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Practical Biochemistry: A Student Companion

Book · July 2015

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Anand Tiwari, PhD

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There is a growing need for a reference book of this type due to
curriculum modularization in various disciplines of Life Science at all levels
and in all areas of Biochemistry. The “Practical Biochemistry: A Student
Companion” is intended to bridge this gap. The author draw on his
teaching and research experience to provide reference book for both
under graduate and graduate students taking Biochemistry as well as
young researchers in life and teachers of Biochemistry. This book has three
parts: Part I deal with identification and quantitative determination of
carbohydrates, lipids, amino acids, proteins and nucleic acids. Part II
includes enzyme assays and Part III covers various bioanalytical techniques.
The book is written in a lucid way with several chemical equations and
illustrations.

Anand Tiwari

Dr. Anand Tiwari is the principal investigator under

Practical Biochemistry:
the DST-SERB Start-Up Research Grant for Young
Scientists. He obtained his Master’s and Doctorate A Student Companion
degree (CSIR-JRF fellowship) in Biochemistry at
Osmania University, India and a postdoctoral stint at
IMCB, Singapore. He has several research publications
in peer-reviewed international journals.

978-3-659-75716-7
 Contents

Preface................................................................................................................... v

Part I : Qualitative & Quantitative Analysis ...............................................................4  

1.1  Carbohydrates .........................................................................................................4  
Experiment  1:  Molish  Test.....................................................................................................5  
Experiment  2:  Iodine  Test .....................................................................................................6  
Experiment  3:  Benedict’s  Test .............................................................................................7  
Experiment  4:  Barfoed’s  Test...............................................................................................8  
Experiment  5:  Seliwanoff’s  Test..........................................................................................9  
Experiment  6:  Osazone  Test .............................................................................................. 10  
Experiment  7:  Athrone  Test............................................................................................... 11  
Experiment  8:  Dinitrosalicylic  acid  Method................................................................ 12  
Experiment  9:  Roe’s  method ............................................................................................. 13  
Experiment  10:  Fehling’s  test............................................................................................ 14  
Experiment  11:  Somogyi-­‐Nelson  method.................................................................... 15  
Experiment  12:  Mucic  acid  test ........................................................................................ 16  
1.2  Amino  acids ........................................................................................................... 17  
Experiment  13:  Ninhydrin  Test  (qualitative)............................................................. 18  
Experiment  14:  Ninhydrin  Test  (quantitative) ......................................................... 19  
Experiment  15:  Isatin  Test ................................................................................................. 20  
Experiment  16:  Xanthoprotic  Test.................................................................................. 21  
Experiment  17:  Pauly’s  diazo  test ................................................................................... 22  
Experiment  18:  Sakaguchi  Test........................................................................................ 23  
Experiment  19:  Millon’s  Test ............................................................................................ 24  
Experiment  20:  Ehrlich  Test.............................................................................................. 25  
Experiment  21:  Nitroprusside  Test................................................................................ 26  
Experiment  22:  Sullivan  and  McCarthy’s  Test ........................................................... 27  
Experiment  23:  Hopkins-­‐Cole  Test................................................................................. 28  
Experiment  24:  Lead  acetate  Test................................................................................... 29  
Experiment  25:  Gerngross  test......................................................................................... 30  
Experiment  26:  Amino  acids  Titration  Curve ............................................................ 31  
1.3  Lipids ....................................................................................................................... 32  

  1  
 Experiment  27:  Ethanol  emulsion  Test......................................................................... 33  
Experiment  28:  Acrolein  Test ........................................................................................... 33  
Experiment  29:  Sudan  IV..................................................................................................... 33  
Experiment  30:  Acid  Value ................................................................................................. 34  
Experiment  31:  Peroxide  Value........................................................................................ 35  
Experiment  32:  Saponification  Value ............................................................................ 36  
Experiment  33:  Iodine  Value............................................................................................. 37  
Experiment  34:  Libermann-­‐Burchard  Method.......................................................... 38  
1.4  Nucleic  acids.......................................................................................................... 39  
Experiment  35:  Diphenylamine  Method ...................................................................... 39  
Experiment  36:  Fiske-­‐Subbarow  Method .................................................................... 40  
Experiment  37:  Bial’s  orcinol  Method........................................................................... 41  
Experiment  38:  Quantitation  of  DNA  by  A260  nm...................................................... 42  
1.5  Proteins .................................................................................................................. 43  
Experiment  39:  Biuret  protein  assay............................................................................. 43  
Experiment  40:  Folin-­‐Lowry’s  Method ......................................................................... 44  
Experiment  41:  Bradford  Method ................................................................................... 45  
Experiment  42:  Microkjeldal  Method............................................................................ 46  
Experiment  43:  Isoelectric  Point  (pI) ............................................................................ 47  

Part II: ENZYMOLOGY .............................................................................................48  

Experiment  44:  Alkaline  Phosphatase  assay.............................................................. 48  
Experiment  45:  Acid  phosphatase  assay...................................................................... 49  
Experiment  46:  β-­‐amylase  assay ..................................................................................... 50  
Experiment  47:  Urease  assay ............................................................................................ 51  
Experiment  48:  pH  Optima  of  Urease............................................................................ 52  
Experiment  49:  Temperature  Optima  of  Urease ...................................................... 53  

Part III: BIOANALYTICAL TECHNIQUES.............................................................54  

Experiment  50:  Molar  attenuation  coefficient........................................................... 54  
Experiment  51:  Ammonium  Sulfate  Precipitation................................................... 55  
Experiment  52:  Dialysis....................................................................................................... 56  
Experiment  53:  Paper  Chromatography  of  amino  acids ....................................... 57  
Experiment  54:  Paper  Chromatography  of  sugars .................................................. 58  
Experiment  55:  Paper  Chromatography  of  purines  and  pyrimidines............. 59  
Experiment  56:  Sanger’s  FDNB  Method  (N-­‐terminal  amino  acid).................... 60  

  2  
 Experiment  57:  Carboxypeptidase  Method  (C-­‐terminal  amino  acid).............. 62  
Experiment  58:  Thin  Layer  Chromatography  (TLC)  of  amino  acids................ 63  
Experiment  59:  TLC  of  plant  pigments ......................................................................... 64  
Experiment  60:  Ion  Exchange  Chromatography....................................................... 66  
Experiment  61:  Gel-­‐filtration  Chromatography........................................................ 67  
Experiment  62:  Agarose  Gel  Electrophoresis ............................................................ 69  
Experiment  63:  Cellulose  Acetate  Paper  Electrophoresis .................................... 71  
Bibliography............................................................................................................... 72  

  3  
 PART I : QUALITATIVE & QUANTITATIVE ANALYSIS
1.1 CARBOHYDRATES

No Reagent Composition and Preparation

1 Molish 5% (w/v) α-napthol in 95% ethanol. 500 mg α-napthol in 10 ml 95%
ethanol.
2 Iodine Lugol’s solution: Dissolve 10 g KI in 100 ml water. Add 5 g I2 crystals to
this solution and mix. Store in brown bottle.
3 Benedict CuSO4.5H2O in Citrate buffer. Dissolve 17.3 g sodium citrate and 10 g
sodium carbonate in 85 ml H2O. Filter it. Dissolve 1.73 g CuSO4.5H2O
in 10 ml H2O followed by addition of carbonate-citrate mixture. Make
up to 100 ml with H2O.
4 Barfoed Dissolve 13.3 g of copper acetate in 200 ml water. Boil and add 1.8 ml
glacial acetic acid. Cool and make up to 200 ml with H2O and filter.
5 Seliwanoff 0.05% resorcinol (m-dihydroxybenzene) in 3 N HCl. Dissolve 50 mg
resorcinol in 33 ml conc. HCl, make to 100 ml with H2O.
6 Osazone Mix 1:2 (w/w) phenylhydrazine and sodium acetate.

  4  
 Experiment 1: Molish Test

Molish test is a group test for all carbohydrates either free or bound to proteins and lipids.

Principle: The reaction is based on the fact that concentrated acid catalyses the
dehydration of sugars to form furfural (from pentoses) or hydroxymethyl furfural (from
hexoses). Either of these aldehydes condenses with two molecules of napthol to
form a purple or violet colored complex at the interface of the acid and test layer. If the
carbohydrate is a poly- or disaccharide, a glycoprotein or a glycolipid, the acid first
hydrolyses it into component monosaccharides, which get dehydrated to form
furfural or its derivatives.

Procedure:
1. Add 2-3 drops of α-napthol solution to 2 ml each of water (blank/negative
control), 2% glucose solution (positive control) and test sample in a test tube and
mix.
2. Hold the test tube in inclined position and gently add 1 ml H2SO4 along the wall
of the test tube (do not mix).
Note:
1. Trioses and tetroses do not have the necessary five carbon atoms for furfural formation
so they do not give positive result for this reaction.
2. Molish test is not a specific test for carbohydrates. Furfurals as such or furfural yielding
substance, some organic acids like citric acid, lactic acid, oxalic acid, formic acid etc,
can give a positive test.
3. A red ring may form if a concentrated sugar solution is used. This may be due to
partial charring of the sugar by the acid.
4. A black ring may form if conc. H2SO4 is not added very slowly. This can be due to the
heat generated during the reaction, which can char the carbohydrates.
5. A green ring may form due to certain impurities in the reagent interacting with α-
napthol and the acid.
6. α-napthol solution is unstable and should be prepared fresh.
7. The test tube should be completely dry.

  5  
 Experiment 2: Iodine Test

Iodine test is used to distinguish mono- or disaccharides from certain polysaccharides like
amylose, dextrin and glycogen.

Principle: Iodine test is based on the fact that polyiodide ions form colored adsorption
complex with helical chains of glucose residues of amylose (blue-black), dextrin (purple)
or glycogen (reddish-brown). Monosaccharides, disaccharides and branched
polysaccharides like cellulose remain colorless. Amylopectin produces an orange-yellow
hue. The reagent used in iodine test is Lugol’s iodine, which is an aqueous solution of
elemental iodine and potassium iodide. Iodine on its own is insoluble in water. Addition of
potassium iodide results in reversible reaction of the iodide ion with iodine to form triiodide
ion, which further reacts with an iodine molecule to form pentaiodide ion. Bench iodine
solution appears brown, whereas, the iodide, triiodide and pentaiodide ions are colorless.
Many details of how exactly the color during this test is developed are still unknown. It is
observed that the helix (coil or spring) structure of the glucose chain is the key to this test.
Further, the resulting color depends on the length of the glucose chains. The triiodide and
pentaiodide ions formed are linear and slip inside the helix structure. It is believed that
transfer of charge between the helix and the polyiodide ions results in changes in the
spacing of the energy levels, which can absorb visible light, giving the complex its color.

Procedure: Add 2-3 drops of Lugol’s solution to 5 ml of solution to be tested.

Note:
1. On heating, the blue color amylose-iodine complex disassociates but is formed again on
cooling because the helical structure of amylose is disrupted, there by amylose loses its
iodine binding capacity and the blue color. The blue color reappears on cooling due to
the recovery of iodine binding capacity due to regaining of helical structure.
2. At very low pH amylose undergoes hydrolysis, so it is not recommended to conduct
iodine test for amylose under these conditions.
3. The blue color amylose-iodine complex can be detected visually with concentrations of
iodine as low as 20 µM at 20 °C.
4. The dextrin-iodine complex disassociates on heating but is not formed again on cooling
because the concentration of residual iodine is too low to form complex with dextrin.
5. Presence of proteins (particularly albumin) in the sample may produce false positive
results as proteins can compete with starch for binding iodine.

  6  
 Experiment 3: Benedict’s Test

Benedict’s test is used to differentiate between reducing and non-reducing sugars.

 
Principle: The carbohydrates having a free or potentially free, aldehyde or ketone group
can act as a reducing agent. Benedict’s reagent appears deep blue in color and consists of
copper sulphate mixed with sodium citrate and a weak alkali, sodium carbonate. When
reducing sugars are heated in the presence of alkali they get converted to enediols, which
are powerful reducing agents. Enediols reduce the cupric ions (Cu2+) present in the
Benedicts reagent to cuprous ions (Cu+), which get precipitated as insoluble red colored
cuprous oxide (Cu2O).
The test is semi-quantitative, since the color of the precipitate indicates approximate
quantity of the sugar present in the sample. For a sample containing reducing sugar the
color of the sample during boiling progress from blue (with no reducing sugar present),
green, yellow, orange, red, and then brick red or brown (with high concentration of
reducing sugars).

The citrate ions form a complex with cupric ions and prevent its precipitation with the
hydroxide ions as cupric hydroxide.
Procedure:
1. Add 0.5-1.0 ml of the test solution to 2 ml of Benedict’s reagent.
2. Keep the test tubes in boiling water bath for 4-10 min.
Note:
1. Sucrose, starch, inositol gives negative result, whereas lactose and maltose give positive
result with Benedict’s reagent.
2. Benedict modified the Fehling’s solution to make an improved single reagent, which is
quite stable. It is very sensitive to even small quantities of reducing sugars (0.1%) and
yields enough precipitate.
3. False positive reaction for urine sample may be obtained due to presence of reducing
substances like uric acid, ascorbic acid (during vitamin supplementation), drugs
(levodopa) etc
4. A greenish precipitate indicates about 0.5 g% concentration; yellow precipitate indicates
1 g% concentration; orange indicates 1.5 g% and red indicates 2 g% or higher
concentration.

  7  
 Experiment 4: Barfoed’s Test
 
Barfoed’s test is used to detect reducing monosaccharides in the presence of disaccharides.
 
Principle: Barfoed’s reagent consists of copper acetate in dilute acetic acid. Since acidic
pH is unfavorable for reduction, monosaccharides, which are stronger reducing agents,
react in about 1-2 min, whereas reducing disaccharides take 7-12 min to first get
hydrolyzed in the acidic solution and then react. A thin red precipitate is formed at the
bottom or sides of the tube. Thus, the difference in reducing strength can be detected.

Procedure:
1. Add 3 ml of Barfoed’s reagent to 1 ml of sample.
2. Keep the test tubes in boiling water bath for 1-2 min only.

Note:
1. The boiling should not exceed 1-2 min, otherwise reducing disaccharides may be
hydrolyzed and give a positive test result.
2. The disaccharide solutions used for this test should not exceed 1% (w/v).
Sugars (1% w/v)
Glucose Fructose Xylose Sucrose Maltose Galactose
Upto 2 min + + + - - -
After 7-12 min + + + + + +
Higher concentration (≥ 5%) gives positive result for disaccharides with Barfoed’s test.

3. Chloride ions interfere with this test. Since urine contains chloride ions Barfoed’s test is
not suitable for detection of reducing sugars in urine.

  8  
 Experiment 5: Seliwanoff’s Test

Seliwanoff’s test is a timed color reaction specific for ketohexoses. It is used to distinguish
ketoses from aldoses.  
Principle: The reagent consists of resorcinol and conc HCl. The acid hydrolyses of
polysaccharides and oligosaccharides yields simpler sugars. Ketohexoses undergo
dehydration to yield 5-hydroxymethyl furfural more rapidly than aldohexoses. The
dehydrated ketose reacts with resorcinol to produce a deep cheery red color complex (not
precipitate). Aldoses may react slightly to produce a faint pink to cherry red color.

