2013
Biochemistry
Laboratory Manual
DR.GYANENDRA AWASTHI
DR.SANTOSH KUMAR
DR.ASHWANI SANGHI
MR.SHIV SHARAN SINGH
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BIOCHEMISTRY LABORATORY MANUAL
DR.GYANENDRA AWASTHI
DR.SANTOSH KUMAR
DR.ASHWANI SANGHI
MR.SHIV SHARAN SINGH
Department of Biochemistry
Dolphin (PG) Institute of Biomedical & Natural Sciences,
DEHRA DUN (UTTARAKHAND)
2013
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© Copyright Reserved
2013
All rights reserved. No part of this publication may be reproduced, stored, in a
retrieval system or transmitted, in any form or by any means, electronic,
mechanical, photocopying, reordering or otherwise, without the prior permission
of the publisher.
ISBN: 978-93-83520-17-6
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Author’s Preface
Living systems are shaped by an enormous variety of biochemical reactions which can be
understand via various biochemical techniques. In the present Manual an effort has been made to
discuss these biochemical techniques in simple and lucid manner so that reader can have
comprehensive understanding of the subject. Unlike other basic science subjects like Chemistry,
Zoology and Botany, Biochemistry practical’s generally require a variety of chemicals and
expensive equipments.
One of the highlight of the present manual is that it covers the practical aspects of different
biochemical techniques for undergraduate and postgraduate students of life sciences. The manual
is divided into seven main sections, each of which subdivided into chapters. First section deals
with buffers, pH and solution preparation mainly. Second and third unit deals with analysis of
biomolecules both qualitatively and quantitatively. The fourth, fifth and sixth unit mainly
concerned with chromatographic, electrophoretic and spectroscopic techniques. The last unit is
regarding demonstration of PCR and ELISA.
The present script is just a compilation of facts and interpretation from different sources. The
Authors does not claim the originality of the subjects. The present manual is the author’s
understandings of the various techniques described and are fully responsible for the errors and
misinterpretations.
Dr.Gyanendra Awasthi
Dr.Santosh Kumar
Head & Reader,
Assistant Professor,
Department of Biochemistry,
Department of Biochemistry,
DIBNS, Dehradun
DIBNS, Dehradun
Dr.Ashwani Sanghi
Mr.Shiv Sharan Singh
Assistant Professor,
Assistant Professor,
Department of Biochemistry,
Department of Biochemistry,
DIBNS, Dehradun
DIBNS, Dehradun
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INDEX
Exp.No.
Name Of The Experiment
Page No.
Section I: Solutions, Buffers & pH
01.
Solution
2–3
02.
Buffers
4–9
03.
pH
10 – 11
Section II: Qualitative Analysis Of Biomolecules
04.
Molisch’s Test
13 – 14
05.
Iodine Test
15 – 16
06.
Benedict’s’s Test
17 – 18
07.
Barfoed’s Test
19 – 20
08.
Seliwanoff’s Test
21 – 22
09.
Bial’s Test
23 – 24
10.
Biuret Test
28 – 29
11.
Ninhydrin Test
30 – 32
12.
Xanthoproteic Test
33 – 34
13.
Millon’s Test
35 – 36
14.
Sakaguchi’s Test
37 – 38
15.
Lipids Solubility Test
41
16.
Acrolein Test
42
17.
Zak Test
43
Section III: Quantitative Analysis Of Biomolecules
18.
Ferricyanide Assay
45 – 46
19.
Lowry’s Assay
47 – 49
20.
Acid Value Determination
50 – 51
21.
Saponification Value Determination
52 – 55
Section IV: Chromatographic Techniques
22.
Ascending Paper Chromatography
58 – 62
23.
Thin Layer Chromatography
63 – 66
Section V: Electrophoretic Techniques
24.
Agarose Gel Electrophoresis
69 – 70
25.
PAGE
71 – 75
Section VI: Spectroscopic Techniques
26. (a.)
Verification of Beer’s Law
77 – 80
26. (b.)
Determinmation of max
Section VII: Laboratory Demonstrations
27.
Polymerase Chain Reaction
82 – 86
28.
ELISA
87 - 91
ABOUT AUTHOR
92
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Section: I
Solutions, Buffers & pH
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Experiment No. 01
AIM: Preparation Of Normal, Molar & Percent Solutions.
Molarity (M) :
This is the most common method for expressing the concentration of a solution in
biochemical studies. The molarity of a solution is the number of moles of the
solute dissolved per L of the solution. A solution which contains 1 mole of the
solute in one L of the solution is called a molar solution. Molarity of a solution can
be calculated as follows:
Weight of a solute in g/L of solution
Molarity =
Mol. Wt. of solute
It may be noted that in case of molar solutions, the combined total volume of the
solute and solvent is one L. Thus for preparing 0.1 M NaOH, one may proceed as
follows:
Mol. Wt. of NaOH = 40
Required molarity of solution = 0.1M
Amount (in g) of NaOH per L of solution = Mol. Wt.of NaOH x molarity
= 40 x 0.1= 4 g
Thus, weigh 4 g of NaOH, dissolve it in a small volume of solvent (water) and
make the final volume to 1 L with the solvent.
Sometime it is desirable to know number of moles of a substance in a reaction
mixture. This can be calculated using a simple relationship:
1 M solution = 1 mole of the substance/L of solution.
= 1 mmole/ml of solution
= 1 µmole/µl of solution
1 mM solution = 1 mmole/L of solution
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= 1 µmole/ml of solution
Normality (N):
The normality of a solution is the number of gram equivalents of the solute per L
of the solution. Therefore,
Amount of a substance in g/L of solution
Normality =
Eq. wt. of substance
For preparing 0.1 N Na2CO3 (Eq.wt. of Na2CO3= 53) solution, dissolve 5.3g Na2CO3
in a final volume of 1 L of solution.
Percentage by Mass or % (w/w):
It is the weight of the component present in 100 parts by weight of the solution.
In a solution containing 10g sugar in 40g of water, then
10x100
Mass % of sugar =
= 20%
(10+40)
Percentage by volume or % (v/v) :
It is the volume of the component in 100 parts by volume of the solution. In a
solution containing 20 ml alcohol in 80 ml of water, the % volume of alcohol will
be
20 x 100
=
20%
(20 + 80)
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EXPERIMENT NO.02
AIM: To Prepare Buffer Solutions.
PHOSPHATE BUFFER: Phosphate salts are known by several names and the
correct phosphate must be used to prepare buffer solutions. One phosphate
cannot be substituted for another phosphate. Check formula of salt to be certain.
Formula
Name of salt
Other names
potassium dihydrogen phosphate
potassium dihydrogen orthophosphate
monobasic
potassium
phosphate
monopotassium
phosphate
acid
potassium
phosphate
potassium biphosphate
K2HPO4
potassium hydrogen phosphate
dipotassium hydrogen orthophosphate
dipotassium
hydrogen
phosphate
dibasic
potassium
phosphate
dipotassium phosphate
K3PO4
potassium phosphate
tribasic
potassium
tripotassium phosphate
KH2PO4
phosphate
Standardization buffers (For pH=7.00): Add 29.1 ml of 0.1 molar NaOH to 50 ml
0.1 molar potassium dihydrogen phosphate. Alternatively: Dissolve 1.20g of
sodium dihydrogen phosphate and 0.885g of disidium hydrogen phosphate in 1
liter volume distilled water.
Standardization buffers (For pH= 4.00): Add 0.1 ml of 0.1 molar NaOH to 50 ml of
0.1 molar potassium hydrogen phthalate. Alternatively, Dissolve 8.954g of
disodium hydrogen phosphste.12 H2O and 3.4023g of potassium di hydrogen
phosphate in 1 liter volume distilled water.
RANGE OF COMMON BUFFER SYSTEMS:
Buffering pH
Range @ 25°C
Buffering System
Hydrochloric acid/ Potassium chloride
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1.0 - 2.2
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Glycine/ Hydrochloric acid
2.2 - 3.6
Potassium hydrogen phthalate/ Hydrochloric acid
2.2 - 4.0
Citric acid/ Sodium citrate
3.0 - 6.2
Sodium acetate/ Acetic acid
3.7 - 5.6
Potassium hydrogen phtaalate/ Sodium hydroxide
4.1 - 5.9
Disodium hydrogen phthalate / Sodium dihydrogen orthophospate
5.8 - 8.0
Dipotassium hydrogen phthalate / Potassium dihydrogen orthophospate
5.8 - 8.0
Potassium dihydrogen orthophosphate / sodium hydroxide
5.8 - 8.0
Barbitone sodium / Hydrochloric acid
6.8 - 9.6
Tris (hydroxylmethyl) aminomethane / Hydrochloric acid
7.0 - 9.0
Sodium tetraborate/ Hydrochloric acid
8.1 - 9.2
Glycine/ Sodium hydroxide
8.6 - 10.6
Sodium carbonate/ Sodium hydrogen carbonate
9.2 - 10.8
Sodium tetraborate/ Sodium hydroxide
9.3 - 10.7
Sodium bicarbonate / Sodium hydroxide
9.60 - 11.0
Sodium hydrogen orthophosphate / Sodium hydroxide
11.0 - 11.9
Potassium chloride/ Sodium hydroxide
12.0 - 13.0
PREPARING A BUFFER SOLUTION: This page gives tabulated info on the
preparation of buffers by mixing adjusters with a known volume of the primary
salt solution, and made up to 200 ml with distilled water.
BUFFERS (pH: 1- 9)
Buffer
A
pH 1.0 - 2.2
: Buffer
B
pH 2.2 - 4.00
: Buffer
C
: Buffer
D
pH 4.10 - 5.90
pH 5.8 - 8.00
: Buffer
E
pH 7.0 - 9.00
:
100 ml 0.1 M 100 ml 0.1 M
100 ml 0.1 M tris
50 ml 0.2 M KCl potassium
potassium
100 ml 0.1 M
(hydroxymethyl)
hydrogen
KH2PO4 + ml of
+ ml of 0.2 M hydrogen
aminomethane +
HCl
phthalate + ml phthalate + ml 0.1 M NaOH
ml of 0.1 M HCl
of 0.1 M HCl
of 0.1 M NaOH
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pH
ml of 0.2M HCl
pH
added
ml of 0.1M HCl
pH
added
1.00
134.0
2.20
99.0
4.10
1.10
105.6
2.30
91.6
4.20
1.20
85.0
2.40
84.4
4.30
1.30
67.2
2.50
77.6
4.40
1.40
53.2
2.60
70.8
4.50
1.50
41.4
2.70
64.2
4.60
1.60
32.4
2.80
57.8
4.70
1.70
26.0
2.90
51.4
4.80
1.80
20.4
3.00
44.6
4.90
1.90
16.2
3.10
37.6
5.00
2.00
13.0
3.20
31.4
5.10
2.10
10.2
3.30
25.8
5.20
2.20
7.8
3.40
20.8
5.30
3.50
16.4
5.40
3.60
12.6
5.50
3.70
9.0
5.60
3.80
5.8
5.70
3.90
2.8
5.80
4.00
0.2
5.90
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BUFFERS (pH: 8 – 13)
Buffer F:
pH 8.0 - 9.10
Buffer
G
pH 9.2 - 10.80
: Buffer
H
: Buffer
I
: Buffer
J
:
pH 9.60 - 11.00
pH 10.90 - 12.00 pH 12.00 - 13.00
100 ml 0.025 M 100 ml 0.025 M
50 ml 0.2 M KCl
100 ml 0.05 M 100 ml 0.05 M
Na2B4O7.10H2O
Na2B4O7.10H2O
+
volume
NaHCO3 + ml of Na2HPO4 + ml of
(borax) + ml of
(borax) + ml of
indicated (in ml)
0.1 M NaOH
0.1 M NaOH
0.2 M NaOH
0.1 M HCl
0.1 M NaOH
pH
ml of 0.1M HCl
pH
added
ml
of
0.1M
pH
NaOH added
8.00
41.0
9.20
1.8
9.60
8.10
39.4
9.30
7.2
9.70
8.20
37.6
9.40
12.4
9.80
8.30
35.4
9.50
17.6
9.90
8.40
33.2
9.60
22.2
10.00
8.50
30.4
9.70
26.2
10.10
8.60
27.0
9.80
30.0
10.20
8.70
23.2
9.90
33.4
10.30
8.80
19.2
10.00
36.6
10.40
8.90
14.2
10.10
39.0
10.50
9.00
9.2
10.20
41.0
10.60
9.10
4.0
10.30
42.6
10.70
10.40
44.2
10.80
10.50
45.4
10.90
10.60
46.6
11.00
10.70
47.6
10.80
48.5
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ACETATE BUFFER SOLUTIONS (pH 3 – 6): Make up the following solutions(1) 0.1M acetic acid
(2) 0.1M sodium acetate (tri-hydrate) (13.6 g/L )
Mix in the following proportions to get the required pH
pH
vol. of 0.1M
acetic acid
vol. of 0.1M
sodium acetate
3
982.3 ml
17.7 ml
4
847.0 ml
153.0 ml
5
357.0 ml
643.0 ml
6
52.2 ml
947.8 ml
PHOSPHATE BUFFER SOLUTIONS (pH 7 – 11): Make up the following solutions(1) 0.1M disodium hydrogen phosphate (14.2g /L)
(2) 0.1M HCl
(3) 0.1M NaOH
Mix in the following proportions to get the required pH
pH
vol. of
phosphate
vol. of 0.1M
HCl
vol. of 0.1M
NaOH
7
756.0 ml
244 ml
8
955.1 ml
44.9 ml
9
955.0 ml
45.0 ml
10
966.4 ml
33.6
11
965.3 ml
34.7
Addition of acid or base to a salt (pH 3 – 11)
Here, the primary salt is a solid and is weighed out in grams. A measured amount
of 0.1M HCl or NaOH is added, then made up to 1 liter to give the relevant buffer
solution.
