Amino Acids

PRACTICAL 5 AMINO ACIDS AND PROTEINS and Proteins

Structure
5.1 Introduction
5.2 Amino Acids - The Building Blocks of Proteins
5.2.1 Properties of Amino Acids
5.2.2 Separation of Amino Acids using Paper Chromatography
5.3 Proteins
5.3.1 Proteins - Basic Concepts
5.3.2 Methods of Protein Estimation
Experiment 1: Studying the Properties of Amino Acids
Experiment 2: Separation of Amino Acids By Paper Chromatography
Experiment 3: Estimation of Nitrogen and Protein Content Using Microkjeldahl’s Method
Experiment 4: Determination of Protein Content Using Biuret Method

5.1 INTRODUCTION
The last practical focused on studying the properties and estimation of carbohydrates.
Moving further, we shall now study the properties of amino acids which we know are
the building blocks of proteins. In this practical, we shall carry out specific tests in
the laboratory and differentiate between the different amino acids. Further, the process
for the separation of amino acids using paper chromatography shall be conducted.
The second part of this practical focuses on proteins. We know that all proteins contain
nitrogen in addition to carbon, hydrogen, oxygen and some other elements like sulphur.
Thus, determination of nitrogen is commonly used to determine the protein content of a
sample. In fact, several methods for protein estimation are available. We shall study
about two such important and practical methods we can use in the laboratory for protein
estimation. These include: kjeldahl’s method and the biuret method. Using these methods
we shall actually estimate the protein content of the given sample in the laboratory.

Objectives
After studying this practical and undertaking the activities given herewith, you will be
able to:

describe the properties of amino acids like tyrosine, tryptophan, arginine, cystine,
phenylalanine etc.,

identify the amino acids on the basis of their properties,

carry out the paper chromatography technique for seperation of amino acids,

estimate the amount of nitrogen and protein present in a given sample, and

determine the protein content in a given sample by biuret method.

5.2 AMINO ACIDS IN THE BUILDING BLOCKS OF

PROTEINS
You have studied that amino acids are the building blocks of proteins. Can you recall the
20 odd amino acids required by our body? Look up the Nutritional Biochemistry Theory
Course, Unit 2. List a few of them in the space provided.
Can you also now illustrate the structure of an amino acid? Draw the structure in the
space provided.

131
Nutritional Amino acids, as you have already illustrated, have a carboxyl group and an amino group
Biochemistry bonded to a common carbon atom and differ from each other in their side chains or R
groups. The R groups vary in structure, size and electric charge which influence the
solubility of amino acids in water. The amino acids are joined together by specific covalent
bonds called peptide bonds.
All amino acids except glycine have a chiral centre and are optically active and have
two possible stereoisomers called enantiomers. You have already studied this term.
Can you define it? Define the term enantiomer in the space provided herewith. Also
graphically represent the L and D isomers.
Enantiomer definition:

L-amino acid D-amino acid

Can you define the difference between D and L isomerism?

You may recall studying in the theory course that all amino acids in proteins belong to the
L series.
Further, we have also studied that amino acids are classified according to their R groups.
In general, they are classified as:
1. Non-polar aliphatic R groups
2. Polar uncharged R groups
3. Positively charge (basic) R groups
4. Negatively charged (acidic) R groups

Give 2 examples of amino acids that belong to each of the above mentioned groups:
Amino acids with non-polar aliphatic R Amino acids with polar charged R
group group

Amino acids with positively charged R Amino acids with negatively charged R
group group

You may also recall studying that amino acid may also be classified as aromatic or non-
aromatic. Amino acids, which have a phenyl (benzene) ring in their structure, are called
aromatic amino acids. Can you list a few aromatic amino acids?
List them in the space provided:

132
The distinctive physical, chemical and biological properties associated with an amino acid are the Amino Acids
result of the R-group. There are 20 major amino acids that differ in their R-group. The R-group can and Proteins
be hydrophobic or polar, aromatic or aliphatic, charged or uncharged as we have seen above. The
different R-groups are responsible for amino acids having different polarities, solubilities and
chromatographic behavior.

Amino acids, you may also recall studying in the theory course, when dissolved in water, exist as
zwitterions. A zwitterion can act as an acid (proton donor) or base (proton acceptor). They exhibit
chemical properties due to COOH and NH2 groups. Let us study some of the properties of amino
acids in the laboratory.

