Biochem Journal 2015

Students name:
School/College
name:
Harshavardhan Mahalakshmi Ganesan

D. Y. PATIL UNIVERSITY, NAVI MUMBAI,
SCHOOL OF BIOTECHNOLOGY AND
BIOINFORMATICS
Class:
BBT-2
Roll number:
BBT-2-14017
Practical II: Biochemistry/Analytical Techniques
Subject:
Index
Exp
no.
Date
Experiment title
Pg
no
06.08.2015
Study of pH meter
13.08.2015
Preparation of buffer solutions
20.08.2015
Qualitative tests for carbohydrates
10
27.08.2015
Lipid solubility test
14
24.09.2015
Lipid TLC
16
08.10.2015
max & Beer-Lamberts law
18
13.10.2015
Paper chromatography of A.A
22
17.10.2015
17.10.2015
10
20.10.2015
Reducing sugar estimn - DNS method 28
11
29.10.2015
Protein estimation by Biuret method
30
12
05.11.2015
Extraction of casein from milk
32
Signature
Qualitative tests for proteins and A.A 24

Starch extraction
26
Certificate
Class: BBT-2
Year: 2015
This is to certify that the work entered in this

journal is the bonafide work of
Mr. HARSHAVARDHAN M GANESAN,
Roll number: BBT-2-14017,
who has worked for III semester
in D.Y.Patil University, Navi Mumbai,
School of Biotechnology & Bioinformatics

during the academic year: 2015-16
__________
Head of the
Department
Date: 16.11.2015
__________
External
examiner
__________
Internal
examiner
School of Biotechnology
& Bioinformatics
3
D. Y. PATIL UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
Programme:
Practical
Title:
B.Tech Biotechnology
Semester:
III
Study of pH meter
Experiment. No.: 1
Date: 06.08.2015
Aim:
To Study the construction & working of pH Meter.
Principle:
Acids dissolve in water forming H+ ions. The greater the concentration

of H+ ions, stronger is the acid, the converse applies to bases (w.r.t OHions). pH can be defined as negative log to the base10 of hydrogen ion
concentration.
pH= -log10 [ H+]
pH is the degree of acidity or alkalinity of a solution on a scale of 1 to

14. The e.m.f developed between the 2 elements depends on the
concentration of hydrogen ions & hence the pH. In order to measure this
e.m.f no current flow should occur in the electrode flow pair, otherwise
chemical reactions at the cell boundaries with results in polarization of
electrodes.
To avoid this, a high impendence (reaction) circuit is used to detect the
e.m.f (potential). This converts the voltage to current which can be
amplified & measured & an electric current circuit which measures e.m.f
developed across the electrode. These two electrodes used in the pH
meter are two half cells.
The glass electrode contains a glass bulb, coated with silica & mineral
salts, that is permeable to hydrogen ions. As a result, potential develops
across the glass membrane. The potential developed is linearly
proportional to the pH of the solution in which the electrode is immersed.
The relationship is expressed by Nernsts equation as:
E=E0-(2.303RT)/nf *log a
Where,
E= Electrode potential at specified concentrations; E0= Standard electrode
potential; R= Gas constant; T= absolute temperature; F= faradays
constant & a= activity of ions.
Working: On placing the probe into the solution, the H+ ions will move
towards the glass electrode replacing some of the metal ions on the glass.
This causes a potential difference to be developed which is picked up by
the silver wire & the same is passed on to the voltmeter where it is
amplified and displayed as pH units. Increase in concentration of H+ ions
increases the voltage and hence displays lower pH.
Requirement:
1. pH meter
2. standard buffer solution(s)
Procedure:
1. STANDARDISATION OF pH METER: Sureties on the pH

meter & allow it to warm up for 15mins with electrodes properly
dipped in distilled water. This prevents any fluctuations while
measuring the voltage or pH.
2. All the test solution & buffer solution added should be brought to
ambient temperature of room in which pH meter is kept. Check
the position of pointer on scale & it should show zero or set zero if
required.
3. The pH meter should be adjusted to measure the pH at room
temperature by adjusting the temperature knob.
4. The standardisation of the pH with 3 standard buffers is important
i.e. pH 4, pH 7 &
pH 9 to ensure that the pH is functioning
properly over the entire range. The electrodes should be washed

thoroughly with D/W & immersed in a beaker containing fresh
distilled water. This protects the electrode from dying & increases
its lasting period.
Result:
The construction and working of pH meter was observed and studied.
Precautions:
1. pH meter bulb should not be allowed to dry, so it should be kept immersed

in water when not in use.
2. After each operation bulb should be washed with distilled water.
3. pH meter should be calibrated using standard buffer solution of pH 4,7 & 9.

