Mechanism of buffer action and

buffer preparation
Maintaining the optimum pH during the biological sample
processing is to maintain the proper functional and
structural aspects of the sample. It is important to
understand the theory behind the buffering action

Related LOs: Buffer composition

> Prior Viewing – IDD-1. Extraction of bacterial protein, IDD-17. SDS-PAGE

> Future Viewing – IDD-32. Buffer preparation for western analysis

 Course Name: Mechanism of buffer action and buffer preparation

 Level(UG/PG): UG

 Author(s): Dinesh Raghu, Vinayak Pachapur

 Mentor: Dr.Sanjeeva Srivastava

*The contents in this ppt are licensed under Creative Commons Attribution-NonCommercial-ShareAlike 2.5 India license
 Learning objectives
1
• After interacting with this Learning Object, the learner
will be able to:
2
1. Define buffer preparation and the mechanism involved
2. Operate to optimize the pH of the buffer
3 3. Assess the troubleshooting steps involved in the
experiments.

5
 Master Layout
1 Mechanism of buffer action (Slide: 5-10)

2 Buffer Preparation (Slide: 11-12)

pH meter standarization (Slide: 13)

3
pH instrument working mechanism (Slide: 14-15)

T4: pH measurement (Slide: 16)

4

5
 Definitions and Keywords
1 1) Buffer : A aqueous solution mixture made in weak base with conjugate
acid or weak acid with conjugate base.

2) Acid buffer: solution mixture containing acid and salt of same acid with
2 strong base

3) Basic buffer: solution mixture containing base and salt of the same
base with strong acid.

4) pH meter: An instrument that measures the differences in ionic

3 strength between the sample and the standard and display it . Mostly
hydrogen ions are detected to calculate the pH

5
1 Step 1: T1:Mechanism of buffer action

2
Acidic
buffer Hcl

3 1)CH3COONa (aq) ————-> CH3COO- (aq) + Na+ (aq)

2) CH3COOH (acetic acid)

B)After Hcl addition

4 CH3COO- (aq) + H+ (aq) ⇌ CH3COOH

Dissociated
sodium acetate From Hcl
5
1 Step 1: T1: Mechanism of buffer action
Description of the action
Show a bottle labeled as “ acidic
2 buffer”, instruct user to click on the
bottle and show a tab as shown in
figure (previous slide).
Now show a bottle labeled as Hcl
3
and the user should click on the
bottle open the lid and take a filler
and add drops of the Hcl to the
acidic buffer bottle . Then the user
should see the reactions as in the
figure simultaneously while adding
the HCl to the solution. Show like
H+ ion in reaction (B) moving
4 towards the CH3COO- ion and
bind to it.
5
Audio Narration
Acidic buffer containing weak acid
with the salt of the weak acid and
strong base for eg sodium acetate
and acetic acid. Sodium acetate
will be in the dissociated form as
sodium ions and acetate ions. ,
acetic acid will be in non
dissociated form, when an strong
acid like Hcl was added the H+
ions from the acid will react with
the dissociated form acetate ion
and form the acetic acid thereby
constantly maintaining the pH of
the buffer.
1 Step 2: T1:Mechanism of buffer action

2 Acidic buffer
NaOH

3 1)CH3COONa (aq) ————-> CH3COO- (aq) + Na+ (aq)

2) CH3COOH (acetic acid)

B)After NaOH addition

4 1)CH3COOH (aq) ————-> CH3COO- (aq) + H+ (aq)