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pH
Salt mixture
Dilute each mixture to 1 liter solution with distilled water
3
10.21g potassium hydrogen phthalate and 223 ml of 0.10M HCl
4
10.21g potassium hydrogen phthalate and 1ml of 0.10M HCl
5
10.21g potassium hydrogen phthalate and 226ml of 0.10M NaOH
6
6.81g potassium dihydrogen phOsphate and 56ml of 0.10M NaOH
7
6.81g potassium dihydrogen phosphate and 291ml of 0.10M NaOH
8
6.81g potassium dihydrogen phosphate and 467ml of 0.10M NaOH
9
4.77g sodium tetraborate and 46ml of 0.10M HCl
10
4.77g sodium tetraborate and 183ml of 0.10M NaOH
11
2.10g sodium bicarbonate and 227ml of 0.10M NaOH
McIlvaine’s buffer (pH 7.20): 173.9 ml of 0.2 M Na2HPO4 and 26.1 ml of 0.1 M
citric acid were mixed to prepare the buffer of pH 7.2 and the final pH adjustment
was done by addition of either of the two solutions simultaneously.
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Experiment No.03
AIM: To Find Out The Strength Of The Given Hydrochloric Acid Solution (Approx.
Strength N/10) By Titrating It Against Sodium Hydroxide Using pH Meter.
APPARATUS: pH meter with glass electrode, reference electrode, beaker, burette,
stirrer etc.
CHEMICALS: HCl, NaOH.
THEORY: When an alkali is added to an acid solution, the pH of the solution
increases slowly. But at the equivalence point, the rate of change of the solution
is very rapid. A plot is drawn between volume of the alkali added and the pH of
the solution. The sharp break in the curve gives the equivalence point, from which
the strength can be calculated using normality equation.
INSTRUMENTATION: In pH meter the glass electrode is incorporated in an
ordinary potentiometric circuit. The potentiometric pH meter differs from a simple
potentiometer to the extent that the galvanometer is replaced by an electronic
circuit that amplifies the current in the cell circuit by a factor of 109 or more.
Before using, the pH meter is first standardised with a buffer solution of known pH.
Then the glass and reference electrode are immersed in an unknown solution and
the pH is read directly on pH scale.
PROCEDURE:
1) Caliberate the pH meter with the glass electrode in the buffer solution of
known pH.
2) Wash the glass electrode and the reference electrode with distilled water
and then rinse with the acid solution.
3) Take 5ml of HCl solution in a beaker. Add sufficient water so as reference
and glass electrodes are completely dipped.
4) Note down the pH of the pure acid solution.
5) Now add 10ml of N/10 NaOH from the burette and note down the pH after
each addition.
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6) Continue adding NaOH solution from the burette and note down the pH
after each addition.
7) Near the equivalence point the change in pH is much more rapid than in any
other region.
OBSERVATION:Volume of acid taken = 5ml
Vol. of alkali added 0.0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0
CALCULATION: Plot a curve with pH values as ordinate and volume of alkali added
as abscissa. The sharp break in curve corresponds to the equivalence point.
Volume of alkali added (ml)
Let the volume of alkali at equivalence point = x mL
Acid
alkali
N1V1
=
N2V2
N1 X 5
=
N/10 X x
N1
=
N/10 X
Strength of HCl solution
= 36.5 X x/ 10 X 5 g/L
RESULT: The strength of given acid solution is ……. g/l
PRECAUTIONS:
1) The pH meter should be caliberated before use.
2) After addition of alkali, the solution should be thoroughly stirred.
3) Electrodes must be immersed properly in the solution.
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Section: II
Qualitative Analysis Of
Biomolecules
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Experiment No.04
AIM: To Detect The Presence Of Carbohydrate In The Given Samples By
Molisch’s Test.
PRINCIPLE: Molisch's test (named after Austrian botanist Hans Molisch) is a
sensitive chemical test for the presence of carbohydrates, based on the
dehydration of the carbohydrate by sulfuric acid to produce an aldehyde, which
condenses with two molecules of phenol (usually α-naphthol, though other
phenols (e.g. resorcinol, thymol) also give colored products), resulting in a red- or
purple-colored compound.
All carbohydrates (larger than tetroses) – monosaccharides, disaccharides, and
polysaccharides – should give a positive reaction, and nucleic acids and
glycoproteins also give a positive reaction, as all these compounds are eventually
hydrolyzed to monosaccharides by strong mineral acids. Pentoses are then
dehydrated to furfural, while hexoses are dehydrated to 5-hydroxymethylfurfural.
Either of these aldehydes, if present, will condense with two molecules of
naphthol to form a purple-colored product, as illustrated below by the example of
glucose:
REAGENTS:
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1) 5% α-napthol in 95% alcohol
2) Concentrated H2SO4
3) 1% solution different carbohydrates.
PROCEDURE: Add 2-3 drops of α- naphthol solution to 2ml of test solution. Very
gently pipette 1ml conc. H2SO4 along the side of the test tube so that the 2
distinct layers are formed. Carefully observe any colour change at the junction of
2 layers. Appearance of purple colour indicates the presence of carbohydrates in
the sample preparation or the test solution.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.05
AIM: To Detect The Presence Of Polysaccharides (Starch & Glycogen) In The
Given Samples By Iodine Test.
PRINCIPLE: The Iodine test is used to test for the presence of starch. Iodine
solution — iodine dissolved in an aqueous solution of potassium iodide — reacts
with the starch producing a purple black/blue black color.
Iodine forms coloured adsorption complexes with polysaccharides. Starch gives
blue colour with iodine, while glycogen gives reddish brown coloured complex.
Hence it is a useful, convenient method for the detection of amylase, amylopectin
& glycogen.
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REAGENTS:
1) Iodine solution: prepare 0.005N iodine solution in 3% (w/v) potassium iodide
solution.
2) Sugar solution: 1% solution of different carbohydrates.
PROCEDURE: 1.0 ml of test solution in a test tube & added a drop of iodine
solution in each test tube. A blank is performed with water. Test tube is shaken
and color is observed. Test tube in which color is developed is heated & change in
color observed now test tube is cooled & change in color observed.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.06
AIM: To Detect The Presence Of Reducing Sugar In The Given Samples By
Benedict’s Test.
PRINCIPLE: Carbohydrates with free or potentially free reducing groups easily
reduce metal like copper (Cu), Ba, Hg (mercury), Iron (Fe) & silver (Ag) in Alkaline
solution when blue alkaline cupric oxide or hydroxide suspended in alkaline
medium is heated it forms blue precipitate of cupric oxide (CuO ) but in presence
of reducing substances, e.g reducing sugars having free or potentially free
aldehyde or ketonic group upon heating blue cupric hydroxide converted into
insoluble brownish red cuprous oxide (Cu2O) suspensions of metal hydroxide,
used in metal reduction test and to precipitate in alkaline medium to check that
organic compound having more than one alcoholic groups are added to give free
metals. This test is more sensitive and reagent does not deteriorate if stored for a
longer time. In this method the sodium citrate functions as a chelating agent by
forming soluble complex ions with Cu++, preventing the precipitation of CuCO3
in alkaline solutions. Presence of reducing sugar results in the formation of red
precipitate of cuprous oxide. Depending on the concentration of sugars, yellow to
green color is developed. All monosaccharides are reducing sugars as they all
have a free reactive carbonyl group. Some disaccharides like maltose have
exposed carbonyl groups and are also reducing sugars but less reactive than
monosaccharides.
D-glucose + 2CuO
D-gluconic acid + Cu2O
(Blue)
REAGENTS:
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(Brick red precipitate)
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1) Benedict’s reagent A: Dissolve 173 gm of sodium citrate & 100gm of anhydrous
Na2CO3 in 600ml of hot H2O. Dilute to the 800ml with water.
2) Benedict’s reagent B: Dissolve 17.3g of CuSO4.5H2O in 100ml hot water. Cool &
l % dilute to 100ml.
Add both reagents with constant stirring. Make the final volume to 1L.
3) Sugar solution: 1% solution of different carbohydrates.
PROCEDURE: Add 0.5-1ml of the test solution or sample extract to 5ml of
Benedict’s reagent. Keep the test tubes in vigorously heated boiling water bath.
Cool the solution. Observe the colour change from blue to green, yellow, orange
or red depending upon the amount of reducing sugar present in the test sample.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.07
AIM: To Differentiate Between Monosaccharides & Reducing Disaccharides By
Barfoed’s Test.
PRINCIPLE: Barfoed's Test is a chemical test used for detecting the presence of
monosaccharides. It was invented by Danish chemist Christen Thomsen Barfoed
and is primarily used in botany. The test is similar to the reaction of Benedict's
solution to aldehydes, except that reduction of copper occurs in acidic medium
rather alkaline medium.
Barfoed's reagent, a mixture of ethanoic (acetic) acid and copper(II) acetate, is
combined with the test solution and boiled. A red copper(II) oxide precipitate is
formed will indicates the presence of reducing sugar. The reaction will be
negative in the presence of disaccharide sugars because they are weaker reducing
agents. This test is specific for monosaccharides . Due to the weakly acidic nature
of Barfoed's reagent, it is reduced only by monosaccharides.
Disaccharides may also react, but the reaction is much slower.The aldehyde
group of the monosaccharide which normally forms a cyclic hemiacetal is
oxidized to the carboxylate. Monosaccharides usually react in about 1-2min while
the reducing disaccharides take much longer time between 7-12min to get
hydrolysed & then react with the reagent.
D-glucose + 2CuO
D-gluconic acid + Cu2O
REAGENTS:
1) Barfoed’s regent: Dissolve 13.3g of copper acetate in 200ml water & 1.8 ml of
glacial acetic acid to it.
2) Sugar solution: 1% solution of different carbohydrates.
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PROCEDURE: 1.0 ml of test solution was taken and to it added 2 ml of Barfoed’s
regent and it was then boiled for 1-2 min and allowed to stand for few minutes.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
Interpretation
RESULT & CONCLUSION:
COMMENTS:
1) This test is not specific for glucose or any other monosaccharides but simply
used to detect reducing sugars.
2) Disaccharides also respond to this test.
3) This test is copper reduction test but it differs from Fehling’s or Benedict’s test
in that reduction is brought about in acid solution.
4) Chloride interferes in this test and therefore unsuitable for detection of sugar
in urine or any other body fluid containing Cl.
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Experiment No.08
AIM: To Detect The Presence Of Ketose Sugars In The Given Samples By
Seliwanoff’s Test.
PRINCIPLE: It is a color reaction specific for ketoses. One can distinguish aldoses
from ketoses based on their ability to form furfurals. When conc. HCl is added,
ketoses undergo dehydration to yield furfural derivatives more rapidly than
aldoses. These derivatives form complexes with resorcinol to yield deep red color.
The test reagent causes the dehydration of ketohexoses to form 5hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol present in
the test reagent to produce a red product within two minutes. Aldohexoses
reacts so more slowly to form the same product. Aldoses generally exist in
solution as pyranoses, whereas ketoses generally exist as furanoses, hence the
ability of ketoses to rapidly dehydrate to yield furfurals:
REAGENTS: A
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1) Seliwanoff’s reagent- 0.05% (w/v) resorcinol in 3 N HCl.
2) 1% solution of different carbohydrates.