Procedure:
1. Add 2 ml of Seliwanoff’s reagent to 1 ml of sample.
2. Keep in boiling water bath for 1 min.
Note:
1. Sucrose and inulin also give this test because these are hydrolysed by acid to give
fructose.
2. High concentration of glucose or other sugar may interfere by producing similar
colored compounds with seliwanoff reagent.
3. Prolonged boiling can transform glucose to fructose by catalytic action of acid and form
cherry red complex.

  9  
 Experiment 6: Osazone Test

Osazone test is used to detect reducing sugars.

Principle: The reagent consists of phenylhydrazine in acetate buffer. Carbohydrates with

free or potentially free carbonyl groups react with phenylhydrazine to form osazone. The
condensation-oxidation-condensation reaction between three molecules of phenylhydrazine
and carbon one and two of aldoses or ketoses to yields 1,2-diphenyhydrazone, which are
known as osazone. Osazone appear as yellow colored crystals of characteristic shape,
solubility, melting point and time of formation. Since both carbons 1 and 2 are involved in
the reaction C-2 epimers produce the same osazone. Ketoses with configurations identical
to aldoses below C-2 give the same osazones e.g. glucose and fructose.

The characteristic features of osazone are given in the following table:

Carbohydrate (osazone) Time of formation (min) Crystalline structure
Fructosazone 2 Needle shape
Glucosazone 5 Needle shape
Galactosazone 20 Thorny ball shape
Maltosazone 30-45 Sunflower/Star shape
Lactosazone 30-45 Cotton ball/Powder puff shape

Procedure:
1. Add 0.3 g of osazone mixture and 5 drops of glacial acetic acid to 5 ml of sample in each
of the test tubes and mix well (warm gently if required to dissolve the contents).
2. Keep the test tube in boiling water bath and observe for formation of crystal at various
time points indicated in the observation table.
3. Observe the shape of the crystal under low magnification.
Note:
1. This is the only test to distinguish lactose from maltose during identification of
unknown sugars.
2. Sucrose is a non-reducing sugar but it forms osazone on boiling for 30 min. Maltose and
Lactose form osazone after boiling the sample for more than 2 hrs.
3. Osazones of monosaccharides are insoluble in hot solution unlike those of disacchrides.
4. After incubating the tubes in boiling water bath for 30 min, the osazone solution should
be allowed to cool gradually to room temperature to facilitate proper formation of the
crystals. Tubes should not be cooled under running tap water or on ice.
5. Sodium acetate and acetic acid together function as acetate buffer and maintain the pH
at 5, which is proper for the formation of osazones. If excess acetic acid is added, the
pH of the reaction reduces further resulting in the hydrolysis of disaccharides to
monosacharides, which then form osazones insoluble in hot solution.

  10  
 Experiment 7: Athrone Test
 
Anthrone method is a group test for carbohydrates. It is a rapid and convenient method for
quantification of carbohydrates either free or bound to proteins and lipids.
 
Principle: If the carbohydrate is a poly- or disaccharide, a glycoprotein or a glycolipid, the
concentrated acid present in anthrone reagent first hydrolyses it into component
monosaccharides. Further the concentrated acid catalyses the dehydration of sugars to
form furfural (from pentoses) or hydroxymethyl furfural (from hexoses). Either of these
aldehydes condenses with two molecules of napthol to form a bluish green complex, which
is quantified by measuring absorbance at 620 nm in a spectrophotometer or using red filter
in a colorimeter.

Reagents: Dissolve 2 g anthrone in 50 ml conc. H2SO4, makeup to 100 ml with conc.

H2SO4.
Procedure:
1. Setup and process the standard (glucose), blank and unknown sample as described in
the table below.
Tube No. 1 2 3 4 5 6 7 8
Blank Glucose Standard Test
[Sugar] µg/ml --- 10 20 40 60 80 100 NA
Working soln (ml) --- 0.1 0.2 0.4 0.6 0.8 1.0 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 ---
Anthrone reagent 4 ml
Boil for 10 min
A620 nm

2. Measure absorbance at 620 nm in spectrophotometer or in colorimeter using red filter.

Use the blank to set absorbance to zero then measure absorbance of each tube.
3. Plot a standard curve using absorbance values of serial dilutions of glucose. (X-axis:
sugar concentration in µg/ml; Y-axis: absorbance at 620 nm). Based on the standard
curve determine carbohydrate content in unknown sample.
Note:
1. Anthrone test is not a specific test for carbohydrates. Furfurals as such and furfural
yielding substance, some organic acids like citric acid, lactic acid, oxalic acid, formic
acid etc, can give a positive test.
2. If the sample contains proteins with large number of tryptophan residues, the reaction
produces red color.

  11  
 Experiment 8: Dinitrosalicylic acid Method

Dinitrosalicylic acid (DNSA) method is used for estimating the concentration of reducing
sugars in a sample.
 
Principle: The carbohydrates having a free or potentially free, aldehyde or ketone group
can act as reducing agents. The DNSA reagent is an alkaline solution of 3,5-
dinitrosalicyclic acid and tartarate salt. On heating the reducing sugars can reduce 3,5-
dinitrosalicyclic acid to 3-amino-5-nitrosalicyclic acid, which appears orange-red in color.
The intensity of the color is proportional to amount of reducing sugar present in the
sample. The orange color developed is compared to standards in a spectrophotometer at
540 nm.

Reagents: Dissolve 30 g potassium sodium tartarate in 50 ml water and add 20 ml 2 M

NaOH. Mix well, add 1 g DNSA powder and make up to 100 ml with H2O
Procedure:
1. Setup and process the standard (glucose), blank and unknown sample as
described in the table below.
Tube No. 1 2 3 4 5 6 7
Blank Glucose Standard Test
[Glucose] µg/ml --- 100 200 300 400 500 NA
Working soln (ml) --- 0.1 0.2 0.3 0.4 0.5 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 ---
DNSA reagent 1 ml
Boil for 10 min
A540 nm
2. Measure absorbance at 540 nm in spectrophotometer. Use the blank to set
absorbance to zero then measure absorbance of each tube.
3. Plot a standard curve using absorbance values of serial dilutions of glucose. (X-
axis: sugar concentration in µg/ml; Y-axis: absorbance at 540 nm). Based on the
standard curve determine carbohydrate content in unknown sample.
Note:
1. Different sugars give different color yields. The method is therefore not suitable for the
determination of a complex mixture of reducing sugars.
2. The DNSA method is simple, sensitive and adaptable to large sample size.

  12  
 Experiment 9: Roe’s method

The Roe’s method is a modified Seliwanoff’s method and is generally used to determine the
concentration of fructose in a given sample.
 
Principle: Ketosugars undergo rapid dehydration to hydroxymethyl furfural than aldo
sugars. The hydroxymethyl furfural formed later condenses with resorcinol to give a pink
colored complex (λmax 520 nm).
Reagents: (i) Standard fructose stock solution (1 mg/ml): weigh 100 mg of fructose and
transfer it into a 100 ml volumetric flask. Make up the volume to 100 ml with distilled
water. (ii) Working standard fructose solution (100 µg/ml): dilute 10 ml of the stock
solution to 100 ml with distilled water in a volumetric flask. (iii) Resorcinol reagent:
resorcinol (0.1% w/v) and thiourea (0.25% w/v) in glacial acetic acid. (iv) Dilute HCl: mix
concentrated hydrochloric acid and distilled water in the ratio of 5:1
Procedure:
1. Setup and process the standard (glucose), blank and unknown sample as described in
the table below.
Tube No. 1 2 3 4 5 6 7 8 9 12
Blank Fructose Standard Test
NA
[Fructose] (µg/ml) --- 10 20 30 40 50 60 70 80

Working soln (ml) --- 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 2.0
Water (ml) 2 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 ---
Resorcinol reagent 1 ml
dil. HCl 7 ml
Heat at 80°C for 10 min
A520 nm
2. Measure absorbance at 520 nm in spectrophotometer. Use the blank to set
absorbance to zero then measure absorbance of each tube.
3. Plot a standard curve using absorbance values of serial dilutions of fructose. (X-
axis: sugar concentration in µg/ml; Y-axis: absorbance at 520 nm). Based on the
standard curve determine carbohydrate content in unknown sample (remember
to take the dilution factor into account).

  13  
 Experiment 10: Fehling’s test

Fehling’s test is used to differentiate between reducing and non-reducing sugars.

 
Principle: The carbohydrates having a free or potentially free, aldehyde or ketone group
can act as reducing agents. Fehling’s reagent appears deep blue in color and consists of
copper sulphate mixed with potassium sodium tartarate and a strong alkali, usually,
sodium hydroxide. On heating the sample in presence of Fehling’s solution,
bistartarocuprate(II) complex oxidizes the aldoses to corresponding aldonic acids, and in
the process the copper(II) ions of the complex are reduced to insoluble yellow or red color
precipitate of cuprous(I) oxide(Cu2O) ions. The ketoses on the other hand are oxidized to
yield shorter chain acids.

The tartarate ions prevent the formation of insoluble Cu(OH)2 from the reaction of
CuSO4.5H2O and NaOH present in the solution by forming bistartaratocuprate(II)
complex. This complex releases cupric ions slowly for reduction thus preventing the
formation of black cupric oxide. If Fehling’s solution is heated in the absence of reducing
sugars it forms black precipitate of cupric oxide.

Reagents: Fehling’s solution A- Dissolve 7.0 g of CuSO4.7H2O in 100 ml H2O. Fehling’s

solution B- Dissolve 24.0 g of KOH and 34.6 g of potassium sodium tartarate in 100 ml
H2O. Fehling’s reagent- Mix equal volumes of A and B solution just before use.
Procedure:
1. Add 1 ml of Fehling’s reagent to 1 ml of test solution and mix well.
2. Place the test tube in boiling water bath for 2-3 min.
Note:
1. Fehling’s test gives positive result for aldo monosaccharides (due to presence of
oxidizable aldehyde group) and also for keto monosaccharides, since they are converted
to aldoses by the base in the reagent.
2. Sucrose does not react with Fehling’s reagent because the anomeric carbon of glucose is
involved in glycosidic bond formation (free form of aldehyde is not present). Lactose
and Maltose are reducing sugars. The reducing sugars e.g. glucose get oxidized because
glucose exists in aqueous solution in three different forms (two cyclic and one non-
cyclic). The non-cyclic is the least common but it is present in small amount. This form
has an aldehydic group at the end of the chain and is the group that is oxidized. As the
aldehyde form gets oxidized, more of the non-cyclic form is produced due to Le
Chatelier's principle.
3. Fehling’s is used to determine dextrose equivalents for the conversion of starch to
glucose syrup.
4. Compounds other than reducing sugars also test positive to this test, e.g. chloroform,
ammonium salts. Formic acid is readily oxidized to carbon dioxide and water and gives
positive Fehling’s test result.
5. Fehling’s can be used to screen for glucose in urine, to detect diabetes. Since cuprous
oxide is dissolved by ammonia, it is not possible to detect small quantities of reducing
sugars in fluids like urine, which are saturated with ammonia.

  14  
 Experiment 11: Somogyi-Nelson method

Somogyi-Nelson method is used for quantitative determination of relatively pure sample of

reducing sugars.

Principle: The carbohydrates having a free or potentially free, aldehyde or ketone group
can act as reducing agents. Alkaline copper tartarate is the active reagent in Somogyi-
Nelson method. When a reducing sugar is heated in an alkaline solution of copper
tartarate, cuprous oxide is produced, which in turn reacts with arsenomolybdate to give
molybdenum blue. The blue color developed is compared to standards in a
spectrophotometer at 620 nm. The presence of sodium sulfate in the reaction mixture
prevents reoxidation of cuprous oxide by atmospheric oxygen.
Reagents: Dissolve 1.5 g copper sulfate in a small volume of distilled water. Add one drop
of sulfuric acid and make up to 10 ml.
Procedure:
1. Add 1 ml alkaline copper reagent to each of the 1 ml standard, blank and sample.
2. Place the tubes in a boiling water bath for 10 min.
3. Let the tubes cool down, then add 1 ml arsenomolybdate reagent.
4. Make up the volume to 10 ml with water.
5. After 10 min, read the absorbance at 620 nm.
Note:
1. Since different reducing sugars produce blue colored product of various intensities, this
method is not suitable for mixture of reducing sugars.
2. Proteins interfere with this reaction. Samples can be deproteinated by zinc hydroxide
precipitation.

  15  
 Experiment 12: Mucic acid test

Mucic acid test is used to detect the presence of galactose.

Principle: Monosaccharides upon treating with strong oxidizing agents such as nitric acid
yield saccharic acids. Nitric acid has the capacity to xidize both aldehyde and primary
alcoholic groups present at C1 and C6 respectively of galactose to yield an insoluble
precipiate (rod shaped crystals) of mucic acid and thus the name of the test. Galactose
containing saccharides test positive to this reaction.

Reagent: conc. Nitric acid. Sugar solution: galactose (2% w/v in distilled water).
Procedure:
1. Add 2 ml of conc. nitric acid slowely to 5 ml of the galactose solution in a small beaker.
2. Heat over a flame (cautiously) till the volume is reduced to about 2-3 ml.
3. Gradually cool the solution to room temperature.

  16  
1.2 AMINO ACIDS

No Reagent Composition and Preparation

1 Ninhydrin 2% (w/v) Ninhydrin in acetone. 1 g ninhydrin in 50 ml acetome
2 Isatin Isatin 1% (w/v) in acetic acid. 0.5 g isatin in 50 ml acetic acid
3 Xanthoprotic Conc. HNO3 and 40% NaOH
4 Sakaguchi 40% NaOH, 1% α-napthol . 40 g NaOH in 100 ml H2O, 1 g α-napthol in
alcohol.
5 Pauly’s diazo Sulfanilic acid (1% w/v) in 1 N hydrochloric acid, sodium nitrite (5%
w/v) in distilled water (prepare fresh), sodium carbonate (10% w/v) in
distilled water.
6 Sodium 2% sodium nitroprusside. 2 g sodium nitroprusside in 100 ml H2O
nitroprusside
7 Ehrlich’s Dissolve 10 g p-dimethylaminobenzaldehyde in 100 ml conc. HCl.
8 Millon’s Acid mercuric sulfate: 10% (w/v) mercuric sulfate in 10% sulfuric acid.
1% sodium nitrite in water (prepare fresh).
9 Sullivan’s 5 N sodium hydroxide, sodium nitroprusside (10% w/v), 6 N HCl,
glycine (2% w/v)
10 Amino acids 0.1% individual amino acids. Dissolve separately 100 mg alanine, proline,
tryptophan, tyrosine, arginine, cysteine and methionine in 100 ml distilled
water

  17  
 Experiment 13: Ninhydrin Test (qualitative)

Ninhydrin test is commonly used to differentiate carbohydrates from amino acids. This test
is also useful in quantitative determination of amino acids in a given sample.
 
Principle: This assay is based on the fact that two molecules of ninhydrin (2,2-
dihydroxyindane-1, 3-dione) react with a free alpha-amino acid to produce a deep blue or
purple color known as Ruhemann’s purple. In this reaction ninhydrin acts as an oxidizing
agent and causes the deamination and decarboxylation of the amino acids at elevated
temperature, which is followed by condensation between reduced ninhydrin molecules,
released ammonia and second molecule of ninhydrin, to form the blue-purple complex (λmax
570 nm). The intensity of the formed complex is proportional to the concentration of amino
acids in the solution.

Procedure: Add few drops of ninhydrin reagent to 1 ml of amino acid solution and mix.
Place the tubes in boiling water bath for 5 min and then allow cooling to room
temperature.
Note:
1. Ninhydrin reacts with proline, a secondary amine, to yield an iminium salt, which is
yellow-orange in color.

