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FACULTY OF APPLIED SCIENCE

BACHELOR IN SCIENCE (HONS) CHEMISTRY (FORENSIC

ANALYSIS)

CHM510 - ANALYTICAL SEPARATION METHODS

Lab Title : Dilution and Molarity

Name : Nik NurFatin Nasuha Bt SaifulAzli
Student ID: 2019619318
Group : AS2534B1
Lecturer : Dr. Wan Rozianoor Bt Mohd Hassan
Experiment 1 - Dilution and Molarity
10/10

i. How would you prepare:

A. 10 mL of a 1:10 dilution of a 1 M of NaCl solution and what would the final

concentration of NaCl?
Dilution factor = 1:10
1
x 1M = 0.1 M
10

M1V1 = M2V2
(1 M) V1 = (0.1 M)(10 mL)
V1 = 1 mL
-> Mix 1 mL of NaCl with 9 mL of water. The final concentration of NaCl will be 0.1 M.

B. 80 mL of a 1:20 dilution of a 1 M NaCl solution?

Dilution factor = 1:20
1
x 1M = 0.05 M
20

M1V1 = M2V2
(1 M) V1 = (0.005 M)(80 mL)
V1 = 4 mL
-> Mix 4 mL of NaCl with 76 mL of water. The final concentration of NaCl will be 0.05 M.

C. 50 mL of a 1:25 dilution of a 1 M NaCl solution?

Dilution factor = 1:25
1
x 1M = 0.04 M
25

M1V1 = M2V2
(1 M) V1 = (0.04 M)(50 mL)
V1 = 2 mL
-> Mix 2 mL of a NaCl with 48 mL of water. The final concentration of NaCl will be 0.04
M.
ii. How would you prepare exactly 6 mL of a 1/20 dilution (assume the concentration of
your starting solution is 1)
Dilution factor = 1:25
1
x 1M = 0.05 M
20

M1V1 = M2V2
(1 M) V1 = (0.05 M)(6 mL)
V1 = 0.3 mL
- > Mix 0.3 mL of 1 concentration of solution with 5.7 mL of water to make 6 mL of 0.05 M
of starting solution.

iii. You are provided with an antibody (Ab) that has a concentration of 600 μg/μL. For
lab, it is necessary to make the following dilution:
A. 10 µL of 600 µg/L Ab + 190 µL of buffer to make a 1:20 dilution at 30 µg/µL.
1
x 600 µg/L = 30 µg/µL
20

B. 20 µL of 1:20 Ab + 40 µL of buffer to make a 1:60 dilution at 10 µg/µL.

M1V1 = M2V2
1
(20 × 600) (20 mL) = M2 (60 mL)

M2 = 10 µg/µL

C. 5 µL of 1:60 Ab + 5 µL of buffer to make a 1:120 dilution at 5 µg/µL.

M1V1 = M2V2
1
(60 × 600)(5 mL) = M2 (10 mL)

M2 = 5 µg/µL
5 µg/µL : 600 µg/µL
1 : 120

D. 10 µL of 1:60 Ab + 90 µL of buffer to make a 1:600 dilution at 1 µg/µL.

M1V1 = M2V2
1
(60 × 600) (10 mL) = M2 (100 mL)
 M2 = 1 µg/µL
1 µg/µL : 600 µg/µL
1 : 600

E. 10 µL of 1:60 Ab + 40 µL of buffer to make a 1:300 dilution at a 2 µg/µL.

M1V1 = M2V2
1
(60 × 600) (10 mL) = M2 (50 mL)

M2 = 2 µg/µL
2 µg/µL : 600 µg/µL
1 : 300

F. 10 µL of 1:60 Ab + 10 µL of buffer to make a 1:120 dilution at a 5 µm/µL.

M1V1 = M2V2
1
(60 × 600) (10 mL) = M2 (20 mL)