PROCEDURE: 2.0 ml of seliwonoff’s reagent was taken in a test tube and 0.5 ml of
test solution was added to this. Test tube was placed in boiling water bath. Test
was performed with different carbohydrates and with water as blank. A cherry
red condensation product will be observed indicating the presence of ketoses in
the test sample. There will be no significant change in colour produced for aldose
sugar.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
Interpretation
RESULT & CONCLUSION:
COMMENT: Prolonged heating will hydrolyse polysaccharides and may interfere
in this test.
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Experiment No.09
AIM: To Detect The Presence Of Pentose Sugar In The Given Samples By Bial’s
Test.
PRINCIPLE: Bial’s test can be used to distinguish pentoses from hexoses. In the
presence of concentrated HCl, pentoses react to give furfural, whereas hexoses
give hydroxymethyfurfural. Orcinol and furfural condense in the presence of ferric
ion to form a colored product. Appearance of green colour or precipitate indicates
the presence of. Hexoses, which give 5-hydroxyfurfural on dehydration, react with
Bial’s reagent to give a brownish colour. Di- and polysaccharides give the same
results but at a much slower rate:
REAGENTS:
1) Bial’s reagent: Dissolve 1.5 gm of orcinol in 100ml of conc. HCl & add 20-30
drops of 10% ferric chloride solution to it.
2) 1% solution of different carbohydrates.
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PROCEDURE: 1.0 ml of sugar solution added to about 2.0ml of bial’s reagent &
heated until boiling, a blue green color indicates presence of a pentose sugar. Test
is performed with different carbohydrates and water as blank.
OBSERVATION TABLE:
Si.No.
Sample
1.
1% Glucose
2.
1% Fructose
3.
1% Ribose
4.
1% Maltose
5.
1% Sucrose
6.
1% Starch
7.
1% Glycogen
8.
Water
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Summary: Carbohydrates Qualitative Analysis
No. Test
Observation
Inference
Reaction
Molisch’s Test
2-3
1
drops
of
beta-
naphthol
solution
is
added to 2ml of the test
solution.
Very
added 1ml of
gently
Conc.
A deep violet
coloration is
produced at the
junction of two
layers.
This is due to the
formation
of
an
unstable condensation
product
of
betanaphthol with furfural
(produced
by
the
dehydration
of
the
carbohydrate)
Presence of
carbohydrates.
H2SO4 along the side of
the test tube.
Iodine test
2
colour
iodine Blue
solution is added to 1ml observed
of the test solution and
4-5
mixed
gently
drops
of
the
is Presence
polysaccharide
of
Iodine forms coloured
adsorption complexes
with polysaccharides
contents
Benedict’s test
To 5 ml of Benedict's
3
solution, add 1ml of the
Formation of a green,
Presence
test solution and shake
red,
or
yellow
reducing sugars
each tube. Place the tube precipitate
in a boiling water bath
If the saccharide is a
reducing sugar it will
of
reduce Copper [Cu]
(11) ions to Cu(1)
oxide, a red precipitate
and heat for 3 minutes.
Remove the tubes from
the heat and allow them
to cool.
Barfoed’s test
5
A deep blue colour is
To 2 ml of the solution to formed with a red
be tested added 2 ml of ppt. settling down at
freshly
prepared the bottom or sides
Barfoed's reagent. Place of the test tube.
test tubes into a boiling
water bath and heat for 3
minutes. Allow to cool.
Presence
of
reducing
sugars
[appearance of a
red ppt as a thin
film at the bottom
of the test tube
within 3-5 min. is
indicative
of
reducing
monosaccharide. If the
ppt formation takes
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If the saccharide is a
reducing sugar it will
reduce Cu (11) ions to
Cu(1) oxide
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more time then it is
a
reducing
disaccharide
A cherry red colored
Presence of ketoses
precipitate within 5
[Sucrose gives a
minute is obtained
positive ketohexose
test
]
Seliwanoff test
6
To 3ml of of Seliwanoff’s
reagent, add 1ml of the
test solution, boil in
A faint red
water bath for 2 minutes
produced
colour
Presence of aldoses
When reacted with
Seliwanoff
reagent,
ketoses react within 2
minutes
forming
a
cherry
red
condensation product
Aldopentoses
react
slowly
forming
the
coloured condensation
product
Bials test
A blue-green product
7
Presence
pentoses
Add 3ml of Bial’s reagent
to 0.2ml of the test A muddy brown to
Presence
solution.
heat
the gray product
hexoses,
solution in a boiling
water bath for 2 minutes
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of
The furfurals formed
produces condensation
products with specific
of
colour
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Differences Encountered In A Real Laboratory
In an actual laboratory setting, there are certain important steps that are not
necessarily applicable in a virtual lab:
1. Always wear lab coat and gloves when you are in the lab. When you enter the
lab,switch on the exhaust fan and make sure that all the reagents required for
the experiment are available. If it is not available, prepare the reagents using
the components for reagent preparation.
2. Care should be taken while handling caustic acids like Conc. Sulphuric acid
[H2SO4], nitric acid [HNO3], Hydrochloric acid [HCl]. These acids should be
opened and used in FUMEHOOD only. Accidental spill of these acids will cause
severe burns and itching. Wash the spilled area with cold water and inform the
lab assistant immediately.
3. When Sodium hydroxide is prepared, make sure that it is handled with care as
the sodium hydroxide solution is caustic in nature.
4. Always check the water level in the water bath and if it is up to the level [nearly
half the volume], switch on the water bath and adjust to the required
temperature. Take care while using the water bath for the boiling step in the
experiment. Hold the test tube using a test tube holder.
5. There should be a proportion between the reagents added and the test
solution to obtain good result within the time mentioned. The droppers used
should not be mixed between the reagents, always use individual droppers for
each reagent.
6. The color formed will depend upon the quality of the reagents. So care should
be taken while preparing the reagents. If commercially available reagents are
used assure that it is not kept open for long time.
7. Clean the test tubes and glass wares with soap and distilled water. Recap the
reagent bottles once the experiment is completed. The water bath and the
exhaust fan should be switched off.
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Experiment No.10
AIM: To Detect The Presence Of Peptide Bonds In The Given Samples By Biuret
Test.
PRINCIPLE: The biuret test will indicate the presence of amino acid residues of
peptides containing two or more amino acid residues and therefore is used to
determine whether or not a protein is present. This test relies on the fact that
amino acid residues form a colored complex with Cu+2 ion in basic medium:
The test is given by those substances which contain at least two carbonyl group
joined either directly through a single atom of carbon or nitrogen. In this test
alkaline CuSO4 reacts with compounds containing two or more peptide bond
giving a violet colored complex. This biuret test is apparently due to co-ordination
of cupric- ion with the unshared electron pair of peptide nitrogen and oxygen of
water to form coloured co-ordination complex which may be represented. All
proteins should give a positive test whereas simple amino acids should give a
negative test.
REAGENTS:
1)
1% CuSO4.5H2O solution
2)
40% NaOH
3)
0.5% protein- solution of bovine serum albumin & casein in NaOH
4)
0.5% amino acid solution
PROCEDURE: 1ml of sample solution was taken in a test tube & 0.5ml of NaOH is
added & mix well. 2-5 drops of CuSO4 solution was added. Observe for the pink or
violet colour shows presence of peptides or proteins in the sample.
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OBSERVATION TABLE:
Si.No.
Sample
1.
0.5% Glycine
2.
0.5% BSA
3.
0.5% Casein
4.
0.5% Urea
Initial Observation
Final Observation
Interpretation
RESULT & CONCLUSION:
COMMENTS:
1)
Dipeptides do not give this test. Two or more peptide linkages being
required.
2)
Presence of MgSO4 in solution to be tested interfere with reaction because
of precipitation of Mg(OH)2.
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Experiment No.11
AIM: To Detect The Presence Of Amino Acids In The Given Samples By Ninhydrin
Test.
PRINCIPLE: This is due to a reaction between amino group of free amino acid and
ninhydrin (triketohydrindene hydrate). Ninhydrin is a powerful oxidizing agent
and in its presence, amino acid undergo oxidative determination liberating
ammonia, CO2, a corresponding aldehyde and reduced form of ninhydrin. The
ammonia formed from amino group react with ninhydrin and its reduced product
(hydridantin) to give a blue substrate diketohydrin (ruhemann’s purple) however,
in case of imino acid like proline, a different product having a bright yellow colour
is formed. Asparagine which has a free amide group reacts to give a brown
coloured product. This test is also given by protein and peptides.
Ruhemann’s Purple
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REAGENTS:
1) Boiling water bath.
2) Ninhydrin: 0.2% solution prepared in acetone.
3) Test solution: prepare solutions containing 0.5% of different amino acids.
PROCEDURE: Add 2-5 drop of ninhydrin solution to 1ml of test solution or sample
preparation mix and keep for 5min in boiling water bath and observe the
development of pink, purple or violet-blue colour. Imino acid like proline and
hydroxyproline give a yellow coloured complex.
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OBSERVATION TABLE:
Si.No.
Sample
1.
0.5% Glycine
2.
0.5% BSA
3.
0.5% Proline
4.
0.5% Asparagine
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.12
AIM: To Detect The Presence Of Aromatic Amino Acids In The Given Samples By
Xanthoproteic Test.
PRINCIPLE: Aromatic amino acids, such as Phenyl alanine, tyrosine and
tryptophan, respond to this test. In the presence of concentrated nitric acid, the
aromatic phenyl ring gets nitrated to give yellow colored nitro-derivatives. At
alkaline pH the color changes to orange due to the ionization of the phenolic
group. Protein containing these amino acid also give a positive response to this
test.
MATERIALS AND REAGENTS:
1) Conc.HNO3
2) NaOH solution (40%, w/v): Dissolve 40gm of NaOH in water and make the final
volume to 100 ml.
3) Test solution: Prepare separate solution containing 0.5% of amino acid like
tyrosine, glycine, tryptophan, phenylalanine, lysine etc.
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PROCEDURE: To 1ml of the amino acid solution taken in a test tube, add few
drops of nitric acid and vortex the contents. Boil the contents over a Bunsen flame
or in water bath, using a test tube holder, for few minutes. Cool the test tube
under running tap water and add few drops of alkali.Note whether the mixture
turns orange red in colour. Appearance of orange red colour denotes presence of
aromatic amino acid.
OBSERVATION TABLE:
Si.No.
Sample
1.
0.5% Glycine
2.
0.5% Trytophan
3.
0.5% Lysine
4.
0.5% Tyrosine
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.13
AIM: To Detect The Presence Of Amino Acids (Containing Hydroxybenzene
Radical) In The Given Samples By Millon’s Test.
PRINCIPLE: Phenolic amino acids such as Tyrosine and its derivatives respond to
this test. Compounds with a hydroxybenzene radical react with Millon’s reagent
to form a red colored complex. Millon’s reagent is a solution of mercuric sulphate
in sulphuric acid.
Hg
+
4HNO3
Hg(NO3)2 +
2NO2+
2H2O
REAGENTS:
1)
Millon’s regent (15%W/V mercuric sulphate in 6N sulphuric acid)
2)
Sodium nitrite (5%W/V) in distilled water ( to be freshly prepared)
3)
1mg/ml solution of glycine, casein & bovine serum albumin.
PROCEDURE: To 1ml of the amino acid solution in a test tube, add few drops of
millon’s reagent and vortex. Boil the contents over a Bunsen flame for 3 – 5
minutes. Cool the contents under running tap water and add few drops of sodium
nitrite solution. A positive reaction will also be obtained for the proteins which
contain tyrosine.
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OBSERVATION TABLE:
Si.No.
Sample
1.
0.5% Glycine
2.
0.5% BSA
3.
0.5% Casein
4.
0.5% Tyrosine
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Experiment No.14
AIM: To Detect The Presence Of Amino Acids (Containing Guanidium Group) In
The Given Samples By Sakaguchi’s Test.
PRINCIPLE: Under alkaline condition, α- naphthol (1-hydroxy naphthalene) reacts
with a mono – substituted guanidine compound like arginine, which upon
treatment with hypobromite or hypochlorite, produces a characteristic red color.
REAGENTS:
1) Amino acids: 0.5% solution of amino acids like glycine, arginine, lysine etc.
2) 0.5% urea solution
3) NaOH 40% (w/v)
4) α naphthol: 1% (w/v) in alcohol
5) Hypobromite solution (To be freshly prepared) : -Take 100 of 5%(W/V) sodium
hydroxide solution in a glass reagent bottle and add 1ml of pre chilled liquid
bromine, using a pro pipette. Shake the contents till bromine dissolves)
PROCEDURE: To 1 ml of prechilled amino acid solution and few drops of NaOH is
mixed and 2 drops of alpha naphthol is added. Mix thoroughly and add 4-5 drops
of hypobromite reagent and observe for the formation of red colour which would
indicate the presence of arginine or a guanidium compound.