2. Ninhydrin test is quite sensitive (~ 0.5 nmol). Ninhydrin is commonly used to detect
fingerprints, since the terminal amines of lysine residues in peptides and proteins shed
off in fingerprints react with ninhydrin.

  18  
 Experiment 14: Ninhydrin Test (quantitative)

Principle: Same as described in experiment 13.

Reagent: Dissolve 0.35 g ninhydrin in 100 ml ethanol. (iso-propanol or 1:1 mixture of
acetone/butanol may be used instead of ethanol). Diluent solvent: Mix equal volumes of
water and n-propanol.
Procedure:
1. Setup and process the standard (amino acid), blank and unknown sample as described
in the table below.
Tube No. 1 2 3 4 5 6 7 8
Blank Amino acid standard Test
[Alanine] µg/ml --- 10 20 40 60 80 100 NA
Working solu (ml) --- 0.1 0.2 0.4 0.6 0.8 1.0 1.0
Ninhydrin 1 ml
Water (ml) 1.1 1.9 1.8 1.7 1.6 1.5 1.4 ---
Boil for 20 min. Cool to room temperature in a cold
water bath.
Diluent 5 ml
A570 nm
2. Measure absorbance at 570 nm (440 nm for proline and hydroxyproline) in
spectrophotometer. The color is stable for 1 h. Use the blank to set absorbance to
zero then measure absorbance of each tube.
3. Plot a standard curve using absorbance values of serial dilutions of alanine. (X-
axis: amino acid concentration in µg/ml; Y-axis: absorbance). Based on the
standard curve determine amino acid content in unknown sample.
Note:
1. The color intensity developed by reaction of amino acids with ninhydrin depends on
type of amino acid.
2. Ninhydrin is also used in solid phase peptide synthesis to monitor deprotection for
amino acid analysis of proteins.
3. Allow all the samples to cool down to room temperature since the same sample at
different temperatures often shows different color intensity. Also, maintain consistent
volume.

  19  
 Experiment 15: Isatin Test

Principle: Imino acids such as proline and hydroxyproline react with isatin (1H-indole-
2,3-dione) under acidic conditions to yield a blue colored adduct.

Procedure:
1. Apply a drop of imino acid solution on a whatmann no 1 filter paper strip (size 25x50
cm) and dry the spot using a hot air gun/hair dryer or hot air oven.
2. Apply a drop of isatin reagent on the dried spot. Repeat the drying procedure with hot
air gun for a few minutes and observe.
3. Appearance of a characteristic blue colored spot on the filter paper confirms the
presence of imino acid. Other amino acids give pink color with this reagent.

  20  
 Experiment 16: Xanthoprotic Test

Xanthoprotic test is used to determine presence of aromatic amino acids like tyrosine and
tryptophan.
 
Principle: This test is based on the fact that aromatic groups in the amino acids are
nitrated by heating with concentrated HNO3 to yield intensely yellow colored nitro
derivative. In the presence of alkali, a salt of the tautomeric form of the nitro compound is
formed, which has an orange color.

Procedure:
1. Add 0.5 ml conc. Nitric acid to 0.5 ml of amino acid solution and mix.
2. Let the tube cool to room temperature (appearance of yellow color indicates aromatic
amino acid).
3. Add NaOH till yellow color is converted to orange color.
Note: In phenylalanine the phenyl group is very stable, therefore it doesnot react with
nitric acid in conditions of this test.

  21  
 Experiment 17: Pauly’s diazo test

Pauly’s diazo test is used to detect the presence of tyrosine or histidine.

 
Principle: Sulfanilic acid upon diazotization in the presence of sodium nitrite and
hydrochloric acid results in the formation of p-phenyldiazosulphonate. The diazonium salt
formed couples with histidine in alkaline medium to form a dark cherry-red color, which
retains its tint on dilution and changes to orange when the solution is made acidic. Tyrosine
also gives a red color but this becomes yellow on dilution and bronze-yellow on
acidification.
One mole of histidine or tyrosine reacts with 2 moles of the diazonium compound to
produce one mole of bis(azobenzenesulphonic acid)histidine or bis(azobenzenesulphonic
acid)tyrosine.

Procedure:
1. Add few drops of pre-chilled sodium nitrite to 1 ml of chilled sulfanilic acid and vortex.
2. Immediately add few drops of pre-chilled amino acid solution and vortex.
3. Add drop wise sodium carbonate solution till color begins to appear.
Note: Histidine gives a negative test with Millon’s reagent.

  22  
 Experiment 18: Sakaguchi Test

The Sakaguchi test detects the presence of guanidinium group of arginine in proteins.

Principle: Sakaguchi reagent is composed of 1-naphthol and a drop of sodium

hypobromite. The guanidine group in arginine reacts with Sakaguchi reagent to form a red
colored complex (due to the formation of an indophenol-like structure). The exact
mechanism of this reaction is not yet known.

Procedure:
1. Add 1 ml NaOH and 2 drops of α-napthol to 2 ml test solution. Mix well.
2. Add 4-5 drops of bromine water and observe for red color.

  23  
 Experiment 19: Millon’s Test

Millon’s test is a specific test for tyrosine.

Principle: The reaction is based on the fact that the hydroxybenzene radical of tyrosine
reacts with Millon’s reagent, which constitutes acid mercuric sulphate and sodium nitrite to
yield a red color complex.

Procedure:
1. Add 12 ml of Millon’s reagent to 2 ml test solution.
2. Keep in boiling water bath for 10 min.
3. Cool under running tap water and add 1 ml of 1% NaNO2 solution, gently heat if
required.
Note: Proteins give a white precipitate with Millon’s reagent on heating. On addition of
sodium nitrate the precipitate turns pink red.

  24  
 Experiment 20: Ehrlich Test

Ehrlich test is a specific test for tryptophan.

 
Principle: This reaction is based on the fact that under acidic conditions the Ehrlich’s
aldehyde reagent (p-dimethyaminobenzaldehyde) undergoes electrophilic substitution
reaction at the indole (a benzyl pyrrole) ring of tryptophan to yield a blue-violet
condensation product.

Procedure: Add 2 ml Ehrlich’s reagent to 0.5 ml test solution and observe the tube for
color change.

  25  
 Experiment 21: Nitroprusside Test

Nitroprusside test is used to detect presence of free –SH groups, cystein in a protein.
 
Principle: Reaction of cystein with sodium nitroprusside, Na2Fe(CN)5NO.2H2O, in
ammonical solution will give a purple or brown product, a complex anion in which an
octohedral ferrous center is surrounded by five tightly bonded cyanide groups. Many
proteins that give a negative test may exhibit a positive test after heat coagulation or
denaturation, indicating the liberation of free –SH groups.

Procedure:
1. Add 0.5 ml sodium nitroprusside to 2 ml test solution.
2. Add 0.5 ml ammonium hydroxide solution.

  26  
 Experiment 22: Sullivan and McCarthy’s Test

Sullivan and McCarthy’s test is used for detection of methionine.

Principle: Addition of sodium nitroprusside to an alkaline solution of methionine followed

by acidification of the reaction yields a red color. This reaction also forms the basis for the
quantitative determination of methionine.
Procedure:
1. Add few drops of sodium hydroxide (5 N) to 1 ml of the test solution.
2. Add few drops of glycine (2%) and 10% sodium nitroprusside solution and vortex.
3. Place the test tube at 40°C for 15 min in a water bath.
4. Incubate the tube in ice cold water for 5 min and add 0.5 ml of 6 N HCl.
5. Vortex the contents and allow to stand at room temperature for 15 min. Observe for
color change.

  27  
 Experiment 23: Hopkins-Cole Test

The Hopkins-Cole reaction, also known as the glyoxylic acid reaction, is a specific test
used for detecting the presence of the indole ring and thus tryptophan in proteins.

Principle: The test is based on the fact that layering concentrated sulfuric acid over a
mixture of protein with Hopkins-Cole reagent (containing glyoxylic acid) results in the
formation of a violet-purple ring at the interface of the liquids.

Reagents: 0.1% amino acid solution. Glyoxylic acid: Expose glacial acetic acid to sunlight
for a few days.
Procedure:
1. Add 2 ml light exposed glacial acetic acid to 2 ml of test solution.
2. Add conc. H2SO4 along the sides of the tube held in slanting position so that two
distinct phases are formed.
Note:  Nitrites, chlorates, nitrates and excess chlorides prevent the reaction from occurring.

  28  
 Experiment 24: Lead acetate Test

The lead acetate test is a specific test for cysteine and cystine.

Principle: In a strongly alkaline environment cysteine release sulfide ions, which react with
lead acetate to form black lead sulfide.

Reagents: 2% (w/v) lead acetate solution in water, 40% NaOH.

Procedure:
1. Add few drops of lead acetate solution to 2 ml of amino acid solution.
2. Add few drops of 10% NaOH.
3. Heat and allow standing for 5 min.
Note:
1. This test is not answered by methionine because the sulfur, involved in thioester
linkage, is not released by treatment with NaOH.
2. Addition of excess lead acetate solution will result in white precipitate.

  29  
 Experiment 25: Gerngross test

Gerngross test is used for detection of tyrosine.

Principle: 1- nitroso-2-napthol reacts with tyrosine in acid medium to give a purple-red

colored complex.
Reagents: Nitrosonapthol (0.1% w/v) in ethanol, conc. Nitric acid, 0.1% (w/v) tyrosine in
distilled water (adding few drops of 6 N hydrochloric acid).
Procedure: Take 1ml of the amino acid solution in a test tube and add few drops of
nitrosonapthol and vortex. Boil the contents over a bunsen flame for 3-5 min. Cool the
contents under running tap water and add 1-2 drops of nitric acid.
Note: Histidine gives a negative gerngross test.

  30  
 Experiment 26: Amino acids Titration Curve

Principle: Amino acids have the general formula [Link] acids can exist
as ampholytes (can act both as an acid, a proton donor or a base, a proton acceptor) or
zwitterions (dipolar ion) in solution, depending on the pH of the solution. The pH at which
an amino acid carries no net charge is known as its isoelectric point (pI) or isoelectric pH.
At isoelectric point all the groups in the amino acid are ionised such that the molecule
carries no net charge. Therefore at its isoelectric point the amino acid will not exhibit
mobility in an electrical field. Solubility and buffering will also be at its minimum.
If a solution of an amino acid at its isoelectric point is titrated with hydrochloric acid
(addition of protons) a certain pH is reached where 50% of the molecules are in cation
form and 50% in zwitterion form. This pH is pK1 (with regard to –COOH group). If
further protons are added to the solution, more molecules become cationic in nature and
solubility increases.
Alternatively, a solution of an amino acid at its isoelectric point is titrated with NaOH
(addition of base/ removal of protons) a certain pH is reached where 50% of the molecules
acquire the anion form and 50% remain in zwitterion form. This pH is pK2 (with regard to
–NH2 group).
The isoelectric pH for monoamino-monocarboxylic amino acids can be calculated from the
equation: pI = (pK1 + pK2)/ 2.
It is therefore evident that the buffering action of amino acid solution is greatest at and
around pK1 or pK2 and minimum at pI.
There are correspondingly more pK values for amino acids having more than two ionizable
groups. The buffering capacity of plasma proteins and hemoglobin is largely due to
histidine residues. Since the pK value of imidazole group of histidine is 6.1 it acts as an
effective buffer at the physiological pH of 7.4.
Reagents: prepare 0.1 M HCl, 0.1 M NaOH, 0.1 M glycine, 0.1 M histidine, 0.05 M lysine
and 0.05 M glutamate solutions.
Procedure:
1. Titrate 10 ml of glycine solution against 0.1 M HCl. Keep track of the pH change at
fixed intervals (evry ml of acid added) using a pH meter. Continue titrate till the pH of
the sample reaches 1.3.
2. Perform a separate titration of 10 ml glycine against 0.1 M NaOH till pH of 12.5 is
attained. Record pH change at regular intervels.
3. Plot the titration curve by taking the volume of acid/alkali added on the X-axis and the
pH attained on the Y-axis. Note the two plateaus on the curve where despite the
addition of acid/alkali the pH of the solution does not change. From the graph calculate
the pKa value. Compare this value with that reported in literature.

  31  
1.3 LIPIDS

  32  
 Experiment 27: Ethanol emulsion Test

Ethanol emulsion test is a general group test for detection of lipids.

 
Principle: The presence of lipids is observed by the appearance of cloudy white layer on
top of the reaction mixture. This test is based on the fact that lipids are dissolve in ethanol
(due to hydrophobic interactions) but on addition of water, lipids spontaneously disperse
to form micelles (small droplets), which form the top layer (being less dense than water
and ethanol) and appear cloudy white (since droplets diffract light). The lipids come out of
the solution because the overall strength of hydrogen bonding interaction between ethanol
and water is much greater than hydrophobic interaction between lipid and ethanol.
Reagents: Ethanol and water
Procedure: Add 2 ml ethanol to few drops of the fat sample and shake well. Then add 2 ml
of water and shake well.
Note: Samples with high lipid content will form a thicker cloudy suspension.

Experiment 28: Acrolein Test

The acrolein test is used to detect glycerol or its fatty acid esters.

Principle: This test is based on the fact that a pungent smelling (odor similar to burnt
cooking grease) compound, acrolein (unsaturated aldehyde) is formed by dehydration of
glycerol, when a fat is strongly heated in the presence of a dehydrating agent such as
potassium hydrogen sulfate.

Reagent: Anhydrous potassium hydrogen sulfate

Procedure: Add 1 g KHSO4 to 5-6 drops of the lipid sample. Heat the test tube slowly
over a bunsen burner till fumes appear. Note the odor of the fumes.
Note: Glycerol may be taken as a positive control for correct identification of odor of
acrolein.

Experiment 29: Sudan IV

The Sudan IV test is used to detect lipids in a given sample.

 
Principle: This test is based on the fact that Sudan stains are non-polar compounds with
high affinity for fats. They are soluble in lipids and insoluble in water. A positive test is
indicated by a scarlet (red-orange) color in the lipid layer. The color change observed is
only physical and does not alter the chemical
structure of the lipid.

Reagents: Sudan IV dye and water

Procedure: Add 10 drops of the sample to 5 ml of
water in the test tube. Transfer a small amount of
Sudan IV stain to the tube (a tooth pick will do).
Stopper the mouth of test tube and mix well.
Observe for any change after 3 min.

  33  
 Experiment 30: Acid Value

The acid value is defined as the number of milligrams of potassium hydroxide required to
neutralize one gram of the oil or fat sample under the standard set of conditions.