Programme:
Practical
Title:
Semester:
Preparation of buffer solutions
Experiment. No.: 2
Aim:
Principle:
III
Date: 13.08.2015
To prepare solutions and buffers
A buffer system is one that can resist a change in pH on addition

of acid or alkalis. It consists of a conjugate acid-base pair. The
buffer system consists of a mixture of weak acid & its
conjugate/base buffer of acetic acid, sodium acetate. A
physiological buffer is carbonate, bicarbonate system in blood.
Buffers are extensively used in biochemical studies since they aid

in maintaining a rear constant pH of media while performing
various lab operations viz. extraction of various bio molecules,
their isolation, purification, selection of an appropriate buffer with
optimal pH is important as it may have an influence on
extractability
stability
&
biological
functioning
of
cell
constituents. For e.g. the activity & stability of extracted enzyme

is dependent on the pH of the system. The pH may vary during an
enzyme reaction due to release or acceptance of proteins which
may affect the activity of the enzyme. Therefore buffers are
necessary to stabilize the H+ ion concentration & remove adverse
effects due to pH change on enzyme under investigation.
Henderson Hasselbach equation can be used to express the

dissociation constant of an acid.
HAH+ + AK= [H+] [A-]/ [HA]
Where,
6
K= dissociation constant of the acid by rearranging,
[H+] = K [HA]/ [A-]
Taking negative logarithm on both the sides;

-log [H+] = -logK (log [HA])/ [A-]
But
-log [H+] = pH
&
-log K = pKa
Therefore
pH = pKa (log [HA]) / [A-]
Therefore
pH= pKa + (log [A-]) / [HA]
This can be written in the general form as:
pH= pKa + log[conjugate base]/[conjugate acid]

Theory:
Solution: Any non-reactive substances mixed together homogenously

form a solution.
One mole is its molecular weight in grams (gram equivalent)
Ex: NaOH : 40 mol wt
1 mol of NaOH = 40 gm of NaOH
1 mol of NaOH in 1L water = 1M NaOH
Q1. How many grams of NaOH would be required to make 50ml of 0.5M
solution?
A. Molarity = (amt of solvent/ mol w.t)
1M = 40gm of NaOH 0.5M = 20gm of NaOH
If 20gm in 1L, then xgm in 50ml
x = 1gm
1gm of NaOH is to be mixed in water, and brought up to 50ml, to
make 0.5M solution.
Q2. Prepare 0.1N & 0.5N Na2CaO3 whose equivalent weight is 53gm.
A. 1N = 53gm in 1L 0.1N = ygm y = 5.3gm
1N = 53gm 0.5N = zgm z = 26.5gm
5.3gm & 26.5 gm of Na2CaO3 is to be mixed in 1L of water to make 0.1N
& 0.5N Na2CaO3 respectively.
7
Q3. Prepare 150ml of 70% alcohol from 95% stock.

A. N1V1 = N2V2
0.95 x x = 70% x 150ml
x = 110.5ml
Or
n
Req conc x total vol/given concn = 110.5ml
150ml xgm = amount of water to be added
Water to be added = 39.5ml
Q4. Prepare 100ml of 0.1M NaOH
A. 1mol = 40gm of NaOH
0.1M in 1L water = 4gm
0.1M in 100ml water = 0.4gm
0.4gm of NaOH is to be added to 100ml of water to make a 0.1M
solution.
Q5. Prepare 50ml of 70% alcohol from 100% stock.
A. N1V1 = N2V2
0.1 x x = 70% x 50ml
x = 35ml
Or
n
Req conc x total vol/given concn = 35ml
50ml xgm = amount of water to be added
Water to be added = 15ml
Q6. What volume of HCl do you need to prepare 0.1N? If concn = 37.5%,
sp.gravity = 1.84 & eq.wt = 49gm.
A. V1 = eq.wt of acid x V2 x N x 100
1000 x sp.gravity x purity
V1 = 36.5gm x 100 x 0.1 x 100
1000 x 1.84 x 37.5%
V1 = 52.9ml
Q7. Prepare a buffer solution of Acetic acid = 0.2M & Sodium acetate =
0.2M with total volume 50ml. Find out the resultant pH.
A. 46.3ml of acetic acid & 3.7ml of sodium acetate for 100ml
Hence, for 50ml 23.15ml of acetic acid & 1.85ml of
sodium acetate. pH = 2.17

