FACULTY OF APPLIED SCIENCE

LABORATORY MANUAL

GENERAL BIOCHEMISTRY

(MIC180)
 CONTENT

INTRODUCTION 1

EXPERIMENT 1: EFFECT OF β-AMYLASE ON STARCH 5

EXPERIMENT 2: QUALITATIVE TESTS FOR AMINO ACID 6

EXPERIMENT 3: COLOURIMETRIC TESTS FOR 11

CARBOHYDRATES

EXPERIMENT 4: THIN LAYER CHROMATOGRAPHY (TLC) 14

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 INTRODUCTION

GOOD LABORATORY PRACTICE (GLP) FOR A BIOCHEMISTRY LABORATORY

1. Read the laboratory manual thoroughly before you come to the laboratory.
Establish the aim of the experiment; be prepared to handle unfamiliar apparatus
efficiently; plan a work schedule allowing time for incubations, etc.
2. If working in groups, discuss the experiment thoroughly with your group members
before the class. This saves you from wasting your time in the laboratory trying
to decide who is to do what.
3. Lab coat should always be worn and confined to the work area. Leave your bags
outside the lab. Keep your personal items, for example, files, books off the
laboratory tables.
4. It is your responsibility to keep your area clean at the end of each lab
class/session.
5. Materials that you are strongly advised to have while working in the lab:
a) Glass marking pencil or pen or small stickers for labelling
b) A small towel
6. Never eat, drink or smoking in the lab.
7. No mouth pipetting or putting other objects in your mouth and no application of
cosmetics in the lab.
8. If you are injured (burned or cut), notify your instructor or any person in charge
immediately

OTHER GUIDES IN BIOCHEMISTRY LAB

1. Treat all apparatus with respect. Faulty equipment should be reported at once.
2. General apparatus, glassware and pipettes must be kept in labelled cupboards
and drawers.
3. When using solutions, solid or liquid chemicals from stock bottles, remove a little
more than is required and discard any excess in the appropriate bottles provided
in the lab.

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4. Never replace solutions or solids in the stock bottles. With your marker, label
every container into which you dispense stock solutions.
5. Never pour inflammable liquids and organic solvent into the sink: use waste
solvent bottles.

WRITING THE LABORATORY REPORT

The purpose of a laboratory report is to document findings and communicate their

significance. There are many ways to write your laboratory report, but nearly all will
have the same elements.

A good laboratory report does more than present data; it demonstrates the writer’s
comprehension of the concept behind the data. Merely recording the expected and
observed results is not sufficient. You should identify how and why differences
occurred; explain how they affect your experiment and show your understanding of
the principles of the experiment. Bear in mind that a format, however helpful,
cannot replace clear thinking and organised writing. You will need to organise your
ideas and express coherently.

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COMPONENTS OF LABORATORY REPORT

a) Title
Title of your experiment.
b) Objective
State the aims of the experiment. It should be in point form and very
focused.
c) Introduction
A good introduction provides important background and/theory of the
experiment.
d) Materials
A simple list of what is required during the experiment including chemical
reagents, apparatus and equipment.
e) Methods
Must be written in third person language and past tense.
f) Results
Use the most suitable method to present the result in the experiment such as
table, graphs and figures.
g) Discussion
Your discussion must explain, analyse and interpret your result. It answers,
“What is the significance or meaning of the results?”
h) Conclusion
A short statement of what you have found out or demonstrated in the
practical. Sometimes, it is appropriate to summarise the quantitative results.
Relate to your conclusion.
i) References
A minimum of three references excluding your lab manual. Written in the
American Psychology 7th Edition (APA 7th Edition) style.

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LABORATORY 1: EFFECT OF β-AMYLASE ON STARCH

β-amylase will hydrolyse starch at the α-1,4 linkage to produce maltose. Maltose is
disaccharide in which glucose is linked to glucose in which posses a reducing group.
The reducing group will reduce DNSA reagent, causing colour changes from yellow to
brown. The β-amylase-starch reduction is to be studied by following a progress curve.

Methods

1. Mix the following reagent in a boiling tube and incubate at 30°C:

Material Volume (ml)
a) Starch 1% 4.0
b) Acetate buffer, 1.0 M, pH 5.0 4.0
c) Distilled water 5.0

2. Place β-amylase in a separate tube and incubate at 30°C.

3. Prepare a series of labelled tubes containing 2.0 ml of DNSA reagent.
4. Label the tubes at tblank, t0, t2, t4, t8, t15, t30 and t60. Keep these tubes on
ice. Add 1.0 ml of the reaction mixture into DNSA tube labelled tblank.
5. To start the enzymatic reaction, add 1.0 ml of β-amylase to the reaction mixture
and mix.
6. Immediately pipette 1.0 ml of the reaction mixture into the DNSA tube labelled
t0.
7. Repeat sampling at times 2, 4, 8, 15, 30 and 60 minutes, by pipetting 1.0 ml of
the reaction mixture into the appropriately labelled DNSA tube.
8. Add further 1.0 ml of water to each DNSA tube to develop the colour and read the
absorbance at 560nm by using a spectrophotometer. Use tblank to zero your
spectrophotometer.
9. Plot a graph of absorbance against time to see the progress of β-amylase-starch
reduction reaction.

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LABORATORY 2: QUALITATIVE TESTS FOR AMINO ACIDS

Introduction

Amino acids are building blocks for proteins. They share the same acid and amino
group, but the side chain differs. When amino acids are linked together to form an
amide, the product is called a peptide, and the newly formed bond is called a peptide
bond. If a few amino acids (up to 20) are linked, the molecule is called a polypeptide.
When amino acids (20-1000) are in the peptide chain, it is called a protein.

The α-amino acid can be detected using ninhydrin which forms a blue to blue-violet
colour as a positive indication. Other colours (yellow, orange, red) are negative. The
test is very sensitive and often used for colourimetric determination of amino acids
solutions or as a visualisation agent in the chromatography of amino acids. During
the reaction, the amino group will liberate ammonia; the ammonia will react with
ninhydrin to form a coloured complex.

Most proteins give positive results in Xanthoprotein Test because of the presence of
the phenyl group (-C6H5). The phenyl group produced coloured nitro compounds in
reaction with nitric acid.

The amino acid with a thiol group (-SH) such as cysteine when heated in the presence
of alkali will produce sulfide ion. These ions can be detected by reaction with lead
cations- a black precipitate plumbum sulphide (PbS) is formed.

In Millon’s Test, if the hydroxyphenyl side group is present, a red colour will be
observed. Tyrosine is the only amino acid which gives a positive test. However, any
molecule with the phenolic –OH will react.

In Hopkins-Cole Test, heterocyclic side chain (indole chain) of tryptophan reacts to

give a purple to violet ring at the interface of the two layers.

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In Sakaguchi Test, ammonia and ammonium ions give positive reactions in this test.

Objective

To conduct a series of qualitative tests for amino acids.

Methods

Test 1: Ninhydrin Test

a) Pipette 1 ml of glycine in a test tube

b) Add 5 drops of ninhydrin
c) Heat the test mixture in boiling water bath for 2 minutes
d) Record changes
e) Repeat using stock solutions of tyrosine, tryptophan, phenylalanine, and
proline.

Test 2: Xanthoproteic Test

a) Pipette 1 ml of glycine in a test tube

b) Add 1 ml of concentrated nitric acid
c) Swirl the test tubes and immerse the test tube in cold water to cool them down
d) Record any changes
e) Add 40% NaOH solution dropwise to change the pH solution to alkaline
f) Record changes
g) Repeat using stock solutions of tyrosine, tryptophan, phenylalanine and
phenol.

Test 3: Millon Test

a) Pipette 1 ml of glycine in a test tube

b) Add 5 drops of Millon reagent
c) Heat the test mixture in a boiling water bath for 10 minutes

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 d) Cool it down to room temperature
e) Add 5 drops of natrium nitric
f) Record changes
g) Repeat using stock solutions of tyrosine and phenylalanine

Test 4: Hopkins-Cole Test

a) Mix 1 ml of glycine with 1 ml of glacial acetic acid in a clean test tube

b) Incline the test tube and slowly add 1 ml of concentrated sulfuric but do not
mix
c) Two layers should form
d) Let the layers stand and note the colour at the interface after 2-3 minutes
e) Repeat using stock solutions of tyrosine and tryptophan

Test 5: Lead Sulphide Test

a) Pipette 1 ml of cysteine in a test tube

b) Add 1 ml of 40% NaOH
c) Swirl the test tubes
d) Heat the test mixture in a boiling water bath for 2 minutes
e) Immerse the test tube in cold water to cool them down
f) Add 1 ml of sodium plumbate (Molecular Formula:Na2O3Pb)
g) Record any changes
h) Repeat using stock solutions of cystine and methionine

Test 6: Sakaguchi Test

a) Pipette 1 ml of arginine in a test tube

b) Add 1 ml of 40% NaOH
c) Add 2 drops of napthol
d) Add 5 drops of bromine
e) Swirl the test tubes

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 f) Record any changes
g) Repeat the procedure by using glycosiamine, methyl guanidine, creatine, and
urea.

Results

Ninhydrin Test

Sample Observation Conclusion

Glycine
Tyrosine
Tryptophan
Phenylalanine
Proline

Xanthoprotein Test
Sample Observation Conclusion
Glycine
Tyrosine
Tryptophan
Phenylalanine
Phenol

Millon Test
Sample Observation Conclusion
Glycine
Tyrosine
Phenylalanine

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Hopkins-Cole Test
Sample Observation Conclusion
Glycine
Tyrosine
Tryptophan

Lead Sulphide Test

Sample Observation Conclusion
Cysteine
Cystine
Methionine

Sakaguchi Test
Sample Observation Conclusion
Arginine
Glycosiamine
Methyl guanidine
Creatine
Urea

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LABORATORY 3: CALORIMETRIC TESTS FOR CARBOHYDRATES

Introduction

Carbohydrate is a class of organic molecules with the general formula Cn(H2O)n. The
basic carbohydrate units are called monosaccharides. There are numerous different
types of monosaccharide which differ in their number of carbon atoms and the
arrangement of the H and O atoms attached at the carbons. Monosaccharides are
linked together by a glycosidic bond to form oligosaccharide and polysaccharide.

Several calorimetric tests have been devised to detect members of this biologically
significant class of compounds. These tests will utilise a test reagent that yields a
colour change after reacting with specific functional groups of the compounds being
tested. The following exercises are reactions that can detect the presence or absence
of carbohydrates in test solutions. They range in specificity to the very general.

Objective

To use the following qualitative tests to distinguish specific sugars from each other.

Methods

Test 1: Benedict’s Test

a) Add 1.0 of sucrose solution to 1 ml of Benedict’s reagent.

b) Heat in a boiling water bath for 5 minutes.

c) Repeat the same procedure with glucose.

A brick-red precipitate indicates a positive test for reducing sugars.

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Test 2: Barfoed’s Test

a) Add 1.0 mL of sucrose solution to 1 ml of Barfoed’s reagent.

b) Heat in a boiling water bath for 10 minutes.

c) While heating, write down the changes in every 3 minutes.

d) Repeat the same procedure with glucose and lactose.

A copious amount of brick-red precipitate indicates a reducing monosaccharide.

Hydrolysis of disaccharides may lead to trace precipitates.

Test 3: Seliwanoff’s Test

a) Add 1.0 mL of glucose solution to 1 ml of Seliwanoff’s reagent.

b) Heat in boiling water bath for 5 minutes.

c) Repeat the same procedure with fructose.

A deep red coloured precipitate indicates ketohexoses. Other sugars give red colour
upon prolonged heating.

Test 4: Bial Test

a) Add 1.0 ml of sucrose solution to 1 ml of Bial’s reagent.

b) Heat in boiling water bath for 2 minutes.

c) Repeat the same procedure with arabinose and glucose.

A blue-green colour indicates pentoses; a yellow-green colour indicates hexoses

and disaccharides are yellow.

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Questions

1. If a sample being tested gives a positive result for Benedict’s test and Barfoed’s
test, then what will the sample be? (1m)

2. Name the suitable test to distinguish:

a) a pentose and a hexose (1m)

b) an aldohexose and a ketohexose (1m)

3. Identify the brick-red precipitate in a benedict test. (1m)

4. Name the bond involved when a disaccharide is hydrolysed. (1m)

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LABORATORY 4 : THIN LAYER CHROMATOGRAPHY

Sample Preparation (ground nut extract)

• Add 4 ml of methanol and 8 ml of chloroform to 5 mg of ground peanuts.

• Mix well.
• Centrifuge at 3000 rpm for 5 minutes.
• Use the top layer as your sample.

Standards

• Olive oil
• Oleic acid
• Cholesterol
• Phosphotidylcholine

Methods

1. Pour solvent into a chromatography tank and seal it, so the atmosphere is
saturated with solvent vapour (done by Lab Staff).
2. Apply a drop of the mixture to be separated onto a sheet of the Thin Layer
Chromatography (TLC). This is the origin of the chromatogram. The spot
should be small and concentrated.
3. Repeat for samples (2-6 spots) and standards (2 spots). Label the spot.
4. Place the chromatography sheet into the tank.
5. When the solvent has nearly reached the top of the TLC, the TLC is removed,
and the position of the solvent front will be marked lightly using a pencil.
6. The chromatogram needs to be developed to make the spots visible by using
a specific reagent.
7. The chromatogram can be analysed by measuring the distance travelled by
the solvent front and the distance from the origin to the centre of each spot.
To calculate the Rf (relative front) value for each spot:
Rf = Distance moved by spot (sample)
Distance moved by solvent (solvent front)
8. Rf value can be used to identify components of a mixture by comparing to the
standard Rf values.

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Questions

1. What is the mobile phase for thin layer chromatography?

2. What is the reagent used to develop thin layer chromatography?

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