Biochemistry

LAB MANUAL
G-BSCN
1 YEAR
ST

1 SEMESTER
ST

BY: JUNAID ALI GONDAL & ANAM NASIR

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 1

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 2
Table of contents
S# Index Page No REMARKS
1. Some Biochemistry Equipment

2. Biochemistry Lab Practices and Safety Rules

3. Hazards and their precautionary measures

4. Properties of organic compounds

5. Molish test

6. Benedicts test

7. BI- uret test

8. Heat coagulation test

9. REFERENCES

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 3

LAB EQUIPMENTS

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 4

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 5
HAZARDS AND THEIR PRECUATIONARY FIRST
MEASURES
OBJECTIVES:
At the end of session learners will be able to:
1. List the lab instructions to be followed.
2. List various hazards and accidents that can be occur during lab work.
3. Give precautionary measures that can prevent such situations.
4. Give first aid in general.
5. Discus safety measure that we can follow for managing accidental
conditions.
6. Solve the given case.

Biochemistry safety rules and procedures:

1. Wash your hands with disinfectant soap when you arrive at the lab and
again before you leave.
2. Absolutely no food, drinks, chewing gum, or smoking is allowed in the
laboratory.
3. Do not put anything in your mouth such as pencils, pens, labels, or
fingers.
4. Do not store food in areas where microorganisms are stored.
5. Purchase a lab coat and safety glasses, bring them to class, and use
them.
6. Avoid loose fitting items of clothing. Wear appropriate shoes (sandals
are not allowed) in the Laboratory
7. Keep your workspace free of all unnecessary materials.
8. Disinfect work areas before and after use with 70% ethanol or fresh
10% bleach.
9. Laboratory Equipment and work surfaces should be decontaminated
with an appropriate disinfectant on a Routine basis, and especially after
spills, splashes, or other contamination.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 6

 10.Label everything clearly.
11.Replace caps on reagents, solution bottles, and bacterial cultures.
12.Do not open Petri dishes in the lab unless absolutely necessary.
13.Inoculating loops and needles should be flame sterilized in a Bunsen
burner before you lay them down.
14.Turn off Bunsen burners when is not in use. Long hair must be
restrained if Bunsen burners are in use.
15.Sterilize equipment and materials.
16.Consider everything a biohazard. Do not pour anything down the sink.
Autoclave liquids and broth cultures to sterilize them before discarding.
17.Dispose of broken glass in the broken glass container.
18.Dispose of razor blades, syringe needles, and sharp metal objects in the
“sharps” container.
19.Report all injuries or accidents immediately to the instructor, no matter
how small they seem.

Safety measures for chemical in lab:

❖ Before begning any activity:
1. Know what is expected.
2. Prepare the clear work environment and wait for permission.
3. Whenever special attention is needed in lab acitivty you will se the
board and listen attentively to instructor.
4. Caution (this means special care must be taken when preceding with
activity).
❖ General safety precuations:

1. Know locations of laboratory safety showers, eyewashstations, and fire

extinguishers. The safety equipment may be located in the hallway near the
laboratory entrance.
2. Know emergency exit routes.
3. Avoid skin and eye contact with all chemicals.
4. Minimize all chemical exposures.
5. No horseplay will be tolerated.
6. Assume that all chemicals of unknown toxicity are highly toxic.
7. Post warning signs when unusual hazards, hazardous materials, hazardous
equipment, or other special conditions are present.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 7

 8. Avoid distracting or startling persons working in the laboratory.
9. Use equipment only for its designated purpose.
10.Combine reagents in their appropriate order, such as adding acid to water.
11.Avoid adding solids to hot liquids.
12.All laboratory personnel should place emphasis on safety and chemical
hygiene at all times.
13.Never leave containers of chemicals open.
14.All containers must have appropriate labels. Unlabeled chemicals should
never be used.
15.Do not taste or intentionally sniff chemicals.
16.Never consume and/or store food or beverages or apply cosmetics in areas
where hazardous chemicals are used or stored.
17.Do not use mouth suction for pipetting or starting a siphon.
18.Wash exposed areas of the skin prior to leaving the laboratory.
19.Long hair and loose clothing must be pulled back and secured from
entanglement or potential capture.
20.No contact lenses should be worn around hazardous chemicals – even when
wearing safety glasses.
21.Laboratory safety glasses or goggles should be worn in any area where
chemicals are used or stored. They should also be worn any time there is a
chance of splashes or particulates to enter the eye. Closed toe shoes will be
worn at all times in the laboratory. Perforated shoes or sandals are not
appropriate.
22.Determine the potential hazards and appropriate safety precautions before
beginning any work.
23.Procedures should be developed that minimize the formation and dispersion
of aerosols.
24.If an unknown chemical is produced in the laboratory, the material should be
considered hazardous.
25.Do not pour chemicals down drains. Do NOT utilize the sewer for chemical
waste disposal.
26.Keep all sink traps (including cup sink traps and floor drains) filled with
water by running water down the drain at least monthly.
27.Do not utilize fume hoods for evaporations and disposal of volatile solvents.
28.Perform work with hazardous chemicals in a properly working fume hood to
reduce potential exposures.
29.Avoid working alone in a building. Do not work alone in a laboratory if the
procedures being conducted are hazardous.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 8