M2 = 5 µg/µL
5 µg/µL : 600 µg/µL
1 : 120

iv. How much 2.0 M NaCl solution would you need to make 250 mL of 0.15 M NaCl
solution?
M1V1 = M2V2
(2 M)V1 = (0.15 M)(250 mL)
V1 = 18.75 mL

v. What would be the concentration of a solution made by diluting 45.0 mL of 4.2 M

KOH to 250 mL.
M1V1 = M2V2
(4.2 M)(45.0 mL) = M2(250 mL)
M2 = 0.756 M
vi. What would be the concentration of a solution made by adding 250 mL of water to
45.0 mL of 4.2 M KOH?
M1V1 = M2V2
(4.2 M)(45 mL) = M2 (250 mL + 45 mL)
M2 = 0.64 M

vii. How much 0.20 M glucose solution can be made from 50.0 mL of 0.50 M glucose
solution?
M1V1 = M2V2
(0.50 M)(50 mL) = (0.20 M) V2
V2 = 125 mL

viii. What is the molarity of solution that has 4.5 mol of solute dissolved in 300 mL of
solution?
Moles of solute
Molarity = Litres of solution
4.5 mol
Molarity = 0.3 L

= 15 M

ix. What is the molarity of a solution of NaOH that has 0.491 g dissolved in 400 mL of
solution?
Mass
No. of moles = Molar mass
0.491 g
= 39.997 g/mol

= 0.01228 mol
0.01228 mol
Molarity = 0.4 L

= 0.0307 M

x. What is the molarity of a solution prepared by diluting 10.00 mL of a 4.281 M of

solution to 50.00 mL?
M1V1 = M2V2
(4.281 M)(10 mL) = M2 (50 mL)
M2 = 0.8562 M
 FACULTY OF APPLIED SCIENCE

BACHELOR IN SCIENCE (HONS) CHEMISTRY (FORENSIC

ANALYSIS)

CHM510 - ANALYTICAL SEPARATION METHODS

Lab Title : Protein Determination

Name : Nik NurFatin Nasuha Bt SaifulAzli
Student ID: 2019619318
Group : AS2534B1
Lecturer : Dr. Wan Rozianoor Bt Mohd Hassan
Experiment 2: Protein Determination 9.5/10

1. Name the dye used in Bradford Assay (1 mark).

= Coomassie brilliant blue dye

2. State the colour change that occurs when proteins combine with the dye reagent (1
mark).
= The colour turns blue from brownish.
0.5

3. In Lowry protein assay, name the bond in protein that binds to copper ions (1 mark).
= Peptide bond

4. A Bradford assay was conducted to determine the total protein concentration in a

sample. A volume of 2 μL of the original sample was diluted to 100 μL with buffer
before performing the assay. The diluted sample gave an absorbance at 595 nm of
0.255. Using the data for the standards below,

Protein A595
concentration
(μg/mL)
25 0.008
125 0.087
250 0.113
500 0.197
750 0.295
1000 0.429
1500 0.608

a) Plot a standard curve based on the data given (3 marks).

 Y-Values
0.7

0.6 y = 0.0004x + 0.0117

Absorbance (595 nm)

0.5

0.4

0.3

0.2

0.1

0
0 200 400 600 800 1000 1200 1400 1600
Protein Concentration (μg/mL)

b) Determine the concentration of protein in the diluted sample (2 marks).

Y = mx + c
(0.255) = 0.0004x + 0.0117
x = 608.25 μg/mL

c) Calculate the total protein concentration in the original sample (2 marks).

= 50 x 608.25 μg/mL
= 30412.5 μg/mL
 FACULTY OF APPLIED SCIENCE

BACHELOR IN SCIENCE (HONS) CHEMISTRY (FORENSIC

ANALYSIS)

CHM510 - ANALYTICAL SEPARATION METHODS

Lab Title : Determination of Enzymes Activity

Name : Nik NurFatin Nasuha Bt SaifulAzli
Student ID: 2019619318
Group : AS2534B1
Lecturer : Dr. Wan Rozianoor Bt Mohd Hassan
Experiment 3: Determination of Enzymes Activity
9.5/10

1. Substitute the solution in the video into the equation below. (1 mark)
Protease + Casein Protease-casein complex Tyrosine

2. State the function of the Folin & Ciocalteau’s in the video. (1 marks)
= A solution that will react with the hydrozine to generate a measurable colour change
that will be directly related to the reactivity of protease asay.