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OBSERVATION TABLE:
Si.No.
Sample
1.
0.5% Glycine
2.
0.5% Lysine
3.
0.5% Urea
4.
0.5% Arginine
Initial Observation
Final Observation
RESULT & CONCLUSION:
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Interpretation
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Differences Encountered In A Real Laboratory:
In an actual laboratory setting, there are certain important steps that are not
necessarily applicable in a virtual lab.
1. Always wear lab coat and gloves when you are in the lab. When you enter the
lab, switch on the exhaust fan and make sure that all the reagents required for
the experiment are available. If it is not available, prepare the reagents using
the components shown in the reagent preparation.
2. Care should be taken while handling reagents like Conc. Sulphuric acid and
Hydrochloric acid. These concentrated acids should be opened and used only in
a FUMEHOOD. These concentrated acids cause severe burns and on inhaling
can cause damage to the lining of the lungs.
3. Reagents like Ninhydrin reagent, sulphanilic acid, isatin reagent, bromin,
Sodium nitroprusside should also be handled with care. Accidental spill of these
reagent will cause burns and itches. Wash the spilled area with cold water and
inform the lab assistant immediately.
4. Make sure that the waterbath is set to the proper temperature before starting
with the experiment.
5. Take care while heating the sample over the flame.
6. In Xanthoproteic test, results can be observed clearly on boiling the contents in
a waterbath.
7. The development of colors will depend upon the quality of the reagents
prepared.
8. Wipe the lab bench after the experiment is completed.
9. Make sure to switch off the waterbath and the exhaust fans before leaving the
lab.
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EXPERIMENT NO.15
AIM: Test For Solubility Of Given Lipid Sample.
PRINCIPLE:
The test is based on the property of solubility of lipids in organic solvents and
insolubility in water. The oil will float on water because of lesser specific gravity.
REAGENTS:
1) Lipid sample
2) Different solvents – water, ethanol, acetone, chloroform & ether
PROCEDURE:
Place 5 drops of and oil or a small sample of your lipid into each of three separate
test tubes. To the first tube add 5 ml. of water, to the second 5 ml. of ethanol, to
the third 5 ml. of acetone, to the fourth 5 ml. of chloroform and to the firth add 5
ml. of ether. Shake each tube well and allow to stand for a few minutes. Observe
whether solution or emulsification has occurred.
OBSERVATION TABLE:
Si.No.
Solvents
1.
Water
2.
Ethanol
3.
Acetone
4.
Chloroform
5.
Ether
Final Observation
RESULT & CONCLUSION:
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Interpretation
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EXPERIMENT NO.16
AIM: Acrolein Test For The Presence Of Glycerol.
PRINCIPLE: When glycerol is heated with potassium bisulphate or concentrated
H2SO4, dehydration occurs and aldehyde Acrolein formed which has characteristic
odour. This test responds to glycerol free or linked as an ester.
CH2 – OH
Heat
CH – OH
CH2 – OH
CH2
CH + 2H2O
KHSO4 or Conc.H2SO4
Glycerol
CHO
Acrolein
MATERIALS:
1. Test compounds ( Oil or fat ,Oleic acid)
2. Potassium bisulphate or conc. H2SO4
PROCEDURE:
1. Place 5 drops of test compound in a clean and dry test tube
2. Add 1 ml of conc. H2SO4 carefully. Or 1.0 g of KHSO4
3. Heat the test tube directly.
4. Note the characteristic pungent odour of Acrolein.
RESULT & CONCLUSION:
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EXPERIMENT NO.17
AIM: Zak Test For The Presence Of Cholesterol.
PRINCIPLE: This test is used for determination of cholesterol in blood.
MATERIALS:
1) 0.2 g cholesterol in 1ml of conc. acetic acid
2) Ferric chloride
3) Conc. Acetic acid
4) Conc. Sulfuric acid
PROCEDURE:
1. Place 0.5 ml of prepared cholesterol solution in a dry test tube.
2. Add 2 ml of colored solution ( mixture of 10% ferric chloride , Conc.
CH3COOH and Conc. H2SO4)
3. Observe appearance deep red which refers to existence of cholesterol.
RESULT & CONCLUSION:
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Section: III
Quantitative Analysis Of
Biomolecules
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EXPERIMENT NO.18
AIM: Estimation Of Carbohydrates (Total And Reducing Sugars, Sucrose And
Starch By Ferricyanide Method (Titrematric Method).
PRINCIPLE:
Alkaline potassium ferricyanide oxidizes sugars. This method is based on
reduction of the residual potassium ferricyanide by KI and the unreacted KI is
volumetrically measured by titration against Na2S2O3. The chemical reactions
involved are as follows:
2K2 [Fe (CN) 6] + 2KOH
CH2OH (CHOH) 4 CHO + ½ O2
2K4 [Fe (CN)6] + H2O + ½ O2
CH2OH (CHOH) 4 COOH
Glucose
Gluconic acid
Excess of ferricyanide reacts with KI
KI [Fe (CN)6] + KI
3ZnSO4 + 2K4 [Fe (CN)6]
K4 [Fe(CN)6]2 + I
K2Zn3[Fe(CN)6]2 + 3K2SO4
Potassium zinc ferrocynide
3I2 + 6OH
5I- + IO3- + 3H2O
5I- + IO3- + 6H+
3I2 + 3H2O
2Na2S2O3 + I2
Na2 S4 O6 + 2NaI
Sodium tetrathionate
MATERIALS AND REAGENTS:
1. Burette
2. Boiling water bath.
3. Potassium ferricyanide: Dissolve 8.25g potassium ferricyanide and 10.6g
anhydrous sodium carbonate in 1 L of distilled water.
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4. Iodine solution: Prepare by dissolving 12.6 g KI, 25 g ZnSO4 and 125 g NaCl in
500 ml distilled water. Filter and store in colored bottle.
5. Sodium thiosulphate solution: Make 0.01 N sodium thiosulphate solutions by
dissolving 2.5069 g of sodium thiosulphate in 1 L of distilled water.
6. Starch indicator solution: Suspend 1 g soluble starch in 20 ml of distilled water
and then add 60 ml of boiling water. Add 20g NaCl to this solution and make
the volume 100ml.
7. 5% glacial acetic acid.
PROCEDURE:
1. Take 5ml of potassium ferricyanide and 5 ml of aliquot of the sample extract in
a test tube, heat for 15 min in boiling water bath and then cool it.
2. Add 5 ml of iodine-solution followed by 3 ml of 5% glacial acetic acid. The
excess iodide is titrated against 0.01 N Na2S2O3 till the colour of the solution
turns pale yellow. Now add starch indicator solution, upon which the colour
will change to blue.
3. Complete the titration till disappearance of blue colour.
4. Run blank taking water instead of sugar solution or sample aliquot and proceed
in the same manner. Volume of Na2S2O3 used for the sample is deducted from
that consumed for the blank.
CALCULATIONS:
The amount of reducing sugars is calculated from the following relationship:
mg of reducing sugar in 5 ml of sample extract = µ (x + 0.05)
Where, µ = 0.338
x= vol. of 0.01 N Na2S2O3 used for sample, i.e.
Vol. of Na2S2O3 used in blank – Vol. used in sample.
RESULT & CONCLUSION:
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Experiment No.19
AIM: To Estimate Protein Quantitatively In The Given Sample By Lowry’S
Method.
PRINCIPLE: The Lowry protein assay is named after Oliver H. Lowry, who
developed and introduced it (Lowry, et al., 1951). The phenolic group of tyrosine
and trytophan residues (amino acid) in a protein will produce a blue purple color
complex , with maximum absorption in the region of 660 nm wavelength, with
Folin- Ciocalteau reagent which consists of sodium tungstate molybdate and
phosphate. Thus the intensity of color depends on the amount of these aromatic
amino acids present and will thus vary for different proteins. Most proteins
estimation techniques use Bovin Serum Albumin (BSA) universally as a standard
protein, because of its low cost, high purity and ready availability.
The –CO-NH- (peptide bonds) in polypeptide chain reacts with copper sulphate in
an alkaline medium to give a blue coloured complex. In addition, tyrosine &
tryptophan residues of proteins cause reduction of the phosphomolybdate &
phosphotungstate components of the Folin-Ciocalteau reagent to give bluish
products which contribute towards enhancing the sensitivity of this method. It is,
however, important to remember that several compounds like EDTA, Tris,
carbohydrates, NH+4, K+, Mg++ ions, thiol reagents, phenols etc. interfere with the
colour development & it should be ensured that such substances are not present
in sample preparations. The incubation time is very critical for a reproducible
assay. The reaction is also dependent on pH and a working range of pH 9 to 10.5 is
essential.
REAGENTS:
1) Reagent A: 2% Na2CO3 solution was prepared in 0.1 N NaOH
2) Reagent B: 1% CuSO4.5H2O (prepared in water)
3) Reagent C: 2% sodium potassium tartarate (prepared in water)
4) Reagent D: 1.0 ml of reagent B and 1.0 ml of reagent C were mixed with 98.0 ml
of reagent A just prior to use.
5) Reagent E: 1N Folin-ciocalteau’s reagent prepared by diluting the commercially
available reagent (2 N) with equal volume of distilled water at the time of use
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6) Reagent F: BSA standard protein solution (1 mg BSA/ mL of distilled water)
PROCEDURE: [Run triplicate determination for all samples.]
1) Different dilutions of BSA solutions are prepared by mixing stock BSA solution
(1 mg/ ml) and water in the test tube as given in the table. The final volume in
each of the test tubes is 1 ml. The BSA range is 0.01 to 0.10 mg/ ml.
2) Add 3.0 ml of freshly prepared reagent D (analytical reagent). Mix the solutions
well.
3) This solution is incubated at room temperature for 10 mins.
4) Then add 0.3 ml of reagent E to each tube and incubate for 30 min. Zero the
colorimeter with blank and take the optical density (measure the absorbance)
at 660 nm.
5) Plot the absorbance against protein concentration to get a standard calibration
curve.
6) Check the absorbance of unknown sample and determine the concentration of
the unknown sample using the standard curve plotted above.
BSA
Water (µL)
(µL)
Sample conc.
(mg/mL)
Reagent D (mL)
Reagent E (mL)
O.D.
660 nm
0
1000
3
0.3
10
990
3
0.3
20
980
3
0.3
30
970
3
0.3
40
960
3
0.3
50
950
3
0.3
60
940
3
0.3
70
930
3
0.3
80
920
3
0.3
90
910
3
0.3
100
900
3
0.3
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The protocol requires that the Folin phenol reagent be added to each tube
precisely at the end of the ten minute incubation. At the alkaline pH of the Lowry
reagent, the Folin phenol reagent is almost immediately inactivated. Therefore, it
is best to add the Folin phenol reagent at the precise time while simultaneously
mixing each tube. Because this is somewhat cumbersome, some practice is
required to obtain consistent results. This also limits the total number of samples
that can be assayed in a single run.
RESULT & CONCLUSION:
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Experiment No.20
AIM: To Determine The Acid Value Of The Given Fats Or Oil Sample.
PRINCIPLE: Different fat sample may contain varying amount of fatty acids. In
addition, the fats often become rancid during storage and this rancidity is
chemical or enzymatic hydrolysis of fats into free acids and glycerol the amount of
free fatty acids can be determined volumetrically by treating the sample with
potassium hydroxide. The acidity of fats and oils is expressed as its acid value or
number which is defined as mg KOH required to neutralize the free fatty acid
present in 1gm of fat or oil. The amount of free acids present or acid value of fat is
a useful parameter which gives an indication about the age and extent of its
deterioration.
MATERIALS AND REAGENTS:
1) Burette
2) Conical flask.
3) Test compounds (olive oil, butter, margarine etc; fresh and samples that have
been stored at room temperature for several days may be used for
comparison)
4) 1% phenolphthalein solution in 95% alcohol.
5) 0.1N potassium hydroxide: Weigh 5.6g of KOH and dissolve it in distilled water
and make the final volume to 1L. Standardize this solution by titrating it with a
known volume of 0.1N oxalic acid (prepare by taking 630mg oxalic acid in
100ml water) using phenolphthalein as indicator till a permanent pink colour
appears. Calculate the actual normality (N2) of KOH solution from equation
N1V1 = N2V2, where N1 and v1 are normality and volume of oxalic acid taken for
titration and V2 is the volume of KOH solution used.