Principle: Oils and fats are esters of fatty acids with glycerol. Fresh sample of oil/fat would
be expected to have very low levels of free fatty acids. When the oil/fat are exposed too
long to oxygen in air, they may spoil and become rancid. This is caused by
microbial/enzymatic activity or due to atmospheric oxygen initiated-free radical propagated
oxidative cleavage of double bonds in unsaturated fatty acids, which produces aldehydes
and carboxylic acids of shorter chain length. Therefore, the amount of free acids associated
with oil/fat provides a fair indication of its age and extent of deterioration. Titration of
fat/oil with potassium hydroxide solution reveals the free carboxylic acids in the sample.
High acid number will indicate that the oil/fat is rancid.
Reagents: (i) 1% (w/v) phenophthalein in absolute ethanol. (ii) Oil/fat solvent: 1:1 ratio of
ether and 95% ethanol (take a small volume of this solvent, add phenolpthalein and see if it
turns pink, if it does, neutralize with acid till pink color disappears). (iii) 0.1 N KOH
solution.
Procedure:
1. Add 50 ml of oil/fat solvent to 10 g of molten sample and mix well.
2. Add few drops of phenolphthalein to 10 ml of the above sample and mix well.
3. Titrate with 0.1 N KOH till end point is reached (the solution turns faint pink and
persists for ≥ 10 s). Perform a blank titration with solvent alone.
Calculation:
Acid value = (56.1 x Z x N) / P
Where, Z= ml alcoholic KOH, N= normality of alcoholic KOH and P= weight of the
sample in g.
Note:
1. Perform titration with fat/oil solvent alone (blank). Subtract the titer value from
previous titer value.
2. Perform the experiment with at least duplicates. The difference between duplicate
determinants should be less than or equal to 0.1 mg KOH/g fat.

  34  
 Experiment 31: Peroxide Value

The peroxide value is defined as the amount of peroxide oxygen per kilogram of fat or oil.

Principle: Oxidative rancidity of unsaturated fats or oils is primarily due to autoxidation

by free radical reaction involving oxygen and generation of peroxide intermediates. The
concentration of peroxide in an oil or fat is its peroxide value, and gives a fair indication of
its age and extent of deterioration. The peroxide value is defined as the amount of peroxide
oxygen per one kilogram of fat or oil. It is expressed either in milliequivalents of
peroxide/kg or millimoles of peroxide. The peroxide value is determined by estimating the
iodine released from potassium iodine by peroxide, by titrating against sodium thiosulfate.
The reaction scheme for hydroxyperoxides in presence of acetic acid is as follows:
Generation of iodine:

The peroxides formed in fat or oil react with iodide to release iodine. The base (OH-)
produced in this reaction is taken up by excess acetic acid present in the reaction mixture.
Titration step:

The iodine liberated in the previous step is titrated with sodium thiosulfate.
The indicator used in this reaction is a starch solution where amylose forms a blue to black
solution with iodine and is colorless where iodine is titrated.
Reagents: (i) Acetic acid- chloroform solution (7.2 ml Acetic acid and 4.8 ml chloroform)
(ii) Saturated potassium iodide solution. Store in dark (iii) Sodium thiosulfate 0.1 N (iv)
1% starch solution.
Procedure:
1. Add 12 ml of the acetic acid-chloroform solution to 2 g of fat/oil sample (molten) in a
100 ml stoppard Erlenmeyer flask and mix well to dissolve the contents.
2. Add 0.2 ml of saturated potassium iodide solution to the flask. Stopper the flask and
mix.
3. Add 12 ml of distilled water to the flask. Stopper the flask and shake vigorously to
liberate the iodine from the chloroform layer.
4. Titrate with 0.1 N sodium thiosulfate. If the color of the test solution is deep red orange,
titrate slowly until the color lightens, if not move to next step.
5. Add 1 ml of starch solution as indicator. Titrate until the blue gray color disappears in
the aqueous (upper layer). Record the volume of titrant consumed.
Calculation:
Peroxide value is expressed as milliequivalents of peroxide oxygen per kg of oil sample
(mEq Kg-1). Peroxide value = (Titer value x N x 1000) / (Weight of oil sample in g)
Where, N = Normality of the sodium thiosulfate solution used.
Note:
1. A precaution that should be observed is to add the starch indicator solution only near
the end point (the end point is near when fading of the yellowish iodine color occurs)
because at high iodine concentration starch is decomposed to products whose indicator
properties are not entirely reversible.
2. Peroxide values of fresh oils are less than 10 milliequivalents /kg, when the peroxide
value is between 30 and 40 milliequivalents/kg, a rancid taste is noticeable.

  35  
 Experiment 32: Saponification Value

Saponification value is defined as the number of milligrams of potassium hydroxide

required to saponify of 1 g fat or oil under defined conditions.

Principle: In the presence of alkali fats are hydrolyzed resulting in formation of salts of
fatty acids (also called soap) and glycerol. The process is known as saponification. As most
of the fat/oil are triacylglycerol (three fatty acids ester linked to glycerol), a comparison of
the fatty acid chain length can be done by this method. On hydrolysis, per unit mass of
fat/oil containing long chain fatty acids will yield relatively fewer numbers of carboxylic
functional groups as compared to short chain fatty acids. If more milligrams of base are
required to neutralize N grams of fat then there are more milligrams of the fat/oil and the chain
lengths are relatively small. Saponification value is defined as the number of milligrams of
potassium hydroxide required to neutralize the fatty acids resulting from the complete
hydrolysis of 1 g fat or oil under the conditions specified.

Reagents: 1. 0.5 M HCl 2. 0.5 N KOH 3. Fat solvent: 1:1 mixture of 95% ethanol and
ether 4. 1% phenolphthalein in alcohol
Procedure:
1. Dissolve 1 g of 1 ml of the given fat or oil in 5 ml fat solvent in a conical flask. Also,
setup a blank. Mix well.
2. Add 25 ml of 0.5 N alcoholic KOH and attach the flask to a reflex condenser.
3. Heat both flasks in boiling water bath for 30 min
4. Cool and titrate the contents of both flasks against 0.5 N HCl containing
phenolphthalein indicator. Record your readings as T ml for test and B ml for blank.
Calculation:
Calculate the saponification value by the formula:

Saponification value (mg KOH/ g) = Z ml x 28.05

Wt. of fat
Where, Z is difference between blank and sample titer value.
Note:
1. The multiplication factor of 28.05 in the above equation is included since 1 ml of 0.5 N
KOH contains 28.05 mg of KOH.
2. The calculated molar mass is not applicable to fats and oils containing high amounts of
unsaponifiable material, free fatty acids (>0.1%), or mono- and diacylglycerols (>0.1%).
3. Since butter has a large proportion of lower fatty acids it has a high saponification
value. Coconut oil also has a comparatively higher saponification value.
4. Saponification value may be used to determine the approximate molecular weight of
fat/oil. Number of moles = mass of oil/relative atomic mass.
5. The test sample goes into solution, which upon shaking results in foaming due to
formation of soap.

  36  
 Experiment 33: Iodine Value

Iodine number is used to determine the amount of unsaturation in fats and oils.

Principle: Iodine is added across the double bonds of unsaturated oils by electrophillic
addition reaction to form saturated halo-products. The extent of halogenation is a measure
of the degree of unsaturation in oils. Iodine number is defined as grams of iodine absorbed
by 100 g of fat. The reagent used in this test is iodine monochloride, which reacts with the
unsaturated fatty acids. Further potassium iodide is used to convert the unreacted iodine
monochloride to free iodine (I2). The liberated iodine is titrated against sodium thiosulfate.

In general, greater the degree of unsaturation the greater is probability of the oil or fat to
become rancid by oxidation. The iodine value is a measure of the degree of unsaturation of
a fat or oil and thus allows its classification into non-drying, drying and semi-drying oils
(though complete information on their drying property is not obtained). Oils having high
iodine value such as linseed oil dry up quickly and are called drying oils while coconut oil
is non-drying oil. Determination of iodine value of an oil or fat can help in detection of
adulteration. The iodine values of a few oils are: Coconut oil: 8.4; Olive oil: 79-83;
Cottonseed oil: 103-111; Lineseed oil: 175-202.
Reagents: Iodine monochloride reagent: In a 500 ml volumetric flask containing 100 ml
glacial acetic acid add 5 ml of iodine monochloride and make up the volume to 500 ml with
acetic acid. 10% w/v potassium iodide (prepare fresh). 0.1 N sodium thiosulfate (prepare
fresh). 1% w/v starch indicator solution: dissolve 1 g starch in 40 ml water (heat gently to
dissolve) and make up the volume to 100 ml. 2% w/v fat sample in chloroform.
Procedure:
1. Pipette out 10 ml of the fat sample into an iodine flask (250 ml). Add 20 ml of iodine
monochloride reagent. Stopper the flask and shake the contents. Leave the flask in dark
at for 30 min. In a separate flask setup reagent blank without fat sample.
2. Add 10 ml of potassium iodide solution to the flask and mix. Rinse the stopper using 40
ml distilled water. The contents of the flask are titrated against 0.1 N sodium thiosulfate
solution taken in a burette until a pale straw color is observed. Add 1-2 ml of starch
indicator. Addition of the indicator results in blue coloration. Continue the titration
until the solution turns colorless. Note the titer value both for fat sample and blank.
Calculation:
Iodine number of fat = (Z ml x 12.7) / (weight of fat sample in g x 100)
Where, Z is difference between blank and sample titer value.
Note: Shake the flask thoroughly throughout the titration to ensure that all the iodine is
expelled from the chloroform layer. Each ml of sodium thiosuphate (0.1 N) is equivalent to
12.7 mg of iodine.

  37  
 Experiment 34: Libermann-Burchard Method

Libermann-Burchard method is used for estimating serum cholesterol of the given sample.

Principle: The test is based on the fact that the sterols with unsaturation in ring A or B
react with concentrated sulfuric acid in the presence of acetic anhydride and undergo
dehydration (at C3), oxidation (to form 3,5-cholestadiene) and isomerisation reactions to
yield a polymer containing a green color chromophore (λmax 640 nm).

Reagents: (i) 1:1 ethanol-acetone mixture (ii) 250 µg/ml cholesterol stock solution in
chloroform (iii) Acetic anhydride- H2SO4 reagent: Add 2 ml conc. H2SO4 to 60 ml acetic
anhydride in a beaker on ice. Gently mix the keeping the mixture chilled. Prepare fresh. If
blue tinge appears, discard the reagent and prepare again.
Procedure:
1. Add 0.2 ml of blood/serum to 10 ml ethanol-acetone solvent in a centrifuge tube. Mix
well.
2. Keep the tube in boiling water bath with gentle agitation until the solvent begins to boil.
Shake the tube for 5 min (proteins precipitate)
3. Cool to room temperature. Centrifuge for 5 min, collect supernatant in a fresh test tube
and evaporate the solvent in a boiling water bath.
4. Cool and redissolve the residue in 2 ml chloroform.
5. To prepare a standard curve set up a series of test tubes with 2 ml of cholesterol in the
range of 0-250 µg/ml. Similarly prepare the reagent blank with 2 ml of chloroform only.
6. Add 2 ml of the reagent to all the tubes and mix. Incubate all the tubes in dark for 15
min at room temperature and measure the absorbance at 640 nm.
7. Likewise prepare various dilutions of the test sample and process the tubes in exactly
the same manner as mentioned earlier. Estimate the concentration of cholesterol of
chloroform using the standard curve.
Note: Serum cholesterol level is highly variable and the normal range also varies widely.
Generally, 150-250 mg per 100 ml is taken as normal range using the chemical method.
High values are usually associated with diabetes mellitus, artherosclerosis, hypothyroidism,
nephrotic syndrome and obstructive jaundice, whereas lower values may indicate severe
liver disease, hyperthyroidism and malnutrition.

  38  
1.4 NUCLEIC ACIDS

Experiment 35: Diphenylamine Method

The diphenylamine method is used to estimate concentration of DNA in a given sample.

Principle: This method is based on the fact that under acidic conditions and heat DNA is
depurinated and only 2-deoxyribose sugar linked to purine residues are dehydrated to yield
a highly reactive compound ω -hydroxylevulinylaldehyde, which in turn reacts with
diphenylamine (DPA) to produce a bluish-green complex (λmax 595 nm). Within the linear
range of the assay the intensity of the colored complex is proportional the concentration of
DNA in the sample.

Reagents: (i) Diphenylamine reagent (prepare fresh): Dissolve 1 g diphenlyamine in 100

ml glacial acetic acid then add 2.5 ml conc. H2SO4. Protect the reagent from light. (ii) 100
µg/ml calf thymus DNA dissolved in distilled water.
Procedure: Setup and process the blank, sample and standards as indicated in the table
below.
Tube No. 1 2 3 4 5 6 7 8
Blank DNA Standard Test
[DNA] µg/ml --- 10 20 40 60 80 100 NA
Working soln. (ml) --- 0.1 0.2 0.4 0.6 0.8 1.0 ---
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 ---
DPA reagent 3 ml
A595 nm
From the standard curve determine the concentration of DNA in unknown sample.
Note:
1. Deoxyadenosine may be preferred over DNA for preparation of standards because it is
possible to accurately measure the concentration of deoxyadenosine from its molar
extinction.
2. Since only the purine-linked deoxyriboses participate in this reaction; this method
measures only half of the total number of nucleotides.

  39  
 Experiment 36: Fiske-Subbarow Method

Fiske Subbarow method is used for quantitative determination of inorganic phosphate.

 
Principle: The assay is based on the fact that phosphorous, under acidic conditions, reacts
with ammonium molybdate to form phosphomolybdic acid, which in turn is reduced by 1-
amino-2-napthol-4-sulfonic acid to quantitatively yield molybdenum blue complex (λmax at
660 nm) the intensity of which is proportional to the phosphate present. Phosphorous acts
as a quantitative catalyst during this reaction.
Reagents: (i) Standard stock solution: 13.6 mg/ml:
dissolve analytically pure KH2PO4 (1.36 g mono-
basic salt) in 30 ml of distilled water and make up
the volume to 100 ml in a volumetric flask with
distilled water. Add a few drops of chloroform,
which acts as a preservative. (ii) Working standard:
dilute 1.0 ml of stock solution to 100 ml with
distilled water to give a phosphorous concentration
of 31 µg/ml. (iii) 2.5% w/v ammonium molybdate
solution. (iv) 5N H2SO4 (v) Amino napthol
sulphonic acid (ANSA) reagent: the powdered
reagent is prepared by mixing 0.2 g of 1-amino-2-
napthol-4-sulphonic acid, 1.2 g of sodium bisulfite
and 1.2 g of sodium sulfite. This mixture is ground
to a fine powder using a porcelin mortar and pestle. Weigh 0.25 g of this powder and
dissolve in 10 ml of distilled water (this solution should be prepared fresh).
Procedure: Setup and process the samples, blank and standards, as indicated in the
observation table. Compute the concentration of the phosphorous in the sample from the
calibration curve.

Tube No. 1 2 3 4 5 6 7 8 9 10 11 12
Blank KH2PO4 standard Test
[Phosphorous] µg/ml --- 3.1 6.2 9.3 12.4 15.5 18.6 21.7 24.8 27.9 31.0 NA
Working soln. (ml) --- 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 --- ---
5 N H2SO4 (ml) 1 ml
Amm. Molybdate (ml) 1 ml
ANSA reagent (ml) 0.1 ml
Water (ml) 6.9 ml
A660 nm

  40  
 Experiment 37: Bial’s orcinol Method

Bial’s test is used for detection of pentoses and pentosans (polymer of pentoses).

Principle: The assay is based on the fact that under acidic conditions pentosans are
hydrolyzed to pentoses. Further, pentoses (like the ribose sugar moiety of RNA) are
dehydrated to yield furfural, which in turn condense with orcinol to form a green colored
complex (λmax 665 nm), whose intensity is proportional to the concentration of pentose
sugar present. The intensity of color developed also depends on the concentration of HCl,
ferric chloride, orcinol and duration of boiling. Usually purine nucleotides are more
responsive than pyrimidine nucleotides to this test.