5.2.1 Properties of Amino Acids

Some of the important properties of amino acids are tabulated herewith:

Properties
1. Solubility Amino acids are soluble in various solvents
(a) In Cold water due to presence of polar groups. Tyrosine is
(b) In Hot water the least soluble in water. It is also insoluble
(c) In Dilute acid like HCl in organic solvents but readily soluble in dilute
(d) In Strong alkali like NaOH solution alkalies and acids. Tryptophan and arginine
(e) In Weak alkali like NH4OH solution are soluble in all solvents except alcohol.
(f) In Alcohol Cystine is poorly soluble. It is only slightly
soluble in water but soluble in solutions of
dilute acids and alkalies. While
phenylalanine is insoluble in cold water, it
is easily soluble in hot water. It is insoluble in
alcohol, but soluble in acids and alkalies.
2. Ninhydrin Test Amino acids on heating with ninhydrin
reagent are quantitatively deaminated to give
a blue colour. When some of the ninhydrin
reacts with its reduction product hydrindantin
and ammonia, a blue coloured substance
(Ruhemann’s purple) is formed.
3. Folin’s Test This test is given due to the aromatic ring in
the aromatic amino acids. It is given by
tyrosine as well as tryptophan.
4. Hofmann’s Test The reaction is due to formation of a
coloured mercury compound with the
hydroxy phenyl group and hence is given
positive by all phenols. Tyrosine is the only
phenolic amino acid, thus this test confirms
tyrosine.
5. Sulphuric acid Test The purple colour that is seen is due to the
formation of ferric salt of tyrosine-sulphuric
acid.
6. Morner’s Test This reaction is due to the presence of
phenolic hydroxyl group in tyrosine.

7. Hopkins-Cole test The reaction is due to the presence of indole

ring in tryptophan. It reacts with glyoxylic
acid to form a coloured complex. The copper
is required for the test since pure tryptophan
gives the test in presence of copper or ferric
ions.
133
Nutritional
Biochemistry 8. Sakaguchi Test This test is given by any substance having a
guanidine group. Arginine has a guanidine
group and thus gives a positive Sakaguchi test.
In an alkaline medium, α-naphthol combines
with the guanidino group of arginine to form a
complex which is oxidized by sodium
hypochlorite to produce a red colour.
9. Xanthoproteic Test This test is due to nitration of phenyl ring.
However, this test is generally given by
phenylalanine when combined in peptide
linkage and not in pure form.
10. Lead Acetate Test On boiling with NaOH, the sulfur present in
cystine (the R group contains S) is released as
Na2S. This reacts with lead acetate to form a
brown or black ppt of lead sulfide. While cystine
(and cysteine) responds to this test, the sulfur
present in methionine is not released by the
above treatment.

We shall carry out these tests in the laboratory and differentiate between the different
amino acids. Activity 1 later in this practical focuses on studying the properties of
amino acids on the basis of their properties like solubility and colour reaction due to their
structure and the side chain (R). Before we move on to the activity, let us also quickly
review another process which we can use in the laboratory for the separation of amino
acids i.e. separation of amino acids using paper chromatography.

5.2.2 Separation of Amino Acids using Paper Chromatography

One of the important techniques used in the laboratory for seperating amino acids is
paper chromatography. We have already described the technique earlier in practical
2. Look up Practical 2, once again and get an overview on chromatography. The theory/
principle behind the chromatography process is once again described herewith for your
reference.

Theory/Principle
Chromatography is a collective term used for those processes which allow resolution of
mixture by affecting the separation of all or some of their components in zones or
phases different from those in which they are originally present. It is a technique used
for the separation of a mixture brought about by dynamic partition or continuous distribution
of dissolved material between two immiscible phases, of which one is mobile and the
other is stationary. The mobile phase flows through the stationary phase. During the
process of movement of mobile phase, small differences in adsorption-desorption, or
partitioning or ion exchange behaviour of different components of the mixture are
multiplied many-fold thus enabling their separation. The ability of chromatography to
separate different solutes depends on the selectivity of the process and the degree to
which the system can distinguish between different solutes.
Paper chromatography is a very useful technique for separating mixtures of metal ions,
anions, amino acids, sugars, dyes, drugs etc. It is based on the principle of partition of a
substance between two immiscible solvents in which one of the solvents is static while
the other is mobile. In paper chromatography, the water held in cellulose fibers acts as
a static phase whereas water or any other solvent acts as a mobile phase.
Paper chromatography can separate different amino acids based on their varying
solubilities in two different solvents. In this method, a sample of an amino acid (or
134 mixture of amino acids) is applied as a small spot near one edge of a piece of
chromatography paper. The edge of the paper is then placed in a shallow layer of Amino Acids
solvent mixture in a chromatography tank. and Proteins

