General Instruction:

1. You must be familiar with the background of the experiment before you attend
laboratory.
2. Each practical session is guided by Demonstrators and they will be
responsible for supervising your work and correcting your record book.
3. If you encounter difficulties during the lab, please feel free to consult any
available demonstrators.
4. If you unable to attend the practical session due to sickness, you must submit
a medical certificate certified by the University Medial Officer. It should be
submitted to the Head of department with an excuse letter.
5. Attendance is 90 % compulsory for practical session to sit for the end of
course examination.

6. Record books should be submitted to you demonstrator in charge within two

days of time from the practical session. Recording of the practical will cover
the followings sections.

Date: - Mention the date of the practical.

Practical no: - Mention the number of the practical.
Title: - Give the topic/ heading of the practical.

Aim: - Give the aim behind the practical.

Theory/ Principle: - Write the theory of the practical.
Required materials: - Give the aim of the practical
Procedure: - This section is a concise summary of what you did, and it should
be in passive form.
Observation: - Mention your observations; for example, colours changes of
solutions, the evolution of heat or gas etc.
Reading/ Results: - All the results should be listed. Tables are an excellent
way of showing many results in a small space.
Calculation: - Expect to complete calculation using data obtain from your
experiment.
Conclusion: - A brief statement of what you have shown in the laboratory.
Discussion: - Be logical and concise (argue your case) answers related to the
experiment.
Apparatus
Equipment will be supplied during the practical session. If you break any apparatus,
you must inform to the technical officer.
a) General equipment

Red litmus paper Blue litmus paper

Mortar and pestle Evaporating dish

Filter paper
Spatula

Stopper
Pipette filler

Brushes
Funnels

Test tube holder Test tube track

Glass rod Thermometer

Watch glass Glass droppers

b) Glass equipment

Conical flask
Beaker

Boiling tube Test tube

Pipette Burette

Volumetric flasks Measuring cylinder

c) Hardware
Bunsen burner Microscope

Hot plate Magnetic stirrer

Water bath Oven

 Clamp Burette holder/ Stand

Filtering ring Boss head

d) Safety equipment

Lab coats
Lab glasses

Gloves Mask
 DEPRTMENT OF HUMAN BIOLOGICAL SCIENCES
FACULTY OF ALLIED HEALTH SCIENCES, UNIVERSITY OF JAFFNA
EXPERIMENT NO: 1

pH AND BUFFERS
1. THEORY
The pH of a solution is negative logarithm of hydrogen ion concentration (or hydronium ion
concentration)
pH = - log H3O+
The pH of a solution can be determined by following methods:
 by using pH indicator papers (Red Litmus, Blue Litmus)
 by using pH meter and
 by using indictors and buffers or indicators and Lovibond comparator

The pH papers are impregnated with organic dyes whose colour is dependent on pH. Wide range
pH paper permit the estimation of pH within 1 pH unit and narrow range pH paper bring the pH
estimation within ±0.2 unit.
Indicators are weak acids. Their ionized forms have different colours. The actual colour in a
solution therefore depends on the solution containing the indicator.
Table: Some useful indicators.
Colour
Indicators pH range
Acid Alkali
Thymol Blue (acid range) 1.2-2.8 Red Yellow
Methyl Orange 3.1-4.4 Red Orange Yellow
Bromophenol Blue 3.0-4.6 Yellow Purple
Congo Red 3.0-5.0 Violet Red Orange
Bromocresol Green 3.6-5.2 Yellow Blue
Methyl Red 4.3-6.1 Red Yellow
Bromoceresol purple 5.0-6.6 Yellow Purple
Litmus 5.0-8.0 Red Blue
Bromothymol Blue 6.0-7.6 Yellow Blue
Phenol Red 6.7-8.3 Yellow Red
Cresol Red 7.2-8.8 Yellow Violet
Thymol blue (alkaline range) 6.0-9.6 Yellow Blue
Phenolphthalein 8.2-10.0 Colourless Pink
 Figure: pH indicator paper

2. pH METER pH indicator paper

The pH meter is used to accurately measure the pH of a solution. The instrument consists of
a glass electrode, a reference with a salt bridge and potentiometer. The glass and the reference
electrodes can be combined together as one electrode system as in figure below. This is called
combined electrode and it is found in most of the recent pH meters.

Figure: Combined electrode.

