Objective:

To characterize carbohydrates present in an unknown solution on the basis of various chemical assays.

Theory:

Carbohydrates are polyhydroxy aldehydes and ketones or substances that hydrolyze to yield polyhydroxy aldehydes and ketones.
Aldehydes (CHO) and ketones ( = CO) constitute the major groups in carbohydrates.

Carbohydrates

are

mainly

divided

into

monosaccharides,

disaccharides

and

polysaccharides.

The

commonly

occurring

monosaccharides includes glucose, fructose, galactose, ribose, etc. The two monosaccharides combine together to form
disaccharides which include sucrose, lactose and maltose. Starch and cellulose fall into the category of polysaccharides, which
consist of many monosaccharide residues.
Molischs Test:
This is a common test for all carbohydrates larger than tetroses. The

test is on the basis that pentoses and hexoses are

dehydrated by conc. Sulphuric acid to form furfural or hydroxymethylfurfural, respectively. These products condense with naphthol to form purple condensation product.

Furfural

-Naphthol

Fehlings Test:

This forms the reduction test of carbohydrates. Fehlings solution contains blue alkaline cupric hydroxide solution, heated with
reducing sugars gets reduced to yellow or red cuprous oxide and is precipitated. Hence, formation of the yellow or brownish-red
colored precipitate helps in the detection of reducing sugars in the test solution.

Benedicts Test:

As in Fehlings test, free aldehyde or keto group in the reducing sugars reduce cupric hydroxide in alkaline medium to red colored
cuprous oxide. Depending on the concentration of sugars, yellow to green color is developed . All monosaccharides are reducing
sugars as they all have a free reactive carbonyl group. Some disaccharides, like maltose, have exposed carbonyl groups and are
also reducing sugars, but less reactive than monosaccharides

Barfoeds Test:

Barfoed's test is used to detect the presence of monosaccharide (reducing) sugars in solution. Barfoed's reagent, a mixture of
ethanoic (acetic) acid and copper(II) acetate, is combined with the test solution and boiled. A red copper(II) oxide precipitate is
formed will indicates the presence of reducing sugar. The reaction will be negative in the presence of disaccharide sugars because

they are weaker reducing agents. This test is specific for monosaccharides . Due to the weakly acidic nature of Barfoed's reagent, it
is reduced only by monosaccharides.

Seliwanoffs Test:

It is a color reaction specific for ketoses. When conce: HCl is added. ketoses undergo dehydration to yield furfural derivatives more
rapidly than aldoses. These derivatives form complexes with resorcinol to yield deep red color. The test reagent causes the
dehydration of ketohexoses to form 5-hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol present in the test
reagent to produce a red product within two minutes (reaction not shown). Aldohexoses reacts so more slowly to form the same
product.

Bials Test:

Bials test is used to distinguish between pentoses and hexoses. They react with Bials reagent and are converted to furfural.
Orcinol and furfural condense in the presence of ferric ion to form a colored product. Appearance of green colour or precipitate
indicates the presence of pentoses and formation of muddy brown precipitate shows the presence of hexoses.

Iodine Test:

This test is used for the detection of starch in the solution. The blue-black colour is due to the formation of starch-iodine complex.
Starch contain polymer of -amylose and amylopectin which forms a complex with iodine to give the blue black colour.

Osazone Test:

The ketoses and aldoses react with phenylhydrazine to produce a phenylhydrazone which further reacts with another two molecules
of phenylhydrazine to yield osazone. Needle-shaped yellow osazone crystals are produced by glucose, fructose and mannose,
whereas lactosazone produces mushroom shaped crystals. Crystals of different shapes will be shown by different osazones. Flowershaped crystals are produced by maltose.

Objective:
To quantify the amount of amino acids by using ninhydrin reaction.