6) Fat solvent (95% ethanol : ether 1:1, v/v)
PROCEDURE:
1) Take 5g of fat sample in a conical flask and add 25ml of fat solvents (reagent
no.6) to it .Shake well and a few drops of phenolphthalein solution and again
mix the content thoroughly.
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2) Titrate the above solution with 0.1N KOH until a faint pink colour persists for
20-30sec.
3) Note the volume of KOH used.
4) Repeat the steps 1-3 with a blank (reagent no.6) which does not contain any fat
sample.
CALCUTATION:
0.1N KOH solution used for blank = xml
0.1N KOH solution used for sample = yml
Titer value for sample
= (y-x) ml
Acid value (mg KOH/g fat)
=
1ml of 1N KOH contains 56.1mg of KOH. Hence a factor of 56.1 is incorporated in
the numerator in the above equation to obtain weight of KOH from the volume of
0.1N KOH solution used during this titration.
RESULT & CONCLUSION:
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Experiment No. 21
AIM: To Determine Of Saponification Value Of The Given Fats Or Oil Sample.
THEORY:
Saponification is
the hydrolysis of
fats or oils
under
basic
conditions
to
afford glycerol
and the salt of
the
corresponding fatty acid. Saponification literally means "soap making". It is
important to the industrial user to know the amount of free fatty acid present,
since this determines in large measure the refining loss. The amount of free fatty
acid is estimated by determining the quantity of alkali that must be added to the
fat to render it neutral. This is done by warming a known amount of the fat with
strong aqueous caustic soda solution, which converts the free fatty acid into soap.
This soap is then removed and the amount of fat remaining is then determined.
The loss is estimated by subtracting this amount from the amount of fat originally
taken for the test.
The saponification number is the number of milligrams of potassium hydroxide
required to neutralize the fatty acids resulting from the complete hydrolysis of 1g
of fat. It gives information concerning the character of the fatty acids of the fatthe longer the carbon chain, the less acid is liberated per gram of fat hydrolysed.
It is also considered as a measure of the average molecular weight (or chain
length) of all the fatty acids present. The long chain fatty acids found in fats have
low saponification value because they have a relatively fewer number of
carboxylic functional groups per unit mass of the fat and therefore high molecular
weight.
PRINCIPLE: Fats (triglycerides) upon alkaline hydrolysis (either with KOH or NaOH)
yield glycerol and potassium or sodium salts of fatty acids (soap).
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The procedure involves reflexing of known amount of fat or oils with a fixed an
excess of alcoholic KOH. The amount of KOH remaining after hydrolysis is
determined by back titrating with standardized 0.5N HCl and amount of KOH
utilized for saponification can thus be calculated.
MATERIALS REQUIRED:
1) Fats and Oils [coconut oil, sunflower oil]
2) Conical Flask
3) 100ml beaker
4) Weigh Balance
5) Dropper
6) Reflux condenser
7) Boiling Water bath
8) Glass pipette (25ml)
9) Burette
REAGENTS REQUIRED:
1) Ethanolic KOH(95% ethanol, v/v)
2) Potassium hydroxide [0.5N]
3) Fat solvent
4) Hydrochloric acid[0.5N]
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5) Phenolphthalein indicator
PROCEDURE:
1) Weigh 1g of fat in a tared beaker and dissolve in about 3ml of the fat solvent
[ethanol /ether mixture].
2) Quantitatively transfer the contents of the beaker three times with a further
7ml of the solvent.
3) Add 25ml of 0.5N alcoholic KOH and mix well, attach this to a reflux
condenser.
4) Set up another reflux condenser as the blank with all other reagents present
except the fat.
5) Place both the flask on a boiling water bath for 30 minutes.
6) Cool the flasks to room temperature.
7) Now add phenolphthalein indicator to both the flasks and titrate with 0.5N
HCl.
8) Note down the endpoint of blank and test.
9) The difference between the blank and test reading gives the number of
millilitres of 0.5N KOH required to saponify 1g of fat.
10) Calculate the saponification value using the formula:
Saponification value or number of fat = mg of KOH consumed by 1g of fat.
Weight of KOH = Normality of KOH x Equivalent weight x volume of KOH in litres
Volume of KOH consumed by 1g fat = [Blank – test]ml
CALCULATIONS:
Volume of 0.5N KOH used for titrating blank= x ml
Volume of 0.5N KOH used for titrating test sample= y ml
Titre value of sample = (x-y) ml
Saponification value =
28.05x titre value
Wt. of sample (g)
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RESULT & CONCLUSION:
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Differences Encountered In a Real Laboratory:
In an actual laboratory setting, there are certain important steps that are not
necessarily applicable in a virtual lab.
1. Always wear lab coat and gloves when you are in the lab. When you enter the
lab, switch on the exhaust fan and make sure that all the reagents required for
the experiment are available. If it is not available, prepare the reagents using
the components shown in the reagent preparation.
2. Care should be taken while handling reagents like potassium hydroxide and
hydrochloric acid. Accidental spill of these reagents will cause severe itching
sensation. Wash the spilled area with cold water and inform the lab assistant
immediately.
3. Caution should be taken while attaching the reflux condensors to the conical
flask.
4. Make sure that the waterbath is set to 100 degree celsius and the reflux
condensors are set up with proper settings before starting with the
experiment.
5. The endpoint point of titration should be carefully observed as the
disappearance of pink colour to white color.
6. After the experiment, switch off the waterbath and carefully remove the reflux
condensors.
7. After completing the experiment, clean the glass wares and wipe the lab bench.
8. Switch off the exhaust fans.
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Section: IV
Chromatographic Techniques
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EXPERIMENT NO.22
AIM: Separation And Identification Of Amino Acids By Ascending Paper
Chromatography.
THEORY:
Chromatography is a common technique for separating chemical substances. The
prefix “chroma,” which suggests “color,” comes from the fact that some of the
earliest applications of chromatography were to separate components of the
green pigment, chlorophyll. In this experiment you will use chromatography to
separate and identify amino acids, the building blocks of proteins.
Chromatography is a common technique for separating chemical substances. The
prefix “chroma,” which suggests “color,” comes from the fact that some of the
earliest applications of chromatography were to separate components of the
green pigment, chlorophyll. You may have already used this method to separate
the colored components in ink. In this experiment you will use chromatography to
separate and identify amino acids, the building blocks of proteins.
The term “paper chromatography” used in this experiment’s title identifies the
composition of the stationary phase. The compositions of the stationary and
mobile phases define a specific chromatographic method. Indeed, many different
combinations are possible. However, all of the methods are based on the rate at
which the analyzed substances migrate while in simultaneous contact with the
stationary and mobile phases. The relative affinity of a substance for each phase
depends on properties such as molecular weight, structure and shape of the
molecule, and the polarity of the molecule.
PRINCIPLE:
In this experiment, very small volumes of solutions containing amino acids will be
applied (this process is sometimes called “spotting”) at the bottom of a
rectangular piece of filter paper. For ready comparison of each trial, it is vital that
each solution be applied on the same starting line. After the solutions have been
applied, the paper will be rolled into a cylinder and placed in a beaker that
contains a few milliliters of the liquid mobile phase. For this separation, a solution
containing n-butanol, water and acetic acid is the optimum mobile phase. As soon
as the paper is placed in the mobile phase, the solution (sometimes called the
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eluting solvent) will begin to rise up the paper. This phenomenon is called
capillary action.
As the mobile phase rises on the paper it will eventually encounter the “spots” of
amino acids. The fate of each amino acid in the mixture now depends on the
affinity of each substance for the mobile and stationary phases. If an amino acid
has a higher affinity for the mobile phase than the stationary phase, it will tend to
travel with the solvent front and be relatively unimpeded by the filter paper. In
contrast, if the amino acid has a higher affinity for the paper than the solvent, it
will tend to “stick” to the paper and travel more slowly than the solvent front. It is
these differences in the amino acid affinities that lead to their separation on the
paper. The affinities of these amino acids for the mobile phase can be correlated
to the solubility of the different amino acids in the solvent (i.e., an amino acid that
is highly soluble in the eluting solvent will have a higher affinity for the mobile
phase than an amino acid that is less soluble in the solvent.).
When the solvent front comes near the top of the filter paper, the paper is
removed from the beaker and allowed to dry. At this point, the various amino
acids are invisible. The acids can be visualized by spraying the paper with a
compound called ninhydrin. Ninhydrin reacts with amino acids to form a blueviolet compound. Therefore, the sprayed filter paper should show a number of
spots, each one corresponding to an amino acid. The further the spot from the
starting line, the higher the affinity of the amino acid for the mobile phase and
the faster its migration.
The relative extent to which solute molecules move in a chromatography
experiment is indicated by Rf values. The Rf value for a component is defined as
the ratio of the distance moved by that particular component divided by the
distance moved by the solvent. Figure 1 represents the migration of two
components. Measurements are made from the line on which the original
samples were applied to the center of the migrated spot. In the figure, dA is the
distance traveled by component A, dB is the distance traveled by component B,
and dsolv is the distance traveled by the eluting solution. In all three cases, the
travel time is the same.
Thus the Rf values for components A and B are
Rf(A) = dA/dsolv Rf(B) = dB/dsolv
Figure 1: Paper chromatography - migration of two components.
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Note that Rf values can range from 0 to 1. In this example, Rf(A) is obviously larger
than Rf(B). Although Rf values are not exactly reproducible, they are reasonably
good guides for identifying the various amino acids. Paper chromatography is
most effective for the identification of unknown substances when known samples
are run on the same paper chromatograph with unknowns.
The separated amino acids are detected by spraying the air dried chromatogram
with ninhydrin reagent. All amino acids give purple or bluish purple colour on
reaction with ninhydrin except proline and hydroxylproline which give a yellow
coloured. The reactions leading to the formation of purple complexes are given
below:
Ninhydrin + Amino acid
Hydrindantin + RCHO + NH3 + CO2
Ninhydrin + Ammonia + Hydrindantin
Purple coloured product + 3H2O
MATERIALS AND REAGENTS
1) Whatman No. 1 filter paper sheet.
2) Micropipette / micro syringe.
3) Hair drier.
4) Sprayer.
5) Oven set at 105oC.
6) Chromatographic chamber saturated with solvent vapours.
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7) Developing solvent: Take butanol, acetic acid and water in the ratio of 4:1:5 in a
separating funnel and mix it thoroughly. Allow the phases completely. Use the
lower aqueous phase for saturating the chamber.
PROCEDURE:
1. Obtain a sheet of filter paper, and draw a faint pencil line about 1 to 2 cm
from one of the long edges and parallel to that edge. This will be the bottom
of the chromatogram.
2. Mark off seven equally spaced points along this line. (They should be
separated by about 2 cm). Your samples will be applied to these spots. The
laboratory contains solutions of four identified amino acids and a sample of a
hydrolyzed protein. In addition, you will be given a numbered unknown that
will contain one or more of the known amino acids.
3. Dip the open end of a clean capillary into the solution to draw up a small
volume of the solution into the tube. Lightly and briefly touch the tube to the
paper and allow the sample to transfer. The spot should be about 2-3 mm in
diameter. Place one spot of each of the four known amino acids on the
separate points that you previously marked on the filter paper. In addition,
apply samples of your unknown to two of the points. Be careful not to
contaminate either the solutions or the spots.
4. Label each spot (with pencil and below the starting line) to indicate its
identity. Finally, it’s a good idea to avoid getting fingerprints on the
chromatographic paper.
5. When you have finished spotting your paper, allow it to dry by waving it in the
air or using a heat lamp or hair dryer. (Don’t get it too hot.)
6. Meanwhile, in the hood, pour about 15-20 mL of the eluting solution (nbutanol and acetic acid) into a clean, dry 600 mL beaker and cover the beaker
with a watch glass or plastic wrap.
7. When the sample spots have dried, roll the paper into a cylinder, with the
short sides almost touching. Use a bit of “Scotch” tape along the top of the
paper to hold the cylinder together.
8. Evenly lower the paper cylinder, sample side down, into the beaker. The
solvent will wet the paper, but the sample spots should not be immersed. In
addition, the paper should not touch the walls of the beaker.
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9. At this point, cover the beaker with a watch glass or plastic wrap and place the
beaker in the hood.
10. When the solvent front gets within about 1 or 2 cm of the top of the paper (in
perhaps about 2 hrs), remove the paper, use a pencil to mark the solvent front
at several points, unroll the cylinder, and let the chromatography paper dry in
the hood.
11. When the paper is dry, spray it with ninhydrin reagent.
12. Allow the paper to dry, perhaps using the hair dryer, heat lamp, or an oven at
o
about 100 C, but don’t overcook it!
