Lab 1 Report sheet

Calculations

1- What is the % (w/v) of 1.0 L solution that contains 5.0 g of NaCl?

2- Find the percent volume (v/v%) of ethanol in a solution prepared by diluting 30.0
mL of ethanol to 250 mL.

3- Carry out the following calculations:

a) How many moles of NaCl (MW = 58.5) are required to prepare 100 mL of a
1.6 M solution?

b) How many grams of NaCl are required, MW NaCl= 58.5 g/mole?

4. How many moles of HCl are present in 50 mL of 3.0 M HCl solution?

5. What are the normalities of (a) 0.213 M HCl, and (b) 0.010 M Ca(OH) ?
2

6. How would you prepare 240 mL of a 1.0% (w/v) solution of glucose from a 6.0% (w/v)
solution? How would you prepare 2.5 L of a 2% (w/v) glucose solution from 5% (w/v)
aqueous glucose?
 Experiment 2
Experimental applications on solution preparation and dilution.

In this laboratory you will be preparing and diluting solution as per given in the lab.
In all steps distilled water will be used as a diluent

Using the following materials, prepare the solution below

Materials

• NaCl
• KCl
• Red buffer
• 10% bromophenol blue stock (3mls)
• H2SO4
• Volumetric flasks (50 mL)
• Digital balance
• Graduated pipettes
• Pipette filler.
• Distilled water
• Test tubes
• Erlenmeyer flask
• Beaker

1- Prepare 4 mls of 0.078% of bromophenol blue, given that the stock

concentration is 10% bromophenol blue. Write down the procedure you
followed in your report sheet.

2- Prepare 50 ml of 20% w/v NaCl?

3- Prepare 50 ml of 136.8 mM NaCl using the stock solution (20 % w/v) you
prepared in the last step, knowing that the molecular weight of NaCl is
58.44 g/mole.

4- You have a 10X red buffer, prepare 10 ml of 1X buffer.

5- Prepare 50 ml of 150 mM KCl knowing that the molecular weight of KCl

is 74.5513?

6- Prepare 50 ml of 0.2 N H2SO4, from a stock of 2 M H2SO4 found in the

fume hood?
 Lab 2 Report sheet
Experimental applications on solution preparation and dilution
Student name:
Student ID: section:

A. Write down the calculation that you used to prepare the solutions (mention
volumes, weight, glassware used and any other steps involved (you can draw
schemes)?

1- Solution 1

2- Solution 2

3- Solution 3

4- Solution 4
 5- Solution 5

6- Solution 6

C. A solution contains 15 g of CaCl2 in a total volume of 200 mL. Express the

concentration of this solution in terms of (a) g per L, (b) % (w/v), (c) M? (MW of
CaCl2 = 110.986 g/mole)?
Practical work
A. Biuret test:

1. Place 15 drops of each sample (unknown) test solution

in a separate test tube.
2. add 5 drops of 20% NaOH and mix.
3. Add 2 drops of 0.2% CuSO4 solution, warm if necessary

B. Heat denaturation:
Take 5 ml of each sample (unknown) test solution in a separate test
tube, heat to boiling.
What do you observe?

C. Ninhydrin Test:
1. Take 2 ml of each sample solution in a separate test tube.
2. Add 0.5 ml of 0.1 % Ninhydrin reagent the test tubes.
3. Place the test tubes in the water bath for 5 minutes and then
allow cooling to room temperature.
4. Observe the formation of a deep blue/purple colour indicates
the presence of amino acids.
Result:
 blue-purple reaction
 yellow or yellow to red color
Interpretation
1. This reaction suggests that amino acids, other
amines, and ammonia are present in the test
material.
2. Negative: not a.a , not protein
3. Positive purple: aa or protein with free amino gp
4. Positive yellow: proline aa or hydroxyproline.
 D. Millon’s test

1. Take 2 ml of each sample solution or

unknown solution is taken in a seperate
test tube.
2. To this, about 2 ml of Millon’s reagent is
added. Observe the formation of red brick
precipitate
3. if red colored precipitate is not observed
immediately. the test tubes are then kept
in the water bath for about 2 minutes The
tubes are then observed for the formation
of the colored precipitate.

Positive result: A positive result in the Millon’s test is demonstrated by the formation of a
red brick or pink colored precipitate. This indicates the presence of tyrosine or tyrosine
containing protein.
Negative result: A negative result in the Millon’s test is demonstrated by the absence of
colored precipitate in the test tube. This indicates the absence of tyrosine or tyrosine-
containing protein.