Reagents: (i) Orcinol reagent: dissolve 300 mg orcinol in 5 ml ethanol and add 3.5 ml of
this just before use to 100 ml of a 0.1% solution of FeCl3.6H2O in conc. HCl. Store the
reagent in brown colored bottle and use it within couple of hours. (ii) 300 µg/ml RNA
stock solution in Tris-EDTA buffer (10 mM Tris-acetate and 1 mM EDTA, pH 7.2).
Procedure: Setup and process the blank, standards and samples as indicated in the table
below.
Tube No. 1 2 3 4 5 6 7 8 9 10 11 12
Blank RNA Standard Test
[RNA] µg/ml --- 30 60 90 120 150 180 210 240 270 300 NA
Working soln (ml) --- 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 --- ---
Orcinol reagent 3 ml
A665 nm
Compute the concentration of RNA in the sample from the calibration curve.
Note:
1. Prolonged boiling may give similar blue-green color due to formation of glucuronates in
the sample. The controls will be useful in ruling out false positive result.
2. Bail’s solution must be prepared fresh.

  41  
 Experiment 38: Quantitation of DNA by A260 nm

UV absorbance at 260 nm is simple and nondestructive method for determination of

DNA/RNA concentration.

Principle: Nucleic acids absorb strongly in the UV region with a λmax of 260 nm. This
physical property is due to the conjugated aromatic nature of the bases. This is also the
basis for this simple, rapid, nondestructive and very sensitive method for determination of
nucleic acids concentration in solution. For a double-stranded DNA solution that has an
absorbance (A260) of one corresponds to a concentration of 50 µg/ml. The Beer-lambert law
is valid at least to an OD = 2. The lower concentration limit of ~ 0.5 µg/ml for dsDNA
corresponds to 0.01 OD units. Some common contaminents that absorb in this range are
RNA, phenols and proteins. Since an absorbance spectrum of RNA is similar to DNA,
RNA contamination is difficult to detect. If the contaminating RNA concentration is in
excess of 0.4 µg/ml (A260 of 0.01 OD) it will affect the final DNA concentration. Protein
contamination, on the other hand, is easier to detect since the absorbance spectra of protein
is distinct from that of DNA, with a λmax of 280 nm. A protein concentration of 0.3 mg/ml
(A260 of 0.01 OD) may be accepted as an upper limit before an effect on the determined
DNA concentration is observed. The ratio of the absorbance at 260 and 280 nm (A260/280) is
used to assess the purity of nucleic acids. For pure DNA, A260/280 is ~1.8 and for pure RNA
A260/280 is ~2. Lower values may be obained with contaminating protein in the sample. It is
not a sensitive method to detect protein contamination in nucleic acids (rather it is a highly
sensitive method to detect nucleic acid contamination in protein). Moreover, the ratio
A260:A280 also depends on pH and ionic strength of the sample.
Materials: DNA sample (purified plasmid or genomic DNA)
Procedure:
Suspend the DNA sample in water (or 1X TE buffer). Set the spectrophotometer to zero
using blank (just water in a quartz cuvette). Dilute the sample to give readings between 0.1
and 1.0 unit at 260 nm.
Calculation:
dsDNA concentration (µg/ml or µM) = 50 µg/ml x A260 x dilution factor

  42  
1.5 PROTEINS

Experiment 39: Biuret protein assay

Biuret assay is used for quantification of proteins already in solution or easily soluble in
dilute alkali.

Principle: The Biuret reagent contains sodium hydroxide, copper (II) sulfate and
potassium sodium tartarate. Under alkaline conditions of the biuret reaction (pH 14),
deprotonation of the amide nitrogen occurs which leads to high
electron density at the nitrogen atom. Further, copper (II) ion
complexes with four peptide nitrogens to yield a tetradentate
violet colored coordination complex (λmax 565 nm). Since
peptide bonds occur with the same frequency per amino acid in
proteins, biuret test can be used to assess the concentration of
proteins. At high pH Cu2+ binding with OH- ion leads to an
insoluble precipitate, this is minimized by addition of potassium
sodium tartarate, which stabilize the cupric ions.
Reagents: Biuret reagent: Dissolve 0.3 g CuSO4 and 0.9 g sodium potassium tartarate in
50 ml of 0.2 N NaOH solution. Add 0.5 g of KI and make up the volume to 100 ml using
0.2 N NaOH. BSA stock solution: 20 mg/ml BSA
Procedure:
1. Setup and process the blank, standards and samples as indicated in the table below.
Tube No. 1 2 3 4 5 6 7 8 9
Blank BSA Standard Test
[BSA] mg/ml --- 1 2.5 5 7.5 10 15 20 NA
Working soln (ml) --- 0.5 1.25 2.5 3.75 5 7.5 10 10
Water (ml) 10 9.5 8.75 7.5 6.25 5 2.5 --- ---
Biuret reagent 6 ml  
Incubate at room temperature for 10 min
A565 nm
Compute the concentration of protein in test sample from the calibration curve.
Note:
1. Proteins with abnormally high percentage of aromatic amino acids are known to
produce higher reading.
2. The color developed in this assay is stable but it is recommended to take the readings of
the samples within 10 min of each other.
3. Biuret assay is influenced by very few interfering agents like ammonium salts.
4. Biuret ((H2N-CO-)2NH) is not a reagent in this test but the
test is so named because the reaction was first observed with
peptide-like bonds in the biuret molecule. A more accurate
name is the alkaline copper reagent (ACR) test.

  43  
 Experiment 40: Folin-Lowry’s Method

The Folin-Lowry’s method is used for quantification of proteins already in solution or

easily soluble in dilute alkali.

Principle: Folin-Ciocalteu reagent uses phosphomolybdotungstate for colorimetric

detection of proteins. The reaction mechanism is not very well understood, but involves
both the formation of coordination complex between copper (II) ions and peptide bonds
under alkaline conditions and the oxidation of phenolic group of tyrosine and trytophan
residues, resulting in the formation of a blue purple color complex (λmax 660 nm).
Reagents: (i) Solution A: 2% Na2CO3 in 0.1N NaOH. (ii) Solution B: 0.5% CuSO4 in 1%
sodium potassium tartarate solution. (iii) Solution C: Solution A and B mixed in the ratio
50:1 just before use. (iv) Commercially available Folin-Ciocalteu reagent diluted in water
in the ratio of 1:1 just before use. (v) 500 µg/ml BSA stock solution: Dissolve 50 mg BSA
in 100 ml water.
Procedure:
1. Setup and process the blank, standards and samples as indicated in the table below.
Tube No. 1 2 3 4 5 6 7 8 9 10 11 12
Blank BSA Standard Test
[BSA] µg/ml --- 50 100 150 200 250 300 350 400 450 500 NA
Working soln (ml) --- 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 --- ---
Solution C 5.5 ml
Mix and incubate at room temperature for 10 min
Folin’s reagent dil. 0.5 ml
Mix and incubate in dark for 30 min
A660 nm
Compute the concentration of protein in test sample from the calibration curve.
Note:
1. The method is sensitive down to about 10 µg/ml and is probably the most widely used
protein assay despite its being only a relative method, subject to interference from Tris
buffer, EDTA, nonionic and cationic detergents, carbohydrate, lipids and some salts.
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.
2. 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.

  44  
 Experiment 41: Bradford Method

The Bradford protein assay is used to determine the concentration of protein in a solution.

Principle: This assay is based on the fact that proportional

binding of Coomassie Brilliant Blue G-250 dye to proteins
under acidic conditions leads to absorbance shift of the dye from
doubly protonated red cationic form (λmax 470 nm) to a stable
unprotonated blue protein-dye form (λmax 595 nm). Within the
linear range of the assay (~5-25 µg/ml), increase in absorption at
595 nm is proportional to the amount of bound dye, and thus to
the concentration of protein present in the sample. Coomassie
Brilliant Blue G-250 dye binds mostly to basic amino acids
(primarily arginine in an approximately proportional manner).
The assay is therefore dependent on the amino acid composition
of the specific protein.
Reagents: (i) Bradford reagent: Dissolve 20 mg of Comassie Brilliant Blue G-250 in 10 ml
of ethanol. Add 20 ml of 85% phosphoric acid and makeup the volume to 200 ml with
water. (ii) 200 ml of 0.1 M phosphate buffer pH 7.5 3. (iii) BSA stock solution: Dissolve 20
mg of BSA in 100 ml of 0.1 M phosphate buffer of pH 7.5
Procedure:
1. Setup and process the blank, standards and samples as indicated in the table below.
Tube No. 1 2 3 4 5 6 7 8 9 10 11 12
Blank BSA Standard Test
[BSA] µg/ml --- 20 40 60 80 100 120 140 160 180 200 NA
Working soln (ml) --- 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 --- ---
Bradford reagent 5 ml
Mix and incubate at room temperature for 5 min
A595 nm
Compute the concentration of protein in test sample from the calibration curve.
Note:
1. Bradford method is rapid and has high sensitivity (about 1 µg), the color developed is
stable but varies with proteins and interfering substances include detergents and
strongly basic buffers. Biuret method has low sensitivity (1-20 mg). Lowry method has
better sensitivity (~5 µg) but is time consuming (40-60 min) and interfering substances
include ammonium sulfate and mercaptans. All the three method are destructive to the
protein sample. Spectrophotometric absorption at 280 nm is a nondestructive method
and is widely used for monitoring column eluents.
2. Commassie G-250 and commassie R-250 are both chemically disulfonated
triphenylmethane compounds. The G and R labels refer to dyes having a greenish or
reddish blue hue. Commassie R-250 lacks two methyl groups present in commassie G-
250. The R-250 dye is used for staining of proteins in gel electrophoresis whereas the G-
250 dye is used in Bradford assay.
3. Known sources of interference, such as some detergents, flavonoids, and basic protein
buffers, stabilize the green neutral dye species by direct binding or by shifting the pH.

  45  
 Experiment 42: Microkjeldal Method

The Kjeldahl method is used to determine the crude protein content of a sample by
estimating the sample’s nitrogen content.

Principle: The Kjeldahl method occurs in three main steps:

1) Digestion: It involves boiling a homogenous organic sample in concentrated sulphuric
acid so that the nitrogen present in the organic sample is reduced to ammonium sulfate.
This chemical decomposition of the sample is complete when the reaction mixture turns
clear and colorless from the initially dark-color. Potassium sulphate is added to increase the
boiling point of the reaction mixture by about 50°C. Titanium dioxide is often added as a
specific catalyst to accelerate the decomposition.
2) Distillation: It involves addition of excess base to the acid digestion mixture, which
converts NH4+ to NH3. The reaction mixture is distilled and the NH3 gas dissolved in a
receiving solution of boric acid.
3) Titration: It involves quantification of the amount of ammonia ions in the receiving
solution by titration with acid. The amount of nitrogen in a sample can be calculated from
this value.
Reagents: (i) 0.1 N anhydrous Na2CO3 (ii) 0.1 N H2SO4: Dilute 2.8 ml conc. H2SO4 in 1
liter water) (iii) 10ml of 1% methyl orange solution (iv) 50 ml Tashiro’s mixture: Dissolve
10 g boric acid in 480 ml hot water, cool and add 2 ml of 0.1% alcoholic solution of
bromocresol green and 4 ml of 0.1% methyl red solution. Make up to 500 ml with water.
Procedure:
Standardization of H2SO4
1. Titrate 10 ml of Na2CO3 solution with H2SO4 solution using 1% methyl orange as the
indicator. Considering Na2CO3 to be exactly 0.1 N, calculate the exact normality of
H2SO4.
Sample Digestion
2. Weigh 5 g of the selected foodstuff and transfer to a Kjeldahl digestion flask.
3. Add 50 ml conc. H2SO4, 2.5 g cupric sulfate and a pinch of potassium sulfate. Digest
this mixture over a direct flame in a fume chamber till a light green/blue color clear
solution is obtained. To remove the carbon deposits formed, heat that area till they
disappear.
Distillation
4. Cool the flask and adjust the volume by using water, to 50 ml shake and transfer the
contents to a distillation jacket containing 10 ml of 40% NaOH and steam distill this
alkali-treated digest in an airtight apparatus, whose delivery tube is dipped in to a
recessive flask containing 5 ml Tashiro mixture. Distill till the level of the solution in the
receiving flask becomes double, which usually takes 15-25 min.
5. Titrate the solution against standardized N/70 H2SO4, whose exact normality has been
predetermined till the color changes from green to purple.
6. Calculate the nitrogen content. To obtain the crude protein content of the foodstuff,
simply multiply the nitrogen value by 6.25.  
Note: Kjeldahl method is not applicable to compounds containing nitrogen in nitro and azo
groups and nitrogen present in rings (e.g. pyridine) as nitrogen of these compounds does
not convert to ammonium sulfate under the conditions of this method.

  46  
 Experiment 43: Isoelectric Point (pI)

Principle: The isoelectric point (pI) protein is the pH at which a protein is ionized equally
as an acid and as a base, so the protein carries no net electrical charge. At its pI the protein
does not migrate in an electrical field. The pI of a protein is a constant and is independent
of the concentration of the protein. The side chain groups of amino acids in the protein
contribute to the ionization of proteins. At a pH below their pI, proteins have a net positive
charge and will move towards the cathode (positive electrode) whereas at a pH above its
pI the proteins show a net negative charge and move toward the anode (negative
electrode).

Reagents: (i) 0.01 N acetic acid (ii) 0.1 N acetic acid (iii) 1% casein in 0.1 N sodium
acetate.
Procedure:
1. Setup the tubes as indicated in the observation table.
2. Add 1 ml of casein solution to each tube. Mix well (avoid frothing).
3. Let the tubes stand undisturbed and observe for appearance of turbidity and precipitate
at 5-10 min interval for half an hour.

Test tube No. 1 2 3 4 5 6 7 8 9

pH 5.95 5.64 5.34 5.04 4.74 4.44 4.12 3.14 3.54
Protein soln. (ml) 1 1 1 1 1 1 1 1 1
Water (ml) 8.38 7.75 8.75 8.50 8.0 7.0 5.0 1.0 7.40
0.01 N CH3COOH (ml) 0.62 1.25 - - - - - - -
0.01 N CH3COOH (ml) - - 0.25 0.50 1.00 2.00 4.00 8.00 -
0.01 N CH3COOH (ml) - - - - - - - - 1.60
Precipitate
Note: The isoelectric point of casein is 4.74. At this pH maximum terbidity or precipiate
may be observed.

  47  
 PART II: ENZYMOLOGY
Experiment 44: Alkaline Phosphatase assay

Principle: The estimation of alkaline phosphatase or orthophosphoric monoester

phosphohydrolase (EC [Link]) level of blood serum is of clinical importance. This enzyme
levels are elevated in bone, liver, kidney and other tissues in diseased condition. Alkaline
phosphate has a role in transport of phosphate groups across the cell membrane. The pH
optimum for serum alkaline phosphatase is between pH 8 and 10. The alkaline phosphatase
activity is measured by incubating serum, the enzyme source, with the substrate, p-
nitrophenyl phosphate dissolved in an alkaline buffer at a specific temperature for a certain
time. The alkaline phosphatase hydrolyses p-nitrophenyl phosphate to release yellow
colored p-nitrophenol with λmax at 405 nm, the concentration of which is determined by
plotting a standard curve for p-nitrophenol (0-50 µg/ml). The enzyme activity units are
expressed as micromoles of p-nitrophenol released per milliliter per minute at specific assay
conditions.