 30.The PEL and the Threshold Limit Values (TLV) will be observed in all
areas. If exposure above a PEL/TLV is suspected for an ongoing process,
please contact EHS immediately.
31.Laboratory employees should have access to a chemical inventory list,
applicable SDSs, Department Laboratory Safety Manual, and relevant SOPs.
32.Access to laboratories and support areas such as stockrooms, specialized
laboratories, etc. should be limited to approved personnel only.
33.All equipment should be regularly inspected for wear or deterioration.
34.Equipment should be maintained according to the manufacturer’s
requirements and records of certification, maintenance, or repairs should be
maintained for the life of the equipment.
35.Designated and well-marked waste storage locations are necessary.
36.No cell phone or ear phone usage in the active portion of the laboratories, or
during experimental operations.
37.Clothing made of synthetic fibers should not be worn while working with
flammable liquids or when a fire hazard is present as these materials tend to
melt and stick to exposed skin.
38.Laboratory coats should not be stored in offices or break rooms as this
spreads contaminates to other areas.
39.Computers and instrumentation should be labeled to indicate whether gloves
should be worn or not. Inconsistent glove use around keyboards/keypads is a
source of potential contamination.
40.Avoid wearing jewelry in the lab as this can pose multiple safety hazards.

❖ Handling a heat source:

1. Turn off heat sources when they are not in use.

2. Point test tubes away from yourself and others when heating substances in
them.
3. Use the proper procedures when lighting a Bunsen burner.
4. To avoid burns, do not handle heated glassware or materials directly.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 9

PORPERTIES OF ORGANIC COMPOUNDS
Objectives:
At the end of lab learners will be able to:
1. Define organic compounds.
2. List the types of organic compounds.
3. Describe the use of organic compounds.
4. Describe the characterstics of organic compounds.
5. Differentiate between organic and inorganic compounds.

Introduction to organic compounds:

Organic chemistry is a branch of chemistry that studies the structure, properties
and reactions of organic compounds, which contain carbon in covalent bonding.
Study of structure determines their structural formula.
Organic compounds are called "organic" because they are associated with living
organisms. ... There are four main types, or classes, of organic compounds found in
all living things: carbohydrates, lipids, proteins, and nucleic acids
organic compound, any of a large class of chemical compounds in which one or
more atoms of carbon are covalently linked to atoms of other elements, most
commonly hydrogen, oxygen, or nitrogen. The few carbon-
containing compounds not classified as organic include carbides, carbonates,
and cyanides.
Types of organic compounds:
These are the compounds that are functional group and are not attached directly to
a benzene ring.
1. Alkanes
2. Alkines
3. Halogenoalkanes (haloalkanes and alkyl halids)
4. Alcohols
5. Aldehydes and ketones
6. Carboxylic acids
7. Acyle chlorides

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 10

 8. Acid anhydrides
9. Esters
10.Amides
11.Nitriles
12.Amines
13.Amino acid and other biochemistry.

Uses of organic chemistry:

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 11

Characterstics of organic compounds:
these are the gnenral characterstics of organic compounds:

• Organic compounds include complex structures and high molecular weights.

• These are soluble in organic solvents and mostly insoluble in water.
• Mostly depend on only three elements: Carbon, Hydrogen and nitrogen.
• These compounds are combustible in nature.
• Most properties of the compounds are decided by the functional group
attached to them.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 12