E. Hopkins-Cole test

1. In two separate test tubes, take 2 ml of

Hopkins-Cole reagent and 2 ml of each
sample liquid are taken.
2. To this, concentrated H2SO4 is added along
the sides of the test tube held at a slanting
position. Two distinct layers of liquid are to
be formed without mixing. (Note: Mouth of
the tube must point away from the face, as
we should be careful while adding the
sulfuric acid.)
3. The test tube should be observed for the formation of a purple colored ring at
the interface of two layers.

Interpretation of result
Positive result: A positive result is represented by the formation of a purple-colored ring
at the junction of two layers. This indicates the presence of tryptophan-containing
proteins.
Negative result: A negative result is represented by the absence of a purple-colored ring
in the test tube. This indicates the absence of tryptophan-containing proteins.
 F. Lead sulfur test

1. In two separate test tubes, place 2 ml of sample solution

add 2 ml of 20% NaOH and boil for a minute.
2. Cool it and add a drop of 20 % lead acetate solution.
3. Observe the formation of black lead sulfide precipitate.
Note: Carry out this test in the hood if possible.
Positive test: Formation of black precipitate indicate the presence
of sulfur-containing amino acid.
Negative test: No formation of black precipitate indicates the
absence of sulfur-containing amino acid.

G. Nitroprusside test:

1. In two separate test tubes, place 2 ml of sample solution.

2. Add 0.5 mL nitroprusside solution and shake thoroughly.
3. Add 0.5 mL of 20% Sodium hydroxid .
4. Observe the color change.

Result interpretation
Positive test: Formation of red color indicate presence of cysteine.
Negative test: No formation of red color indicates absence of
cysteine.
Experimental procedure:

Materials

- Unknown sample
- Micropipette (P-1000, P-20)
- Tips
- 6 Eppendorf’s
- Cuvette
- Distilled water
- BSA standard 5 (125 µg/ml)
- BSA standard 4 (250 µg/ml)
- BSA standard 3 (500 µg/ml)
- BSA standard 2 (1000 µg/ml)
- BSA standard 1 (1500 µg/ml)
- Spectrophotometer

Method
1. Label each Eppendorf with the concentration of the standard/
sample on the cap or the lateral side. Then:
Add 20 µl of each standard to 1000 µl Bradford reagent in an
Eppendorf and mix by flipping upside down (do not shake
vigorously and cause bubbles, it will interfere with your
measurements).

Assay range (sample volume), 100 µg/ml-1,500 µg/ml (20 µL).

Ration of sample to Bradford reagent is 1:50
(20 µl sample: 1000 µl Bradford reagent)

Eppendorf Bradford reagent Standard/sample

1 1000 µl 20 µl standard 5 (125 µg/ml)
2 1000 µl 20 µl standard 4 (250 µg/ml)
3 1000 µl 20 µl standard 3 (500 µg/ml)
4 1000 µl 20 µl standard 2 (1000 µg/ml)
5 1000 µl 20 µl standard 1 (1500 µg/ml)
6 1000 µl 20 µl Unknown sample

2. Incubate for 5-10 minutes at room temperature.

3. Switch the instrument at least 5-10 minutes before use to allow it to stabilize.
4. Select the λ max.
5. Place the blank solution (or water) in the cuvette, so that the instrument is
zeroed.
- Make sure the clear faces of the cuvette are in the light path and that the
outside of the cuvette is dry and clean.
 - Handle cuvettes only by the top edge of the ribbed sides (finger print affects
the measurement). All solutions should be free of bubbles.

6. Place the sample (standard/unknown) in the allocated place in the

spectrophotometer.
7. Measure absorbance of standards and unknowns at 595nm, using water as a
blank (when measuring by spectrophotometer, use 800 µl of the
sample/standard or more in plastic cuvettes, using less volume will result in bad
absorbance numbers- sample cuvette must be half/two third full)
8. Measure absorbance of the unknowns at 595nm similar to standard procedure.
9. Record your measurements.

 For standard samples: you can use one plastic cuvette multiple times without
washing if you work from low to high concentration. Otherwise, you must use
a new cuvette each time or wash the cuvettes for reuse to get the blue out.

