Laboratory Manual

Biochemistry
(BSC-102)

Student Name USAMA JAVED

Student ID S20BME24

Batch Spring 2021

Lab Instructor:
Mr. Tehseen Nawaz
Dr. Arooj Safiq
 INDEX
S.No. Date Experiment Remark Sign.
16/11/21 Introduction to lab safety and lab
1 techniques.
23/11/21 To calibrate a micropipette and measure
2 its accuracy.
23/11/21 To prepare molar and percentage
3 solutions of different compounds and their
interconversions.
30/11/21 To learn how to prepare the serial
4 dilution
30/11/21 To estimate the concentration of
5 spectrophotometry of a solute in solution.

6
7 .

8
9
10 .

11
12
13
14
15
16
1 LAB EXPERIMENT

Object:
Introduction to lab and safety techniques and learning about lab equipment’s

Introduction:
In this lab of biochemistry, we were being instructed about the code of conduct every
student has to maintain in the lab safety and precautionary measures that should be
followed by every individual within the lab.

Lab Safety
Few safety and precautionary measured are necessary to be followed by every
individual in the lab.
 First to prevent any sort of contamination in the lab.it is necessary to wear proper
and complete lab attire which includes a lab coat, safety glasses gloves, etc.
 Disposing of all the used equipment after coming out of the lab is necessary so
that one can be safe himself and save others from the dangerous chemicals
he/she might have been using in the lab.
 Do not use any unnamed chemicals, or a chemical-containing bottle with no label
as you might not be knowing about the effect or harm it could during an
experiment.
 Moreover, one should know the basic rule while handling chemicals like to dilute
acid, acid should be poured drop by drop into the water rather than the other way
round.
 Do not use mouth suction for pipetting or starting a siphon.
 Wash exposed areas of the skin prior to beforethe laboratory.
 Long hair and loose clothing must be pulled back and secured from
entanglement or potential capture.
 No contact lenses should be worn around hazardous chemicals – even when
wearing safety glasses.
 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 thenteringlosed toe shoes will be
 worn at all times in the laboratory. Perforated shoes or sandals are not
appropriate.
 Determine the potential hazards and appropriate safety precautions before
beginning any work.
 Procedures should be developed that minimize the formation and dispersion of
aerosols.
 If an unknown chemical is produced in the laboratory, the material should be
considered hazardous.
 When an accident happens in the lab one must immediately report it regardless
of the fact even thouis such that the necessary action against the accident could
be carried out immediately.
 Eyes should be immediately washed if a chemical gets into the eyes of an
individual accidaccidentallyeriment involving mixtures containing harmfufumeses
should always be performed using a gas mask and under the lumeshood.
 When smelling a certain chemical the chemical or mixture should never be
brought near one’s noose.
 Do not utilize fume hoods for evaporations and disposal of volatile solvents.
 Encloseis d the experiment process to avoaccan identsent.
 2 LAB EXPERIMENT
OBJECTIVE:
To calibrate a micropipette measurers’ its accuracy.
INTRODUCTION:
In this biochemistry lab, one of the maiobjectsct that an experimenter
hasis to achiis eve the abilimeasureasures and transfer sa mall volume of liquid with
the accuracy for obtaining the required motive of the experiment. In this experime,nt we
use a device known has micropipette.
A pipette mainly comprises of a narrow tube used to transfer solution or liquid,
preparing solution with the various concentration and also for diluting solution. Pipette
are various types but we use one in the laboratory was micropipette. These helps
mainly in the transferring small amount of liquid of ranging from 1ml to 0.1µl. For
avoiding contact of pipette with liquid itself we use disposable tips made up of
polypropylene.

