D. Y.

PATIL DEEMED TO BE UNIVERSITY, NAVI MUMBAI

SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
Plot – 50, Sector – 15, CBD Belapur, Navi Mumbai – 400 614

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Date :

D. Y. PATIL DEEMED TO BE UNIVERSITY, NAVI MUMBAI

 SCHOOL OF BIOTECHNOLOGY AND BIOINFORMATICS
PRACTICAL MANUAL

Programme:B.Tech (Biotechnology) Semester-I

Practical Title:- Applied Chemistry

Experiment 1:-Oxidation-Reduction titrations

Aim:-Estimate the strength of the given sample of KMnO 4 solution in g/litre. Prepare a
standard solution of N/10 oxalic acid.

Principle:- A solution of oxalic acid acidified with dilute sulphuric acid is titrated slowly
with potassium permanganate at a temperature between 60-70°C until a permanent pink
color gets developed in the solution. The reaction is as follows:

2KMnO4 + 3H2SO4 K2SO4 + 2MnSO4 + 3H2O + 5[O]

[H2C2O4 + (O) 2CO2 + H2O] ×5

2KMnO4 + 3H2SO4 + 5H2C2O4 K2SO4 + 2MnSO4 + 10CO2 + 8H2O

-
Or 2MnO4 + 5C2O42- + 16H+ 2Mn2+ + 10CO2 + 8H2O

Requirements:-
a. Chemicals required:- Oxalic acid, KMnO4 solution, dilute H2SO4.
b. Glassware required:- Burette, Burette stand, pipette, beaker, conical flask,
standard volumetric flask, glass funnel, tripod stand, wire guaze, gas burner, digital
balance, glass rod etc.
Indicator used: KMnO4 (Self indicator)
End Point:Colourless to permanent pink.
Procedure:-
I. Preparation of N/10 oxalic acid
1. Weigh out accurately 1.675g of pure oxalic acid crystals in a watch glass or a butter
 paper.
2. Transfer it into a 100 ml standard volumetric flask.
3. To this add minimum quantity of distilled water and dissolve the crystals of oxalic
acid by shaking gently and then make the solution up to the convenient mark on it
by adding more distilled water.
4. Shake the flask up and down to ensure thorough mixing.
II. Titration of KMnO4 with oxalic acid
1. Rinse the burette with potassium permanganate solution and fill it up to a
convenient mark.
2. Pipette out 10ml of the standard solution of N/10 oxalic acid into a conical flask
and add to it a test tube of dilute sulphuric acid.
3. Heat the flask on a wire guaze to 60-70°C, i.e., to a temperature which is just
unbearable to touch by hand.
4. Note the initial reading of the burette by noting the upper meniscus.
5. Run the potassium permanganate solution slowly in small amounts at a time into
the hot solution and shake till the end point is reached.
6. The end point is a change of colorless solution to a just permanent pink solution.
7. Note the final reading of the burette by noting the upper meniscus only.
8. Repeat the titration to get a set of three concordant readings.

Observation Table:-Titration of KMnO4with oxalic acid

Burette reading (ml) Final volume

Sr.No. Volume of Oxalic acid Initial Burette Final burette of KMnO4 (ml)
(ml) reading (ml) reading (ml)
1.
2.
3.
4.

Calculations:-Apply law of equivalence, N1V1=N2V2

Result:- The strength of a given sample of KMnO4 solution is ……...g/litre.

Experiment No:- 2
Aim:- To prepare a standard N/30 of ferrous ammonium sulphate and determine the
strength of given ferrous ammonium sulphate in g/litre using potassium dichromate as an
intermediate solution.

Principle:- In the presence of dilute sulphuric acid K2Cr2O7 oxidizes ferrous ion into ferric
ion according to the following reaction:
6[ FeSO4.(NH4)2SO4.6H2O] + K2Cr2O7 + H2SO4 3Fe 2(SO4)3 + Cr2SO4)3 + K2SO4 +
6(NH4)2SO4 + 43H2O
Ferrous ion reacts with potassium ferricyanide solution an external indicator producing a
blue colour of Turnbull’s blue (ferri ferro cyanide).
3Fe2+ + 2[Fe(CN6)]3- Fe3[Fe(CN)6]2
At the end point no blue colour is produced as all the ferrous ions present in the solution
have completely oxidized to ferric ions.