The solvent mixture contains several components, one of which is usually water and
another of which is a more non-polar solvent. As the solvent mixture moves up the
paper by capillary action, the water in the mixture binds to the hydrophilic paper (cellulose)
and creates a liquid stationary phase of many small water droplets. The non-polar
solvent continues to move up the paper forming a liquid mobile phase. Since amino acids
have different R-groups, they also have different degrees of solubility in water vs. the
non-polar solvent. An amino acid with a polar R-group will be more soluble in water
than in the non-polar solvent, so it will dissolve more in the stationary water phase and
will move up the paper only slightly. An amino acid with a hydrophobic R-group will be
more soluble in the mobile non-polar solvent than in water, so it will continue to move up
the paper. Different amino acids will move different distances up the paper depending
upon their relative solubilities in the two solvents, allowing for separation of amino acid
mixtures.
The movement of amino acids can be defined by a quantity known as Rf value, which
measures the movement of an amino acid compared to the movement of the solvent. At
the start of the chromatography, the amino acid is spotted at what is called the origin.
The chromatography is then performed, and the procedure is stopped before the solvent
runs all the way up the paper. The level to which the solvent has risen is called the
solvent front. The Rf value of an amino acid is the ratio of the distance travelled by
the amino acid from the origin to the distance travelled by the solvent from the
origin.

distance travelled by the substance

Rf =
distance travelled by the solvent front

Since Rf value for an amino acid is constant for a given chromatography system, an
unknown amino acid can be identified by comparing its Rf value to those of known amino
acids.
Certain technical aspects are important when performing paper chromatography. First,
it is necessary to keep the applied amino acid spot very small. The spot tends to spread
out as it moves up the paper, so starting with a big spot will produce a large smear by the
end of the procedure, making it difficult to measure an accurate Rf value. Second, the
chromatogram paper must be kept very clean. Fingerprints or other types of contamination
will interfere with the chromatography and give poor results. Finally, since amino acids
are colourless, something must be done to detect the amino acids at the completion of
the chromatography. One of the simplest methods for this involves spraying the paper
with ninhydrin. When heated, ninhydrin reacts with amino acids to produce a blue-
purple colour (yellow in the case of proline), making the amino acids spots visible for
analysis.
With this basic understanding, we are now equipped to carry out the exercise relating to
separation of amino acid using paper chromatography. The activity 2 in this practical will
give you good practice and experience with paper chromatography.
But before we move on to the activities, let us quickly also review the literature for
proteins.

5.3 PROTEINS
The discussion below focuses on the basic concepts of proteins with regards to structure
and properties and also describes the methods commonly used for protein estimation in
the laboratory. We begin our study with the basic concepts.

135
Nutritional 5.3.1 Proteins _ Basic Concept
Biochemistry
Proteins, as we already knows by now are products of amino acids. Each molecule of
protein is composed of many molecules of amino acids joined by peptide bond as can be
seen in Figure 5.1.

Figure 5.1: Peptide bond formation

Proteins contain nitrogen in addition to carbon, hydrogen, oxygen and some other elements
like sulphur. Thus determination of nitrogen is commonly used to determine the protein
content of a sample. Proteins contain about 16% nitrogen and multiplying the nitrogen
content by a factor of 6.25 gives us the amount of crude protein present in the given
sample.
Several methods for protein estimation are available. Let us get to know about these
methods.

5.3.2 Methods of Protein Estimation

Several methods for protein estimation are available. These methods are based on several
analytical characteristics of the protein. Laboratory estimation of protein is based on any
of the following principles:
i) Nitrogen estimation based on the principle of Kjeldahl’s method,
ii) Estimation of tyrosine in protein (as in Lowry’s method) or both phenyl alanine and
tyrosine in protein (Folin’s method).
iii) Biuret method - most commonly used method in clinical practice and teaching
laboratories.

We have studied some of the tests in qualitative methods and the principle is the same.
The Biuret method for example can be used for both qualitative and quantitative estimation
of proteins. Let us learn about these methods in details. We shall, however, focus on the
kjeldahl’s method and the biuret method here in this section.

A. Nitrogen Estimation-Kjeldahl’s Method

Nitrogen in the protein is estimated by a method referred to as Kjeldahl’s method.
Although over a period of time many other methods have emerged for determination of
organic nitrogen, this method still remains an old favourite because it is reliable and has
very well standardized procedures.

The method involves the digestion of the weighed sample with concentrated sulphuric
acid, selenium oxide, potassuim sulphate and copper sulphate in a long necked special
digestion flask known as kjeldahl’s flask. Potassium sulphate increases the boiling
point of the mixture. Since the digestion results in fuming of the sulphuric acid, the
procedure has to be carried out in a fume hood and the procedure takes several hours.
The sample is digested until the solution in the digestion flask becomes clear after
which it is transferred to an appropriate volumetric flask (usually 50 or 100 ml flask)
and the volume is made up to the mark with distilled water. Aliquots of this sample are
used for distillation in the kjeldahl’s apparatus and nitrogen in the protein is estimated.
Most proteins contains 16% nitrogen. Thus, percent nitrogen content is multiplied by
136 6.25 to get crude protein content.
 Amino Acids
and Proteins

Figure 5.2: The kjeldahl’s apparatus

The experiment involves decomposition of ammonium sulphate by a fixed volume of

alkali (NaOH). The reaction is highlighted in Figure 5.3. Ammonium sulphate on
decomposition releases ammonia which is collected in a flask of boric acid solution
Tashiro’s indicator. With boric acid ammonia forms a complex ‘ammoium borate’. As
ammonia is distilled into the boric acid, the Tashiro indicator slowly changes colour from
pink to green. This is because the colour of the indicator is pH dependent - it is pink in
acidic medium and green in alkaline medium. When the green coloured solution containing
the boric-acid-ammonia complex is retitrated against H2SO4, the solution slowly becomes
acidic again and the indicator becomes pink. This point when colour changes from green
to pink is taken as the end point.