 If your unknown colour is more intense than the higher standard, you should
dilute it in water• It is advisable to make several different dilutions of your
unknown sample (1 in 2 or 1 in 4 dilution) so that it falls within the standard
range of the assay. When analysing the data, you have to multiply the
concentration you got after dilution with the dilution factor:
 concentration of the unknown= concentration after dilution x dilution factor.
 YOU DO NEED TO RUN STANDARDS EVERY TIME! The reagent’s
response will change even after one day.

Data analysis:
Draw a standard curve, and find the linear equation for your curve, R2 value, and
calculate your unknown concentration.

• Create a calibration/standard curve graph of bovine serum albumin (BSA) protein

concentration vs. absorbance using Bradford method.
1. Enter the recorded measurement you obtained in the lab as in the image below,
then click insert, choose scatter and click on the second option (scatter with
smooth line and marker).
2. Double click on the line generated and choose the +, choose trendline, and then
click on the linear option, then more options
3. On the right panel click on: Display equation on chart and display R-squared
value on chart. R2 value should ideally be above 0.9.
4. Edit y and x-axis and the plot title,
5. Print the form and submit it with your report. From the absorbances of the
unknowns, find the protein concentration using the linear equation generated.

2.

3.

1.

4.

5. 6.

7.
8.

9.
Unknown absorbance = 0.978
y = 0.0006x + 0.6258 , R² = 0.9767
0.978= 0.0006x + 0.6258
0.978-0.6258 = 0.0006x
X= 587 microgram/ml
sustained slow release of amino acids into the blood stream, sometimes lasting for several
hours. Often casein is available as hydrolyzed casein, whereby it is hydrolysed by a
protease such as trypsin. Hydrolysed forms are noted to taste bitter and such supplements
are often refused by infants and lab animals in favour of intact casein

Procedure:
Material:
1. 100 mL of Milk (10% w/v)
2. Acetic acid (CH3COOH) (10% v/v), dropper.
3. Ethanol (95%v/v) (flammable)
4. Diethyl ether (highly flammable)
5. Thermometer 100oC (ethanol or mercury thermometer).
6. Hot plate.
7. Graduated cylinder.
8. Beaker.
9. Volumetric flask.
10. funnel
11. Spatula.
12. Glass rod.
13. Muslin cloth
14. Filter paper
15. Watch glass.

Method:
1. Weigh an empty 250 ml beaker,then place 100 ml of milk and weigh the milk
2. warm the milk to 40°C on a hot plate.
3. Once temperature is reached, remove the beaker containing milk from the hot plate
and start adding acetic acid slowly (drop by drop) to the milk while stirring with a
stirring rod.
Note: never transfer the acetic acid all at once.
4. keep adding acetic acid drop by drop until milk clots starts to form and the casein
in precipitated (~2 ml CH3COOH). This point means that you have reached a pH
of 4.6-4.8
5. . Leave to stand for 5 min and filter/decant through muslin cloth (you may need to
wait for 10-15 minutes for the filtration to be completed).
6. Collect the casein precipitate and transfer into a beaker using spatula. And suspend
the precipitate in 50 ml mixture of equal volume of ethanol and ether (1:1), mix by
 glass rod so that the extra fat may come down to the solvent system. Note: Be very
careful not to spill the precipitate.
7. filter the precipitate again using filter paper. Wait until the solution is totally
filtered.
8. Once all the solvent has been removed from casein. Remove the powder and spread
out on a watch glass (or petri dish, weighing boat) until it is completely dry (usually
overnight).
Note: label your watch glass/weighing boat with your group name and section
number.
9. Weigh your sample and calculate the percent yield you obtained (Normal Range 3-
5 %)

𝑔𝑟𝑎𝑚 𝑐𝑎𝑠𝑖𝑒𝑛
%𝑐𝑎𝑠𝑒𝑖𝑛 = 𝑋 100
𝑔𝑟𝑎𝑚𝑠 𝑜𝑓 𝑚𝑖𝑙𝑘

5
%𝑐𝑎𝑠𝑒𝑖𝑛 = 𝑋 100
105

% casein= 4.7%
 Experiment 7
Xanthine oxidase with allopurinol-IC50 protocol
Introduction:

Xanthine oxidase (XO) is a form of xanthine oxidoreductase, a type of enzyme that generates
reactive oxygen species. These enzymes catalyse the oxidation of hypoxanthine to xanthine and
can further catalyse the oxidation of xanthine to uric acid. These enzymes play an important role
in the catabolism of purines in some species, including humans.