Requirements:-
a. Chemicals required: N/30 ferrous ammonium sulphate solution, unknown ferrous
ammonium sulphate solution, standard potassium dichromate solution, dilute
sulphuric acid solution, potassium ferricyanide solution (external indicator).
b. Glasswares required:Burette, pipette, volumetric flask, conical flask, beaker,
burette stand, glass funnel, a white tile or a porcelain plate, glass rod, dropper.
Indicator used: Potassium ferricyanide (External indicator)
End Point: Prussian blue to light green ( no change in the colour of the indicator).

Procedure:-
I. Preparation of N/30 Ferrous ammonium sulphate solution.
1. The equivalent weight of ferrous ammonium sulphate solution is 392.
2. For preparing 250ml of N/30 ferrous ammonium sulphate solution weigh accurately
3.26g ferrous ammonium sulphate crystals.
3. Transfer the weighed crystals of ferrous ammonium sulphate into 100 ml standard
volumetric flask containing 5ml of dilute H2SO4.
4. Dissolve the solid by shaking and make up the solution up to the convenient mark
by adding more distilled water.
II. Titration of K2Cr2O7 with N/30 Ferrous ammonium sulphate solution.
1. Rinse the burette with K2Cr2O7solution and fill the burette up to the convenient
mark.
2. Place a number of drops of potassium ferricyanide solution on a white tile with the
help of a glass rod or a dropper.
3. Pipette out 10ml of N/30 ferrous ammonium sulphate solution in a conical flask. To
it add half test tube of dilute sulphuric acid.
4. Run potassium dichromate solution from the burette into the conical flask in 1ml
lots and stir the reaction mixture.
5. Take out a drop of a reaction mixture from the titration flask with the help of a
glass rod and mix it with one of the drops of ferricyanide indicator on the tile.
[Note: As far as possible take out least number of drops of the reaction mixture
every time to maintain accuracy]. A deep blue color will appear. This shows the
presence of ferrous ions in the solution. The presence of a blue color indicates that
the end point has not yet reached. [Note: If no blue color is produced, the end point
is passed].
6. Continue the addition of dichromate solution in 1ml lots and after each addition
take out a drop of a reaction mixture and mix it with one of the drop of indicator.
Repeat the process till no blue color is obtained. [Note: the glass rod should be
washed every time after testing the titration mixture and then put it in the beaker].
7. As the addition of K2Cr2O7solutionis continued and the drops of the reaction
mixture are mixed with the indicator drops, the blue color will be replaced by
greenish blue, then light greenish color and finally when the reaction is over (i.e.,
when all the ferrous ion has changed into ferric ions) no change in the color of the
indicator will be produced.
8. The first titration is the rough one, it gives an idea about the range in which the end
point lies. Suppose a greenish color is produced at 23ml and at 24ml no color is
produced. Then the end point lies in between 23ml and 24ml i.e., 23.5ml.
9. In the second titration add 23ml of K 2Cr2O7solutionwithout taking out any drop
 from the titration mixture for testing, then add dichromate solution in small aliquots
(0.2ml) then drop wise and note the last drop which produce no change in the color
of the indicator drop.
10. Repeat the titration to yield three concordant readings.
III. Titration of K2Cr2O7 solutionwith unknown ferrous ammonium sulphate
solution.
In a similar manner repeat the titration with unknown ferrous ammonium sulphatesoluiton.

Observation Table:- For the titration of K 2Cr2O7 with N/30 Ferrous ammonium
sulphate (FAS) solution.
Burette reading (ml) Final volume
Sr.No. Volume of FAS (ml) Initial Burette Final burette of K2Cr2O7
reading (ml) reading (ml) (ml)
1.
2.
3.
4.

Observation Table:- For the titration of K 2Cr2O7 with unknown Ferrous ammonium
sulphate (FAS) solution.
Burette reading (ml) Final volume
Sr.No. Volume of unknown Initial Burette Final burette of K2Cr2O7
FAS (ml) reading (ml) reading (ml) (ml)
1.
2.
3.
4.

Calculations:- Apply law of equivalence, N1V1=N2V2=N3V4

Result:- The strength of a given unknown ferrous ammonium solution is …………..g/litre.
Experiment No:- 3
Aim:- To determine the strength of given K2Cr2O7 solution with the help of N/30 ferrous
ammonium sulphate solution using potassium ferricyanide solution as an external
indicator.