MATERIALS:
 Micropipette
 Analytical balance
 Beakers
 Waters
 Polypropylene tips

METHODS:
In this experiment first we follow some steps
1) In firs step we attach the disposable polypropylene tip to the end of the pipette.
2) We adjust the volume with the help of adjusting knob to 1000µ.
3) Now single press the knob and dip the knob in the beaker which contains water
and release the plungers slowly as the water level rise in the pipette.
4) Now go towards analytical balance and place the container in the analytical
balance cover all the sides with glass plate and press the button tear and values
becomes zero.
 5) Second phase bring the pipette dose to the container in the analytical balance
and drop the water in the container slowly by pressing the plunger & knob.
6) And cover the outer surface with glass plates.
7) Note down the readings.
8) Repeat this process thrice time and note all the readings to check the accuracy
of pipette it should give a reading f 1gm in 1000µ

OBSERVATION AND CALCULATION:

Volume (µl) Weight (gm)
1000µ 0.95
1000µ 0.97
1000µ 0.96
Taking average:
mean value ¿( a+b+ c)/3
mean value= (0.95+0.97+0.96) / 3
mean value= 0.96gm

RESULT:
Form the above readings it can be concluded that the micropipette used of
high accuracy and the slight change in readings might due to human error and handling.
The mean average value is 0.96gm.
 3 LAB EXPERIMENT
OBJECTIVE:

To prepare molar and percentage solutions of different compounds and

their interconversions.

INTRODUCTION:

In this lab we prepare molar solution of different compounds. We move

towards mole. A mole is defined as that the amount of substance which contains
Avogadro’s number of atoms if the substance is atomic or Avogadro’s number of
molecules if the substance is molecular. Mole of carbon atoms= 6.022 x10power 23
atoms of carbon atom. A mole of hydrogen atom means 6.022 x10 power23 atoms of
hydrogen whereas a mole of hydrogen molecule means 6.022 x10 power23 molecules
of hydrogen atoms.
A molar solution is defined as aqueous solution that contains 1 mole
(gram molecular weight) of a compound dissolved in 1 liter of a solution. In other words,
the solution has a concentration of 1 mol/L or a molarity of 1 (1M).
Formula:
Molarity (M) = moles of solute (n) / liters of solution (v)
M= molar concentration
n = moles of solute
v = liters of solution
A molal solution is a solution that contains 1 molecular weight of solute in
a kilogram of solvent. It is the strength or concentration of a solution, especially the
amount of dissolved substance in a given volume of solvent. It is a concentration of a
solution expressed in moles or molality (m).
Molality (m) = moles of solute (n) / liters of solvent (v)
m= molar concentration
n = moles of solute
v = kg of solvent
Normality:
 It is defined as the number of gram equivalents per liter volume of the solution.

METHODS:

In this lab we work on following topics Molarity molar solution and

interconversion
1): First of all, we will take 39.997g of NaOH using analytical balance because molar
mass of NaOH is 39.997g/mol.
2): Then we will take a beaker and we will take distilled water in that beaker.
3): We will add the solute i.e. NaOH in the distilled water and mix the solute in the
solvent till it dissolved fully.
4): We will take 800ml of water in another beaker and we will add the solution of above
NaOH in this beaker so that total volume of the final solution could become 1dmᵌ.
5): In this way we can successfully prepare one molar solution of NaOH.

OBSERVATION:

One molar solution of NAOH

NAOH = 39.997g/mol
mole = 1 mole
1 molar solution = 39.997 g/mol / 1 liter of water/1000m
1 molar solution = 1/1
One molar solution of HCL
HCL = 36.458 g/mol
mole = 1 mole
1 molar solution = 36.458 g/mol / 1 liter of water/1000ml
1 molar solution = 1/1
One molar solution of CH3COONA
CH3COONA = 82.0343 g/mol
mole = 1 mole
1 molar solution = 82.0343 g/mol / 1 liter of water/1000ml
1 molar solution = 1/1

RESULT:

We prepared one molar solution of NAOH, HCL, CH3COONA.