Programme:
Practical
Title:
Semester:
III
Qualitative tests for carbohydrates
Experiment. No.: 3
Date: 20.08.2015
Aim:
To detect the presence and type of carbohydrate(s), in the given

environmental sample, using qualitative tests
Theory:
Carbohydrates are compounds consisting of carbon, hydrogen and

oxygen; with hydrogen and oxygen in the ratio of 2:1. Carbohydrates
include sugars and starches and can be divided into three types namely
monosaccharides and disaccharides and polysaccharides formed by
condensation of monosaccharides. Monosaccharides are polyhydroxy
aldehydes and ketones.
Principle:
Molischs test:- (General test for Carbohydrates):

Conc. H2SO4 hydrolyses the glycosidic bonds of polysaccharides which
are then dehydrated to furfural and its derivatives. These products then
combine with sulphonated naphthol to give a purple colored complex.
This reaction is given generally by all carbohydrates.
Iodine test for Polysaccharides:

Iodine forms colored absorption complexes with polysaccharides. Starch
gives blue color with Iodine, while Glycogen reacts to form reddish
brown complex. Hence it is a useful, convenient and rapid test for
detection of amylase, amylopectin and glycogen.
Benedicts test (for reducing sugars):
It is a test for detecting reducing compounds. It is a Modified form of
Fehlings test where alkaline CuSO4 containing Cu(OH) is converted to a
reddish precipitate of Cu2O when heated in the presence of a reducing
10
agent such as Glucose or Maltose.

Na2CO3 + 2H2O NaOH + H2CO3
2NaOH + CuSO4 Cu(OH)2 + Na2SO4
Cu(OH)2 CuO + H2O (Cupric Oxide)
Glucose + 2CuO (Gluconic acid oxidised) + Cu2O (reduced red ppt
cuprous oxide)
Requirement:
Procedure:
1.
2.
3.
4.
5.
6.
7.
8.
9.
Conc. H2SO4
Molischs reagent
Benedicts reagent
Barfoeds reagent
Fehlings A & B
Seliwanoffs reagent
Bials reagent
Sample solution to test for presence of carbohydrates
Iodine solution: 0.005N I2 in 3% KI solution
Molischs Test: (for carbohydrates)

1. Take 2ml of test solution in a test tube.
2. Add 2 drops of Molischs reagent naphthol reagent
mix well and wait for 2-3 minutes.
3. Take 1ml of conc. H2SO4 solution allowing it to flow
from the side of the tube.
4. At the junction of the 2 reddish violet purple ring appears
in the presence of the sugar.
Iodine Test: (for polysaccharides)

To 1ml of sample extract or test solution in a test tube add 4-5
drops of iodine solution to it and mix the contents and observe for
color formation.
Benedict s test: (for reducing sugars)
1. Take 2ml of Benedicts solution in the test tube
2. Add 1ml of sugar sample and mix the solution well
3. Boil the solution in boiling water bath for 5 to 10 minutes
and allow to cool
4. Observe the color of precipitate.
11
Barfoeds test: (for mono/disaccharides)

1. Take 2ml of Barfoeds solution in the test tube
3. Place the solution in boiling water bath for 5 minutes and
allow to cool
Fehlings test: (for reducing sugars)
1. Take 1ml of Fehlings A & B solution in the test tube
3. Place the solution in boiling water bath for 5 to 10 minutes
and allow to cool
Seliwanoffs test: (for mono/disaccharide & aldose/ketose)
1. Take 2ml of Seliwanoffs reagent in the test tube
3. Place the solution in boiling water bath for 1 to 2 minutes
and allow to cool
Bials test: (for pentose/hexose)
1. Take 2ml of Bials reagent in the test tube
2. Add 4 to 5 drops of sugar sample and mix the solution well
3. Place the solution in boiling water bath for 2 minutes and
allow to cool
12
Type
Test
Observation
Molischs test:
GT
-naphthol + conc H2SO4 +
Carbohydrat
Purple ring
sample
GT
Iodine test:
No change in
Polysacchari
Iodine solution + sample
colour
des - absent
Reagent + sample {boiling
Reducing
Red ppt
water bath for 5-10 minutes}