Principle:- In the presence of dilute sulphuric acid K2Cr2O7 oxidizes ferrous ion into ferric
ion according to the following reaction:
6[ FeSO4.(NH4)2SO4.6H2O] + K2Cr2O7 + H2SO4 3Fe 2(SO4)3 + Cr2SO4)3 + K2SO4 +
6(NH4)2SO4 + 43H2O
Ferrous ion reacts with potassium ferricyanide solution an external indicator producing a
blue colour of Turnbull’s blue (ferri ferro cyanide).
3Fe2+ + 2[Fe(CN6)]3- Fe3[Fe(CN)6]2
At the end point no blue colour is produced as all the ferrous ions present in the solution
have completely oxidized to ferric ions.

Requirements:-
a. Chemicals required: N/30 ferrous ammonium sulphate solution, unknown ferrous
ammonium sulphate solution, standard potassium dichromate solution, dilute
sulphuric acid solution, potassium ferricyanide solution (external indicator).
b. Glasswares required: Burette, pipette, volumetric flask, conical flask, beaker,
burette stand, glass funnel, a white tile or a porcelain plate, glass rod, dropper.
Indicator used: Potassium ferricyanide (External indicator)
End Point: Prussian blue to light green ( no change in the colour of the indicator).

Procedure:-
I. Preparation of N/30 Ferrous ammonium sulphate solution.
1. The equivalent weight of ferrous ammonium sulphate solution is 392.
2. For preparing 250ml of N/30 ferrous ammonium sulphate solution weigh accurately
3.267g ferrous ammonium sulphate crystals.
3. Transfer the weighed crystals of ferrous ammonium sulphate into 100 ml standard
 volumetric flask containing 5 ml of dilute H2SO4.
4. Dissolve the solid by shaking and make up the solution up to the convenient mark
by adding more distilled water.
II. Titration of K2Cr2O7 with N/30 Ferrous ammonium sulphate solution.
1. Rinse the burette with K2Cr2O7solution and fill the burette up to the convenient
mark.
2. Place a number of drops of potassium ferricyanide solution on a white tile with the
help of a glass rod or a dropper.
3. Pipette out 10ml of N/30 ferrous ammonium sulphate solution in a conical flask. To
it add half test tube of dilute sulphuric acid.
4. Run potassium dichromate solution from the burette into the conical flask in 1ml
lots and stir the reaction mixture.
5. Take out a drop of a reaction mixture from the titration flask with the help of a
glass rod and mix it with one of the drops of ferricyanide indicator on the tile.
[Note: As far as possible take out least number of drops of the reaction mixture
every time to maintain accuracy]. A deep blue color will appear. This shows the
presence of ferrous ions in the solution. The presence of a blue color indicates that
the end point has not yet reached. [Note: If no blue color is produced, the end point
is passed].
6. Continue the addition of dichromate solution in 1ml lots and after each addition
take out a drop of a reaction mixture and mix it with one of the drop of indicator.
Repeat the process till no blue color is obtained. [Note: the glass rod should be
washed every time after testing the titration mixture and then put it in the beaker].
7. As the addition of K2Cr2O7solutionis continued and the drops of the reaction
mixture are mixed with the indicator drops, the blue color will be replaced by
greenish blue, then light greenish color and finally when the reaction is over (i.e.,
when all the ferrous ion has changed into ferric ions) no change in the color of the
indicator will be produced.
8. The first titration is the rough one, it gives an idea about the range in which the end
point lies. Suppose a greenish color is produced at 23ml and at 24ml no color is
produced. Then the end point lies in between 23ml and 24ml i.e., 23.5ml.
 9. In the second titration add 23ml of K 2Cr2O7solutionwithout taking out any drop
from the titration mixture for testing, then add dichromate solution in small aliquots
(0.2ml) then drop wise and note the last drop which produce no change in the color
of the indicator drop.
10. Repeat the titration to yield three concordant readings.

Observation Table:- For the titration of K 2Cr2O7 with N/30 Ferrous ammonium
sulphate (FAS) solution.
Burette reading (ml) Final volume
Sr.No. Volume of FAS (ml) Initial Burette Final burette of K2Cr2O7
reading (ml) reading (ml) (ml)
1.
2.
3.
4.

Calculations:- Apply law of equivalence, N1V1=N2V2

Result:- The strength of a given unknown K2Cr2O7solution is …………..g/litre.

Experiment 4:- Chemical Kinetics

Aim:-To study the order of a reaction for the hydrolysis of ethyl acetate at room temperature in
the presence of hydrochloric acid.