Theory:
Amino acids are known as the building blocks of all proteins. There are 20 different amino acids commonly found in proteins.
Amino acids are comprised of a carboxyl group and an amino group attached to the same carbon atom (the carbon).
They vary in size, structure, electric charge and solubility in water because of the variation in their side chains ( R groups).
Detection, quantification and identification of amino acids in any sample constitute important steps in the study of proteins.
The general structure of an amino acid is shown below:

Alpha amino acids react with Ninhydrin involved in the development of color which is explained by the following five steps.
1.

alpha-amino

acid

Ninhydrin

--->

Reduced

ninhydrin

+Alpha

amino

acid

+H 2O

This is an oxidative deamination reaction that elicit two hydrogen from the alpha amino acid to produce an alpha imino acid. Also
the

ninhydrin

reduced

and
2.

loses

an

alpha-amino

oxygen
acid

atom
+

with
H2O

the

formation
--->

of

water

alpha-keto

molecule.

acid

+NH3

The rapid hydrolysis of NH group in the alpha imino acid will cause the formation of an alpha- keto acid with an ammonia
molecule.

This

alpha-keto
3.

acid

further

alpha-keto

involved
acid

in
+

the
NH 3

decarboxylation
--->

reaction

aldehyde

of
+

step.
CO2

Under a heated condition to form an aldehyde that has one less carbon atom than the original amino acid. A carbon
dioxide molecule is produced along with aldehyde. These first three steps produce the reduced ninhydrin and ammonia that

are required for the production of color .The overall reaction for the above reactions is simply explained in Reaction (4) as
follows:
4.

alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BLUE) + 3H2O

In summary, ninhydrin, which is originally yellow, reacts with amino acid and turns deep

purple. It is this purple color that is

detected in this method. Ninhydrin will react with a free alpha-amino group, NH2-C-COOH. This group is present in all amino
acids, proteins or peptides. Whereas, the decarboxylation reaction will proceed for a free amino acid, it will not happen for
peptides and proteins. Theoretically only amino acids produce color with ninhydrin reagent. However, one should always check
out the possible interference from peptides and proteins by performing blank tests especially when such solutions are
readily available. For example, one can simply add the ninhydrin reagent to a solution of only proteins and see if there
is any color development. There is no excuse for failing to perform such a vital test when the sample mixture contains both
proteins and amino acids. There are also reports that chemical compounds other than amino acids also respond positively to
this reaction.
The ninhydrin reaction, one of the most important method of detecting amino acids, both technically and historically, has been
conventionally used to detect their microgram amounts. When amino acids with a free alpha amino groups are treated with an
excess of ninhydrin, they yield a purple colored product. Under appropriate conditions, the color intensity produced is proportional
to the amino acid concentration.

The primary amino groups react with ninhydrin to form the purple colour dye now called Ruhemann's purple (RP) was discovered by
Siegfried Ruhemann in 1910. Iminoacids like

proline, the guanidino group of arginine, the amide groups of asparagine, the indole

ring of tryptophan, the sulfhydryl group of cysteine, amino groups of cytosine and guanine, and cyanide ions also react with
ninhydrin to form various chromophores that can be analyzed.
The overall reaction can be written as follows:

Primary amines also react with ninhydrin, but do not liberate of CO2. [ Caution : Ninhydrin is a very reactive oxidizing agent,
so should be handled with care].

Several other convenient reagents are available which can react with the alpha amino group to form colored or fluorescent
derivatives. These include fluorescamine, dansyl chloride, dabsyl chloride, etc., used in the detection of trace amounts of amino
acids at the nanogram level.

In the quantitative estimation of amino acid using Ninhydrin reagent, the absorbance of the Ruhemann's purple formed by the
reaction at 570nm is measured. For imino acids, the absorbtion happens at 440nm. The principle behind the colorimetric estimation
is given below:

Principle of the Colorimeter:

Measurement of Absorbance (A):
Unknown compounds may be identified by their characteristic absorption spectra in the ultraviolet, visible or infrared regions.
Enzyme-catalysed reactions frequently can be followed by measuring spectrophotometrically the appearance of a product or
disappearance of a substrate. A spectrophotometer /colorimeter is an instrument for measuring the absorbance of a solution by
measuring the amount of light of a given wavelength that is transmitted by a sample.