Observation:
CT
Red ppt
within 2-5
water bath for 5 minutes}
minutes

water bath for 2 minutes}

water bath for 1-2 minutes}
Result:
Monosaccha
rides present
Colour
change but
Hexose
not blue
present
green
Seliwanoffs test:
CT
sugar may
be present
Barfoeds test:
Bials test:
CT
es may be
present
Benedicts test:
GT
Inference
Deep red
Keto hexose
present
The given sample (B) is a ketohexose monosaccharide reducing sugar.
13

Programme:
Practical
Title:
Semester:
III
Lipid solubility test
Experiment. No.: 4
Date: 27.08.2015
To identify the solvent in which butter, ghee, palmitic acid, oleic acid,
vegetable oil & coconut oil gets dissolved
Aim:
Fatty acid samples, distilled water, acetone, chloroform & ethylether
Requirement:
Procedure:
1. Add 0.5 ml of each of the seven solvents in seven test tubes.

2. Add 0.5 ml of a particular lipid sample (say butter) to all the seven
test tubes containing solvents. Mix well and observe for solubility.
3. Incubate the tubes at 55oC for about 10 minutes & observe the
solubility again & record the observations.
4. Repeat the same with other lipid samples.
Observation & Result:

Solvents
Fatty acids
Distilled
Acetone
water
Chloroform
Ethyl ehter
RT
IT
RT
IT
RT
IT
RT
IT
Butter
Ghee
Palmitic acid
M*
Oleic acid
Veg. oil
Coc. Oil
Stearic acid
M*
Key: {RT: Room temperature , IT: Incubated temperature (55 oC), I: Immiscible, M:
Miscible, M*: Sparingly Miscible}
14
15

Programme:
Practical
Title:
Semester:
III
Separation of various components in different lipid factions by TLC
Experiment. No.: 5
Date: 24.09.2015
Aim:
To identify the components of a lipid mixture using TLC
Principle:
In Chromatography techniques, the separation of components of a

mixture takes place because of the partition co-efficient of these
components between two immune-soluble phases, separation is due to
small variation in physical and chemical properties which result from
structural differences of chemically related group of compounds under
investigation. They therefore have a relatively lesser or greater affinity for
mobile or stationary phase of chromatographic system and the separation
is based on the difference in there affinities for stationary and mobile
phase. These differences are due to the two physical phenomenon
exhibited by compounds.
1)
Adsorption
2)
Partition
On heat activated layer of silica gel a mixture of unknown lipids is

spotted and run to determine its constituent lipid by their comparison with
the standard lipid spots on the basis of Rf.
Rf = Distance travelled by solute (lipid)
Distance travelled by solvent front
Requirement:
1.
2.
3.
4.
5.
6.
7.
Activated TLC plates.

Mixture of unknown lipids.
Standard lipid samples.
Capillary/micropipette.
Solvent system: Hexane: di-ethyl-ether: g.a.a (80:20:1)
Saturated solvent chamber.
Oven at 110 C.
16
1. A clean grease-free plate is placed on top of a tray and slurry of
Procedure:
silica gel is prepared and spread evenly over it by means of an

applicator.
2. The plates are dried and activated in a hot air oven at 110C for 45
minutes.
3. The plates are then spotted with small amounts of standard lipid
solutions and unknown samples using capillaries.
4. The plates are then placed in a glass chamber saturated with
solvent system.
5. When the solvent front reaches at least two third of silica gel
layer, the plates are then removed and solvent front is marked.
After drying for 5 minutes, they are then sprayed with 50%
H2SO4 solution (to develop colour) and heated at 120oC for 30
minutes.
6. Spots are marked and the Rf value is measured and compared with
the standard.
Observation table:
Sample
Distance
travel by
sample(cm)
Distance travel Rf = (dist

by solvent (cm) solute)/(dist
solvent)
Colour
of
sample
Oleic Acid
2.6
0.37
Purple
Palmitic
acid
Steric acid
Unknown
sample 2
2.5
0.35
Purple
Result:
Unknown sample 2 contains Oleic acid among other chemicals.
Conclusion:
Only Oleic acid, among the three standards, was visible. Hence, we
cannot comment on the presence/absence, of Palmitic acid or Steric acid,
in the given sample.
17

Programme:
Practical
Title:
III
Determination of max & verification of Beer Lamberts law
Experiment. No.: 6
Aim:
Semester:
Date: 08.10.2015
To determine max of a given coloured solution and to verify BeerLamberts law.
Principle:
Lamberts law: Talks about the proportional-relationship between the
path length (L) of light and absorbance (A). A L (1)
Beers law: Talks about the inverse-proportional-relationship between
amount of transmitted light (T) and concentration (C). T 1/C
It is noteworthy that absorbance decreases with increase in amount of

transmitted light, hence A 1/T A C .........(2)
From (1) & (2) A CL
Beer Lambert Law:

It states that when a monochromatic light passes through a coloured
solution, the amount of light transmitted decreases exponentially:
With the increase in concentration of the coloured substance.
With the increase in length of medium through which light passes.
The following quantitative relationship can be drawn:

i) If Io, is the intensity of incident light and I is the intensity of
transmitted light then I/Io is called the Transmittance.
ii) If Io is taken as 100 then I is represented as percent transmission.
Absorbance A CL A = CL
Where C is the concentration (g/l), L is length in cm through which light
18
passes & is called extinction coefficient.

When C=1 mol/L and L=1cm, then measured Extinction = A
This means absorbance is directly proportional to concentration of
solution. Therefore the graph of absorbance against concentration gives a
straight line passing through origin.
Requirement: Colorimeter, 1% K2Cr2O7, 1% CuSO4, D/W, cuvets, filters: 450nm,

470nm, 510nm, 520nm, 540nm, 570nm, 600nm and 670nm.
Procedure:
A) Determination of max:
1. Take 1% K2Cr2O7 and 1% CuSO4.
2. Adjust the colorimeter to100% transmittance / zero absorbance with
distilled water as a blank.
3. Read the absorbance of K2Cr2O7 & 1% CuSO4 over entire range of
wavelength from 450nm to 670nm using colorimeter.
4. For every wavelength repeat the blank adjustment using D/W.
5. Note down the absorbance at the corresponding wavelength.
6. Plot a graph of absorbance against wavelength and join the
consecutive points. The wavelength showing maximum absorbance or
optical density is max.
B) Verification of Beers Law:
1. Prepare a series of dilution of K2Cr2O7 and measure the absorbance of
the K2Cr2O7 dilutions of different concentrations at max.
2. Plot the graph of absorbance against concentration & obtain a straight
line passing through origin.
Observation table:
Sr.
No.
1
2
3
4
5
Concentra
tion of
K2Cr2O7
(%)
0.2
0.4
0.6
0.8
1.0
Volume of
K2Cr2O7
(ml)
Volume of
D/W (ml)
Diluent
(ml)
Total
volume
(ml)
Absorban
ce
0.6
1.2
1.8
2.4
3
2.4
1.8
1.2
0.6
0
10
10
10
10
10
13
13
13
13
13
0.30
0.75
0.87
1.67
1.80
19
Sr.No.
1
2
3
4
5
6
7
8
K2Cr2O7
Wavelength
Absorbance
450
1.49
470 max
1.58
510
0.65
520
0.21
540
0.07
570
0.00
600
0.00
670
0.00
CuSO4
Wavelength
450
470
510
520
540
570
600
670 max
Absorbance
0.01
0.02
0.02
0.01
0.02
0.06
0.10
0.21
Result:
max value of K2Cr2O7 & CuSO4 is 470nm & 670nm respectively
Conclusion:
It was observed that the absorbance increases with increase in

concentration of K2Cr2O7 and hence the Beer-Lamberts law stands
verified.
20
21

Programme:
Practical
Title:
Semester:
III
Paper chromatography of amino acids
Experiment. No.: 7
Date: 13.10.2015
Aim:
To identify the presence of proline, glyceine and alanine in the given

environmental sample using paper chromatography.
Principle:
Chromatography is a technique by which substances in a mixture are separated

based on their differential partition coefficients in two immiscible solvents (a
polar and a nonpolar solvent). In ascending paper chromatography the Whatman
filter paper serves as a support for the stationary hydrophilic polar solvent. The
hydrophobic solvent serves as the mobile phase which ascends up the paper
chromatogram by capillary action. Thus a mixture of amino acids spotted on the
filter paper is separated based on their partition coefficients in the stationary and
mobile phases. Those amino acids which have a higher affinity for the
hydrophilic stationary phase move slower (polar amino acids) than those which
have a higher affinity for the mobile hydrophobic phase. The separated amino
acids can be detected using Ninhydrin reagent and identified by their unique Rf
(Relative to front) values. This technique can be used to determine the
composition of amino acids in a protein.
Rf = Distance travelled by solute (amino acid)
Distance travelled by solvent front
Requirement:
1. Whatmans filter paper no. 1,