Principle:- Ethyl acetate is hydrolysed to give ethyl alcohol and acetic acid as given below:
H+
CH3COOCH2CH3 + H2O CH3COOH + C2H5OH
The reaction is catalyzed by hydrogen ions. In a dilute aqueous solution of ester, concentration of
water being in excess, is very high and practically remains constant during the reaction. The
concentration of H+ which catalyze the reaction also remains constant. Thus, the rate of reaction
is dependent only on the concentration of the ester i.e.

dx
=k [CH3COOCH2CH3]
dt
Following the first order kinetic equation of first order:
2.303 a
k= log
t a−x
Where, k is rate constant of the reaction;‘t’ is time; ‘a’ is initial concentration of reactant (ethyl
acetate); x is concentration of the reactant (ethyl acetate) reacted at time t; (a-x) is the
concentration of reactant (ethyl acetate) remain unreacted at time t.
Since acetic acid is produced as a result of hydrolysis, the kinetics of reaction can be followed by
withdrawing a fixed volume of the reaction mixture from time to time, and titrating with standard
alkali. The titre value is equivalent to the sum of the acid used as the catalyst, which remains
constant throughout, and of the acetic acid produced during the reaction. The difference of titre
values at any time after the commencement of the reaction and that at the commencement gives
the acetic acid formed, and hence the amount of ethyl acetate hydrolysed at that instant.

Requirements:-
a. Chemicals required: 0.1N NaOH solution, 0.5N HCl solution, ethyl acetate,
phenolphthalein indicator and ice.
 b. Glasswares required: Burette, pipette, conical flask, beaker, burette stand, water bath,
thermostat, stop watch.
Indicator used: Phenolphthalein
End Point:Colourless to pink.
Procedure:-
1. Take 100ml of 0.5N HCl in a 250ml conical flask.
2. To another flask add 5ml of ethyl acetate and cover it.
3. Keep both the flasks in waterbath to attain room temperature (about 5 minutes).
4. Rinse and fill the burette with 0.1N NaOH solution.
5. To another conical flask add one piece of ice and two drops of phenolphthalein indicator
and keep it ready for titration.
6. When temperature equilibrium has been reached in both the flasks, pipette out 5ml of
ester and add it into the conical flask containing acid, shake well. This ia called reaction
mixture.
7. Immediately pipette out 5ml of this reaction mixture and add it into the flask containing
ice and phenolphthalein indicator. Note the time of mixing as zero time (T0). Titrate the
solution as rapidly as possible with 0.1N NaOH. The end point is colourless to pink. Note
the reading. The titre value gives the amount of HCl in the sample at the start of the
reaction.
8. Make similar titrations of further 5ml samples at successive intervals of 10,20,30,40 &50
minutes from the zero time against 0.1N NaOH and record the readings.
9. Place the reaction vessel containing the remaining reaction mixture in water bath at about
50°C for at least 1hour to complete the reaction. Then after cooling the flask to the room
temperature, titrate 5ml of the reaction mixture as explained before. Record the reading.
This reading is considered as T∞.
Observation Table:
S.No. Time(t) Burette reading (ml) T∞-Tt log T∞-Tt log (T∞-Tt) k ( from
minutes table)
T∞-Tt

Calculations: Apply first order kinetic equation,

2.303 a
k= log
t a−x
Plot a graph against time‘t’ and log (T∞-Tt) and determine k from slope.

T∞-Tt

Result: The rate constant for hydrolysis of ethyl acetate is……………..