Materials: (i) Enzyme source: collect 5 ml blood in a centrifuge tube and allow it to clot.
Centrifuge the sample at 10,000 rpm for 10 min at 4°C to collect the serum (ii) Inactivated
enzyme (negative control): autoclaved serum (iii) Assay buffer: 0.1 M bicarbonate buffer
of pH 10 (iv) Substrate: 1 mM solution of p-nitrophenyl phosphate in bicarbonate buffer
of pH 10.
Procedure:
1. Setup the enzyme assay as indicated in the observation table.
Tube No. 1 2 3 4 5 6
Blank (Inactivated enzyme) Test (active enzyme)
Assay buffer (ml) 1 1 1 1 1 1
Substrate (ml) 0.5 0.5 0.5 0.5 0.5 0.5
Vortex
Enzyme extract (ml) 0.5 0.5 0.5 0.5 0.5 0.5
Mix and incubate at 37°C in a water bath for 30 min
Incubate tubes in an ice bucket
A410 nm
2. Measure the micromoles of p-nitrophenol released at 410 nm in a spectrophotometer.
Calculate the enzyme activity in terms of the number of micromoles of p-nitrophenol
released per milliliter of the reaction mixture per minute under defined assay conditions
using following formula:

  48  
  
Experiment 45: Acid phosphatase assay

Principle: Acid phosphatases or orthophosphoric monoester phosphohydrolase (EC

[Link]) hydrolyses aliphatic, aromatic or heterocyclic phosphoric acid. The pH optimum
for alkaline phosphatses is between pH 4 and 6. RBC, platelets, WBC and prostrate cells
secrete this class of non-specific enzymes. The acid phosphatase activity is measured by
incubating the enzyme source, potato, with substrate, p-nitrophenyl phosphate dissolved in
an acid buffer at a specific temperature for a certain time. The acid phosphatase hydrolyzes
p-nitrophenyl phosphate to release colorless p-nitrophenol. The enzyme is inactivated and
thus the reaction stopped by addition of alkali to the reaction mixture. At alkaline pH the
released p-nitrophenol turns yellow in color (λmax 405 nm), the concentration of which is
determined by plotting a standard curve for p-nitrophenol (0-50 µg/ml). The enzyme
activity units are expressed as micromoles of p-nitrophenol released per milliliter per
minute at specific assay conditions.

Materials: (i) Enzyme source: Homogenize 20 g peeled and finely cut potato (Solanum
tuberosum) in a blender with 80 ml cold water for 5 min. Centrifuge the homogenate at 5000
rpm for 15 min (preferably cold). Transfer clear supernatant to a fresh tube (ii) Inactivated
enzyme (negative control): autoclave 1 ml of enzyme extract (iii) Assay buffer: citrate
buffer 100 mM, pH 4.8 (iv) Substrate: 15 mM p-nitrophenyl phosphate dissolved in citrate
buffer. 0.2 N sodium hydroxide.
Procedure: Setup the enzyme assay as indicated in the observation table.
Tube No 1 2 3 4 5 6
Blank (inactivated enzyme) Test (active enzyme)
Assay buffer (ml) 0.5 0.5 0.5 0.5 0.5 0.5
Substrate (ml) 0.5 0.5 0.5 0.5 0.5 0.5
Vortex
Enzyme extract (ml) 0.1 0.1 0.1 0.1 0.1 0.1
Mix and incubate at 37°C in a water bath for 10 min
0.2 N NaOH (ml) 3.9 3.9 3.9 3.9 3.9 3.9
Vortex
Incubate tubes in ice bucket
A410 nm

Use the following equation to calculate the enzyme activity in U/ml.

Note:
1. The various factors included in the equation include total assay volume (5 ml), time of
assay (10 min), volume of enzyme extract (0.1 ml), millimolar extinction coefficient of
p-nitrophenol at 410 nm (18.3) and dilution factor (10).
2. If A410nm of the assay mixture after incubation is below 0.1, the incubation time may be
increased to 20 min. If the color developed is too intense, the assay may be repeated
with a diluted enzyme extract (substitute the modified dilution factor in the equation).

  49  
 Experiment 46: β-amylase assay

Principle: β-amylase or 1,4- glucan maltohydrolase (EC [Link]) catalyses the hydrolysis of
α-1→4 glycosidic linkage from the non-reducing end of starch (amylose and amylopectin)
and glycogen to yield maltose units. β-amylase does not hydrolyze α-1→6 glycosidic
linkages present in branch chain of amylopectin and glycogen. The activity of the enzyme
is measured colorimetrically by estimating the amount of maltose released using 3,5-
dinitrosalicyclic acid (DNS) method.
Materials: (i) Enzyme source: collect 10 ml saliva in a beaker. Filter through a 0.2 µ filter.
Dilute 1 ml of filtered saliva with 9 ml of water (ii) Inactivated enzyme preparation: use
autoclaved enzyme as the heat inactivated enzyme (iii) Assay buffer: prepare a 0.02 M
citrate buffer of pH 6 (iv) Substrate: 1% soluble starch solution in assay buffer. (v)
Dinitrosalicylic acid (DNSA) reagent: In a 100 ml volumetric flask dissolve 1 g of DNSA
in 20 ml of 2 N NaOH. Add 50 ml water. Add 30 g sodium potassium tartarate and
dossolve. Makeup the solution to 100 ml with water (vi) Standard maltose solution: 100
mg/ml maltose in water.
Procedure:
Prepare a standard graph of maltose as follows:
Tube No. 1 2 3 4 5 6 7
Blank Maltose Standard
[Maltose] (µg/ml) --- 0.1 0.2 0.4 0.6 0.8 1.0
Working soln (ml) --- 0.1 0.2 0.4 0.6 0.8 1.0
Water (ml) 1 0.9 0.8 0.7 0.6 0.5 ---
DNSA reagent 1 ml
Boil for 10 min
Cool the tubes under running water
A540 nm

Setup the enzyme assay as indicated in the observation table.

Tube No. 1 2 3 4 5 6
Blank (inactivated enzyme) Test (active enzyme)
Substrate (ml) 1 1 1 1 1 1
Enzyme extract (ml) 1 1 1 1 1 1
Incubate at 37°C for 3 min
DNSA reagent (ml) 2 2 2 2 2 2
Mix and incubate in a boiling water bath for 5 min
Cool the tubes under running water
A540 nm or green filter

Amylase activity is expressed in terms of mg maltose liberated in 3 min at 37°C by 1 ml of

enzyme extract. Specific activity of the enzyme is expressed as amylase activity/mg of
protein.

  50  
 Experiment 47: Urease assay

Principle: Urease or urea amidohydrolase (EC [Link]) is an important enzyme in protein

metabolism, that catalyses the degradation of urea into ammonia and carbon dioxide.

The amount of ammonia formed is directly proportional to the amount of urease present in
the assay. Therefore, the activity of urease can be determined by measuring the amount of
ammonia liberated in the reaction. The reagent used for estimating ammonia contains
hypochlorite, phenol and nitroprusside, which under alkaline conditions react with
ammonium ions to forma blue colored indophenol anion (λmax 630 nm) by Berthelot
reaction. Sodium nitroprusside acts as a catalyst for reaction.
Materials: (i) Enzyme source: Homogenize 10 g germinating green gram (moong dal/
Vigna radiata) in 100 ml phosphate buffer (0.2 M, pH 7) using a mortar and pestle.
Centrifuge at 10,000 x g at 4 °C for 10 min (ii) Inactivated enzyme (negative control/
blank): Autoclave 10 ml of the enzyme extract. Decant the clear supernatant to a fresh
tube (iii) Assay buffer: 0.2 M phosphate buffer pH 7. Substrate: 1% urea solution in 0.2 M
phosphate buffer of pH 7 (iv) Phenol reagent: dissolve 50 g phenol in 1 liter of water.
Dissolve 0.25 g sodium nitroprusside to phenol reagent just before use (v) 1% sodium
hypochlorite solution (vi) Ammonium standard: 10 µg/ml solution of ammonium nitrate in
distilled water.
Procedure:
1. Prepare a standard graph of maltose as follows:
Tube No. 1 2 3 4 5 6 7
Blank Ammonia Standard
[Amm. sulphate] (µg/ml)
--- 1.0 2.0 4.0 6.0 8.0 10
Working soln (ml)
--- 0.1 0.2 0.4 0.6 0.8 1.0
Water (ml)
1 0.9 0.8 0.6 0.4 0.2 ---
Phenol reagent 1 ml
Hypochlorite reagent 1 ml
Incubate at 50°C for 5 min
A630 nm

2. Setup the enzyme assay as indicated in the table below.

Reagent Blank (inactivated enzyme) Test (active enzyme)
Assay buffer (ml) 1.0 1.0
Substrate (ml) 1.0 1.0
Enzyme extract - 0.5
Mix and incubate at 37°C for 30 min
7 N Sulfuric acid 1.0 1.0
Phenol reagent 1 ml
Hypochlorite reagent 1 ml
Incubate at 50°C for 5 min
A630 nm

Construct a standard curve for ammonia (X-axis: ammonia concentration in µmoles, Y-

axis: absorbance at 630 nm). Calculate the ammonia liberated by the enzyme reaction from
the calibration plot after deducting the blank value from the test. Urease enzyme unit is
denoted by µmoles of ammonia released per minute per ml of enzyme extract under
defined conditions.

  51  
 Experiment 48: pH Optima of Urease

Principle: The optimum pH of an enzyme is the pH at which it is most active. Each

enzyme has an optimum pH, on both sides of which the velocity may drastically decline,
resulting in a bell shaped curve. The concentration of H+ affects the charge on the amino
acid residues in the enzyme affecting the activity, structural stability and solubility of the
enzyme. These effects are particularly important in vicinity of the active site, which may
influence substrate binding and catalytic activity. Optimum pH may vary depending on the
temperature, concentration of substrate, presence of ions etc. Usually enzymes have the
optimum pH between 6 and 8. Extremes of pH usually result in denaturation of the
enzyme.
Materials: (i) 100 mM phosphate buffer of pH 6.0, 6.5, 7.0 and 8.0 (ii) 7 N sulfuric acid.
Other materials are same as above.
Procedure:
1. Aliquot 1 ml of 100 mM phosphate buffer of pH 6.0, 6.5, 7.0 and 8.0 in individual tubes.
Add 1 ml of 1% fresh urea solution followed by addition of 0.5 ml of the enzyme extract.
2. Vortex and incubate at 37°C for 30 min.
3. Terminate the reaction by adding 1 ml of 7 N sulfuric acid to the tubes.
4. Perform ammonia estimation for the assay tubes.
5. The assay mix may be diluted appropriately, if required. Plot the enzyme activity on X-
axis and pH on Y-axis, to determine the optimal pH for urease activity.

  52  
 Experiment 49: Temperature Optima of Urease

Principle: The optimum temperature of an enzyme is the temperature at which maximum

amount of the substrate is converted to the product per unit time. The velocity of enzyme
reaction increases when temperature of the medium is increased (as more number of
molecules have sufficient energy to break the activation energy barrier); reaches a
maximum and then falls. Usually the rate of reaction of most enzymes will double by a rise
in 10°C. But at higher temperatures (> 50°C) enzymes denaturate and lose their tertiary
structure resulting in a decrease of enzyme activity.
Materials: (i) Variable temperature water bath (ii) 7 N sulfuric acid. Other materials are
same as experiment 47.
Procedure:
1. Aliquot 1 ml of 100 mM phosphate buffer (use the optimal pH as determined in
previous experiment) into 5 test tubes. Add 1 ml of 1% fresh urea solution followed by
addition of 0.5 ml of the enzyme extract to each tube.
2. Vortex and incubate at various temperatures (25, 37, 45, 60 and 70°C) for 30 min.
3. Terminate the reaction by adding 1 ml of 7 N sulfuric acid to the tubes.
4. Perform ammonia estimation for the assay tubes.
6. The assay mix may be diluted appropriately, if required. Plot the enzyme activity on X-
axis and temperature on Y-axis, to determine the optimal temperature for urease
activity.

  53  
 PART III: BIOANALYTICAL TECHNIQUES
Experiment 50: Molar attenuation coefficient

Molar attenuation coefficient (ε) is defined as the absorbance of one molar solution of pure
absorbing material in a 10 mm path length cell, under specified conditions of wavelength
(λmax) and solvent.

Principle: The alternative terms for molar attenuation coefficient (recommended by

IUPAC) are molar absorption coefficient and molar extinction coefficient. Experimentally
absorbance value of a known concentration of the pure analyte is obtained by recording
the absorbance value at its λmax using a spectrophotometer. Molar absorption coefficient
values of a biomolecule can be determined by using the Beer Lambert’s equation: A= εcl.
Where, A= absorbance, c= concentration of the analyte and l = path length. The units of ε
are M-1cm-1.
Reagents: 0.5 mM L-tyrosine in 0.1 N NaOH.
Procedure: Set the UV-Vis spectrophotometer in absorbance mode and set the absorbance
at wavelength 293.5 nm to zero using the blank (0.1 NaOH). Record the absorbance of the
tyrosine solution at 293.5 nm.
Calculation: Calculate the molar absorption coefficient of L-tyrosine by substituting the
experimental value of absorbance in the following equation-

(Recorded at λ293.5 nm)/ (5 X 10-4 M) (1 cm)

  54  
 Experiment 51: Ammonium Sulfate Precipitation

Ammonium sulfate precipitation method is widely used for purification of protein from a
crude extract.

Principle: Ammonium sulfate is an iorganic salt that is used to precipitate proteins from
solution, a process that is called salting out. Solubility of a protein depends on the
proportion and distribution of polar hydrophilic groups and nonpolar hydrophobic groups
in the molecule. The polar water molecules interact with the polar groups of proteins
tending to increase solubility. The added ammonium sulfate reduceds the effective
concentration of water available for the protein thereby leading to increased protein-
protein interactions followed by aggregation or precipitation of the protein.
Generally, an inverse relationship is observed between amount of salt required for
precipitation and the molecular weight of the protein. Proteins, which hold on to water at
higher concentrations of salt, can be separated from one, which are precipated under the
same condition.

Reagents: (i) Saturated ammonium sulfate solution (ii) Solid ammonium sulfate (iii) Hen
egg
Procedure:
1. Collect the egg white portion of an egg in a 100 ml beaker. Add equal volume of
distilled water.
2. In a 15 ml centrifuge tube 2 ml of the dilute egg white solution and add equal volume of
saturated ammonium sulfate solution. Mix gently (avoid frothing).
3. Centrifuge at 2500-3000 rpm for 10 min. Decant the supernatant into a fresh tube.
4. Add solid ammonium sulfate to saturate the solution. Mix gently.
Note: The precipitate observed at 50 and 100% saturation of ammonium sulfate is that of
globulins and albumins, respectively.

  55  
 Experiment 52: Dialysis

The net movement of molecules from a region of high concentration to a lower

concentration through a semi-permeable membrane is called dialysis.

Principle: The pore size of the dialysis membrane restricts large molecules to freely diffuse
across the membrane. The small molecules freely diffuse across the membrane till
equilibrium is reached between the sample (inside the dialysis tube) and buffer solution.
Dialysis is often used in the laboratory for desalting protein samples. During dialysis small
molecules equilibrate across the semi-permeable membrane (freely move in both direction,
in and out across the membrane); hence dialysis in certain cases is used to introduce small
molecules to the sample.