2. Standard amino acids, protein hydrolysate containing mixture of amino acids
(glyceine, alanine & proline).
3. Solvent system butanol acetic acid, water ( 4:1:5) in solvent chamber.
4. 0.2 % Ninhydrin
5. Oven set at 80oC
22
Procedure:
1. Add the required amount of solvent system in the solvent chamber and allow
the chamber to saturate.
2. Spot the standard amino acids in W1 equidistant from each other and 2 cm
from the bottom of the filter paper. Also spot the mixture of amino acids
whose components have to be identified.
3. The spots can be concentrated by spotting for a second time after sufficient
time is given for the initial spot to dry.
4. Transfer the chromatography paper into the solvent chamber such that the
bottom of the Whatman paper just touches the solvent system and the amino
acid spots do not go into the solvent.
5. Allow the solvent system to rise up by capillary action over the amino acid
spots. The amino acids in the mixture will separate as per their partition
coefficients. Remove chromatogram from chamber and mark the solvent
front. Allow the filter to dry. And spray it with Ninhydrin.
6. Keep the filter paper in an oven for 5 minutes.
7. Determine distance travelled by solvent (y cm) and distance travelled by
standard amino acids and amino acids in mixture (x cm) using a scale. Record
results in tabular form.
8. Identify the amino acids in the mixture by comparing Rf values and color of
spots in mixture and Rf values and color of standard amino acids (Proline
and hydroxyproline are yellow in color) and calculate Rf values as x/y.
Observation table:
Sample
Distance travelled by
Sample (A)
Distance travel by
Solute(B)
Rf=(A)/(B)
Proline
1.5cm
6cm
0.25
Glyceine
0.7cm
6cm
0.11
Alanine
1.3cm
6cm
0.21
Unknown
0.8cm & 1.6cm
6cm
0.13 & 0.26
Result:
The unknown sample contains proline & glceine.
Conclusion
Only proline and glyceine were found in the unknown sample as their Rf values
corresponded with that of standards and hence alanine was absent.
23

Programme:
Practical
Title:
Semester:
III
Qualitative tests for proteins and amino acids
Experiment. No.: 8
Date: 17.10.2015
Aim:
To test for the presence of amino acids and proteins using Ninhydrin
and Biuret reagents.
Principle:
Proteins are polymers of amino acids linked together by peptide bonds

(CO-NH). There are 20 different amino acids having the common general
structure, where R can vary. Based on their R groups, amino acids are
classified as Polar, Hydrophilic (acidic/ basic/ or uncharged polar) amino
acids and nonpolar amino acids. During protein synthesis in the
ribosomes amino acids are linked together by condensation between the
amino group of one amino acid and the carboxyl group of another amino
acid.
Ninhydrin test for amino acids:
To 1ml of sample solution (adjusted to neutrality) 5 drops of Ninhydrin
solution is added. The solution is placed in a boiling water bath for 2
minutes and observed for appearance of purple, violet (cysteine) or
yellow (proline and hydroxyl proline) color.
Biuret test for polypeptides:
A general test where Alkaline copper sulphate reacts with compounds
containing two or more peptide bonds, to give a violet/pink product. This
is due to the formation of co-ordination complex of cupric ions with
unshared pair of electrons of peptide nitrogen as oxygen of water.
Requirement:
1.
2.
3.
4.
Test solution
0.2% ninhydrin
biurets reagent
boiling water bath
24
Procedure:
Ninhydrin test for amino acids:
To 1ml of sample solution (adjusted to neutrality) 5 drops of Ninhydrin
solution is added. The solution is placed in a boiling water bath for 2
minutes and observed for appearance of purple, violet (cysteine) or
yellow (proline and hydroxyl proline) color.
Biuret test for polypeptides:

To 1ml of sample solution (adjusted to neutrality) 0.1ml of NaOH
solution is added. To this, 2 to 5 drops of copper sulphate is added and
observed for appearance of violet/pink color.
Observation:
Ninhydrin test: Blue-violet color.

Biuret test:
Purple
Result:
The environmental sample was found to contain proteins as both

ninhydrin & biuret tests yielded positive results.
25

Programme:
Practical
Title:
III
Starch extraction
Experiment. No.: 9
Aim:
Semester:
Date: 17.10.2015
To quantify the amount of starch present in 1gm of potato sample.
Requirement:
1.
2.
3.
4.
5.
Finely chopped potato (5gm)

Water
Pestle-mortar
Muslin cloth
W1
Procedure:
1. Take potato sample (5gm) in pestle-mortar and add 10ml water.