Experiment No.5
Aim:- To identify the functional groups in the given organic compound.
Test for Carboxylic Acids
S.No . Experiment Observation Inference
1. Preliminary test
Physical test Solid crystalline white Acid may be present
powder
Colour Colourless
Odour Odourless
Solubility
2. Flame test
Take a pinch of the substance on a Non-smoky flame Aliphatic compound is
spatula & heat it on the flame. present.
Take a pinch of the substance on a Smoky flame Aromatic compound is
spatula & heat it on the flame. present.
3. Detection of functional group
General test:- Brisk effervescence with Carboxylic acid may be
1. To the aqueous solution of the evolution of CO2 present.
the substance add a pinch of
NaHCO3.
Test for Oxalic acid Pink colour of KMnO4 Oxalic acid is present.
(White, crystalline, water soluble). disappears.
1. To 2ml of neutral solution
of acid add few drops of
CaCl2. A white precipitate
of calcium oxalate is
obtained. Dissolve this ppt.
in dil.H2SO4 and add 2-3
drops of dil.KMnO4
solution.
Test for Salicylic acid Violet coloration. Succinic acid is present.
(White solid, water insoluble).
1. To 1ml of aqueous solution
 of water add few drops of
FeCl3 solution.
2. Oil of winter green test: A characteristic smell of
oil of winter green
Substance + 1ml of methyl alcohol and (methyl salicylate) is
conc. H2SO4. Heat, cool and pour the obtained.
contents into a beaker containing 20ml
of water.
Test for formic acid A greenish blue colour is Formic acid is present.
(Colourless, pungent smelling, water produced.
soluble).
1. Compound+ NaHSO3,
warm+ Sodium
nitroprusside.
(Distinction from acetic acid). A sweet smell of ethyl
formate. Formic acid is present.
2. Compound + 2ml of ethyl
alcohol+ conc. H2SO4, heat.
Test for Acetic acid A fruity smell of ethyl Acetic acid is present.
( Colourless, water soluble, smell like acetate is produced.
vinegar).
1. Compound + 2ml absolute
alcohol+ conc. H2SO4, heat.
2. Neutral solution of acid+ A wine red colour
changes to brown ppt. on
FeCl3solutoin.
heating.
(Formic acid also gives this test).

Result: The following compounds are present in the given organic compound: Oxalic acid,
Succinic acid, Formic acid and Acetic acid.
Test for Alcohols
S.No. Experiment Observation Inference
I. Preliminary test
Physical test
a. Colour
b. Odour
c. Solubility
II. Detection of functional l group A red colour is produced. Alcohol group is
General test: present.
Compound + drops of ceric
ammonium nitrate solution.
Test for Methyl alcohol A black stain is produced. Presence of methyl
(Colourless liquid, water soluble, has alcohol is confirmed.
characteristic odour).
1. To 1ml of the compound
add a trace of solid K2Cr2O7
and few drops of conc.
H2SO4and heat. A pungent
odour of formaldehyde is
obtained. Place a filter paper
strip moistened with
ammonical AgNO3 near the
mouth of the test tube.
Test for ethyl alcohol A fruity smell of ethyl Presence of ethyl
(Colourless liquid, water soluble,has acetate is produced. alcohol is confirmed.
characteristic odour).
1. 1ml of the compound+ 1ml
of acetic acid and heat. Add
few drops of conc. H2SO4.
 2. To 1ml of the compound
add a trace of solid K2Cr2O7
and few drops of conc.
H2SO4and heat. A pungent
odour of formaldehyde is
A black stain is produced.
obtained. Place a filter paper
strip moistened with
Tollen’s reagent near the
mouth of the test tube.

Result: The given organic compound is Methyl alcohol and ethyl alcohol.
Test for ketones
S.No. Experiment Observation Inference
I. Preliminary test
Physical test
a. Colour
b. Odour
Solubility
II. Detection of functional l group A yellow ppt. is obtained. Aldehyde or ketone may
General test: be present.
Compound+ 2ml of 2,4 dinitrophenyl
hydrazine.
Test for Acetone
(Colourless, pleasant smelling, water
soluble).
1. Compound + sodium A wine red colour is Acetone is present.
nitroprusside solution + produced.
NaOH drop by drop.

Result: The given organic compound is Acetone.

Test for aldehydes
S.No. Experiment Observation Inference
I. Preliminary test
Physical test
a. Colour
b. Odour
Solubility
II. Detection of functional A yellow ppt. is Aldehyde or ketone may
l group obtained. be present.
General test:
Compound+ 2ml of 2,4
dinitrophenyl hydrazine.
Test for aldehyde
1. Substance + 3- The formation of a Presence of an aldehyde
4ml freshly black ppt. of metallic is confirmed.
prepared silver or silver mirror.

Tollen’s
reagent. Warm
the tube gently
in a water bath
at 50°C.

Result: The given organic compound is an aldehyde.

Test for amides
S.No. Experiment Observation Inference
I. Preliminary test
Physical test
a. Colour
b. Odour
c. Solubility
II. Flame test Ammonicalodour
Take a pinch of the Amide may be present.
substance on a spatula
& heat it on the flame.
III. Detection of functional Ammonia gas is Amide is present.
group evolved.
General test
1. Compound +
2ml NaOH
and heat.
Test for urea
1. Biuret test
Fuse a small amount of
the urea in a test tube
until it just melts and
ammonia is evolved. Appearance of violet The presence of urea is
After some time when it colour. confirmed.
resolidifies dissolve it in
little of water followed
by biuret reagent drop
 by drop.