Materials: (i) 10 mg/ml BSA and 50 mg/ml ammonium sulfate (ii) 5% w/v barium chloride
solution (iii) Dialysis tubing (molecular weight cut off 12 kDa) (iv) Plastic clamps (v)
Magnetic stirrer and magnetic beads.
Procedure:
1. Seal one end of a 10 cm long dialysis tube with a plastic clamp and fill it with 5 ml of
BSA-salt solution, then seal the other end with another plastic clamp. Place the dialysis
tube in a 2 liter glass beaker filled with distilled water. Gently stir overnight at 4°C
using a magnetic stirrer.
2. Next day collect an aliquot of the water and test for the presence of sulfate ions by
addition of barium chloride solution. On addition of barium chloride the solution may
turn milky-white or yield a white precipitate (barium sulfate is formed by reaction of
barium chloride with sulfate ions).
3. If the water tests positive for sulfate ions replace the water with fresh distilled water
and allow equilibrating. Repeat this step till the water tests negative for sulfate ions.
This indicates the ammonium sulfate salt is almost completely separated from the
protein-salt solution.
Note: The molecular weight of BSA is ~66 kDa and that of ammonium sulfate is 132 Da.
Since the molecular weight cut-off of the dialysis tube is about 12 kDa molecules of size
greater than 12 kDa will be retained with in the dialysis tube, where as molecules with size
less than 12 kDa can freely exchange with the external solution.

  56  
 Experiment 53: Paper Chromatography of amino acids

Paper chromatography is particularly useful in separating mixture of compounds having

similar polarity.

Principle: Paper chromatography is widely used technique for isolation, identification and
quantitative determination of biomoleules such as sugars, amino acids and nucleic acids in
a sample. It is a type of partition chromatography. The analytes are distributed between
two phases, a stationary phase (usually water held in the fibers of paper) and the mobile
phase (developing solvent or moving liquid). The analytes are separated due to differential
affinity towards the mobile and stationary phase. Usually the analyte with higher solubility
in the mobile phase solvent will migrate rapidly than the less soluble components.

The results of a developed paper chromatogram are represented by Rf value of the

analytes, which indicates the relative movement of the analyte to that of the solvent front.
The Rf value (abbreviation for retention factor, retardation factor, retardation force or
relative front) depends on various conditions such as temperature, distance travelled by the
solvent/analytes, concentration of the analytes, mobile phase solvent composition, quality
of the paper and others. The Rf values of adjacent spots of analytes in a paper
chromatogram should differ by a minimum of 0.05.
Materials: (i) Chromatographic chamber (ii) Whatmann No.1 paper (iii) 10 µl Capillary
tubes (iv)Hot air dryer (v) atomizer (vi) gloves (v) Developing solvent- n-butanol: acetic
acid: water ([Link]). Mix 60 ml butanol, 20 ml and 20 ml water (vi) Spray reagent- Add 1 ml
acetic acid to 200 mg of ninhydrin in 99 ml of acetone (vii) Standard amino acids- 2 mg/ml
solution each of glycine, alanine and proline.
Procedure:
1. Draw a pencil line at least 5 cm away from the narrow end (this is the origin line) of
Whatmann No. 1 filter paper (cut according to size of your chromatography chamber,
which could be a glass beaker). Label the spots where samples will be loaded. Load the
standard and unknown amino acid samples with the help of a capillary tube (10 µl),

  57  
 such that the diameter of the sample spot does not exceed 3-4 mm. Dry the spots using a
hair dryer.
2. Place the spotted paper in a chromatographic chamber containing the developing
solvent (if the size of the chromatography chamber is big, you need to pre-saturate the
chamber with the developing solvent). The origin line should be 2 cm above the solvent
level.
3. When the solvent front is about to approach the top end, remove the chromatogram
from the chamber and immediately mark the solvent front using a pencil. Air-dry the
chromatogram at room temperature.
4. Spray the chromatogram with ninhydrin reagent. Alpha-amino acids yield purple color.
Proline gives yellow color. Outline the spots with a pencil. Measure the distance in
centimeters from the origin to the center of the outlined spot and calculate the Rf value
for the amino acids.
Rf = distance moved by the amino acid (cm) / distance moved by the mobile phase (cm)
5. Compare the Rf values of unknown with that of the standard amino acids to identify the
unknown amino acid.
Note: The samples may be reapplied at the same spot after drying to obtain a concentrated
spot.

Experiment 54: Paper Chromatography of sugars

Reagents: (i) Solvent system- pyridine: ethylacetate: acetic acid: water ([Link]). Mix 50 ml
pyridine, 50 ml ethyl acetate, 30 ml acetic acid and 10 ml water (ii) Spray reagent. Dissolve
1 g aniline and 1.6 g phthalic acid in 500 ml methanol (iii) Standard sugars: 2 mg/ml
solution each of glucose, fructose and lactose.
Procedure:
1. Draw a pencil line at least 5 cm away from the narrow end (this is the origin line) of
Whatmann No. 1 filter paper (cut according to size of your chromatography chamber,
which could be a glass beaker). Label the spots where samples will be loaded. Load the
standard and unknown sugar samples with the help of a capillary tube (10 µl), such that
the diameter of the sample spot does not exceed 3-4 mm. Dry the spots using a hair
dryer.
2. Place the spotted paper in a chromatographic chamber containing the developing
solvent (if the size of the chromatography chamber is big, you need to pre-saturate the
chamber with the developing solvent). The origin line should be 2 cm above the solvent
level.
3. When the solvent front is about to approach the top end, remove the chromatogram
from the chamber and immediately mark the solvent front using a pencil. Air-dry the
chromatogram at room temperature.
4. Dip the chromatogram in aniline hydrogen phthalate reagent and incubate for 10 min at
110°C in an oven. Hexose sugars appear as brown colored spots while pentose sugars
form pink spots. Disaccharides react slowly as compared to monosaccharide’s with
spray reagent. Outline the spots with a pencil. Measure the distance in centimeters from
the origin to the center of the outlined spot and calculate the Rf value for the amino
acids.
Rf = distance moved by the amino acid (cm) / distance moved by the mobile phase (cm)
5. Compare the Rf values of unknown with that of the standard amino acids to identify the
unknown amino acid.

  58  
 Experiment 55: Paper Chromatography of purines and pyrimidines

Reagents: (i) Solvent system. Mix 65 ml isoprpanol and 16.7 ml of conc. HCl and make up
the volume to 100 ml with water (ii) Standard purines and pyrimidines: 2 mg/ml solution
each of adenine, guanine, cytosine and uracil in 0.1 N HCl (warm if required).
Procedure:
1. Draw a pencil line at least 5 cm away from the narrow end (this is the origin line) of
Whatmann No. 1 filter paper (cut according to size of your chromatography chamber,
which could be a glass beaker). Label the spots where samples will be loaded. Load the
standard and unknown sugar samples with the help of a capillary tube (10 µl), such that
the diameter of the sample spot does not exceed 3-4 mm. Dry the spots using a hair
dryer.
2. Place the spotted paper in a chromatographic chamber containing the developing
solvent (if the size of the chromatography chamber is big, you need to pre-saturate the
chamber with the developing solvent). The origin line should be 2 cm above the solvent
level.
3. When the solvent front is about to approach the top end, remove the chromatogram
from the chamber and immediately mark the solvent front using a pencil. Air-dry the
chromatogram at room temperature.
6. The separated bases on the chromatogram are visualized as fluorescent spots under a
UV lamp (shortwave 260-300 nm) or UV cabinet. Outline the spots with a pencil.
Measure the distance in centimeters from the origin to the center of the outlined spot
and calculate the Rf value for the amino acids.
Rf = distance moved by the amino acid (cm) / distance moved by the mobile phase (cm)
4. Compare the Rf values of unknown with that of the standard purines and pyrimidines to
identify the unknown bases.

  59  
 Experiment 56: Sanger’s FDNB Method (N-terminal amino acid)

Determination of the N-terminal amino acid by Sanger’s method is used to arrange the
individual peptide fragments after complete sequencing of the protein.

Principle: Sanger’s reagent is 1-fluoro-2,4-dinitrobenzene (FDNB). This method is

important because the first round of Edman degradation is often contaminated by
impurities and therefore does not give an accurate determination of the N-terminal amino
acid.
The Sanger’s method for N-terminal amino acid analysis consists of three stages:
1. The Sanger’s reagent, FDNB, selectively labels the N-terminal amino acid by reacting
with the primary amino group of the N-terminal amino acid under mild alkaline
condition to yield a yellow colored dinitrophenylated (DNP) peptide derivative.
2. Hydrolysis of the peptide under acidic conditions.
3. Extraction of the N-terminal DNP-amino acid with peroxide-free ether.
(Note: This is critical step since FDNB also reacts with ε-amino, thiol, imidazole and phenolic groups at
neutral and acidic pH to yield other DNP-derivatives (DNP-arginine, O-DNP-tyrosine, ε-DNP-lysine,
and imidazole-DNP-histidine). These derivatives are more polar and are partitioned to the aqueous phase,
whereas N-terminal DNP-amino acid is extracted into the ether phase.)
4. Identification of N-terminal DNP-amino acid by chromatography and comparison with
reference standards.

Materials: (i) Hot air oven (ii) Shaker (iii) Water bath (iv) Centrifuge (v) Pre-coated
silica gel G (vi) Glass hydrolysis vial (vii) TLC developing tank with lid (viii) FDNB (5%
w/v) in ethanol, sodium bicarbonate solution (0.4% w/v) (ix) Solvent system- benzyl
alcohol: ethanol (9:1) (x) Peroxide free ether (xi) Sodium bicarbonate (xii) Conc. HCl and
6 M HCl (xiii) acetone (xiv) Pure protein, whose N-terminal amino acid is to be detected
(xv) standard DNP derivative of various amino acids (xvi) 5% solution of 1-fluoro-2,4-
dinitrobenzene (FDNB) in alcohol.
Procedure:
1. Add 10 mg sodium carbonate to 10 mg of protein in a test tube and suspend in 2 ml
water. Add 4 ml of FDNB solution and incubate at room temperature on a shaker for 2
h. If precipitation is observed in the tube it indicates acidic condition. The pH in the
tube should be in the range 8-9. Add adequate amount of sodium bicarbonate to
redissolve the precipitate.
2. Extract the suspension thrice with peroxide free ether (to eliminate dinitrophenol).
3. Adjust the pH to 1 using conc. HCl and extract thrice with ether. Pool the extracts in a
hydrolysis vial and allow it to dry.
4. Add 0.2 ml acetone to the hydrolysis vial and allow dry. Acid hydrolyze the sample by
adding 1 ml of 6 M HCl to the vial, seal it and incubate in an oven at 110°C for 18 h.

  60  
5. Cool the vial to room temperature, open it and resuspend the contents in 1 ml distilled
water. Again extract thrice with 2 ml ether. Pool the extracts, dry the contents by
evaporation and then resuspend in a minimum volume of acetone.
6. Spot 10µl of the extract on to a pre-coated silica gel-G TLC plate along with standards.
Develop the chromatogram in benzyl alcohol: ethanol (9:1) solvent system. Identify the
yellow colored N-terminal amino acid by comparing the Rf values.
Note: A yellow spot of dinitrophenol may be observed on the chromatogram. On exposure
to HCl fumes the yellow spot is decolorized while color of DNP-amino acid spot doesnot
change.

  61  
 Experiment 57: Carboxypeptidase Method (C-terminal amino acid)

A carboxypeptidase (EC number 3.4.16-18) is an exopeptidase that hydrolyzes the C-

terminal peptide bond, resulting in the removal of L-amino acid, one residue at a time, from
the C-terminal of the polypeptide chain.

Principle: Carboxypeptidase A sequentially removes C-terminal acidic or neutral amino

acids whereas carboxypeptidase B have a strong preference for positively charged amino
acids (arginine and lysine). Neither peptidase functions if proline is the penultimate or C-
terminal amino acid. Tripeptides and dipeptides may be resistant to hydrolysis.
Carboxypeptidase Y is the enzyme of choice nowadays because it has broad specificity and
ability to work in the presence of detergents and urea. In this enzyme-based detection
method, the protein sample is incubated with carboxypeptidase A and aliquots removed
form the reaction mixture at timed intervals and released free amino acids analyzed using
paper chromatography. The released amino acids are visualized by using ninhydrin
reagent. The released amino acids may be identified by comparing their Rf values with
those of reference standards.

Materials: (i) Carboxypeptidase A (ii) Water bath (iii) Whatmann No. 1 paper (iv) 10%
trichloroacetic acid (TCA) (v) Chromatographic chamber (vi) 10 µl capillary tubes (v)
Hot air dryer (vi) atomizer (vii) gloves (viii) Developing solvent- n-butanol: acetic acid:
water ([Link]). Mix 60 ml butanol, 20 ml and 20 ml water (ix) Spray reagent- Add 1 ml
acetic acid to 200 mg of ninhydrin in 99 ml of acetone (x) Standard amino acids- 2 mg/ml
solution.
Procedure:
1. Add 2 ml carboxypeptidase A to 2 ml of the protein solution (whose C-terminal amino
acid composition is to be identified) in a test tube and mix well.
2. Incubate the tube at 37°C and analyzed the released C-terminal amino acid by taking
200 µl aliquots every 5 min intervel upto 30 min. Also set up a negative control wherein
water is used instead of the enzyme.
3. Add 200 µl of 10% TCA to each of 0.2 ml digested protein samples, to eliminate the
undigested protein and to terminate enzyme reaction.
4. Centrifuge the tubes at 5,000 rpm for 10 min. Spot 50 µl of each supernatant
individually on a Whatmann No. 1 chromatography paper. Run an ascending
chromatogram as described previously.
5. Calculate the Rf values of the amino acids detected in all the reaction mixtures. Identify
them by comparing their Rf values with those of standard amino acids. Decipher their
sequence based on the order of their appearance in the reaction mixture as a function of
time.

  62  
 Experiment 58: Thin Layer Chromatography (TLC) of amino acids

TLC offers better resolution and speed than paper chromatography.

Principle: Thin layer chromatography (TLC) was originally developed to resolve lipids. It
is widely used today for both analytical and preparative (small amount) analysis of a
variety of compounds. This technique is easy to perform, inexpensive, has good speed of
separation and fine sensitivity. The principle of separation of the mixture of analytes by
TLC depends on their differential solubility in the stationary (liquid bound to the solid
matrix) and mobile (developer solvent) phases. Silica gel and alumina are commonly used
stationary phase, which are coated as a uniform layer (0.25 - 0.5 mm thick) onto glass,
aluminium or plastic plates. The results of a developed TLC are represented by Rf value of
the analytes, which indicates the relative movement of the analyte to that of the solvent
front. The Rf value for an analyte is constant for a given set of experimental conditions.
Also the resolved components may also be isolated from the plate by scratching the spot
and eluting the components from the powder obtained. Specific reagents may be sprayed
on the developed chromatogram so as to produce color spots to help in their detection.
Ninhydrin is a suitable locating reagent for amino acids. In this experiment, individual
amino acids are also run as reference standards.
Materials: (i) TLC plates (commercially available) (ii) Atomizer (iii) Locating reagent-
0.3% ninhydrin in butanol (iv) Developing solvent- butanol: glacial acetic acid: water in
[Link] ratio.
Procedure:
1. Activate the TLC plate by heating at 105°C for 30 min.
2. Saturate the TLC chamber with a solvent containing developing solvent.
3. Prepare known standard solutions of individual amino acids (5 mg/100 ml) in water and
load them as separate spots on the reference line, 1 inch above the bottom of the TLC
plate.
4. Position the plate vertically in the chamber so that the spot do not dip into the solvent.
5. Perform the chromatographic run by leaving the assembly undisturbed.
6. When the solvent has travelled about three-fourth of the length of the plate, take it out
from the chamber.
7. Dry the plate in an oven at 200°C.
8. Spray ninhydrin reagent over the dried plates with the help of an atomizer.
9. Dry the plates gain in the oven observe the color of the spots and calculate the Rf values
of all the components of the mixture using standard spots as the reference. Identify the
components accordingly.