Preparation of M1V1=M2V2 and interconversion

M1V1=M2V2:
The M1V1 equals M2V2 equation is used to calculate dilution. M1 is the molarity and V1
is the volume of the concentrated solution. M2 is the molarity and V2 is the volume of
the of the diluted solution
Volume by volume percentage (v/v%):
Volume/volume percentage (v/v percent) is a measure of
the concentration of a substance in a solution. It is expressed as the ratio of the volume
of the solute to the total volume of the solution multiplied by 100.
Volume percent is defined as:
v/v % = [(volume of solute)/(volume of solution)] x 100%

Weight by volume percentage (w/v%):

Weight/Volume Percentage Concentration is a
measurement of the concentration of a solution. weight/volume percentage
concentration is usually abbreviated as w/v (%). To calculate w/v % concentration:
    mass of solute (g) /   
w/v (%) = × 100
volume of solution (mL)

Weight by weight percentage (w/w%):

This percent by weight formula represents any solution
expressed as %w/w, meaning weight by weight; here, we only consider the weight of
any solution's components. For example: if 100g solution contains 30g HCl and 70g
water, we express it as hydrochloric acid 30% w/w.

Methods:
1: We analyze the total volume we have to prepare.
2. We will take a one molar solution from the stock solution.
3: We will dissolve water in solution for obtaining the correct molarity of solution i.e.
dilution. Whenever we will add water in the solution its molarity will decrease
4: The amount of volume of water we should add can be found by using this relation of:
M1V1=M2V2
After subtracting V1 from V2 total volume of water we should add and in this way
solution of required molarity can be prepared.

Observation:

In this lab we have some given data

1. 0.03 M of NaCl(70ml)
2. 0.17M HCl(55ml)
3. 0.54M of CH3COONa (83ml)

1):0.03 of NaCl(70ml)

Given Data:
M2= 0.03M
V2= 70ml
M1= 1
V1=?
Formula:
M1V1=M2V2
V1=M2V2 / M1
V1=0.03 x 70 /1
V1=2.1ml
Water amount V2-V1= 70-2.1=67ml/d/w

2):0.17M of HCl(55ml)

Given Data:
M2= 0.17M
V2= 55ml
M1= 1
V1=?
Formula:
M1V1=M2V2
V1=M2V2 / M1
V1=0.17 x 55 /1
V1=9.35ml
Water amount V2-V1= 55-9.35=45.65ml/d/w

3):0.54M of CH3COONa(83ml)

Given Data:
M2= 0.54M
V2= 83ml
M1= 1
V1=?
Formula:
M1V1=M2V2
V1=M2V2 / M1
V1=0.54 x 83 /1
V1=44.82ml
Water amount V2-V1= 83-44.82=38.18ml/d/w

Discussion:

we learned about different terms used for the preparation of solutions. We

understand the concept of Moles, Molar Solution, Molarity, and percentage solution. We
also learned a way through which we can prepare one molar solution and also after this
lab we are able to prepare the solution of the required molarity from the stock solution.
Result:

Solution of required molarity has been prepared. in this experiment we have some
given data such as M1, M2, V2 and we find V1.
4 LAB EXPERIMENT (a)
Objective:
To learn how to prepare the serial dilution.

Introduction:
Serial dilution is a sequence of repeated dilutions used to produce a
less dense or concentrated solution from a dense or concentrated one. Serial
dilution is a laboratory technique that involves performing a stepwise dilution
process on a solution with an associated dilution factor. This procedure is used in
the laboratory to reduce the number of cells in a culture to make the process
easier. The cell count or density gradually falls as the serial number increases in
each step of serial dilution. Calculating the overall dilution over the entire series
makes it easier to calculate the cell numbers in the primary solution.
The concentration of the solution is lowered by adding more amount of covalent
this process is known as Dilution. The method of dilution is to fold dilution. In this
method reducing the concentration of a solution by a factor of two is known as
Two Fold dilution. A series of two-fold dilutions is known as two-fold serial
dilutions. In this dilution, the amount of solvent should be very accurate in order
to deliver use a micropipette.