13. When the chromatographic paper has fully dried, outline the spots, mark the
centers of each of the spots, and note their colors. (Not all amino acids give
the same color with ninhydrin).
14. Measure and record the distances the solvent and each of the amino acids
traveled from the origin.
15. Use these distances to calculate Rf values for each sample.
Comparison of the spots should enable you to identify the amino acid(s) present
in your unknown sample.
CALCULATION:
Rf value can be calculated using the formula:
RESULT & CONCLUSION:
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EXPERIMENT NO.23
AIM: Separation And Identification Of Amino Acids By Thin Layer
Chromatography.
THEORY:
Thin layer chromatographic (TLC) technique readily provides qualitative
information and with careful attention to details, it is possible to obtain
quantitative data. Thin layer chromatography is a technique used to separate and
identify compounds of interest. A TLC plate is made up of a thin layer of silica
adhered to glass or aluminum for support. The silica gel acts as the stationary
phase and the solvent mixture acts as the mobile phase. In the ideal solvent
system the compounds of interest are soluble to different degrees. Separation
results from the partition equilibrium of the components in the mixture.
In the simplest form of the technique, a narrow zone or spot of the sample
mixture to be separated is applied near one end of the TLC plate and allowed to
dry. The strip or plate is then placed with this end dipping in to the solvent
mixture, taking care that the sample spot/zone is not immersed in the solvent. As
the solvent moves towards the other end of the strip, the test mixture separates
into various components. This is called as the development of TLC plates. The
separation depends on several factors; (a)solubility: the more soluble a
compound is in a solvent, the faster it will move up the plate. (b) attractions
between the compound and the silica, the more the compound interacts with
silica, the lesser it moves, (c) size of the compound, the larger the compound the
slower it moves up the plate.
The plate is removed after an optimal development time and dried and the
spots/zones are detected using a suitable location reagent. An important
characteristic used in thin layer chromatography is Rf value.
The plate is removed after an optimal development time and dried and the
spots/zones are detected using a suitable location reagent. An important
characteristic used in thin layer chromatography is Rf value.
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Chromatographic Separation of Amino acids:
The present experiment employs the technique of thin layer chromatography to
separate the amino acids in a given mixture.
All 20 of the common amino acids [standard amino acids] are a-amino acids. They
have a carboxyl group and an amino group bonded to the same carbon atom (αcarbon). They differ from each other in their side chains, or R groups, which vary
in structure, size, and electric charge. The interaction of the amino acids with the
stationary phase like silica varies depending on their 'R' groups. The amino acid
that interacts strongly with silica will be carried by the solvent to a small distance,
whereas the one with less interaction will be moved further. By running controls
[known compounds] alongside, it is possible to identify the components of the
mixture.
Since amino acids are colourless compounds, ninhydrin is used for detecting
them. To identify this, after development, the TLC plate is sprayed with ninhydrin
reagent and dried in an oven, at 105°C for about 5 minutes. Ninhydrin reacts with
α- amino acids that results in purple coloured spots [due to the formation of the
complex - Rheuman's purple]. Rf values can be calculated and compared with the
reference values to identify the amino acids. [The Rf value for each known
compound should remain the same provided the development of plate is done
with the same solvent, type of TLC plates, method of spotting and in exactly the
same conditions].
MATERIALS REQUIRED:
REAGENTS:
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1. 2% solution of individual amino acids.
2. Solvent mixture of normal butanol, acetic acid and water in the ratio 12:3:5 by
volume.
3. Ninhydrin reagent.
Requirements:
1. TLC plate.
2. TLC chamber.
3. Capillary tubes.
4. Reagent spray bottle.
5. Conical flasks.
6. Beakers.
Procedure:
1. Pour the solvent mixture in to the TLC chamber and close the chamber.
2. The chamber should not be disturbed for about 30 minutes so that the
atmosphere in the jar becomes saturated with the solvent.
3. Cut the plate to the correct size and using a pencil (never ever use a pen)
gently draw a straight line across the plate approximately 2 cm from the
bottom.
4. Using a capillary tube, a minute drop of amino acid is spotted on the line.
5. Allow the spot to dry.
6. Spot the second amino acid on the plate [enough space should be provided
between the spots].
7. Repeat the above step for spotting the unknown acid.
8. Place the plate in the TLC chamber as evenly as possible and lean it against
the side(immerse the plate such that the line is above the solvent). Allow
capillary action to draw the solvent up the plate until it is approximately 1 cm
from the end.
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9. Remove the plate and immediately draw a pencil line across the solvent top.
10. Under a hood dry the plate with the aid of a blow dryer.
11. Spray the dry plate with ninhydrin reagent.
12. Dry the plates in hot air oven at 105°C for 5 min. [Ninhydrin will react with the
faded spots of amino acids and make them visible as purple coloured spots.]
13. After some time, mark the center of the spots, then measure the distance of
the center of the spots from the origin and calculate the Rf values.
Rf value can be calculated using the formula:
The Rf values with butanol-acetic acid- water solvent are as follows: alanine 0.24,
glutamic acid 0.25, glycine 0.2, leucine 0.58, valine 0.4, lysine 0.58, tyrosine 0.42.
RESULT & CONCLUSION:
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Differences Encountered In A Real Laboratory:
In an actual laboratory setting, there are certain important steps that are not
necessarily applicable in a virtual lab.
1. Always wear lab coat and gloves when you are in the lab. When you enter the
lab, switch on the exhaust fan and make sure that all the reagents required for
the experiment are available. If it is not available, prepare the reagents using
the components shown in the reagent preparation.
2. Care should be taken while handling reagents like Ninhydrin reagent. This
reagent is a strong oxidizing agent and should not be inhaled or spilled on
hands or other body parts. Accidental spill of this reagent will cause severe
itching sensation. Wash the spilled area with cold water and inform the lab
assistant immediately.
3. Hold the TLC plates by their side. Ensure that you do not touch the developing
part of the TLC plate, because your finger prints will also get developed causing
the result to be unclear.
4. Make certain that the spots applied to the plate are above the surface of the
eluting solvent.
5. Before applying the second spot make sure that the previously applied spot is
dried.
6. Spot the components with proper space in between.
7. Ensure that the chamber is saturated with the solvent vapour before you place
the TLC plate in it.
8. Give enough time for the solvent to advance up the plate.
9. The top of the solvent must not advance up to or beyond the edge of the
plates.
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Section: V
Electrophoretic Techniques
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Experiment No.24
AIM: To Conduct Agarose Gel Electrophoresis.
PRINCIPLE: DNA molecules are negatively charged at neutral or alkaline pH and
migrate towards anode when an electric field is applied. Charge/mass ratio in
nucleic acid is unity, thus migration largely occurs on the basis of molecular size of
DNA molecules.
MATERIALS AND REAGENTS:
1. Mini gel Horizontal Agarose Gel electrophoresis unit. Comprises of:i) Gel casting plate
ii) Electrophoretic tank
iii)Comb
iv)Electrophoretic leads
2. Adhesive tape
3. Power pack
4. U.V Transilluminator with camera
5. Micropipette
6. Gloves
7. Tris acetate buffer (TAE) stock solution (5X): A five fold concentrated TAE buffer
stock solution. Consist of:
i) Tris base
: 24.2g
ii) Glacial Acetic acid : 5.71ml
iii)0.5M Acetic acid : 10.0ml
Adjust pH to 8.0 and add water to make 1lilre. Dilute 5 times to obtain working
washing buffer (1X).
8. 0.8% Agarose in 1X TAE Buffer: Dissolve 0.4g agarose in 50ml of 1X TAE buffer
by boiling and maintaining it at 50oc to be used.
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9. Gel loading solution: 1% glycerol and 0.025% bromophenol blue in water.
10. Ethidium bromide: Dissolve 10mg ethidium bromide per ml. of 1x TAE buffer.
11. Isolated DNA sample
PROCEDURE:
1. Take a clean dry gel casting plate and make gel mould using an adhesive tape
along the sides of the plate to prevent running off the material to be poured
on the plate.
2. Pour 1% agarose solution kept at 50oC onto casting plate, immediately place
the comb about 1cm from one end of the plate ensuring that teeth of the
comb do not touch the glass plate.
3. Allow a firm layer of gel formation. Remove the comb and tape surrounding
the plate carefully and transfer the gel plate to the electrophoretic tank such
that wells are towards the cathode.
4. Pour 1X TAE buffer into the tank until the gel is completely submerged;
connect the electrodes to the power supply.
5. Load the isolated DNA preparation into well with the help of the micropipette.
6. Turn on the power supply and run at 100v. Monitor the progress of
bromophenol blue (tracking dye) during electrophoresis.
7. Turn OFF the power supply when tracking dye has reached the opposite side
of the gel.
8. Transfer the gel from the casting plate onto a UV- Transparent thick plastic
sheet and place it in staining tray containing ethidium bromide solution stain
for 20-30min.
9. For destaining the gel, place it in water for 15-20min.
10. Now place the gel along with UV transparent sheet on a UV transilluminator
and view the gel in UV light for presence of orange coloured bands.
11. Gel should be photographed to keep the permanent store.
RESULT & INTERPRETATION:
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EXPERIMENT NO.25
AIM: To Perform Poly Acrylamide Gel Electrophoresis (PAGE).
PRINCIPLE: Electrophoresis is the migration of charged molecule in solution
through a support matrix in response to an electric field. Rate of migration
depends on the strength of the field; on the net charge, size and shape of the
molecules and also on the ionic strength, viscosity and temperature of the matrix.
As an analytical tool, electrophoresis is simple, rapid and highly sensitive.
Polyacrylamide gels are prepared by copolymerization of acrylamide monomer
(CH2=CH-CO-NH2) with a cross linking agent in the presence of the catalyst
accelerator and chain initiator mixture. The relative proportion of acrylamide
monomer to cross-linking agents determine the porosity of the gel. Separation of
protein is carried out using gels ranging from 5-20% of acrylamide. Discontinuity
of the buffer pH and gel concentration is employed to effect band sharpening at
the end of electrophoresis. Polyacrylamide is the medium of choice where high
resolution electrophoresis on the basis of charge and molecular size is required.
Other advantage includes its minimal adsorption capacity, lack of electro osmosis,
preparation of zymogram etc. PAGE is also used in specialized electrophoretic
system such as SDS-PAGE and isoelectric focusing. Sodium dodecyle sulphate
(SDS) is used to induce uniform negative charges on the protein molecule which
itself is charged. In SDS-PAGE the protein molecules are preferentially separated
on the basis of their molecular weight where as in native PAGE separation of
molecule depends on the charge as well as the mass of the entity.
The extent of purification and number of protein components are monitored by
SDS-PAGE. It is performed using 5% stacking gel and 12% resolving gel having pH
of 6.8to 8.8 respectively following methods of Laemmli (1994). Sample is
prepared by mixing protein solution and denaturing sample buffer at 1:1 ratio and
Tris-glycine buffer of pH 8.3 is used as electrode buffer. Separation is performed
at 15mA fixed current until the tracking dye reaches near the end of gel.
Gel is stained by silver staining following the methods of Blum et al, (1987).
Proteins are fixed in gel by overnight treatment with a solution containing 50%
methanol, 12% acetic acid, 0.15% HCHO. Gel is washed with double distilled water
and is incubated in freshly prepared sensitizing solution (0.125% glutaraldehyde,
0.2% sodium thiosulphate, 7% sodium acetate). After 40 minutes the sensitizing
solution is drained off and the gel is rinsed with double distilled water. Gel is
treated with 0.25% silver nitrate solution for 30 minutes, washed and is
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incubated in freshly prepared developing solution (3% sodium carbonate, 0.015%
HCHO) till the bands appear. Immediately after appearance of bands developing
solution is removed and the gel is immersed in stop solution containing 1.5%
EDTA. Longer incubation with developing solution may lead to background
staining.
1. NATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS (NATIVE-PAGE): NativePAGE is done using anionic system of Davis (1964).
REAGENTS USED:
• Acrylamide-bis-acrylamide solution (29.2:0.8): Dissolved 29.2 g acrylamide and
0.8 g N,N’-methylene-bis-acrylamide in distilled water and made up the volume
to 100 ml. Filtered this solution through Whatman filter paper and stored in a
brown bottle at 4oC.
• Resolving gel buffer stock (1.5 M Tris-HCl, pH 8.8): Dissolved 18.17 g of Tris
base in 60 ml distilled water and adjusted the pH of the solution to 8.8 with 1 N
HCl and the final volume was made to100 ml with distilled water. It is stored at
room temperature.