  63  
 Experiment 59: TLC of plant pigments

Principle: In 1902 Mikhail Tswett isolated the colorful pigments in plant chloroplasts. The
principal pigment in higher plants is chlorophyll a. Other pigments like chlorophyll b,
xanthophylls and carotene play a secondary role by transferring the energy absorbed by
chlorophyll a, for use in photosynthesis.
Thin–layer chromatography (TLC) is an example of partition chromatography, which
involves the distribution of an analyte between a liquid phase immobilized on a solid
support and a mobile liquid phase. Greater the attraction between the analyte and
immobile phase, slower the analyte migrates. The converse is true with the mobile phase.
For the separation of plant pigments on TLC, a mixture of non-polar solvents is used
(petroleum ether and acetone). The more non-polar a pigment, the greater its solubility in
non-polar solvent and thus greater the distance the pigment migrates. The separated
pigments can be identified by comparing their Rf values of to that of reference standards.

Reagents: (i) Sample- Spinach (Spinacia oleracea) leaves (ii) silica gel-G (TLC grade), (iii)
developing solvent system- Petroleum ether: acetone (7:3 v/v) (iv) TLC plates
(commercially available) (v) TLC chamber (a 500 ml beaker covered with aluminum foil).
Procedure:
1. Weigh 1 g of acid washed sand and 500 mg each of fresh spinach leaves and anhydrous
magnesium sulfate. Transfer to a mortar and using a pestle grind till a fine dry powder
is obtained.
2. Transfer the powder to a test tube and add 2 ml of acetone. Stopper the test tube and
shake vigorously for one minute. Allow the mixture to stand for 10 min.
3. Add developing solvent to the TLC chamber such that the bottom of the solvent
completely covers the chamber to a depth of about 0.5 cm. Cover the chamber so that
evaporation does not change the composition of the solvent mixture.
4. Using a pencil mark four dots equidistant from each other and parallel (approximately 2
cm) to the bottom of the plate. Spot the extract onto the silica gel plate using a capillary
tube (TLC applicator). To obtain a concentrated dot of the extract repeat several times

  64  
 allowing the extract to dry between consecutive applications. The extract dots should be
as small as possible and dark enough to see the color intensity.
5. Place the TLC plate in the TLC chamber. The TLC plate should sit at the bottom of the
TLC chamber and the solvent surface should be below the extract dots. Cover the TLC
chamber.
6. The different color pigments can be observed separating from each other as the solvent
moves up the TLC plate. Run the chromatogram until the solvent front reaches one
centimeter from the top edge of the plate.
7. Remove the TLC plate from the chamber and immediately mark the solvent front using
a pencil. Quickly measure the pigment distances.
The reference Rf values of various plant pigments are indicated in the table.
Pigments Rf
Chlorophyll a 0.68
Chlorophyll b 0.54
Chlorophyll c 0.03
β-carotene 0.94
Fucoxanthin 0.51
Lutein 0.43
Violaxanthin 0.22

Note: Some pigments particularly β-carotene fade due to bleaching in the presence of light.

  65  
 Experiment 60: Ion Exchange Chromatography

Ion exchange chromatography is used to separate polar and ionic molecules based on their
affinity to the ion exchanger.

Principle: Ion exchange chromatography is a technique widely used in protein

purification, water analysis and quality control. Polar and ionic molecules can be separated
based on their affinity to the ion exchanger. The ion exchanger resin acts as the stationary
phase displaying ionic functional groups that interact with analyte ions of opposite charge.
This type of chromatography is subdivided into anion exchange and cation exchange
chromatography based on the counter ion on the stationary phase exchanged with the
analyte ion.
The capacity of an ion exchange resin is a measure of its ability to remove ions from
solution. The theoretical number of exchangeable ions per unit weight or volume of resin is
defined as the total exchange capacity (TEC) of the resin. Complete regeneration of a resin
is usually not performed (expensive) therefore TEC is of little practical consequence.
Instead the number of exchangeable ions available under a given set of conditions is
measured which is known as operating capacity of the resin. Capacity is commonly
expressed in terms of the number of equivalents of ionic species found in a liter or kilogram
of resin. The determination of exchange capacity of a cation exchanger resin involves
displacement of H+ ions of an ion-exchange matrix with sodium ions followed by titration
of the acid with standardized alkali using phenolphthalein indicator.
Reagents: (i) 4 N sodium hydroxide (ii) Hydrochloric acid (4 N) (iii) 1 M Sodium
chloride (iv) Standardized 0.1 N sodium hydroxide (v) Phenolphthalein indicator (vi) Ion-
exchange resin (cation exchanger): Dowex-50 H+ (200-400 mesh).
Procedure:
1. Wash 20 g of the resin once with 100 ml of 4 N NaOH and thrice with 100 ml distilled
water. Wash the resin once with 100 ml of 4 N HCl followed by several washes of
distilled water to remove acid. Filter the resin through a sinister funnel, followed by a
wash with 100-150 ml of methanol. Dry the resin overnight at room temperature.
2. Incubate 20 ml of 1 M NaCl with 1 g of the dried resin (H+ form) in a 100 ml flask for
45 min (shake intermittently).
3. Filter the resin using a glass funnel plugged with glass wool and collect the filtrate in a
150 ml conical flask. Rinse the beaker twice and the resin once with 5 ml each of
distilled water and collect the filtrate in the conical flask.
4. Add 2-3 drops of phenolphthalein indicator to the filtrate in the conical flask and titrate
the contents against standardized 0.1 N NaOH (in a glass burette) until a pale pink
color appears. Note the volume of NaOH consumed. Perform a titration with 20 ml of 1
M sodium chloride solution and note the blank titer value.
Calculations: The exchange capacity of resin is expressed in terms of milliequivlents (mEq)
per gram of resin. Actual titter value (X) = titer value of resin (ml) – blank titer value (ml)

One ml of 0.1 NaOH is equivalent to 4 mg by weight. One milliequivalent (mEq) of

NaOH = 40 mg.
Note:
1. Chemically Dowex resin is sulfonated polystyrene cross-linked with divinyl benzene.
2. Dowex-50 is commercially available as H+ or Na+ form. The later form cannot be
directly employed for exchange capacity determination. It has to be converted to the H+
form.

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 Experiment 61: Gel-filtration Chromatography

Gel-filtration chromatography is used to resolve analytes by their molecular size.

Principle: Gel-filtration chromatography (also called molecular-sieve chromatography) is a

versatile and popular technique. It is a type of size-exclusion chromatography, where an
aqueous solution is used to transport analytes through the column, resolving them by their
physical property of molecular size. It is widely used in fractionation of proteins and other
water-soluble polymers. The column is packed with inert particles (which form gel beads)
of uniform pore size and acts as the stationary phase. A solution of the analytes of various
molecular sizes is allowed to pass through the column under the influence of a mobile
phase (continuous solvent flow). Analytes with molecular size larger than the pore size
cannot enter inside the gel beads and hence elute rapidly in a single zone. On the other
hand, small molecular size analytes capable of diffusing in and out of the gel beads are
retained for a longer time in the column bed. Analytes of intermediate size migrate at a rate
between those of the large and small molecules. Hence, the order of elution of the analytes
is directly correlated to their molecular size.

Materials: (i) 10 ml plastic disposable syringe (ii) Sephadex G-10 (iii) Glass wool (iv)
Screw clip (v) Silicone tube (vi) burette stand with clamp (vii) Sample: Add 2 mg each
blue-dextran and potassium dichromate in 1 ml water and mix. Blue-dextran is a high
molecular weight polysaccharide (~ 2000 kDa).
Procedure:
1. Prepare the slurry by allowing 10 g of sephadex G-10 to swell in 50 ml water for 3-4 h
(stir intermittently). Decant the solution to remove the fines. Wash 3-4 times with
distilled water and decant the fines.
2. Fit a screw clipped silicone tubing (4-5 cm long) at the tapered end of the column to
regulate flow rate. Plug the bottom of the syringe with glass wool. Pack the column by
pouring the slurry into the syringe gently (avoid trapping air bubbles and packing

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 should be uniform without any banding in the matrix). Allow the gel to settle under
gravity. Equilibrate the column by passing 20-25 ml of distilled water (maintain at least
2-3 cm water level above the matrix through out).
3. Gently apply 0.3 ml of the sample onto the surface of the gel matrix. Run the column
with distilled water at a flow rate of 20 ml/hr. Collect 1 ml fractions in eppendorf tubes.
4. Record the absorbance of the fractions collected (at 630 nm for blue dextran and 340
nm for potassium dichromate). Plot an elution profile graph (X-axis fraction v/s Y-axis
absorbance).
Note: The term gel permeation chromatography should be use only when an organic
solvent is used as a mobile phase. It is used to analyze the molecular weight distribution of
organic-soluble polymers.

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 Experiment 62: Agarose Gel Electrophoresis

Agarose gel electrophoresis is used for resolving DNA/RNA molecules.

Principle: Agarose gel electrophoresis is widely used for resolving DNA and RNA
molecules. Agarose is extracted from seaweed and is a linear polymer of D-galactose and
3,6-anhydro-L-galactose. Agarose gels are prepared by boiling agarose in electrophoresis
buffer and then cooling to 50°C and pouring into a mold. The porosity of agrose gel matrix
decreases with an increase in agrose concentration. DNA is negatively charged at netural
pH and migrates towards the anode under electric field. The mobility of DNA molecules
depends on its molecular size and conformation as well as the agarose concentration. The
rate of migration of DNA molecules in the gel is inversely proportional to the logarithm of
their molecular weights. Generally the superhelical circular form has the greates mobility
followed by the rod-like linear form. The extended circular form migrates more slowly.
Nucleic acids can be visualized as an intense bright orange band on the gel by staining with
an interchelating dye, ethidium bromide (EtBr). A band containing less than about 10 ng
of DNA might not be visible after EtBr staining. Agarose gel has a low resolving power
(the limit of resolution being generally around 750 kb) but has a broad range of separation
(usually 1% agarose gel is used for DNA fragments 50 bp to 20 kb in size). Bromophenol
Blue and xylene cyanol dyes are incorporated in the loading buffer and act as size markers
(migrate at approximately 300 and 3000 bp respectively).

The repeating unit consists of D-galactose (β1→4)-linked to 3,6-anhydro-L-galactose (in

which an ether bridge connects C-3 and C-6). These units are joined by (α1→3) glycosidic
links to form a polymer 600 to 700 residues long. A small fraction of the 3,6-
anhydrogalactose residues have a sulfate ester at C-2.

Materials: (i) Horizontal electrophoresis apparatus with power pack (ii) 10-50 µl
micropipettes (iii) UV transilluminator (iv) Gloves (v) Running buffer stock solution
(10X) Tris-acetate (TAE): Dissolve 48.4 g Tris-base, 11.4 ml glacial acetic acid and 20 ml
of 0.5 M EDTA, pH 8 in 1 liter water. Store at 4°C (vi) EtBr Stain: Dissolve 100 mg
ethidium bromide in 10 ml of 1X TAE buffer (vii) 1% agarose in 1X TAE buffer: Boil the
agarose slurry by till it turns transparent. Add 1 µl of ethidium bromide stain and mix (vii)
Gel tracking dye (6X): Add 1 ml glycerol and 2 mg bromophenol blue in 9 ml water. (viii)
Standard DNA markers: Commercially available DNA ladder.
Procedure:

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1. Pour a suitable volume of 1% molten agarose solution into a gel-casting unit (seal the
open ends with adhesive tape). Insert the comb such that its teeth do not touch the
bottom and is at least 1 cm away from one the nearest end. Allow the gel to set at room
temperature and then remove the comb carefully.
2. Transfer the gel tray along with the gel into the elctrophoresis tank (remember to
remove the adhesive tape). Position the gel such that the loading wells lie towards the
cathode.
3. Fill the tank with appropriate volume of 1X TAE buffer. Load 10-20 µl of DNA
samples (mixed with loading dye) and DNA marker in separating well.
4. Run at 10-25 mAmp till the dye front reaches the other end of the gel.
5. Switch off the power supply and observe for DNA band/s in a UV transilluminator.
6. The approximate molecular weight of the sample DNA can be determined by
comparing its migration with that of the bands of the DNA ladder.

  70  
 Experiment 63: Cellulose Acetate Paper Electrophoresis
 
Principle: Cellulose acetate paper electrophoresis is a simple, rapid and reliable method
widely used to detect common clinically important hemoglobin. This method is called
hemoglobin electrophoresis. The hemoglobin variants (differing in their amino acid
composition) are separated at distinctive rates due to difference in their surface charge.
Under alkaline conditions (pH 8.4-8.6) hemoglobin molecules are negatively charged and
thus migrate towards the anode (positive) during electrophoresis. Hemoglobin variants will
not separate if the amino acid substitution does not result in a change in the overall charge
on the protein or if the amino acid substitution is at a site buried in the three-dimensional
structure of the protein. Under normal conditions, HbA, the adult hemoglobin, consists of
two alpha and two beta chains, whereas HbF, the fetal hemoglobin, consists of two alpha
and two gamma chains. This technique is sensitive enough to separate HbA from HbF and
to detect Hb A2 variants.

Reagents: (i) Electrophoresis buffer: Tris/EDTA/Borate (TEB) pH 8.4. Dissolve 10.3 g

Tris, 0.6 g EDTA and 3.2 g boric acid in 1 L water. Store at 4°C (ii) Fixing and staining
solution: Dissolve 5 g Ponceau S and 7.5 g trichloroacetic acid in 1 L water (iii)
Destaining solution: 5% (v/v) acetic acid (iv) Haemolyzing reagent: Dissolve 0.5% (v/v)
Triton X-100 in 100 mg/l potassium cyanide.
Procedure:
1. Blood sample preparation: Collect the blood samples that in the presence of an
anticoagulant agent, EDTA (1.5 mg/ml). Centrifuge the blood at 1,200 g for 5 min.
Discard the supernatant. Wash the cells thrice with saline. Dilute 20 µl of cells with 150
µl of the haemolyzing reagent.
2. Setup of electrophoresis unit: Soak cellulose acetate strips (of size 2.5 cm Χ 12 cm) in
TEB buffer for 5 min. Remove the strip using a pair of forceps and gently blot between
two filter papers. If opaque white spots appear on the strip put them back in the buffer
and then blot again. Position the strip on an electrophoresis support in a horizontal
electrophoresis tank and then place a wick of filter paper over either end of the strip.
3. Load (about 5 µl) of the sample at about one-third of the distance along the strip that is
about 4 cm from the cathode end.
4. Carry out the electrophoresis at 350 V for about 30 min.
5. Remove the strips from the tank and place the strips in 10% trichloroacetic acid for 5
min to denaturate the proteins, then transfer to Ponceau S and fix and stain for 5 min.
Washing with destaining solution thrice.
6. Blot the strips with clean filter paper to remove excess liquid and allow the strip to dry.

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