The Reaction involved includes:

Protein + H2SO4 (NH4)2SO4 + CO2 + H2 O
(conc. sulphuric acid) (ammonium sulphate)

(NH4)2SO4 + 2NaOH 2NH3 + Na2SO4 + 2H2O

(ammonium sulphate) (sodium hydroxide) (Ammonia) (Sodium sulphate)

Figure 5.3: Reaction involving decomposition of amonium sulfate

The amount of nitrogen can be calculated for this value using the equation:

1000 ml of 1N H2SO4 = 1000 ml of 1N NH3 = 1000 ml of 1N (NH4)2S04 = 1000 ml

of 1 N Nitrogen = 14 g of Nitrogen (based on molecular weight calculations)

The Kjeldahl’s method is commonly used in estimation of protein content of foods and
has been extensively used for protein estimation of various foodstuff. We shall look at
the method in details in activity 3 in this practical and try to find the amount of ammonium
sulphate in the sample from which protein can be calculated.
Next, let us learn the oldest colorimetric method for protein estimation i.e. the biuret method.

B. Biuret Method
The Biuret reaction was one of first colorimetric assays developed for protein estimation.
It is most often used in applications requiring a fast but not a highly accurate value.
The principle of biuret method is described next.

Principle/Theory
If a strongly alkaline solution of Biuret is heated with very dilute copper sulphate, a violet
colour is obtained. The substances which contain two carbamyl (-CONH2 )groups joined
together either directly or through a single carbon or nitrogen atom and those which contain
137
Nutritional two or more peptide links give a blue or purple coloured complex with alkaline copper
Biochemistry solution. Proteins respond positively since there are pairs of -CONH2 groups in the
molecule. The reaction is due to coordination of cupric anion with the unshared electron
pair of peptide nitrogen and the oxygen of the water. A coordination complex is formed
which is purplish coloured and can be colorimetrically measured. When protein solution
is made strongly alkaline with NaOH or KOH and very dilute CuSO4 is added, a purplish
to pink violet colour is obtained, the intensity of the colour depending upon the
concentration of protein or the number of peptide bonds undergoing the reaction.
The biuret reaction is shown in Figure 5.4.

NH2 O O
NH2
C O C NH NH C
C O
NH Cu ++
NH
NH2
NH2 C NH NH C
C O
O O
NH2
Urea Biuret Coloured coordination complex
Figure 5.4: The Biuret reaction

The detailed procedure involved in biuret method is described in activity 4. So with this
knowledge, we can start with the experiments given in this practical.

There are 4 experiments in this practical. Organize your time in such a manner that you
can carry out these experiments one by one.

138
 Amino Acids
EXPERIMENT
and Proteins

STUDYING THE PROPERTIES OF AMINO ACIDS 1

Date: ..........................
Aim: To study the properties of amino acids like tyrosine, tryptophan, arginine, cystine, phenylalanine.

Now conduct the various tests indicated in the format herewith and note down your observations and inference
in the space provided:

Test Observation Inference

Tyrosine
1. Solubility
Test the solubility of the amino
acid in the following solvents
(a) Cold water
(b) Hot water
(c) Dilute acid like HCI
(d) Strong alkali like NaOH
solution
(e) Weak alkali like NH4OH
solution
(f) Alcohol

2. Ninhydrin Test
Ta k e 5 m l of tyrosine
solution add 0.5 ml of 0.1%
ninhydrin solution. Boiling for
1-2 minutes and allow to cool.

3. Folin’s Test
To 1-2 ml of tyrosine solution add
equal amounts of phenol reagent
(folin-ciocalteau reagent-25 g
sodium tungstate + 6.2 g sodium
molybdate + 12.5 ml phosphoric
acid + 25 ml conc. HCI + 37 g
LiSO4 + a few drops of bromine)
and 10 ml of a saturated solution
of sodium carbonate.

4. Hofmann’s Test (Millon’s test

for tyrosine)
To 3 ml of tyrosine add a few
drops of Million’s reagent. Heat.
139
Nutritional
Biochemistry 5. Sulfuric Acid Test
Take a small amount of
tyrosine in a small 10 ml
beaker and add 3-5 drops of
conc H 2 SO 4' Keep on a
boiling water bath for 20
minutes. Cool. Wash the
contents into another small
beaker with distilled water.
Slowly add solid CaCO3 with
stirring until the reaction of
the solution is no longer acid.
Filter. Concentrate the
filtrate, and add it to a few
drops (avoid an excess) of
very dilute neutral ferric
chloride.