2. Crush & grind the sample.
3. Filter the contents using 2 layers of muslin cloth.
4. Collect the filtrate in a 100ml beaker.
5. Allow the starch to settle (15mins).
6. Weigh the petri-plate with W1.
7. Collect the starch in W1 .
8. Air dry the starch for 16 hours and calculate the weight.
Calculation:
W1 Weight of petri-plate & W1 = 97gm
W2 Weight of petri-plate, W1 & starch = 97.320gm
W3 = W2 W1 = 0.32gm
If W3 gm in 5gm, then X gm in 1gm
Xgm = 0.064gm per gram of potato
Result:
0.064gm of starch was to be present in 1gm sample of potato.
26
27

Programme:
Practical
Title:
Semester:
III
Estimation of reducing sugar by DNS method
Experiment. No.: 10
Date: 20.10.2015
Aim:
To estimate the amount of sugar preset in the sample by DNSA

method.
Principle:
Maltose is a reducing sugar which will reduce 3,5 - dinitro salicylic acid
(DNSA) to 3-amino, 5-nitro salicylic acid (ANSA) which is an orange
colored solution and can be estimated at 540nm.
Requirement:
20 mg % of maltose stock solution, unknown sugar, DNSA reagent &

D/w.
Procedure:
1. Prepare varying concentrations of standard maltose solution ranging

from 40 g/ml to 200 g/ml with an interval of 40 g/ml.
2. In the test tube labelled UK, pipette out 2ml of maltose solution of
unknown concentration. Add DNSA (1ml) and mix.
3. Mix contents of each tube and heat for 10 minutes in a water bath.
4. Cool the tubes and add 7ml of D/W in all tubes. Mix contents of each
tube by vortexing.
5. Record the absorbance at 540 nm against the blank.
6. Draw a standard graph by plotting the concentration (g/ml of
maltose on X- axis against the absorbance on the Y-axis.
7. 7) Determine concentration of maltose in given sample.
Calculation:
Given concentration: 200 g/ml
Required concentration: 40 g/ml
Total volume required: 2ml
(RxT)/G = 40 g/ml x 2ml = 0.4ml

200 g/ml
28
Observation table
Sr
Standard Conc.
Amount
Amount
Amount
D/W
O.D at
No.
(g/ml)
of stock
of D/W
of DNSA
(ml)
540nm
(ml)
(ml)
(ml)
0.00
2.0
0.00
40
0.4
0.8
0.07
80
0.8
0.6
0.09
120
1.2
0.4
0.14
160
1.6
0.2
0.16
200
2.0
0.0
0.21
Unknown
2.0
0.0
0.20
If 120g/ml 0.14 OD then z 0.20 OD z = 171.4g/ml

Concentration of z by graph = 170g/ml
& by calculation = 171.4g/ml
Result:
The concentration of maltose, in the given sugar sample, by graph &

calculation, comes out to be 170g/ml & 171.4g/ml respectively.
29

Programme:
Practical
Title:
Semester:
III
Estimation of protein by Biuret method
Experiment. No.: 11
Date: 29.10.2015
Aim:
To quantify the amount of proteins present in the given

environmental sample using Biuret test
Principle:
The test is a general test for compounds having a peptide bond. Alkaline
CuSO4 solution reacts with compounds containing two or more peptide
bonds to give a violet colored complex. The intensity of colour contained
is directly proportional to number of peptide bonds present in protein. The
concentration of an unknown sample can be estimated using a series of
standard concentrations.
Requirement:
Biuert reagent, standard stock solution of Bovine Serum Albumin (BSA)

of 10 mg/ml concentration, unknown sample, colorimeter & D/W.
Procedure:
1. Prepare a set of known standard varying concentrations of BSA from

0 to 5mg/ml with the interval of 1mg/ml, using the stock BSA and
distilled water given in table as given in the observation table.
2. In other test tubes labelled x & y, pipette out 2ml of given protein
sample and add 3ml Biuret reagent (for x and dilute it 1:1 with D/W
for y)
3. Incubate at room temperature for 10 minutes and then read against
blank at 540 nm.
4. Plot a graph of O.D (Y- axis) versus standard concentration of protein
(X-axis) and calculate concentration of protein in given sample by
extrapolating OD of sample onto X-axis.
30
Observation table:
Amount of
Stock BSA
(ml)
0
Distilled
water (ml)
Required
Standard
concn(mg/ml)
0
O.D at
540nm
Biuret
Reagent
(ml)
3
0.2
1.8
0.07
0.4
1.6
0.15
0.6
1.4
0.23
0.8
1.2
0.33
0.38
0.19
0.10
Sr
No.
0.00
Calculation:
For x:
For y:
3mg/ml BSA 0.23 OD & x 0.19 OD
3mg/ml BSA 0.23 OD & y 0.10 OD
x = 2.47mg/ml
y = 1.30mg/ml
Concentration of x by graph = 2.5mg/ml
Concentration of y by graph = 1.3mg/ml
& by calculation = 2.47mg/ml
& by calculation = 1.3mg/ml
Result:
The protein concentration in the unknown sample x & y was found to