The following chemical reactions are catalyzed by xanthine oxidase:

hypoxanthine + H2O + O2 xanthine + H2O2

xanthine + H2O + O2 uric acid + H2O2

Xanthine oxidase (XO) serves as an important

biological source of oxygen-derived free radicals that
contribute to the oxidative damage of living tissues.
Free radical: are atoms or groups of atoms that have a
single unpaired electron. They are unstable molecule
that is made during normal cell metabolism. Free
radicals can build up in cells and cause damage to other
molecules, such as DNA, lipids, and proteins. This
damage may increase the risk of cancer and other
diseases.

Gout-‫النقرص‬

XO is involved in the medical condition known as gout, which is

characterized by hyperuricemia that leads to uric acid deposition
in the joints resulting in painful inflammation. Hyperuricemia,
which is present in 5–30% of the general population, seems to be
increasing worldwide and is considered an important risk factor
in serious disorders, e.g. renal failure.
Uricosuric drugs which increase the urinary excretion of uric acid, or XO inhibitors which block
the terminal step in uric acid biosynthesis, can lower the plasma uric acid concentration, and are
generally employed for the treatment of gout.

Allopurinol is an inhibitor of XO and used clinically in the treatment of gout as well as febuxostat
drug.

Assay procedure:

1. The xanthine oxidase activity will be assayed using xanthine as substrate and utilizing
a plate reader.
2. In a 96 well plate, add 50 μl of allopurinol (concentration as written in the table), 95
μl of 1X Phosphate buffer and 50 μl of xanthine substrate (0.6mM). (Final volume per
well is 200 μl) (prepare the blank and enzyme only wells as per the numbers in the
table below)

Well no. 1 2 3 4 5 6 7 8 9

Phosphate 150µl 145µl 95 µl 95 µl 95 µl 95 µl 95µl 95µl 95µl

Buffer
Xanthine 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl 50µl 50µl 50µl
substrate
(0.6mM)
Allopurinol ----- ----- 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl 50µl
40ng/ml 400ng/ml 2µg/ml 6µg/ml 40µg/ml 50µg/ml 100µg/ml
Xanthine ----- 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5µl 5µl
oxidase
enzyme

3. Preincubate for 10 min at 25°C

4. Start the reaction by the addition of 5 μl ml of XO enzyme (0.1U/ml in phosphate
buffer).
5. Incubate the reaction at 25°C for 30 min and measure the absorbance against
phosphate buffer as blank at 295 nm using Biotek plate reader.
 6. Select the xanthine oxidase protocol from the software and make sure the settings are
correct and validated (set the plate layout according to the wells used)

Calculation:
Calculate % inhibition for each concentration of allopurinol using endpoint absorbances.
Make sure to blank out your Absorbance reading-by subtracting the absorbance of blank.
(show your calculation):
𝐴1−𝐴 (𝑏𝑙𝑎𝑛𝑘)
 %𝑖𝑛� 𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 100 − [
𝐴˳−𝐴 (𝑏𝑙𝑎𝑛𝑘)
] ∗ 100

Where A1 is the activity of the enzyme in presence of plant extract, A˳ is the absorbance in
absence of the plant extract (activity of the enzyme alone).

For example; allopurinol (40 ng/ml  the final conc. in the well is 10 ng/ml as the inhibitor
is diluted by 1 in 4 in each well) and the % inhibition is calculated as follow
Blank Abs=0.18075, A˳=4.473 and A1=2.4159.

2.4159−0.18075
%𝑖𝑛� 𝑖𝑏𝑖𝑡𝑖𝑜𝑛 = 100 − [ ] ∗ 100
4.473 −0.18075

= 100 -52.07
= 47.92%
Draw graph using excel, by following the steps below:
How to determine the IC50?

IC50 is the half maximal inhibitory concentration

• For example: maximum inhibitory concentration was found to be 97% as in the table below:

• Then IC50 for this example will be the concentration which gives an inhibition value of 48.5%

Apply the value on the equation obtained

• y = 10.547ln(x) + 50.15

• 48.5= 10.547ln(x) + 50.15

• Ln(x)= 48.5−50.15
10.547

• Ln(x)= -0.15644
• X= 𝑒−0.15644

• X=0.855181 µg/ml is the IC50 in this

example.
How to find anti-ln
• On calculator: press shift then  press ln

• On excel: find the exponential function and open a

bracket then select the cell which has the number
and close the bracket again, press enter to obtain the
value.
 Experiment 7
Xanthine oxidase with allopurinol-IC50
Student name: Section no.:
1. Group no.:
2. report mark/10
3.
4.
5. ………./ 10
6.
7.