• Stacking gel buffer stock (1.0 M Tris buffer, pH 6.8): Dissolved 12.11 g of Tris
base in 60 ml distilled water and adjusted the pH to 6.8 and its volume is made
up to 100 ml with distilled water. It is stored at room temperature.
• Ammonium persulphate solution (1.5% w/v): The solution is prepared fresh by
dissolving 15.0 mg in 1.0 ml water.
• Reservoir buffer: 3.0 g Tris base and 14.4 g glycine are dissolved in 1000 ml
distilled water. The pH of the solution is adjusted to 8.3.
• Staining solution: Dissolved 250 mg Coomassie brilliant blue R-250 dye in a
solution containing 125 ml methanol and 25 ml glacial acetic acid. It’s volume is
adjusted to 250 ml with distilled water. It is filtered to remove any undissolved
material and stored at room temperature.
• Destaining solution: Mixed 50 ml methanol with 40 ml glacial acetic acid and
made up its volume to 100 ml with distilled water.
• TEMED: As supplied by the manufacturer
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• Sample preparation: Sample was prepared by mixing the purified protein with
the sample buffer (1.0 M Tris-HCl, pH 6.8 containing 5% glycerol and 0.02%
bromophenol blue).
PROCEDURE: Properly cleaned and dried glass plates were tightly held with the
spacer bars on both sides. Resolving (12%) and stacking gel (2.5%) solutions for
polymerization were prepared as given in Table 3.1.
Table: Composition of different recipe of gels for Native-PAGE
Native Gel Recipe (for 3.0 % to 15% PAGE)
Gel
%
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.5
10.0
12.5
15
Water
ml
6.4
6.25
6.0
5.9
5.75
5.6
5.4
5.25
5.1
4.9
4.5
4.1
3.3
2.4
Lower Tris
ml
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
Acrylamide
(30%) + bis
ml
Acrylamide
(8%)
1.0
1.17
1.34
1.5
1.67
1.8
1
2.0
2
2.17
2.34
2.5
2.92
3.3
4.1
4.92
APS (10%)
µL
85
87
87
80
75
75
70
70
62
45
45
45
45
45
TEMED
µL
6.2
6.2
6.2
6.2
5.0
5.0
5.0
3.7
3.7
3.7
2.5
2.5
2.5
2.5
The solution of resolving gel is poured into a vertical slab and a few drops of
distilled water are layered on top of the gel solution to ensure the production of a
flat gel surface. The gel is allowed to polymerize for half an hour.
After polymerization of resolving gel, the water layer is removed and soaked off
with filter paper. The stacking gel solution is then poured and immediately the
comb is fixed at the top to make the wells for sample application. The stacking gel
is allowed to polymerize for half an hour. The comb is removed and the wells are
cleared thoroughly with reservoir buffer using a syringe so that no unpolymerized
acrylamide is left in the wells. The spacer fixed on the lower side is removed and
the lower and upper chambers of the apparatus are filled with reservoir buffer in
such a manner that no air bubble is formed between gel and buffer system. After
this, pre-electrophoresis is carried out at 10 mA for 15 min.
Protein samples are dissolved in sample buffer 1.0 M Tris-HCl (pH 6.8) containing
5% glycerol and 0.02% bromophenol blue and loaded into the wells using a
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Hamilton syringe. The electrodes are connected to electrophoretic power supply
unit and run at 10 mA till the dye reached the end of stacking gel.
Coomassie Staining: After the electrophoresis, the gel is taken out and stained
using coomassie brilliant blue R-250 staining solution with constant shaking for 8
h to visualize protein bands. After staining, the gel is transferred to destaining
solution. The gel is destained with gentle shaking on a gel rocker by changing the
destaining solution several times till the gel background is clear. After destaining,
the gels are photographed and preserved in destaining solution with 10% glycerol
in dark and cool place.
Silver Staining: The protein bands in the gels are also stained by silver staining as
described by Blum et al, (1987). The gel is removed from the chamber and
transferred to the fixative solution (50% methanol and 7% acetic acid in water) for
2 h. The gel is soaked in Hypo solution (20 mg of sodium thiosulfate in 100 ml of
distilled water) for 1 min and then rinsed with distilled water three times for 1
min each. It is then transferred to staining solution (100 mg of AgNO3 in 100 ml of
distilled water and 75 µL of formaldehyde) and stained for 20 min. After proper
washing with distilled water, the gel is developed in a solution of 100 ml of
distilled water containing 2.0 g of Na2CO3 and 50 µL of formaldehyde. After the
development, the reaction is stopped by 0.1% citric acid.
2. SDS-PAGE (sodium dodecyl sulfate- poly acrylamide gel electrophoresis)
SDS-PAGE is carried out by the method of Laemmli (1970) with slight
modifications. acrylamide-bis-acrylamide solution, resolving gel buffer, stacking
gel buffer, ammonium persulphate, staining and destaining solutions, and
bromophenol blue are the same as used for Native- PAGE. The following
additional solutions were prepared for SDS-PAGE:
SDS (10%, w/v): Dissolved 1 g SDS in 10 ml of distilled water.
Reservoir buffer: 3.0 g Tris base, 14.4 g glycine and 1 g SDS are dissolved in
distilled water and its pH was adjusted to pH 8.3. The volume was made to 1 L
with distilled water.
Sample buffer (2x): It is prepared by mixing 2.5 ml of 1M Tris-HCl buffer (pH 6.8),
2.0 ml glycerol (20%), 0.4 g SDS, 1.0 ml β-mercaptoethanol and 0.4 ml of 1%
bromophenol blue and volume is made to 10.0 ml with distilled water.
Sample preparation: Purified enzyme solution is mixed with equal volume of
sample buffer (2x), boiled for 5 min and cooled prior to loading.
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Molecular weight markers: A pre-stained mixture of SDS-PAGE molecular weight
markers viz. BSA (66 kDa), ovalbumin (46 kDa), pepsin (34.7 kDa), trypsinogen (24
kDa) and lysozyme (14.3 kDa) is used as supplied.
PROCEDURE: SDS-PAGE was performed using 12% resolving and 2.5% stacking
gel, the compositions of which are given in Table 3.2. The gels are prepared as
described for native PAGE. The sample containing 100-150µg protein was loaded
in to the wells. The standard SDS-PAGE molecular weight markers are coelectrophoresed. The electrophoresis is carried out and gels are processed for
visualization of protein bands as described for native-PAGE. Molecular mass of
the purified enzyme protein is calculated using Gel Documentation system.
Table: Composition of different Recipe of gels for SDS-PAGE
Native Gel Recipe (for 3.0 % to 15% PAGE)
Gel
%
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.5
10.0
12.5
15
Water
ml
6.4
6.25
6.0
5.9
5.75
5.6
5.4
5.25
5.1
4.9
4.5
4.1
3.3
2.4
Lower Tris
ml
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
2.5
Acrylamide
(30%) + bis
ml
Acrylamide
(8%)
1.0
1.17
1.34
1.5
1.67
1.81
2.02
2.17
2.34
2.5
2.92
3.3
4.1
4.92
APS (10%)
µL
85
87
87
80
75
75
70
70
62
45
45
45
45
45
TEMED
µL
6.2
6.2
6.2
6.2
5.0
5.0
5.0
3.7
3.7
3.7
2.5
2.5
2.5
2.5
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Section: VI
Spectroscopic
Spectroscopic Techniques
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EXPERIMENT NO.26
AIM: (a) Verify Beer’s Law And Apply It To Find The Concentration Of The Given
Unknown Solution.
(b) To Determine ʎmax (Wave Length Of Maximum Absorption) Of Solution Of
KMNO4 Using A Spectrophotometer.
THEORY:
When an electromagnetic radiation is passed through a sample, certain
characteristic wavelengths are absorbed by the sample. As a result the intensity
of the transmitted light is decreased. The measurement of the decrease in
intensity of radiation is the basis of spectrophotometer. Thus the
spectrophotometer compares the intensity of the transmitted light with that of
incident light.
The absorption of light by a substance is governed by certain laws.
According to the Beer Lambert’s law the intensity of the incident light is
proportional to the length of thickness of the absorbing medium and the
concentration of the solution,
Log Io/I = A = ε . c
Io = Intensity of incident light
I = Intensity of transmitted light
A = Absorbance
L = Thickness of the medium
c = Concentration in mol L-1
ε = Molar absorption coefficient
The molar absorption coefficient is the absorbance of a solution having unit
concentration (C = 1M) placed in a cell of unit thickness (l= 1cm). Absorbance is
also called Optical Density (OD)
The absorbance (OD) of a solution in a container of fixed path length is directly
proportional to the concentration of a solution. i .e
A = ε.c
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A plot between absorbance and concentration is expected to be linear. Such a
straight line plot, passing through the origin, shows that Beer – Lambert’s law is
obeyed. This plot, known as calibration curve can be employed in finding the
concentration of a given solution.
REAGENTS: Distilled water, standard solution of KMnO4, tissue paper.
APPARATUS: UV – visible spectrophotometer, beaker.
SPECTROPHOTOMETER:
A spectrometer is a device which detects the percentage transmittance of light
radiation when light of certain intensity and frequency range is passed through
the sample. Thus the instrument compares the intensity of the transmitted light
with that of the incident light.
There are many spectrophotometers available for the visible range extending
from 3800- 7800 Ao.
Setting of the Spectrophotometer
1) Spectrophotometer should initially read zero on transmittance scale (T). if it
does not read zero, set it mechanically with adjusting knob.
2) Connect the instrument to the mains and put on the power switch.
3) Adjust the wavelength knob to the required wavelength region on scale.
4) Choose the position of wavelength switch correspondingly either to 340 –
400 nm or 400-960nm.
5) Adjust the meter needle on zero transmittance scale and 100 on O.D scale.
Working of the Spectrophotometer
6) Open the lid of the cell compartment and insert a cuvette containing the
blank solvent (distilled water). Close the lid.
7) Adjust the needle to 100% transmittance or zero optical density.
8) Remove the cuvette and close the lid tightly again. Empty the cuvette and
rinse it with the standard solution of KMnO4 (0.01IM). Fill it with standard
solution.
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9) Now place the cuvette containing the standard solution in the cell
compartment. Note the O.D and transmittance.
10) Now change the wavelength by 20nm and note absorbance (OD) and
transmittance for each wavelength.
11) Plot a graph between wavelength measurement on the x-axis and
absorbance (OD) on the y-axis.
Verification of Beer’s law
12) Fix the wavelength at ʎmax position.
13) Prepare KMnO4 solution with concentration 0.2%, 0.5%, 1.0%, 1.5%, 2.0%,
2.5%, and 3.0% etc. (20ml each)
14) Note down the absorbance (OD) of series of solution of KMnO4 prepared
above by the method described above.
15) Plot a graph between OD against concentration. (If a straight line is
obtained Beer’s law is verified)
16) Now find out the OD of the unknown solution of the KMnO4. Find out the
concentration of this solution from the graph.
OBSERVATION & TABLE:
(i) Determination of ʎmax
Wavelength (nm)
Absorbance (OD)
(ii) Verification of Beer’s law
S.NO.
Concentration (C) (moles / L)
Absorbance (OD)
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CALCULATION:
(i) A curve is plotted between wavelength and absorbance (OD).
(ii) A curve is plotted between O.D and concentration and if a straight line is
obtained as shown by equation (i), Beer’s law is verified.
(iii) From the graph of O.D versus concentration, the concentration of the
unknown solution can be found out. For example, in the fig x is the O.D of
unknown solution then its concentration will be 1.0%.
RESULT & CONCLUSION:
(i)
ʎmax for KMnO4 = ………nm
(ii)
KMnO4 solution obeys Beer’s law
(iii)
Concentration of the unknown solution = ………mg/L
PRECAUTIONS:
i) Always use dilute solutions for getting calibration curve.
ii) Cuvette should be cleaned properly and must be wiped with tissue paper.
iii) Do not leave any finger marks on the cuvette.
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Section: VII
Laboratory Demonstrations
(PCR & ELISA)
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Experiment No.27
AIM: To Amplify A Specific DNA Fragment By Polymerase Chain Reaction.
INTRODUCTION: PCR is an in vitro method of enzymatic synthesis of specific DNA
fragment developed by Kary Mullis in 1983. It is very simple technique for
characterizing, analyzing & synthesizing DNA from virtually any living organism.
PCR is used to amplify a precise fragment of DNA from a complex mixture of
starting material called as template DNA.
A basic PCR requires the following components:
• DNA template that contains the region to be amplified.
• 2 primers complementary to the 3’ end of each of the sense & antisense strand
of the DNA.