2) H+ + OH- --------------->H2O

From
acetic From NaOH
5 acid
1 Step 2: T1:Mechanism of buffer action
Description of the action
Show a bottle labeled as “ acidic
2 buffer”, let the user to click on the
bottle and show a tab as shown in
figure (previous slide).
Now show a bottle labeled as
3
NaOH and the user should click on
the bottle open the lid and take a
filler and add drops of the NaOH to
the acidic buffer bottle. Then the
user should see the reactions as in
the figure simultaneously while
adding the NaOH to the solution.
Show like OH- ion in reaction (B)
4 moving towards the H+ ion and
bind to it.
5
Audio Narration
Acidic buffer containing weak acid
with the salt of the weak acid and
strong base for eg sodium acetate
and acetic acid. Sodium acetate
will be in the dissociated form as
sodium ions and acetate ions.
acetic acid will be in non
dissociated form, when an strong
acid like NaOH was added the H+
ions from the acetic acid
dissociate and will react with the
hydoxide ion from the sodium
hydroxide and form water, thereby
constantly maintaining the pH of
the buffer.
1 Step 2: T1:Mechanism of buffer action

Calculating the pKa, [H+] and pH

1) if we know the value for Ka , hydrogen ion concentration and pH of the buffer
can be calculated
2 Ka for acetic acid is 1.74 x 10-5 mol dm-3.
2) to calculate the pH of a buffer solution containing 0.20 mol dm-3 of acetic
acid and 0.50 mol dm-3 of sodium acetate.
Substitute in the equation as follows

3 Ka= [CH3COO-][H+]/[CH3COOH]
1.74 x 10-5 =0.50 *[H+]/0.20
[H+]=6.9 * 10-6 mol dm-3.
3) To calculate pH, substitute the [H+] value in the equation below
pH = -log10 [H+]
pH=-
\
log10 6.9 * 10-6 mol dm-3
pH = 5.1
4
4) To calculate ka value

Ka=[H+][A-]/[HA]

5
1 Step 3: T1: Mechanism of buffer action
Description of the action
Instruct the user to go through the
2 calculation and the formulas as in
the previous slide with the audio
narration accompanying it.
Animate like user performing the
3
calculation
4 methodology.
5
Audio Narration
The ka value can be found by
the formula 4 and for the
acetate buffer the Ka is already
known, substitute the values
like concentration of sodium
acetate, acetic acid, ka to find
the pH, go through the
calculation thoroughly along
with the mechanism of buffer
action to understand the
importance of pH calculation
and constant maintenance of
the pH. Following example will
illustrate the buffer preparation
1 Step 4: T2: Buffer Preparation

Tween 20

2 Nacl
Tris
base
Kcl

Audio Narration
Description of the action

3 Pop-up note “ Preapare TBST buffer”. Let user takes out Nacl, Kcl,
Tris Base, tween 20 from the rack and keep it next to balance.
Instruct user to weigh 8g of Nacl, 0.2g of Kcl, 3g of tris base and
600ul of tween 20. let user pick the bottle, uncap it, weigh the
Prepare
TBST buffer
can be used
as distain
solution and
required amount with help of spatula on a paper over the balance. also can be
Display a gradual increase in reading with quantity addition. if the used for
washing the
4 gram exceeds user should remove some quantity or if it less add to membrane.
get the required amount. After weighing transfer the quantity to
beaker. Now take out 1000ul pipette, set it for 600ul, take out
tween20 bottle, uncap it, pipette and transfer 600ul into the beaker.
All events must happen when the user clicks on the hand.
5
1 Step 5: T2: Buffer Preparation

Tween 20

2 Nacl
Tris
base
Kcl

Audio Narration
Description of the action

3 Now instruct the user to take methanol bottle, open the cap,
take 1000ml measuring cylinder, measure 900ml. Let user
remove the excess methanol if level crosses 900ml mark.
Transfer it to beaker. Now take the beaker, shake it to make
The pH of the
TBST buffer need
to set to 7.6 .