Result: The given organic compound is urea.

Experiment 6:-Estimation of vitamin C- Iodometric Method

Aim:-To determine the amount of vitamin-C in sample volumetrically by iodometric method.

Principle:-Vitamin C (Ascorbic acid) is oxidized to dehydroascorbic acid by iodine. The excess

of iodine left behind is back titrated with standard sodium thiosulphate solution from the amount
of iodine consumed the concentration of vitamin-C can be determined. Vitamin C sample make
in 4% oxalic acid to prevent reduction back to ascorbic acid.

Requirements:-1% vitamin C sample, 0.1N I2 solution, standard 0.1 N Na2S2O3 solution, starch
indicator

Procedure:-
I. Standardization of Iodine solution

Titrate 20 ml of 0.1 N I2 solution against standard 0.1N Na2S2O3 solution using starch as an
indicator. Towards the end point the flask solution changes its color from blue to colorless.
Let this reading be ‘A’ ml.
II. Estimation of Vitamin C

Vitamin C sample given in 100 ml flask is diluted with distilled water upto 100 ml mark and
used for estimation.
1. To 100 ml of diluted vitamin C solution in a flask add exactly 20 ml of 0.1N I 2 solution
with constant stirring.
 2. Keep the flask in dark for 5 minutes and then titrate against the standard 0.1 N Na 2S2O3
solution using starch as an indicator towards the end till color of the flask solution
changes from blue to colorless.
3. Let the reading be ‘B’ ml.

Result:

Standard stock solution-100 mg Ascorbic acid in 100 ml of 4% oxalic acid (1mg/ml)

Working standard solution- 10 ml of stock to 100 ml with 4% oxalic acid. (100mg/ml)

Ascorbic acid + I2 Dehydroascorbic acid

Excess KI
Na2S2O3 + I2 Na2S2O3 + 2NaI
Experiment 7:-Standardization of pH meter and preparation of a buffer

pH meter
Aim:-To study the working of pH meter

Principle:-The EMF developed between electrode depends on the concentration on H + ions and
hence the pH. The order to measure this EMF, no current must flow in the electrode pair,
otherwise resultant chemical reaction at the all boundaries will result in polarization of electrode.
To avoid this, a high impedance (resistance) circuit is used to detect the EMF (potential
developed). This converts voltage to current which can be amplified and measured.

Theory and construction- The pH meter consist of an electrode pair which is sensitive to H +
ions concentration and an electric circuit which measures EMF developed across the electrode.
The two electrode used in pH meter are two half cells. The glass electrodes contain a glass bulb
constructed of this glass membrane i.e. permeable to hydrogen ions. As a result, potential
developed is linearly proportional to pH of the solution in which electrode are immersed. The
relationship is expressed by Nearnst equation
2.303 RT
E=E °− ×loga
nF
Where,
E = electrode potential of specified concentration
E0 = Standard electrode potential
R = gas constant
T = absolute temperature
F = Faraday’s constant
A = activity of the ion

Nearnst equation shows that the electrode response depends on temperature and number
of changes on ions. The size of potential due to H+ ions is given by the equation:
2.303 RT
E= log ¿¿
F
The polar concentration of H+ ions inside and outside the glass electrode.
Potential developed by half cell can’t be measured in absolute terms that can only be
compared with that of another half cell. The saturated calomel electrode is most frequently used
as half cell and has a standard potential of +0.242Y (relative to standard H+ electrode)
Calomel electrode thus is a reference electrode. EMF of complete cell forms by linking
two half cell (electrode) is
Eref = Eglass
Where,
Eref= at room temperature is 0.252Y and
Eglass= depends on pH of the solution
Precautions:-
1. Switch on the pH meter , allow it to warm up for 15 minutes with the electrode properly
dipped in distilled water. This prevents any fluctuation while measuring the voltage or
pH.
2. All the test solution and buffer solution should be brought to ambient temperature of
room in pH meter is kept.
3. Check the position of the pointer on scale and it should show zero. Set zero is if required.
4. The pH meter should be adjusted to measure the pH at room temperature by adjusting the
temperature knob.
5. It is important to measure pH with two standard buffers to ensure that pH meter is
functioning properly ever the entire range.
6. The electrode should be washed thoroughly with distilled water using wash bottle after
every sample measurement.
 7. After completing work on pH meter, the electrode should be washed thoroughly with
distilled water and immersed in a beaker containing fresh distilled water. This protects
electrode from drying and increases its lasting period.