Materials:

 Blue Bromophenol
 Distilled water
 Beakers
 Micropipette
 Test tubes
 Test tubes stand

Methods:
In this lab, we work on the following topics dilution and serial
dilution, and two-fold serial dilution.

1. Take five test tubes and fill them with 5ml distilled water in each of them.
2. Label all the test tubes with D1, D2, D3, D4, D5.
3. Take 5ml blue bromophenol put in one test tube D1, D1 making a solution of 1:2
ratios, and shake it.
4. After that take 5ml solution from D1 with help of a micropipette and put it in the
second test tube and label by D2 and shake it. Making a solution of 1:4 ratios.
 5. After that take 5ml solution from D2 with help of a micropipette and put it in the
second test tube and label by D3 and shake it. Making a solution of 1:8 ratios.
6. After that take 5ml solution from D3 with help of a micropipette and put it in the
second test tube and label by D4 and shake it. Making a solution of 1:16 ratios.
7. After that take 5ml solution from D4 with help of a micropipette and put it in the
second test tube and label by D5 and shake it. Making a solution of 1:32 ratios.

Observation:

Solution Concentration Dilution ratio

Blue Bromophenol 60 µg/ml 1:1
D1 30µg/ml 1:2
D2 15 µg/ml 1:4
D3 7.5 µg/ml 1:8
D4 3.75 µg/ml 1:16
D5 1.87µg/ml 1:32

Result:
It was concluded that each successive solution being formed in a two-fold dilution
condition 1; to the previous one.

Discussion:
In this lab we learn about the concepts of the dilution and serial dilution.
We performed two-fold dilution of a chemical blue bromophenol and we came know
that every time solvent concentration is increased and the solute concentration
decreases on every step.
4 LAB EXPERIMENT (b)
Objective:
To estimate the concentration of spectrophotometry of a solute in solution.

Introduction:
A spectrophotometer has been used in the lab to measure easily the
absorption of light by a solution. The “spectrum” refers mainly to multiple light colors
apparent in an analysis of white light using a prism. Spectroscopy is used to determine
the multiple wavelengths of colors of light. These devices have the tendency to measure
absorbance at various specific wavelengths. It comprises a monochromatic which splits
incident light into its component wavelength to reach the sample. The method is useful
as the extinction coefficient of a compound varies with different wavelengths.
To measure the extinction co-efficient using Beer-Lambert Law.
According to the law:
A=Ɛcl
A = absorbance at particular wavelength.
Ɛ = molar extinction co-efficient component at specific (M-1 cm-1).

c = molar concentration of the specific (M).

l = path length of solution is cm.
Ɛ is known as absorbing species, the absorbance of that solution can be used to
calculate the concentration of absorbing species in solution.

Materials:
 Blue Bromophenol
 Micropipette
 Distilled water
 Test tubes
 Beakers
 Test tube stand
 spectrophotometer

Methods:
In this lab, we work on the following topics dilution and serial dilution and
two-fold serial dilution and color absorption in the spectrophotometer.
1. Already we perform by using blue bromophenol and distilled water a two-fold
series dilution in test tubes labeled D1, D2, D3, D4, D5 whose ratios 1:2 D1, 1:4
D2,1:8 D3, 1:16 D4, 1:32 D5 to stock solution.
 2. Make another solution of unknown concentration and label D6 on test tubes.
3. Use the spectrophotometer at a specific wavelength value 590nm and cuvet filled
with distilled water and placed in a spectrophotometer and calibrate it to zero.
4. Measure the absorbance of each of the samples one by one placed in
spectrophotometer gradually and note all the values of D1 to D6.

Observation:
Serial number Absorbance or optical Concentration
density
D1 0.209 ABS 30 µg/ml
D2 0.105 ABS 15 µg/ml
D3 0.053 ABS 7.5 µg/ml
D4 0.044 ABS 3.75 µg/ml
D5 0.0071 ABS 1.87 µg/ml
D6 0.06 ABS Unknown
Graph:
Attached

Result:
Base on the above calculation of graph and observation with the help of
spectrometry we concluded that when the concentration is decreasing then
optical density decreases.