a proper mix as shown in slide 11. Animate the powder

getting into the solution. Now set the pH to 7.6 by using pH
meter.
4

5
1 Step 6: T3: pH meter standarization

2
STD 1
STD 2

3 Description of the action

Display standard pH bottles and pH instrument and
deionized water, discard placed on a table. Instruct user
to caliberate the instrument. Let user ON the instrument.
Audio Narration
Before the pH reading, pH
instrument need to be
calibrated with standards.
Initially for the pH rod is dipped in water, when user Once with STD 1 at pH 4
clicks on read button, display must show a reading “7”. and with STD 2 at pH 9. the
Now show like taking out the rod and washing it with
4 deionized-water, let user cleans the rod with tissue. Now pH meter senses the
pick the STD 1 , uncap it, dip the cleaned rod into the hydrogen ion concentration
solution, user must click read button with display with the standard inside the
showing “4”. now clean the rod and repeat the step to electrode and the differences
note down the reading for STD 2 and now the display are displayed as pH
should show “9”
5
1 Step : T4:pH instrument mechanism working

2
Glass
electrode

3
4

Nernst equation
5
1 Step : T4:pH instrument mechanism working

Description of the action Audio Narration

Show the figures Glass electrode consists of internal reference

one by one so that electrode (silver/silver chloride electrode) and the
the user can glass bulb at the end which is sensitive to the
2 understand the hydrogen ion concentration. This is actually a
principle of working galvanic cell Both sides of the bulb will be
of pH electrode and protonated during the pH measurement which
audio narration causes the potential difference, this protonation
should go occurs till the equilibration is achieved. The

3 simultaneously. potential difference is governed by the nernst

equation and the difference the measure of pH .

5
1 Step 7: T4: pH measurement

2
NaOH

3
HCl
Description of the action
Instruct user to set the pH for TBST pH at 7.6. Now take the Audio Narration
TBST bottle, uncap it, dip the cleaned pH rod into the solution.
User need to click on read button. Initially display must show a
reading 6. now instruct user to add NaOH to adjust the pH. Now Prepare TBST
allow the user to click on NaOH bottle so that drops of NaOH buffer of pH 7.6.
should be added with filler, user need to mix the solution with
4 glass rod, click on read button and the reading should anywhere
near 6.1- 6.3. let user keeps adding the NaOH drop till the pH
display shows 7.6 and later transfer the beaker solution to
1000ml measuring cylinder to makeup the volume to 1000ml by
clicking on methanol and adding it to that. All action should
happen when the user clicks the hand.
5
 Slide 5- Slide Slide - Slide - Slide -
10 11-12 13 14-15 16
Tab 01 Tab 02 Tab 03 Tab 04 Tab 05 Tab 06 Tab 07

Name of the section/stage

Interactivity
area
Animation area
Slide 16:
Button 01
Show like the user added excess NaOH and the pH reaching to 9 instead of 7.6
Button 02

Instruction Button 03

Instruct the user to take Hcl and add to the solution show like the pH decreasing
in 8.6,8.4,8.1, 7.8 and 7.6

Instructions/ Working area

Credits
APPENDIX 1
Questionnaire:

Question1:

What is a buffer?
a) Solution that maintains ionic strength
b) Solution that helps to store two different sample together
c) Solution that maintains pH
d) Solution that has acid/base

Question 2:

Acidic buffer contains

a) Salt of strong base and acid along with the acid
b) Acid with strong base
c) Salt of the acid with water
d) Water and acid

Question 3:

Acetate buffer has

a) Sodium acetate and acetic acid
b) Acetic acid and water
c) Water and sodium acetate
d) Sodium acetate and sample
APPENDIX 1
Questionnaire:
Question 4:

Formula to find pH of the buffer

a) –log[H+]
b) log[H+]
c) 1/[H+]
d) 1/[H-]

Question 5:
pH is measured using

a)Ammeter
b)Voltmeter
c)Litmus paper
d) electrodes
APPENDIX 2

Links for further reading

 Reference websites:
http://www.chemguide.co.uk/physical/acidbaseeq
ia/buffers.html
APPENDIX 3

Summary
Buffers need to be prepared with required amount of reagents that can maintain the
ionic strength of the solution. pH is important for a buffer preparation. All the
reagents prepared is to be properly labeled, prepared fresh for the analysis purpose