Objectives:

Results: Fill in the table below manually or using excel.

Well no. 1 2 3 4 5 6 7 8 9

Phosphate 150µl 145µl 95 µl 95 µl 95 µl 95 µl 95µl 95µl 95µl

Buffer
Xanthine 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl 50µl 50µl 50µl
substrate
(0.6mM)
Allopurinol ----- ----- 50 µl 50 µl 50 µl 50 µl 50 µl 50 µl 50µl
40ng/ml 400ng/ml 2µg/ml 6µg/ml 40µg/ml 50µg/ml 100µg/ml
Xanthine ----- 5 µl 5 µl 5 µl 5 µl 5 µl 5 µl 5µl 5µl
oxidase enzyme

Final ----- Zero

concentration
of allopurinol
per well
(µg/ml).
Absorbance (30 0.52 0.43 0.35 0.3 0.19 0.17 0.07
minutes)
0.02 0.49
%Inhibition

1. Calculate the final concentration of allopurinol per well (µg/ml).

Stock concentration

- 40ng/ml :
 - 400ng/ml

- 2µg/ml:

- 6µg/ml:

- 40µg/ml:

- 50µg/ml

- 100µg/ml

2. Calculate the %xanthine oxidase inhibition for each allopurinol concentration (show
detailed calculation).

Allopurinol conc. (1):

Allopurinol conc. (2)= % inhibition =

Allopurinol conc. (3)= % inhibition =

 Allopurinol conc. (4)= % inhibition =

Allopurinol conc. (5)= % inhibition =

Allopurinol conc. (6)= % inhibition =

Allopurinol conc. (7)= % inhibition =

3. Fill the table given above and plot the final concentration (i.e diluted) vs % inhibition and
determine the IC50 value (show detailed calculation of IC50).
(Print the figure with Y and X axis labelled and add it to the report)

4. From the figure shown below calculate the IC50 for this xanthine oxidase inhibitor?
Practical part:

1. Molisch’s test:
Procedure
1. In a test tube, add 2 ml of test solution ( unknown)

2. Add 2 drops of Molisch’s reagent and mix by gentle shaking

3. Tilt the tube and carefully pour 1 ml conc. H2SO4 along the side of
the tube (no mixing)
4. Violet ring at the junction of the two liquids indicates a positive
result.
Note: The appearance of purple or violet ring confirms the presence
of carbohydrate.

Interpretation: The formation at the junction of two layers of a purple or violet ring or zone
indicates the presence of carbohydrates.
Precautions:

(i) The solution of alpha-naphthol is unstable and should be made fresh.

(ii) Conc.H2SO4 should be carefully added along the sides of the test tube, causing the contents of
the tube to be minimally disturbed.

2. Iodine test:
1. To two ml of your test solution ( unknown), add
few drops of diluted HCl
2. Add 2 drops of iodine solution
3. A blue color indicates positive result
4. Heat the colored solution and observe the
disappearance of the color

Iodine test give A+e result ( blue color complex) with starch as a
polysaccharides.
 3. Benedect’s test
1. In a tube add 5 ml of benedict reagent
2. Add 8 drops of your test solution (unknown) and mix
3. Heat the tube in boiling water bath for 5 min
4. Positive result is indicated by the change in color or the formation of a precipitate
yellow to brick red color, depending upon the conc. of the glucose or reduced sugar
in the solution.

Note: The appearance of red precipitate confirms the presence of carbohydrates.

4. Barfoed’s test
1. Add 1 mL of the test solution (unknown) to a test tube.
2. Add 5 ml Barfoed’s reagents to test tube and mix
3. Place the test tubes in a water bath and observe the time when a precipitate
appears.

Thin red precipitates indicate the presence of a reduction of monosaccharide at the bottom or
sides of the tube.

Positive Barfoed’s test for monosaccharides: glucose, fructose.

Note:The boiling should not be prolonged beyond 1-2min, otherwise the disaccharide reduction
will respond to this test as well.
 5. Seliwanoff’s test
1. Add 1 mL of the test solution (unkown) to about 2 mL of
the Seliwanoff reagent
2. Heat the test tube in boiling water bath for 10-20 min
or just heat to boiling.
A positive reaction is indicated by a deep red color and formation of
brown precipitate.
(The presence of ketohexose indicates the appearance of a red colour
(fructose).