• Thermostable DNA polymerase like Taq, Vent, Pfu etc.
• Deoxynucleotides phosphates (dATP, dCTP, dGTP, dTTP), the building blocks
from which the DNA polymerase synthesizes a new DNA strand.
• Buffer solution which provides a suitable chemical environment for optimal
activity & stability of DNA polymerase.
• Bivalent Mg/Mn ions, which are necessary for maximum Taq polymerase
activity & influence the efficiency of primer to template annealing.
PRINCIPLE: The purpose of a PCR is to amplify a specific DNA or RNA fragment.
PCR comprises of 3 basic steps:
1. Denaturation
2. Annealing
3. Primer extension
Initialization step: this step consists of heating the reaction mixture to 94-96°C
for 1-9 minutes to break the hydrogen bonds in DNA strands.
Denaturation step: this step is the first regular cycling event & consists of
heating the reaction mixture to 94-98°C for 20-30 seconds. As a result the
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template DNA denatures due to disruption of the H-bonds between
complementary bases of the DNA strands, yielding single strand of the DNA.
Annealing step: in this step the reaction temperature is lowered to 50-65°C for
20-40 seconds allowing annealing of the primer to the ss-DNA template.
Typically the annealing temperature is 3-5°C below the Tm (melting
temperature) of the primers used. Stable DNA-DNA H-bonds are only formed
when the primer sequence very closely matches the template sequence. The
polymerase binds to the primer- template hybrid & begins DNA synthesis.
Elongation step: in this step the temperature depends on the DNA polymerase
used. Taq polymerase has its optimum activity at 75-80°C. Commonly a
temperature of 68-72°C is used with this enzyme. The DNA polymerase
synthesizes a new strand of DNA, complementary to the DNA template strand
by incorporating dNTPs that are complementary to the template in 5’-3’
direction, condensing the 5’ –phosphate group of the dNTPs with the 3’
hydroxyl group at the end of the nascent DNA strand. The extension time
depends both upon the DNA polymerase used & on the length of DNA
fragment to be amplified. The DNA polymerase will polymerize a thousand
bases per minute at its optimum temperature. Under optimum conditions, i.e.,
if there are no limitations due to limiting substrates or reagents, at each step,
the amount of DNA target is doubled, leading to exponential amplification of
the specific DNA fragments.
Final elongation: this single step is occasionally performed at a temperature of
70-74°Cfor 5-15min after the last PCR cycle to ensure that any remaining ssDNA is fully extended. Denaturation, annealing & extension steps are repeated
20-30 times in an automated thermocycler that can heat & cool the reaction
mixture in tubes within a very short time. This results in exponential
accumulation of specific DNA fragments, ends of which are defined by 5’ ends
of the primers. The doubling of the number of DNA strands corresponding to
the target sequence allows us to estimate the amplification associated with
each cycle using the formula:
Amplification= 2n , where n= no. of cycles.
Final hold: this step may be employed for short term storage of the reaction
mixture at 4°C for an indefinite time.
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MATERIALS REQUIRED:
Glasswares, Ethidium Bromide, Thermocycler, Electrophoretic Apparatus, UVTransilluminator, Vortex Mixer, Micropipette & Tips, Crushed Ice etc.
Name of the items
Storage
10x assay buffer
-20 °C
Control PCR product
-20 °C
2.5mM dNTP mix
-20 °C
1 kb DNA loader
-20 °C
Forward primer (100ng/µl)
-20 °C
Reverse primer (100ng/µl)
-20 °C
Taq DNA polymerase
-20 °C
Template DNA
-20 °C
Molecular biology grade water
RT
25mM MgCl2
-20 °C
Agarose
RT
50X TAE
RT
6X gel loading buffer
2-8°C
Mineral oil (optional)
RT
PCR tubes
RT
PROCEDURE:
1. Preparation of master mix for PCR - To a PCR tube add all the following
ingredients in the following order:
Sr. no.
Ingredients
1.
Molecular bio. Grade water
2.
10x assay buffer
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Volume in µl
31.5
5
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3.
Template DNA
1
4.
Forward primer (100ng/µl)
1
5.
Reverse primer (100ng/µl)
1
6.
25 mM MgCl2
5
7.
2.5 mM dNTP mix
5
8.
Taq DNA polymerase
0.5
Total volume
50
2. Tap the tube for 1-2 sec. to mix the contents thoroughly.
3. Add 25 µl of mineral oil in the tube to avoid evaporation of the contents.
4. Place the tube in the thermocycler block & set the program to get DNA
amplification.
Note: it is not essential to add mineral oil if the thermocycler is equipped with a
heating lid.
PCR AMPLIFICATION CYCLE:
Carry out the amplification in a thermocycler for 25-30 cycles using the following
reaction conditions.
Initial denaturation at 94°C for 10 minutes
Denaturation at 94°C for 30 sec
Annealing at 58°C for 30 sec
25-30 CYCLES
Extension at 72°C for 45 sec
Final extension at 72°C for 10 min
Cooling at 4°C
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AGAROSE GEL ELECTROPHORESIS:
Electrophoresis of the amplified product will be carried out as per the procedure
given in Experiment No.23.
RESULT & INTERPRETATION:
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Experiment No.28
AIM: To Perform Sandwich ELISA.
INTRODUCTION: ELISA, also called Enzyme Linked Immunosorbent Assay,
employs antigens or antibodies conjugated to enzymes in such a way that the
immunological and enzymatic activity of each component is maintained. These
assays are very sensitive and give accurate results. The estimation of results can
be made either visually or spectrophotometrically. Various formats of ELISA are
available. This method is used for quantitation of antibody.
PRINCIPLE: Sandwich ELISA involves the attachment of a constant dilution of
antibody to the solid phase. After incubation, un-adsorbed antibodies are washed
away. Following that, un-adsorbed reactive sites are blocked on the plate. To that,
antigen at a single dilution or as a dilution range is then added. After incubation
unbound antigen are washed away. Bound antigen is then detected by the
addition of enzyme labeled secondary antibody specific for the "trapped" or
"captured" antigen. After incubation and washing away of unreacted conjugate,
substrate is added and the intensity of the colour is measured.
MATERIALS REQUIRED: ELISA Reader, Distilled water, Glasswares (Conical Flask,
Measuring cylinder, Pipette), Micropipette, Tips
Name of the items
storage
Antigen
-20ºC
Antibody
-20ºC
Sample I
-20ºC
Sample II
-20ºC
Wash buffer
RT
Blocker
4ºC
(dissolve 100mg in 5ml of 1X PBS)
Substrate (freshly prepare)
4ºC
Substrate buffer
4ºC
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Conjugate
4ºC
Coating buffer
4ºC
Stopping solution
RT
Hydrogen peroxide
4ºC
1x PBS
RT
ELISA moduls
RT
WORKING SOLUTION PREPARATION:
1. Blocking Solution
To prepare 2% blocking solution, take 5ml of 1X PBS and add 100mg of blocker
provided and mix well.
Note: Prepare freshly everytime before each experiment
2. Substrate
With the given substrate quantity, add 1ml of substrate buffer and mix well by
repeat pipetting. To this 1 ml again add 69ml of substrate buffer. Aliquot this
70ml stock solution into 7 separate 10ml storage tubes and wrap it with
aluminium foil and store at -20°C for subsequent usage. This will avoid the loss of
effectiveness of the substrate stock solution at the time of thawing for the
subsequent usage of each test. Take 10ml of the substrate stock solution and mix
with 40µl of hydrogen peroxide. (Prepare this step freshly before each test).
PROCEDURE:
1. Coating Of The Antibody
Dilute the antibody with the coating buffer provided at 1: 100 dilutions
and add 100µl to each well of an ELISA plate. Leave it overnight at 4°C for
passive adsorption of the antibody to the ELISA plates. Use 2 strips/rows
(16 wells) for each test for which you would require 1600µl of diluted
antibody. Make at least 1800 µl of diluted antibody to account for minor
pipetting errors.
2. Washing
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The concept of ELISA involves separation of bound and free reagents with the
washing step. The un-adsorbed antibody molecules need to be removed by
washing thrice. Washing is done by adding 300 µl of washing buffer to each
well, shaking it mildly and then discarding it vigorously into the sink. This
procedure should be repeat thrice during each washing step. After washing,
flick the plates and dry on a stack of dry filter papers to avoid any bubble
formation that would interfere in subsequent reagent additions.
3. Blocking
After coating and removal of unbound antibody, the remaining sites on the
ELISA plates has to be blocked to avoid direct binding of antigen or conjugate
which would lead to false positive reactions. Hence add 300µl of the blocker
solution to all the wells and incubate the plate at 37°C for 45 minutes.
NOTE: Use higher volumes (300 µl) of blocker for more efficient blocking of the
reactive sites on the sides of the wells also.
4. Washing
After incubation, washing is done as explained earlier.
5. Antigen Addition
After washing the strips, add 1001-11 of the antigen provided at a series of
double dilutions starting from 1:100 to 1: 1600 in phosphate buffered saline (1X
PBS), into the duplicate wells as:
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WELLS
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A
ANTIGEN 1:100
ANTIGEN 1: 1 00
B
ANTIGEN 1 :200
ANTIGEN 1 :200
C
ANTIGEN 1 :400
ANTIGEN 1 :400
D
ANTIGEN 1 :800
ANTIGEN 1 :800
E
ANTIGEN 1:1600
ANTIGEN 1:1600
F
Negative Control
Negative Control
G
SAMPLE 1 (1:100)
SAMPLE 1 (1:100)
H
SAMPLE2(1: 100)
SAMPLE2(1: 100)
Blocking buffer (100µl) can be added to wells F1 and F2 instead of antigen that
would serve as negative control.
Two samples can be used at a dilution of 1: 100 to wells G 1 and G2 and H1 and
H2 to determine the antigen content in those sample using the standard curve
generated from the standards (Wells A to E -1 and 2). Incubate the plate at
37°C for 45 minutes.
6. Washing
After incubation, washing is done as explained earlier.
7. Conjugate incubation
After washing the wells, add 100µl of the diluted conjugate provided (1: 1000 in
phosphate buffered saline) into all the 16 wells of the ELISA plate. Incubate the
plate at 37° for 45 minutes. And then wash.
8. Substrate addition
Add 100µl of the substrate solution into the 16 wells of the plate and incubate
the strips in dark at room temperature for 10minutes.
9. Stop The Reaction And Reading Optical Density
The appearance of yellowish brown colour indicates that the antigen antibody
reaction has occurred. Stop the reaction by adding 25µl of the stop solution.
Read the optical density (OD) values of the plate in an ELISA reader at 490 nm
wavelength.
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RESULTS & INTERPRETETION:
Add 100µl antibody with coating buffer
(incubate for overnight at 4°C)
Add 300 µl of washing buffer
Add 300 µl of blocking solution
(incubate at 37°C for 45min)
Wash
Add 300 µl of antigen
(incubate)
Wash
Add 100 µl of dilute conjugate
(incubate)
Wash
Add100 µl of substrate solution
(incubate)
Observe the result
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ABOUT THE AUTHORS
Dr Gyanendra Awasthi is presently working as reader and Head, Department of
Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences,
Dehradun. He is teaching Biochemistry to UG and PG students since last ten years. He
did his M.Sc. degree in Biochemistry from Lucknow University and Ph.D. from
H.N.B.Garhwal University Srinagar and also qualified CSIR-NET in life sciences .He
has published 2 books more than 20 research papers in
national and international
journals of repute.
Dr Santosh Kumar is presently working as Assistant professor, Department of
Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences, Dehradun.
He is teaching Biochemistry to UG and PG students since last seven years. He did his
M.VSc. degree in Animal Biochemistry from NDRI; Karnal. He has published more than
05 research papers in national and international journals of repute.
Dr Ashwani Sanghi is presently working as Assistant professor in Department of
Biochemistry in Dolphin (P.G.) Institute of Biomedical and Natural Sciences,
Dehradun. He is teaching Biochemistry to UG and PG students since last 06 years. He
did his M.Sc. degree in Biochemistry and Ph.D. from Kurkushestra University
Kurkushestra. He has published more than 06 research papers in national and
international journals of repute.
Mr Shiv Sharan is presently working as assistant professor Department of Biochemistry
in Dolphin (P.G.) Institute of Biomedical and Natural Sciences, Dehradun. He is teaching
Biochemistry to UG and PG students since last ten years. He did his M.Sc. degree in
Biochemistry from Allahabad Agriculture deemed university Allahabad and pursuing Ph.
D. from Uttrakhand Technical University, Dehradun. He has published more than 05
research papers in national and international journals of repute.
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