6. Morner’s Test
(formaldehyde -sulphuric
acid test)
To 3 ml of Morner’s reagent
(1 ml of formalin + a 100 ml
solution of equal amounts of
distilled water and conc
H2SO4) add a little tyrosine
solution. Heat gently.

Tryptophan
1. Solubility
Test the solubility of the
amino acid in the following
solvents
(a) Cold water
(b) Hot water
(c) Dilute acid like HCI
(d) Strong alkali like NaOH
solution
(e) Weak alkali like
NH4OH solution
(f) Alcohol

2. Ninhydrin test
(write the procedure here in
the space provided)

140
 Amino Acids
3. Folin’s test and Proteins
(write the procedure here in
the space provided)

4. Hopkins- Cole test

To about 3-4 ml of
tryptophan solution add 3 ml
of glyoxylic acid solution.
Mix. Carefully add 5 ml of
conc. sulphuric acid down
the side of the tube. Add a
drop of copper sulphate
solution.

Arginine
1. Solubility
Test the solubility of the
amino acid in the following
solvents
(a) Cold water
(b) Hot water
(c) Dilute acid like HCI
(d) Strong alkali like
NaOH
solution
(e) Weak alkali like
NH4OH solution
(f) Alcohol

2. Ninhydrin test
(Same as before. Write the
procedure in the space
provided)

3. Sakaguchi test
Take 2-3 ml of arginine
solution. To this add 1-2
drops of α-naphthol solution
and 2 ml of sodium

141
Nutritional
Biochemistry Cystine
1. Solubility
Test the solubility in the following
solvents:
(a) Cold water
(b) Hot water
(c) Dilute HCI
(d) KOH solution
(e) NH4OH solution
(f) Alcohol

2. Ninhydrin test
Same as before.

3. Lead Acetate test

To 5 ml of cystine solution add a
few drops of 10% solution of lead
acetate. Mix well. Make the solution
strongly alkaline by adding 40%
KOH or NaOH. Boil for a few
minutes and let it stand for some
time.

Phenylalanine
1. Solubility
Test the solubility in the following
solvents:
(a) Cold water
(b) Hot water
(c) Dilute HCI
(d) KOH solution
(e) NH4OH solution
(f) Alcohol

2. Ninhydrin test
Same as before

3. Xanthoproteic test
To 2-3 ml of phenylalanine solution,
add 1 ml of conc. HNO3. Cool.
Make it alkaline with strong NH3
142
Now conduct the various tests indicated in the format herewith and note down your Amino Acids
observations and inference in the space provided: and Proteins

Results
The given solution contains amino acids ..………….....…………… …………………..

Give the structure of these amino acids.

Write the reaction involved in the tests used to determine these.

Submit your experiment for evaluation.

..................................
Counsellor signature

143
Nutritional
EXPERIMENT
Biochemistry

2 SEPARATION OF AMINO ACIDS BY PAPER

CHROMATOGRAPHY
Date: .......................... In Experiment 1 you learnt methods of identification of amino acids on basis of their
properties like solubility and colour reactions due to their structure and R groups. Let us
also study the separation of amino acids by using chromatography.

Aim: To separate and identify the unknown amino acids in a given mixture by paper
chromatography.

Theory/Principle
You have already studied the principle of chromatography in Section 5.2 above. Based
on your understanding of the topic, write the principle here in the space provided.

Materials: Petridishes (200 mm diameter) with lid, Whatman Chromatographic paper,

brown paper, 500 ml separating funnel, capillary tubes, small beaker, durham tubes, a
pair of scissors stoppered glass tubes, cotton for making wick.

Reagents
The following reagents will be required for carrying out the experiment:
1. Amino acid solution-
Weigh abour 3-4 mg of each amino acid and dissolve it in dil HCl (1-2 drops). Make
the volume upto 10 ml.
2. Chromatographic solvent
Take a separating funnel. To this add 500 ml of freshly shaken mixture of equal
volumes of water and butanol. Add 60 ml of glacial acetic acid. Mix. Shake. Discard
the lower layer. Take the upper layer.
3. Ninhydrin solution
1% ninhydrin in acetone.