3. In an enzymatic reaction, 1.0 mL of acethylcholinesterase was reacted with 2.0 mL

of acethylthiocholine iodide. 2.0mL of phosphate buffer was then been added in.
Incubation for 10 minutes took place before 1.0 mL of acetic acid and 1.0 mL of
dithiobisnitrobenzoic acid was added up to the mixture. The mixture was further
incubated for 10 minutes and 1.0 ml of the sample was placed in cuvette for absorbance
reading at 405 nm.

Using the data for the product standards below,

405 nm Choline
(μmol)
0.00 0
0.067 0.05
0.113 0.1
0.197 0.2
0.395 0.4
0.729 0.8

a) Plot a standard curve based on the data given. (3 marks)

 Absorbance 405 nm VS Choline (μmol)
0.8
y = 0.9014x + 0.0173
0.7

0.6
Absorbance 405 nm

0.5

0.4

0.3

0.2
2.5
0.1

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
Choline (μmol)

b) Determine the activity of the enzyme if the absorbance of enzymatic activity gave a
reading of 0.552 (3 marks).
Y = mx + c
0.552 = 0.9014x + 0.0173
x = 0.593 μmol

0.593 μmol x 7 mL
=
10 min x 1 mL x 1 mL

= 0.4151 μmol min-1 mL-1

c) Calculate the specific enzyme activity if the total protein content is 10 mg/mL and
the enzyme has been diluted 5X before enzymatic reaction takes place.(2 marks)
0.4151 𝑥 5
=
10 mg/mL

= 0.2076 μmol min-1

 FACULTY OF APPLIED SCIENCE

BACHELOR IN SCIENCE (HONS) CHEMISTRY (FORENSIC

ANALYSIS)

CHM510 - ANALYTICAL SEPARATION METHODS

Lab Title : Gel Electrophoresis

Name : Nik NurFatin Nasuha Bt SaifulAzli
Student ID: 2019619318
Group : AS2534B1
Lecturer : Dr. Wan Rozianoor Bt Mohd Hassan
Experiment 4: Gel Electrophoresis
10/10

1. Define gel electrophoresis. (2 marks)

= Gel electrophoresis is a technique used to separate biological molecules by size. The

separation of these molecules is achieved by placing them in a gel with small pores and
creating an electric field across the gel. The molecules will move faster or slower based on
their size and electric charge.

2. Describe how 1 % agarose gel was prepared. (3 marks)

= Weight 1.0 g of agarose and put it into an Erlenmeyer flask.. Mix agarose powder with
TBE (boric acid) buffer and wash out the weighing boat with some buffer to ensure that no
agarose left in the weighing boat. Microwave the agarose mixture for 1-3 minutes until the
agarose is completely dissolved. Let the agarose solution to cool down to 50℃ for 10
minutes. Put the agarose in 64 microliter of loading dye. Pour the agarose gel into the tank.

3. State the number of wells in the agarose gel as shown in the first video. (1 mark)

= 3 wells

4. State the function of ethidium bromide. (1 mark)

= Ethidium bromide is used to visualize DNA in agarose gel electrophoresis experiment.

5. State the role of DNA ladder. (1 mark)

= DNA ladder is used as a reference to determine the size of unknown DNA molecules that
were separated based on their mobility in an electrical field through the gel.

6. Identify which part of DNA molecule contain negative charge. (1 mark)

= Phosphate backbone
7. Explain the function of restriction enzyme. (1 mark)

= Restriction enzyme is used to ensure that DNA can be specifically cut into fragments and
then can be separated by fragment size on the agarose gel.