC4H4O6·4H2O) – stabilizes the chelate complex.

What is the principle - or basis - of the Biuret Test for proteins?

The Biuret test is based on the ability of Cu (II) ions to form a violet-coloured chelate
complex with peptide bonds (-CONH- groups) in alkaline conditions.

Lone electron pairs from 4 nitrogen atoms in the peptide bond coordinate a copper (II) ion to
form the chelate complex.
The chelate complex absorbs light at 540 nm so appears violet. Hence a color change from blue to
violet indicates that proteins are present.

The greater the concentration of peptide bonds, the greater the color intensity. If the concentration
of peptide bonds is low – such as when short-chain peptides are present - the color change is
from blue to pink.

Why would amino acids give a negative result in the Biuret

Test for Proteins?
As 2 peptide bonds are required for the formation of the chelate complex, single amino acids - no
peptide bonds present - and dipeptides - only 1 peptide bond present – give a negative result.

How can the Biuret Test be extended to quantitatively

measure the concentration of protein?
According to the Beer-Lambert Law, the absorption of the sample is directly proportional
to the concentration of the species – in this case peptide bonds. Hence absorption
spectroscopy using a spectrophotometer is a quantitative method which can be used
to determine the concentration of total protein, following the Biuret test.
Ethano EmulsionTest for Fats/Oils Explanation

Ethanol Emulsion Test for Fats and Oils

The Ethanol Emulsion Test is a food test which determines the presence of a broad group of naturally
occurring compounds known as lipids. Lipids consist of fats and oils. Other lipid tests include the Grease
Spot Test and the Sudan Stain Test. The Grease spot test is performed on fats - lipids which are solid at
room temperature. Sudan stain colours lipids red, but is a less common bench reagent than ethanol. The
Ethanol Emulsion Test is the most common test amongst the three.

Procedure

Solid sample
1. Crush the food sample and place in a dry test tube.
2. Add ethanol to about 2 cm3 above the level of the sample and shake thoroughly.
3. Allow the solid to settle (about 3 min) to allow the lipid to be extracted.
4. Decant the ethanol into another test tube.
5. Add 2 cm3 of deionized water to the second test tube
6. Make observations.

Liquid sample
1. Add a few drops of the liquid food sample to a dry test tube.
2. Add 2 cm3 ethanol and shake it thoroughly
3. Add 2 cm3 of deionized water.
4. Make observations.

Observations & Interpretation

Observation Interpretation
Solution remains colourless. No emulsion is formed. Lipids are not present

A layer of cloudy white suspension forms at the top of

the solution. (Upon close inspection you can see the Lipids are present
tiny globules of fat suspended in the solution. This an
emulsion. Foods with high lipid content have a ‘higher’
layer than foods with less).

Principle of the Ethanol Emulsion Test

The solubilities of lipids and ethanol are exploited in this test.

Lipids are non-polar organic compounds. Hence they are soluble in organic solvents such as ethanol
(alcohol), but insoluble in water.

Ethanol is an organic substance and so dissolves other organic substances; it is frequently used as an
organic solvent.

However ethanol is also miscible in water due to the presence of the hydroxyl (-OH) functional groups and
the shortness of its chain (2C). The hydroxyl group participates in hydrogen bonding with water.

The hydrophobic interaction of the carbon in the short chain with water is not great and is overcome by
the hydrogen bonding.

Ethanol extracts the lipid from the crushed solid sample. As ethanol is miscible with lipids no change is seen
upon its addition to the solid and liquid samples.

The lipid spontaneously comes out of solution when water is added and is dispersed as micelles (small
droplets) throughout the solution of ethanol and water.( This happens as hydrophobic portion of the lipid
molecules project inwards and excludes the aqueous environment; the hydrophilic portion (-COOH) group
faces the aqueous environement.)

A layer is formed at the top as lipids are less dense than water. The droplets diffract light, appearing cloudy
white.

CONCLUSIVE TEST

A positive test shows conclusively that lipids are present - and not the other major biological molecules.