Procedure
Now carry out the procedure following the steps given herewith:
1. Take a circular Whatman No 1 filter paper.
2. Take a Petri dish the diameter of which is slightly smaller than the diameter of the
paper.
3. From the centre of the paper draw a circle of 2 cm diameter.
4. Mark the spots with a pencil for spotting on the circle.
5. With the help of a fine capillary tube spot the amino acid standards and unknown
mixture on the marked spots. This process is called spotting. Allow the spot on the
paper to dry completely.
6. Insert a cotton wick in the central hole of the filter paper.
7. Put some solvent in the Petri plate and place the paper on top of it ensuring that the
wick dips into the solvent in the centre of the petridish. Cover the petridish.
8. Leave this arrangement for 3-3½ hours so that the solvent reaches nearly to the
144 end of the circular paper.
9. Remove the lid and mark the solvent front with a lead pencil. Amino Acids
10. Dry the paper in air by clipping it on to a thread line. and Proteins
11. For visualization of amino acid, spray ninhydrin over the paper and dry the paper
again (for 10 minutes).
12. Areas where the amino acid has moved will get a yellow, pink or purple colour.
Mark the areas with a pencil.
13. For identification of amino acids, estimate and measure the distance between the
point of application of amino acid and the centre of the amino zone acid, as well as,
the distance between the point of application and the solvent front.
14. Ratios of the two measurements give the Rf value of standard amino acid and that
of an unknown amino acid to identify the amino acids.

Precautions
1. Hold the paper with the extra strip kept at one side. Handling of whatman paper
with hand should be avoided.
2. The spotting of amino acid should not be more than 1 mm in diameter since large
spots lead to diffused and large amino acid patterns with no sharp outline.
3. The petridish should be saturated with solvent vapours.
4. Solvent front should be marked immediately after removing teh chromatogram, since
it disappears rapidly.
5. The outline of each amino acid should be immediately marked with pencil, Do not
mark with ball point or ink pen.

Observations and Result

Attach the chromatograph prepared in the space provided on next page.

Observation: Circular paper chromatography

Calculations
Calculate the Rf value as indicated:

distance travelled by the substance

Rf value =
distance travelled by the solvent front
Rf value of amino acid 1
Distance travelled by the solute = ..........................................................
Distance travelled by the solvent front = ................................................
Therefore Rf value = .............................................................................

Rf value of amino acid 2

Distance travelled by the solute = ..........................................................
Distance travelled by the solvent front = ................................................
Therefore Rf value = .............................................................................

Rf value of amino acid 3

Distance travelled by the solute = ..........................................................
Distance travelled by the solvent front = ................................................
Therefore Rf value = ..............................................................................
Result
Record your results here as indicated:
The Rf of the amino acids in the mixture are
1.
2. 145
3.
Nutritional Thus, Amino acid 1 is .................................................
Biochemistry
Amino acid 2 is ........................................................
Amino acid 3 is ........................................................

Circular paper Chromatography

Submit your experiment for evaluation.

................................
146 Counsellor signature
 Amino Acids
EXPERIMENT
and Proteins

ESTIMATION OF NITROGEN AND PROTEIN

CONTENT USING MICRO KJELDAHL’S METHOD 3
Aim: To estimate the amount of ammonium sulphate in the given solution by Micro Date: ..........................
Kjeldahl method.

Reagents
Collect the following reagents to conduct the experiment:
40% NaOH
Tashiro’s indicator - 2% boric acid + methyl red + methylene blue
Phenolphthalein
Standard sulphuric acid (0.1N)
Anhydrous sodium carbonate
Methyl orange

Principle/Theory
Write the principle behind the kjeldahl’s method in the space provided herewith. You
can look up section 5.3 for information on this methodology.

Look at the Kjeldahl’s apparatus and draw the diagram. This will enable you to
remember the distillation process. 147
Nutritional
Biochemistry

The Kjeldahl’s apparatus

Procedure
You would realize that this proceedure is carried out in two steps. In the first step the
standardization is done followed by the sample titration in the second step. Carry out the
experiment following the proceedure enumerated herewith:-
1. Standardization of H2SO4-
Prepare a solution of 0.1 N H2SO4 by diluting 2.8 ml of the concentrated acid to 1000
ml in a volumetric flask. This is approximately 0.1N. Standardize the acid against 0.1
N Na2CO3 solution. Accurately weigh 0.530 g of anhydrous Na2CO3. Transfer to a
100 ml volumetric flask and make up the volume to the mark with distilled water.
Pipette 10 ml of this solution in a conical flask and titrate against the diluted H2SO4
prepared using 1% solution of methyl orange as an indicator. Calculate the exact
normality of H2SO4 and dilute it accordingly to obtain exactly 0.0143N H2SO4.
2. Sample titration-
Take the given ammonium sulfate solution into a 100 ml volumetric flask and dilute to
the mark with distilled water. Mix well. Set up the steam distillation apparatus and
pass steam through the apparatus for cleaning it. After the apparatus is cleaned,
pipet 10 ml Tashiro’s mixture into a 100 ml conical flask and place it at the delivery
end of the apparatus making sure that the delivery tube is dipping in the solution. Add
10 ml of the dilute sample from the funnel in the apparatus and few drops of
phenolphthalein. After this wash the funnel with about 5 ml distilled water to ensure
that all the ammonium sulphate has reached the distillation flask. Then add 10 ml of
40% NaOH and immediately close the stopper. Now heat the flask to pass the
steam through this mixture. The liberated NH3 from ammonium sulphate is collected
in the Tashiro’s mixture in the receiving flask. Continue distillation until the level of
the solution in the receiving flask becomes approximately double. Remove the
receiving flask and rinse the tip of the delivery tube with distilled water. Titrate the
solution in the flask against standard H2SO4 till the color changes from green to pink.