Discussion:
In this lab, we have come across the concept of spectroscopy, its construction,
working, purpose.in today’s lab, we used a device that is used to determine the
wavelength of the chemical substances. we determine the optical density of five
different serial dilutions through spectrophotometry.
5 LAB EXPERIMENT
Objective:

To identify the macromolecules in a given sample.

Introduction:

Macromolecules are large, complex molecules. Macromolecules are made up of basic

molecular units. They include proteins (polymers of amino acids), nucleic acids
(polymers of nucleotides), carbohydrates (polymers of sugars), and lipids (with a variety
of modular constituents). They are usually products of smaller molecules, like proteins,
lipids, and carbohydrates.
In this lab, we identify these molecules by following tests.
1. Biuret test
2. Iodine test
3. Sudan IV test
Biuret test:
 The biuret test is the chemical test used for detecting the presence of peptide
bonds. Biuret is a compound formed by heating urea at 1800°C which results in the
condensation of 2 molecules of urea.
When biuret is treated with dilute copper sulfate in alkaline condition at a purple-colored
compound is formed.

Iodine test:
The iodine test is a chemical test used for detecting the presence of starch.
The iodine test is a chemical test used to distinguish mono- or disaccharides from
certain polysaccharides like amylase, dextrin, and glycogen. This test has a variation
termed starch-iodine test that is performed to indicate the presence of glucose.
Carbohydrates, mainly starch is present in the sample could be treated by adding drops
into the sample of iodine. Iodine is initially yellowish-brown in color in presence of starch
turns into bluish-black in color.
Sudan IV test:
The Sudan IV test is a chemical test used for detecting lipids. The Sudan IV test
involves adding a few drops of Sudan IV to the test tube solution. Sudan IV is a dye that
will stain lipids. If no lipids are present, then the dye will sink to the bottom of the test
tube.
Materials:

1. 4 unknown samples
2. 4 test tubes
3. Iodine reagent (Lugol’s iodine)
4. Sudan IV dye reagent
5. Biuret reagent

Procedure:

In this lab, we work on identifying the macromolecule of the given sample.

1. First we have fours samples are present in four test tubes labeled A, B, C, D.
2. All test tubes have an unknown chemical.
3. We took a sample containing protein in test tube B.
4. We took a sample containing starches in test tube C.
5. We took a sample containing oil(fats) in test tube A.
6. We took a sample containing distilled water in test tube D.
7. We added 4-5 drops of biuret reagent in test tubes and D and mixed solutions
gently. We observed and note changes in color in both test tubes
8. We washed test tube D thoroughly with tap water and again filled it with some
distilled water.
9. Now we added 4-5 drops of Lugol’s iodine in the test tubes and D and mixed
solutions gently. we observed and note the change in color in both test tubes.
10. We washed test tube D thoroughly with tap water and again filled it with some
distilled water.
11. Now we added 4-5 drops of Sudan IV reagent in the test tubes and D and
mixed solutions gently. we observed and note the change in color in both test
tubes.
12. We washed test tube D thoroughly with tap water and again filled it with some
distilled water
 13. Note all the readings and changes in colors of the test.

Observation:

Serial no. Sample Biuret Iodine Sudan IV Deduction

test test test
1 A
2 B
3 C
4 D
Result:

From the above observation and tests, we performed the result are
 Sample A contains.
 Sample B contains
 Sample C contains.
 Sample D contains no macromolecules.
Discussion:

In this lab, we learned about proteins, lipids, and starches. We can identify a particular
macromolecule in a sample by using these tests. We understood the principle of the
Biuret test, iodine test, and Sudan IV test. We can easily distinguish between
polysaccharides and monosaccharides.

6 LAB EXPERIMENT