 MOLISH TEST

Object:
This is the quantitative group test for carbohydrates and is given by all
carbohydrates weather bond of such substances is lipid and portein.
Reagent:
Molish reagent is prepared by dissolving of x napthol in 95% of methyl alcohol
and making the volume upto 100ml.
Principle:
The principle of Molisch's test is the dehydration of sulphuric acid into furfural.
When a sample containing carbohydrate molecules is treated with sulphuric acid and
concentrated hydrochloric acid, one hydroxyl group gets eliminated from the sugar
molecule. The hydroxyl group is eliminated in the form of water. Molisch’s test is a
chemical test which is used to check for the presence of carbohydrates in a given
analyte. This test is named after Czech-Austrian botanist Hans Molisch, who is
credited with its discovery. Molisch’s test involves the addition of Molisch’s reagent
(a solution of ∝-naphthol in ethanol) to the analyte and the subsequent addition of a
few drops of concentrated H2SO4 (sulphuric acid) to the mixture.
The formation of a purple or a purplish-red ring at the point of contact between the
H2SO4 and the analyte + Molisch’s reagent mixture confirms the presence of
carbohydrates in the analyte.
Procedure:
2-3 drops of Molisch’s reagent must be added to a small amount of the analyte in a
test tube and mixed well. Now, a few drops of concentrated sulphuric acid must be
added drop-wise along the walls of the test tube to facilitate the formation of a layer
and avoid mixing. The development of a purple ring at the layer formed by the
concentrated acid is a positive indicator for Molisch’s test. If no purple or reddish-
purple colour arises, the given analyte does not contain any carbohydrate.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 13

Observation:

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 14

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Equation:

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Result:
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Discussion:
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BY: JUNAID ALI GONDAL & ANAM NASIR pg. 16

BENEDICT’S TEST
Object:
This is the test for reducing carbohydrates.
Reagents:
Benedicts reagent
➢ Copper sulphate(crystalline 17.3g)
➢ Sodium citrate(173g)
➢ Sodium carbonate (anhydrous 100.0g)
➢ Water to make g.s to 1000ml
Sodium citrate and sodium carbonate are dissolved in 750ml of water by
heating.CuSO4 is separately dissolved in 100ml of water then add both the
solutions and make up the volume.
Principle:
When Benedict’s solution and simple carbohydrates are heated, the solution
changes to orange red/ brick red. This reaction is caused by the reducing property
of simple carbohydrates. The copper (II) ions in the Benedict’s solution are
reduced to Copper (I) ions, which causes the color change.

The red copper(I) oxide formed is insoluble in water and is precipitated out of
solution. This accounts for the precipitate formed. As the concentration of reducing
sugar increases, the nearer the final colour is to brick-red and the greater the
precipitate formed. Sometimes a brick red solid, copper oxide, precipitates out of
the solution and collects at the bottom of the test tube.

Sodium carbonate provides the alkaline conditions which are required for the redox
reaction. Sodium citrate complexes with the copper (II) ions so that they do not
deteriorate to copper(I) ions during storage.
Complex carbohydrates such as starches DO NOT react positive with the
Benedict’s test unless they are broken down through heating or digestion (try
chewing crackers and then doing the test). Table sugar (disaccharide) is a non-
reducing sugar and does also not react with the iodine or with the Benedict
Reagent. Sugar needs to be decomposed into its components glucose and fructose
then the glucose test would be positive but the starch test would still be negative.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 17

Procedure:

Benedict’s solution is a deep-blue alkaline solution used to test for the presence of
the aldehyde functional group, – CHO.

Anhydrous sodium carbonate = 100 gm

Sodium citrate – 173 gm
Copper(II) sulfate pentahydrate = 17.3 gm
One litre of Benedict’s solution can be prepared from 100 g of anhydrous sodium
carbonate, 173 g of sodium citrate and 17.3 g of copper(II) sulfate pentahydrate.

Procedure of Benedict’s Test

1. Approximately 1 ml of sample is placed into a clean test tube.

2. 2 ml (10 drops) of Benedict’s reagent (CuSO4) is placed in the test tube.
3. The solution is then heated in a boiling water bath for 3-5 minutes.
4. Observe for color change in the solution of test tubes or precipitate
formation.

Observation:

If the color upon boiling is changed into green, then there would be 0.1 to 0.5
percent sugar in solution.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 18

 If it changes color to yellow, then 0.5 to 1 percent sugar is present.
If it changes to orange, then it means that 1 to 1.5 percent sugar is present.
If color changes to red,then 1.5 to 2.0 percent sugar is present.
And if color changes to brick red,it means that more than 2 percent sugar is
present in solution.
Positive Benedict’s Test: Formation of a reddish precipitate within three
minutes. Reducing sugars present. Example: Glucose
Negative Benedict’s Test: No color change (Remains Blue). Reducing sugars
absent. Example: Sucrose.
Result:
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Discussion:
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BY: JUNAID ALI GONDAL & ANAM NASIR pg. 19

BIURET TEST
Object:
This is a test for peptide linkage in protein. Since all protein contain peptide
linkages and they respond to this test. This test is found negative in amino acids.
Reagent:
Sodium hydroxide(NaOH)= 10% solution.
Copper sulphate(CuSO4)= 0.5% in water sample protein.
Principle:
A Biuret test is a chemical test used to determine the presence of a peptide bond in
a substance. It is based on the biuret reaction in which a peptide structure
containing at least two peptide links produces a violet color when treated with
alkaline copper sulfate. In presence of an alkaline solution, blue-colored copper II
ion can form a complex with the peptide bonds since the peptide has unshared
electron pairs in nitrogen and oxygen of water. The colored coordination complex
is formed between Cu2+ ion and carbonyl oxygen (>C=O) and amide nitrogen
(=NH) of the peptide bond. Once this complex has been formed, the solution turns
from blue to purple. The deeper the purple color, the higher is the number of
peptide-copper complexes. The reaction occurs in any compound containing at
least two H2N-C, H2N-CH2-, H2N-CS- or similar groups joined together directly
or through a carbon or nitrogen atom. One copper ion is probably linked to 6
nearby peptide linkages by co-ordinate bonds. The intensity of the color is directly
proportional to the number of the peptide bonds present in the protein molecule
that is reacting and also the number of the protein molecules present in the reaction
system.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 20

Procedure:
1. Take 3 clean and dry test tubes.
2. Add 1-2 ml of the test solution, egg albumin, and deionized water in the
respective test tubes.
3. Add 1-2 ml of Biuret reagent to all the test tubes.
4. Shake well and allow the mixtures to stand for 5 minutes.
5. Observe for any color change.

Observation:

Observation Interpretation

No color change, i.e., the solution Proteins are absent (negative biuret
remains blue test)

The solution turns from blue to Proteins are present (positive biuret
deep purple test)

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 21

Equation :

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 22

Result:
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Discussion:
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BY: JUNAID ALI GONDAL & ANAM NASIR pg. 23

HEAT COAGULATION TEST
Objective:
Heat coagulation test.
Theory:
Heat coagulation test of protein is a biochemical test performed to determine the
presence of proteins like albumin and globulin in protein. Coagulation of proteins
as a response to heat is a common phenomenon. The heat coagulation of proteins
occurs in one of the two stages; denaturation and agglutination or the separation of
the denatured protein in a particular form. The heating of coagulable proteins at
their isoelectric pH, a series of changes occur to the proteins; dissociation of
subunits or the quaternary structure, uncoiling of the polypeptide chains ultimately
leading to the matting together of uncoiled polypeptide chains. Processes like
coagulation and flocculation are superficial visible manifestations of changes that
occur in proteins during the denaturation process.
Coagulation of proteins is an irreversible process that is maximum at the isoelectric
pH of the proteins. Heat coagulation of proteins is an important clinical test for the
detection of proteinuria. It is simple and less time-consuming. Both qualitative and
quantitative estimation of proteins can be done with the heat coagulation method.
The quantitative analysis of coagulation can be performed by measuring the
coagulum formed on the test tube.
Principle:
The principle of heat coagulation test is the change in the structure of proteins as a
result of heat and change in pH. Heating a protein in acidic medium results in
denaturation of protein due to the breaking of certain bonds responsible for the
tertiary and quaternary structure of proteins. However, in the case of coagulable
proteins, when these proteins are heated at their isoelectric pH, the polypeptide
chains become uncoiled and matt with each other to form an insoluble mass. The
mass formed doesn’t dissolve back to the liquid. The process of coagulation is
maximum at the isoelectric point, and the mass of the coagulum might differ with
the particle size and the concentration of proteins in the sample. For the heat
coagulation test of albumin and globulin, chlorophenol red is used which adjusts
the pH of the sample to the isoelectric point of albumin. The reagent for this test
also contains acetic acid, which helps in the breaking of peptide bonds present in
the protein molecule, facilitating coagulation.
Reagents:
• Chlorophenol red indicator

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 24

• 1% acetic acid
• Sample

Procedure:

1. About two-thirds of the test tube is filled with the given sample.
2. To the test tube, 1-2 drops of chlorophenol red indicator is added drop by drop
and mixed properly.
3. When a purple color is observed on the test tube, 1% acetic acid is added drop
by drop until the color changes to pale pink.
4. The test tube is then inclined slightly so as to heat the upper portion of the
fluid.
5. The tube is observed for the formation of the coagulum.

Observation:

• Positive result: A positive result of the heat coagulation test is represented by

the formation of a dense coagulum at the upper part of the solution. The lower
part of the solution acts as a control.
• Negative result: A negative result of the heat coagulation test is represented by
the absence of coagulum at the upper layer. This indicates the absence of
albumin and other proteins in urine.

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 25

Result:
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Discussion:
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GOOD LUCK!

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Cell: 03400568646 Email: jagondal123@yahoo.com

BY: JUNAID ALI GONDAL & ANAM NASIR pg. 26