Calculate the amount of ammonium sulfate in the given solution.

Precautions
1. The apparatus should be airtight to prevent leakage of NH3.
2. The tip of the delivery tube should be immersed in Tashiro’s mixture in the receiving
flask.
3. After completion of distillation, remove the receiving flask first to prevent back suction.

Results and Observations

Record your observations in the format given in this section. Follow the reactions and the
quations spelled out for your convenience herewith and fill up the gaps as indicated.The
equations in this section require a lot of concentration and systematic study. So please
be very attentive and alert while writing your observations and results.
1. Standardization of H2SO4
148
Normality of Na2CO3 solution = 0.1 N
 Volume of 0.1 N Na2CO3solution = 10 ml Amino Acids
Volume of 0.1 N H2SO4 required and Proteins

Buret reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3

Titer value = ……..(a) ml

Calculations
Now carry out the calculations as indicated:
N1V1 = N2V2
Where N1 = Normality of Na2CO3 solution = …………..
V1 = Volume of 0.1 N Na2CO3solution = …….. ml
N2 = ( to be calculated)
V2 = …… (a) ml (i.e. the titre value)

Now write the values for V1, N1 and V2 and calculate N2.
N2 = N1 × V1 = ….. × ….. = ……….. (b) N = ....................N H2SO4
V2 ………

2. Sample titration:
Given Unknown sample taken in the volumetric flask and volume made to 100 ml
with distilled water
Volume of dilute (NH4)2SO4 solution for distillation = 10 ml
Volume of ……….. (b) N H2SO4 required

Buret reading (ml)

S.No.
Initial Final Difference
Pilot
1
2
3
Titer value = ……….(c) ml

1000 ml of 1 N H2SO4 = 1000 ml of 1N NH3 = 1000 ml of 1N (NH4)2SO4 = 1000 ml

of 1 N Nitrogen = 14 g of Nitrogen (based on molecular weight calculations)
therefore, 1000 ml of 1N H2SO4 = 14 g Nitrogen
0. 1 ml of 1N H2SO4 = 14 mg Nitrogen
0. 1 ml of 0.1 N H2SO4 = 1.4 mg Nitrogen

0. 1 ml of …… (b) N H2SO4 = …….(b) × 1.4 = ………..(d) mg Nitrogen

0.1

... ........... (c) ml of …… (b) N H2SO4 = c × d = ………..(e) mg Nitrogen

10 ml of dilute solution contains …….. e mg nitrogen

149
Nutritional 100 ml of dilute solution contains = 100 × ……(e) = ………(f) mg of nitrogen
Biochemistry 10

Results
The unknown sample contained …………. mg nitrogen.

Submit your experiment for evaluation.

................................
Counsellor signature

150
 Amino Acids
EXPERIMENT
and Proteins

DETERMINATION OF PROTEIN CONTENT USING

BIURET METHOD 4
The Biuret reaction was one of first colorimetric assays developed for protein estimation. Date: ..........................
It is most often used in applications requiring a fast but not a highly accurate value.

Let us carry out this experiment and learn the oldest colorimetric method for protein
estimation.

Aim: To plot a standard curve for albumin and determine the protein content in the given
solution.

Apparatus
We would require the following appratus to carry out the experiment:
Test tubes Borosil glass (6x3/4 inch) Funnel
Volumetric flasks 100 ml Glass marker/permanent marker
Conical flasks Labels
Pipettes 1 ml, 5 ml, 10 ml Spectrophotometer or colorimeter
Beakers Single pan balance
Measuring cylinder Butter paper for weighing

Reagents
Collect the following reagents to conduct the experiment:
1. 0.2 N NaOH solution - Dissolve 8 g of NaOH pellets in 1 litre of distilled water.
2. Biuret reagent - Dissolve 90 g of Rochelle salt (sodium potassium tartarate)
in 500 ml of 0.2 N sodium hydroxide solution. Add 10 g of copper sulphate and
10 g KI and volume is made to 2 L with 0.2 N alkali.
3. Working standard - Dissolve 200 mg albumin in 100 ml distilled water (2 mg/ml).

Principle
We have already studied about the principle of biuret method earlier in section 2.3. Look
up the section now and write down the principle here in the space provided based on your
understaning about the method.

151
Nutritional Procedure
Biochemistry
Now carry out the proceedure as enumerated herewith:
1. Preparation of standard albumin: A standard solution of 2 mg albumin/ml is
prepared in distilled water. For this weigh 200 mg of albumin and transfer This
amount into a 100 ml volumetric flask. Make up the volume to the mark with
distilled water.
2. Colour development for standard solution:
– Take different volumes of standard albumin solution (0.5, 1.0, 1.5, 2.0, 2.5,
3.0 ml) in different test tubes
– In each case, make up the volume to 3 ml with distilled water.
– Add 3 ml of biuret reagent to each test tube and keep the tubes at room
temperature for 30 minutes for colour development after shaking.
3. Preparation of unknown solution: Take the unknown solution and dilute the
given solution to 100 ml mark and take one volume (e.g. 2.0 ml) in duplicate
similar to that prepared for the standard. Add all other reagents just as you did for
the standard solution.
4. Preparation of blank: Pipet 3 ml of distilled water. (Do not add the standard
solution in the blank). Now repeat the above procedure for colour development.
5. Measurement of O.D: Read the absorbance of all solutions at 540 nm after
setting the colorimeter to 100% transmittance with the blank solution.
6. Standard Curve: Plot a graph between concentration albumin in standard solution
and O.D.
7. Calculate the amount of protein with the help of the standard curve.

Plotting and Calculations

I. Preparation of Standard Albumin

200 mg of albumin was dissolved in distilled water and the volume made to 100 ml.
therefore, the strenght of the standard albumin solution = 2 mg/ml
Since 1.0 ml of the standard albumin solution = 2.0mg albumin
... 0.5 ml of the the standard albumin solution contains = …… mg albumin
... 1.5 ml of the the standard albumin solution contains = …… mg albumin
... 2.0 ml of the the standard albumin solution contains = …… mg albumin
... 2.5 ml of the the standard albumin solution contains = …… mg albumin
... 3.0 ml of the the standard albumin solution contains = …… mg albumin

II. Colorimetric Reading for Standard Solution

Record the colorimetric reading for standard solution here in the format provided. For
concentration of albumin write the amount calculate above in point I. Note the O.D
reading in column V.

I II III IV V VI
Volume Conc. of Distilled Biuret Optical Optical Density
of albumin Water Reagent Density at for 2.0 mg
Std. Sol. (mg) (ml) (ml) 540 nm albumin

0.5

1.0

1.5

2.0

2.5

152 3.0
Mean OD for 2.0 mg albumin = . + …….. + ……. + ……. + ……. +… = (A) =……mg. Amino Acids
6 and Proteins
Now prepare the standard curve for albumin (on a graph paper) with concentration of
albumin (figure included in item II above) on x-axis and the optical density (figures in
item V above) on y-axis. Stick the graph in the space provided on page.

III. Colorimetric Reading for Unknown Solution

a) Optical density of unknown albumin sample, 1 = ………
2 = ………
... mean optical density of unknown sample = (B) ………… .

IV. Determination of Albumin Content

a) From Standard Curve
On the standard curve prepared earlier, plot the OD of the unknown solution on the Y-
axis. Now check the corresponding concentration of albumin (in the unknown solution)
for this OD on the X-axis. Say the value obtained is E.

So 2.0 ml of unknown solution contains E ml of albumin

... 100 ml of unknown solution will contain = E × 100 = F = …..mg.
2

Write the calculations in the space provided:

Observed Value = F = ………….

Expected value = D =…………(Take it from the counsellor)

Calculate the % error (based on optical density) using the formula given herewith:
= D – F × 100
D

153
Nutritional
Biochemistry

154 Standard curve between concentration of standard albumin solution and OD

 b) From Optical Density Amino Acids
and Proteins
Mean O.D for 2.0 mg albumin = A ……… mg
.
. . If OD of albumin is A…….., then concentration of albumin is = 2.0
if OD of the unknown solution is B ………, then concentration of unknown
albumin solution = 2.0 × B = C = …….mg
A
So C …….. mg is present in 1ml of unknown
.
. . 100 ml of unknown contains = C x 100 = …….. × 100 = F ……… mg.

Observed Value = F = ………….

Expected Value = D =…………(Take it from the counsellor)

Calculate the % error (based on optical density) using the formula given herewith:
= D – F × 100
D

Result
The given solution contains ……….................................................... mg of protein.

Now, also answer the review questions given next. These will help you consolidate your
understanding on this topic.

Review questions

1. Why is the nitrogen content multiplied with 6.25 to obtain the protein value?
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................
155
Nutritional
2. Why do proteins give biuret test?
Biochemistry
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................
3. Why do you add NaOH during distillation of ammonium sulphate?
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................
......................................................................................................................

Submit your work for evaluation.

.................................
Counsellor Signature

156