be 2.47mg/ml & 1.30mg/ml respectively.
31

Programme:
Practical
Title:
Semester:
III
Extraction of casein from milk
Experiment. No.: 12
Date: 05.11.2015
To calculate the percentage of casein in the given milk sample
Aim:
Principle:
Requirement:
1. Add 50ml of milk in 100ml beaker and heat it to 55oC.
Procedure:
2. Add acetate buffer to the milk, while stirring, till it gets turbid.
3. Collect the precipitate by filtering the contents using a muslicclothe & funnel.
4. Wash with 25ml 1:1 ethylalcohol: ethylether.
5. Collect the precipitate by filtering the contents using a W1 &
funnel and air dry.
6. Measure the weight & calculate the percentage of casein.
Calculation:
Weight of petri-plate & W1 : 93.18gm & Weight of petri-plate, casein & W1 : 102.59gm
Casein % = Weight of casein x 100
Total volume
Result:
(102.59gm - 93.18gm) x 100 18.82%

50gm
Total protein content in percentage is 18.82%
32
33

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Students name:

Harshavardhan Mahalakshmi Ganesan

Preparation of buffer solutions

Qualitative tests for carbohydrates

Lipid solubility test

max & Beer-Lamberts law

Paper chromatography of A.A

Reducing sugar estimn - DNS method 28

Protein estimation by Biuret method

Extraction of casein from milk

Qualitative tests for proteins and A.A 24

This is to certify that the work entered in this

School of Biotechnology & Bioinformatics

D. Y. PATIL UNIVERSITY, NAVI MUMBAI

To Study the construction & working of pH Meter.

Acids dissolve in water forming H+ ions. The greater the concentration

pH= -log10 [ H+]

pH is the degree of acidity or alkalinity of a solution on a scale of 1 to

1. STANDARDISATION OF pH METER: Sureties on the pH

pH 9 to ensure that the pH is functioning

properly over the entire range. The electrodes should be washed

The construction and working of pH meter was observed and studied.

1. pH meter bulb should not be allowed to dry, so it should be kept immersed

D. Y. PATIL UNIVERSITY, NAVI MUMBAI

Preparation of buffer solutions

To prepare solutions and buffers

A buffer system is one that can resist a change in pH on addition

Buffers are extensively used in biochemical studies since they aid

constituents. For e.g. the activity & stability of extracted enzyme

Henderson Hasselbach equation can be used to express the

K= dissociation constant of the acid by rearranging,

[H+] = K [HA]/ [A-]

Taking negative logarithm on both the sides;

pH = pKa (log [HA]) / [A-]

pH= pKa + (log [A-]) / [HA]

This can be written in the general form as:

pH= pKa + log[conjugate base]/[conjugate acid]

Solution: Any non-reactive substances mixed together homogenously

Q3. Prepare 150ml of 70% alcohol from 95% stock.

D. Y. PATIL UNIVERSITY, NAVI MUMBAI

Qualitative tests for carbohydrates

To detect the presence and type of carbohydrate(s), in the given

Carbohydrates are compounds consisting of carbon, hydrogen and

Molischs test:- (General test for Carbohydrates):

Iodine test for Polysaccharides:

agent such as Glucose or Maltose.

Molischs Test: (for carbohydrates)

Iodine Test: (for polysaccharides)

Barfoeds test: (for mono/disaccharides)

-naphthol + conc H2SO4 +

Iodine solution + sample

Reagent + sample {boiling

water bath for 5-10 minutes}

Reagent + sample {boiling

water bath for 5 minutes}

Reagent + sample {boiling

Reagent + sample {boiling

The given sample (B) is a ketohexose monosaccharide reducing sugar.

D. Y. PATIL UNIVERSITY, NAVI MUMBAI

Lipid solubility test

Fatty acid samples, distilled water, acetone, chloroform & ethylether

1. Add 0.5 ml of each of the seven solvents in seven test tubes.

Observation & Result:

D. Y. PATIL UNIVERSITY, NAVI MUMBAI