Result:-

Buffer solution
Aim:-Preparation of buffer solution (acetate buffer)

Principle:-A buffer solution is one that resists pH change on addition of acid or alkali. Such
solutions are used in many biochemical experiments where the pH needs to be accurately
controlled.
From Handerson- Haselbatch equation,
[ salt ]
pH = pKa+log 10
[ acid ]
pH of a buffer solution depends on two factors. One is the pKa value and other is
ration of salt to acid. The ratio is considered to be same as the amount of salt and acid mixed
together over the pH range 4-10, where the concentration of hydrogen and hydroxyl ions is very
low and can be ignored.
In case of acetate buffer, it is a mixture of acetic acid and sodium acetate
CH3COOH CH3COO- + H+
CH3CHOONa CH3COO- + Na+
Since acetic acid is only weakly dissociated, the concentration of acetic acid is
almost the same as the amount put in the mixture likewise the concentration of acetate ion can be
considered to be the same as the concentration of sodium acetate placed in the mixture since the
salt is completely dissociated.
Some of the commonly used laboratory buffers are- Bicarbonate buffer,
phosphate buffer, tris buffer, acetate buffer, barbiturate buffer, citrate buffer.

Requirements:-
 1. Sodium acetate (0.1 mol/liter)
2. Acetic acid (0.1 mol/liter)
3. Hydrochloric acid (0.1 mol/liter)

Procedure:-
1. Acetate buffer Ph = 3.4
Amount of CH3COOH = 46.3 ml
Amount of CH3COONa =3.2 ml
Mix the contents and dilute with 100 ml with distilled water.

2. Acetate buffer ph = 5.6

Amount of CH3COOH = 4.8 ml
Amount of CH3COONa = 45.2 ml
Mix both the contents and dilute to 100 ml with distilled water.

3. Prepare the above buffer solutions and record the pH with the pH meter.

Result:-
Experiment 8:- Preparation of Sol

Theory: A sol is a colloid made out of very small solid particles in a continuous liquid medium.

Sols are quite stable and show the Tyndall effect. Examples include blood, pigmented ink, cell
fluids, paint, antacides and mud.
Artificial sols may be prepared by dispersion or condensation. Dispersion techniques include
grinding solids to colloidal dimensions by ball milling and Bredig's arc method. The stability of
sols may be maintained by using dispersing agents.
Sols are commonly used as part of the sol–gel process.
A sol generally has a liquid as the dispersing medium and solid as a dispersed phase.
Properties of a Colloid (applicable to sols)

 Heterogeneous Mixture
 Size of colloid varies from 1 nm - 100 nm
 They show the Tyndall effect
 They are quite stable and hence they do not settle down when left undisturbed

Aim: To prepare starch sol

Requirements: Starch powder, 250ml beaker, glass rod, tripod, wire gauze, burner, mortar,
distilled water.

Procedure:
1. Clean a 250ml beaker thoroughly with hot water.
2. Take 50ml of distilled water in the beaker and boil.
3. Grind 1g of starch mortar with 5ml of water to form a thin paste.
4. Gradually pour the paste into boiling water bath. Boil the mixture for (2-3 mins.)and then
allow the solution to cool. The solution thus obtained is a sol.

Result:
Precautions:
1. The apparatus should be clean.
2. Make a thin paste.
3. Add slowly to boiling water.

Aim : To prepare a ferric hydroxide sol

Reaction : FeCl3 + 3H2O → Fe(OH)3 + HCL

Requirements : 3% Fecl3, 250ml conical flask, test tube, wire gauze, tripod stand, burner,
dropper, Etc.

Procedure :
1. Take 100ml d/w and boil it in a conical flask.
2. Take 3% FeCl3 solution and add this drop-wise with the help of a dropper into the boiling
water with constant stiring.
3. Continue till deep wine red sol. of FeCl3 is obtained.
4. Cool the solution and keep it undisturbed for 15 mins.
5. We obtain the required Fe(OH)3 sol.