The instrument has been designed to indicate the hydrogen ion concentration as pH values on
a pH scale of the pH meter. The values range from 1 to 14.
2.1 Standardization of the Instrument

The instrument has to be standardized first before making any pH measurement. This is usually
done by using three buffer solutions of known pH values as follows (standardization method
differ with pH meter).

1. Clean the electrode with a jet of distilled water from a wash bottle and dip the electrode
in a beaker of distilled water.
2. Turn the function swish to pH position and allow the instrument to warm up for 10 min.
3. Select three buffers. The first with a pH of 7.0. Select a second and third buffers with
the pH of 4.0 and 9.0, respectively. (buffer number and type depending on pH meter)
4. Press mode until the pH mode indicator is displayed.
5. Rinse electrode(s) and place into buffer.
6. Press F2 then cal to begin calibration. The date and time of the last calibration will be
displayed.
7. When ready (when stable reading is observerd) is displayed next to the reading, indicating
electrode stability, the reading will flash. Press yes. For manual calibration use ▲ or ▼ to
select the correct value, then press yes to accept each digit. After accepting each digit, press
yes to store buffer value. The meter display freezes for three seconds. The meter
automatically switches to buffer two, indicated by the P2 on the display.
8. Repeat steps 5 through 7 for each buffer.
9. After entering the final buffer value, press measure. The electrode slope will be displayed.
SLP appears in the lower field while the actual electrode slope, in percent, appears in the
main field. After the third buffer point, the meter automatically displays the calibration
slope and advances to the measure mode. Measure is displayed above the main field.
10. Rinse electrode(s) and place into sample. Record pH directly from the main meter display.
Temperature is displayed in the lower field when the ready prompt displays.
11. Remove electrode from the second buffer solution, rinse with distilled water, and place in
a beaker of distilled water.
2.2 Measurement of pH
1. Dip the electrode in a beaker containing the sample of unknown pH. Make sure that the
bulb is completely immersed in the solution.
2. Read the pH value on the pH scale.
3. Rinse the electrodes carefully and replace in distilled water.

As test solution use the followings:

a) N/1000 NaOH
b) 0.001M Acetic acid

2.3 Points to care

a) Electrodes should always be stored in distilled water to prevent drying out. This may cause
irreparable damage to the electrode.
b) Care should be taken in handling the electrodes while rinsing and changing solutions as the
bulb of the electrode is fragile.
c) Please ensure that the bulb and stem of the electrode in solution are rinsed thoroughly
between each reading in order to prevent carryover of chemicals and ions.
d) Avoid bringing the electrode into contact with organic solvents.

3. PREPARATION OF A BUFFER SOLUTION

You are provided with 0.2 M solutions of sodium dihydrogen phosphate and disodium
hydrogen phosphate. Using the above solutions prepare 20 ml of 0.1 M phosphate buffer
solution at pH 7.0. Calculate the ratio of conjugate base to acid using the Henderson-
Hasselbalch equation. The pKa for this buffer system is 6.8.
Measure the pH of your buffer solution with a pH meter. If the measured pH of your buffer
deviates from pH 7.0 by more than 0.1 unit, add drop wise either 0.1 M HC1 or 0.1 M NaOH
(as required) bring your buffer to pH 7.0.
Procedure
 Take 20ml of buffer solution into the beaker by using measuring cylinder.
 Take four test tubes and add 5ml buffer soilution into each tubes and label it. (as 1 - 4)
 Add 1ml and 2ml of 0.1M HCl into each test tubes (1 & 2)
 Add 1ml and 2ml of 0.1M NaOH into each test tubes (3 & 4)
 Take two test tubes and add 5ml distilled water into each tubes and label it. (as 5 & 6)
 Add 1ml 0.1M HCl and 1ml of 0.1M NaOH into each test tubes (5 & 6)
 Mix and measure the pH of each mixture.
 Note the pH value and pH changes of solutions

Record your results.

4. EXERCISE
1. Calculate the pH of 0.1M solution of sodium acetate at 250C. The dissociation constant of
acetic acid is 1.8×10-5 and the ionic product of water is 10-14 at this temperature.
2. The concentration of H2CO3 in blood plasma is about 0.00125M using Henderson-
Hasselbalch equation, calculate the concentration of HCO3- in the plasma when the pH is
7.4 and also when it is 7.1 H2CO3 in blood has a pKa value of 6.1.