Light and Spectrum profile:

Light can be categorized according to its wavelength. Figure 1 shows the relationship between the wavelength of light and the
common types of electromagnetic radiation. Light in the short wavelengths of 200 to 400 nm is referred to as ultraviolet (UV). Light
in the longer wavelengths of 700 to 900 nm is referred to as near infrared (near IR).

Visible light falls between the wavelengths of 400 and 700 nm. All the colors visible to human eye falls under this wavelength range.
Any solution that contains a compound that absorbs light in the visible region will appear colored to the eye. The solution is colored
because specific wavelengths of light are absorbed as they pass through the solution. Then, the only light that the eye will perceive
are the wavelengths of light that are transmitted (not absorbed).

Beer-Lambert Law:
The Beer-Lambert Law states that the amount of light absorbed is proportional to the number of molecules of absorbing substance
in the light path, ie., absorption is proportional both to the concentration of the sample solution and to the length of the light path
through

the

solution.

This

relationship

can

be

expressed

as

follows:

Absorbance, A= x c x l
l

length

of

the

c = concentration of the sample (in Moles/liter),

light

path

through the solution (in cm) and

= molar extinction coefficient
To

determine

the

absolute

concentration of a pure substance,

a standard curve is constructed
from the known

concentrations

and using that standard curve, the

absorbance
unknown

reading

of

concentration

the
was

determined. The determination of

unknown concentration from the
standard curve is done by drawing
a line parallel to the X- axis from
the point on the Y axis that corresponds to the absorbance of the unknown. This line will be made to intersect the standard curve
drawn, and is extended vertically such that it meets the X-axis and the concentration of unknown is read from the

X-axis. A typical

standard curve is depicted in the figure.

Objective:
To separate and identify the amino acids in a mixture by thin layer chromatography.

Chromatography:
Chromatography is by far the most useful general group of techniques available for the separation of closely related compounds in
a mixture. Here the separation is effected by differences in the equilibrium distribution of the components between two immiscible
phases, viz., the stationary and the mobile phases. These differences in the equilibrium distribution are a result of nature and
degree of interaction of the components with these two phases. The stationary phase is a porous medium like silica or alumina,
through which the sample mixture percolates under the influence of a moving solvent (the mobile phase). There are a number of
interactions between the sample and the stationary phase and these have been well exploited to effect the separation of
compounds.

Thin layer chromatography [TLC]:

Thin layer chromatographic (TLC) technique readily provides qualitative information and with careful attention to details, it is
possible to obtain quantitative data. Thin layer chromatography is a technique used to separate and identify compounds of interest.
A TLC plate is made up of a thin layer of silica adhered to glass or aluminum for support. The silica gel acts as the stationary phase
and the solvent mixture acts as the mobile phase. In the ideal solvent system the compounds of interest are soluble to different
degrees. Separation results from the partition equilibrium of the components in the mixture.
In the simplest form of the technique, a narrow zone or spot of the sample mixture to be separated is applied near one end of the
TLC plate and allowed to dry. The strip or plate is then placed with this end dipping in to the solvent mixture, taking care that the
sample spot/zone is not immersed in the solvent. As the solvent moves towards the other end of the strip, the test mixture
separates into various components. This is called as the development of TLC plates. The separation depends on several factors; (a)
solubility: the more soluble a compound is in a solvent, the faster it will move up the plate. (b) attractions between the compound
and the silica, the more the compound interacts with silica, the lesser it moves, (c) size of the compound, the larger the compound
the

slower

it

moves

up

the

plate.

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable location
reagent. An important characteristic used in thin layer chromatography is Rf value.

The plate is removed after an optimal development time and dried and the spots/zones are detected using a suitable location
reagent. An important characteristic used in thin layer chromatography is Rf value.

Chromatographic Separation of Amino acids:

The present experiment employs the technique of thin layer chromatography to separate the amino acids in a given mixture.

All 20 of the common amino acids [standard amino acids] are a-amino acids. They have a carboxyl group and an amino group
bonded to the same carbon atom (the - carbon). They differ from each other in their side chains, or R groups, which vary in
structure, size, and electric charge. The interaction of the amino acids with the stationary phase like silica varies depending on their
'R' groups. The amino acid that interacts strongly with silica will be carried by the solvent to a small distance, whereas the one with
less interaction will be moved further. By running controls [known compounds ] alongside, it is possible to identify the components
of the mixture.

Since amino acids are colourless compounds, ninhydrin is used for detecting them. To identify this, after development, the TLC plate
is sprayed with ninhydrin reagent and dried in an oven, at 105C for about 5 minutes. Ninhydrin reacts with - amino acids that
results in purple coloured spots [ due to the formation of the complex - Rheuman's purple] [http://amrita.vlab.co.in/?
sub=3&brch=63&sim=156&cnt=1]. Rf values can be calculated and compared with the reference values to identify the amino acids.
[The Rf value for each known compound should remain the same provided the development of plate is done with the same solvent,
type of TLC plates, method of spotting and in exactly the same conditions].

1. Introduction: The purpose of this experiment is to isolate a protein (casein) from a natural
source (milk) and to demonstrate that the product obtained is indeed a protein by performing a
number of tests on your product.
This experiment is in not in your laboratory textbook. Review the structure and chemistry of
proteins and amino acids in your lecture textbook.
Background
Protein Solubility: Proteins are macromolecules which are soluble (suspended?) in water.
Proteins are least soluble in water at their isoelectric points and are more soluble at higher or
lower pH's. The solubilitiy at pH's different than the isoelectric point appears to be due to the
presence of an excess of cationic groups or anionic groups on the surface of the protein. If the

protein is, for instance, negatively charged at a pH larger than the isoelectric point, when two
proteins bump into each other, the net negative charge repels them and they do not aggregate as
one would expect from large molecules. However, at the protein's isoelectric point there is no net
charge. When a negative end of one protein bumps into a positive end of another protein,
electrostatic attraction causes the two proteins to stick together. Other proteins run into the
"dimer" and join the group. Eventually, enough proteins aggregate together that the protein
precipitates. In this experiment, casein( pI = 4.6) will be precipitated from milk (pH = ca. 7) by
the addition of glacial acetic acid.
Protein Testing:
Biuret Test: The Biuret Test is a general test for proteins. When a protein reacts with
copper(II) sulfate (blue), the positive test is the formation of a violet colored complex.

The Biuret Test works for any compound containing two or more of the following groups.

Ninhydrin Test: The Ninhydrin Test is a test for amino acids and proteins with a free
-NH2 group. When such an -NH2 group reacts with ninhydrin, a purple-blue complex is
formed.

Xanthproteic Test: Phenyl rings containing an activating group can be nitrated

producing a yellow product.

The production of a yellow colored product upon the addition of nitric acid is a test for
the presence of tyrosine or tryptophan in a protein. The addition of strong base will
deepen the color to orange. The yellow stains on the skin caused by nitric acid are the
result of the xanthoproteic reaction.
Heavy Metal Ions Test: Heavy metal ions precipitate proteins from their solutions by
cross-linking free amino groups and carboxylate groups.

Ions commonly used for testing for the presence of proteins include Zn2+, Fe3+, Cu2+, Sb3+,
Ag1+, Cd2+, and Pb2+.
Among the metal ions, Hg2+, Cd2+, and Pb2+ have very high toxicity. They cause serious
damage to proteins (especially enzymes) by denaturing them. Victims who have
swallowed Hg2+ or Pb2+ ions are often treated with an antidote of a food rich in protein.
The protein can combine with the mercury and lead ions and minimize absorption of
these ions. Milk and raw egg white are used most often. The precipitated protein
complexes are then immediately removed from the stomach by an emetic.
2. Experimental Procedures
A. Isolation of Casein:
a. To a 250-mL Erlenmeyer flask, add 50.0 g of milk (never pour liquids above a
balance!!!) and heat (hot plate!) the flask in a water bath (a 600 mL beaker containing
about 200 mL of distilled water). Stir the solution constantly with a stirring rod. When the
bath temperature has reached about 40o C, remove the flask from the water bath and add
about 10 drops of glacial acetic acid while stirring to precipitate the casein.
Filter the mixture into a 100-mL beaker by pouring the mixture through a cheese cloth
which is fastened with a rubber band over the mouth of the beaker. (Be sure that the
cheese cloth has a small indentation.) After liquid stops coming through the cheese cloth,
remove the cheese cloth from the beaker and gently squeeze the cloth to remove most of
the remaining water. The filtrate in the beaker may be discarded down the drain. Using a
spatula, scrape the precipitate from the cheese cloth back into the Erlenmeyer flask.
Add 25 mL of 95% ethanol to the precipitate in the flask. Stir the mixture for 5 minutes to
dissolve most of the fats and water in the mixture. Allow the precipitate to settle and then
carefully decant the alcohol solution into another beaker. Discard the alcohol solution in
the flammable waste container. Add 25 mL of a 1:1 mixture of ether-ethanol (caution: no
flames anywhere!) to the precipitate to remove remaining fat and water. Stir the mixture
for 5 minutes and filter the mixture by swirling the flask to create a slurry and then
rapidly pouring it into a Buchner funnel under vacuum filtration in a hood. Add about 5
mL of 1:1 ether-ethanol to the flask, swirl the flask and rapidly pour the slurry with the
remaining precipitate over the precipitate in the Buchner funnel. Draw air through the

precipitate for at least a minute to dry the casein. Weigh the casein and calculate the
percent casein in milk.
B. Chemical Analysis of Proteins: For each of the first three sets of tests you will test five
different samples. Prepare five clean, labeled test tubes. For each set of tests, place 15 drops of
2% glycine in Tube 1, place 15 drops of 2% gelatin in Tube 2, place 15 drops of 2% albumin in
Tube 3, place 15 drops of 2% tyrosine in Tube 4, and place 15 drops of water in Tube 5 along
with one quarter of a full spatula of the casein you prepared earlier.
a. Biuret Test: To each of the test tubes, add 5 drops of 10% NaOH solution and two
drops of dilute CuSO4 solution while swirling. The development of a purplish violet color
is evidence of the presence of proteins.
b. Ninhydrin Test: To each of the test tubes, add 5 drops of of ninhydrin reagent and heat
the test tubes in a boiling water bath for about 5 minutes.
c. Xanthoproteic Test: To each of the test tubes, add 10 drops concentrated HNO3 while
swirling. Heat the test tubes carefully in a warm water bath. Additionally, add a couple of
drops of concentrated nitric acid to a dry piece of hard boiled egg white on a watch glass.
Allow the acid to sit on the egg white for at least 5 minutes.
d. Heavy Metal Ion Test: To each of 3 clean, labeled test tubes, add 2 mL. milk. Add a
few drops of each of the following metal ions to the corresponding test tubes: Pb2+ as
Pb(NO3)2 in test tube 1; Hg2+ as Hg(NO3)2 in test tube 2; and Na1+ as NaNO3 in test tube
3. Dispose of the lead and mercury samples in the appropriate waste containers.
e. Unknown: Determine which one of your group's two unknowns is a nitrogen
compound and if it is an amino acid or a protein.
3. Be able to answer the following:
a. How was the casein precipitated?
b. How was the fat removed from the precipitated casein?
c. How was the water removed from the precipitated casein?
d. How does a Buchner funnel differ from a Hirsch funnel? What are the relative
advantages of each?
e. Which test(s) differentiate between proteins and amino acids?
f. Which test(s) could be used to differentiate between an amino acid and a
monosaccharide?
g. Many stains on one's hands can be removed by washing. Why can't nitric acid stains be
removed by washing?

Estimation of
blood
glucose
by
Glucose
oxidase
method

66666666666666666666666
66666666666666666666666

66666666666666666666666
66666666666666666666666
6666
Objectives:

To understand the importance of measuring blood glucose level.

To understand the principles of enzymatic estimation of glucose.