Result :
Experiment. No.9: Preparation of solutions - Molar, Normal, PPM, Percent

Aim: To prepare solutions - Molar, Normal, PPM, Percent for use in biological experiments.

Solution: It is a homogenous mixture of two or more non-reacting substances and has uniform
properties such as chemical composition, density, refractive index etc. It has two components:
i) Solute
ii) Solvent
Solute: It is the dissolved substance in the solutions.
Solvent: It is the medium in which the solute is dissolved.
Solutions of biochemistry practicals are generally used for
i. Extraction of biomolecules from tissues.
ii. Separation and qualitative estimations
iii. Purification
iv. Physio-chemical characterizations.
Composition of a solution can be expressed in two ways: Quantity and Concentration.
Quantity: It is the amount of any substance (solute) present in a solution/solvent irrespective of
the amount/volume of the solvent/solution.
Concentration: It is the quantity of the solute present in an exact or a specific amount of solvent
or that of solution.
Mole: It is defined as the molecular weight of a compound in grams.
Modes of expressing concentration of a solution:
1. Molarity (M): The molarity of a solution is the number of moles of the solute dissolved per
litre of the solution.
Molarity = Weight of a solute in g/L of solution
Mol. Wt. of solute

Example: To prepare 0.1M NaOH [Mol. Wt of NaOH = 40] Find amount

of NaOH per litre of solution.
Therefore, Amount (in g) of NaOH per L of solution
= Mol. Wt. NaOH x molarity
= 40 x 0.1
=4g
Thus, weigh 4 g of NaOH, dissolve it in a small volume of solvent (water) and make the final
volume of 1L with the solvent.

2. Molality (m): A solution which contains 1mole of the solute dissolved in 1kg of the solvent is
called a molal solution.
Molality = Weight of a solute in g/kg of solvent
Mol. Wt. of solute

Example: To prepare 1m Na2CO3 solution. Find amount of Na2CO3 dissolved.

Required molarity = 1m
Mol. Wt of Na2CO3 = 106
Therefore amount of Na2CO3 needed = 106 g of Na2CO3 in one kg of water.

3. Normality (N): The normality of a solution is the number of gram equivalents of the solute
per L of the solution.
Normality = Amount of a substance in g/L of solution
Eq. wt. of substance

Example: To prepare 0.1N Na2CO3 (Eq. wt. of Na2CO3 = 53). Find amount of Na2CO3
dissolved to make 1 N solution.
Dissolve 5.3 g of Na2CO3 in a final volume of 1L of solution.
4. Mass percentage or % (w/w): It is the weight of the component present in 100 parts by
weight of the solution.

Example: In a solution containing 10g of sugar in 40 g of water, then

Mass% of sugar = 10x100
(10+40)
= 20%

5. Percentage by volume or % (v/v): It is the volume of the component in 100 parts by volume
of the solution.
Example: In a solution containing 20ml alcohol in 80ml of water, the % volume of alcohol will
be 20x100
(20 + 80)
= 20%

6. Parts per million (ppm): This is generally employed for those solutions in which a substance
is present in a very small quantity. It represents gram of a solute per million grams of solution or
the gram of a solute per million ml of the solution.
ppm = mass of the component
total mass of the solution
or ppm = g or ml of solute or substance
total mass of the solution
Thus, 1 ppm of solution of NaCl in water represents
1ppm = 1mg of NaCl/L of solution
= 1 mg NaCl/1000 ml of solution
= 1μg NaCl/ml of solution

Types of solutions
1. Stock solutions: Stock solution of a substance is the one having a concentration many folds
higher than that actually required in the experiment. Stock solutions are prepared of the
substances that are to be used frequently and are stable at higher concentration for several days
and can be used after appropriate dilution just before use. The use of a stock solution thus cuts
down on the amount of pipetting and at the same time reduces variability between a number of
similar incubation mixtures, assay mixtures etc.
The volume of the stock solution needed to prepare a solution of required volume containing
desired concentration of the compound can be calculated by: N1V1 = N2V2
where N1 = concentration of the solution to be prepared
V1 = volume of the solution to be prepared
N2 = concentration of the stock solution
V2 = volume of the stock solution
2. Standard solutions and saturated solutions:
When the solution contains the solute in an amount in excess of that which can be uniformly
dissolved at a given temperature and the solute is in equilibrium with the excess of undissolved
solute, the solution is said to be saturated.
Solution of known quantity – as reference is standard solutions.

Result: