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
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Learning objectives
•
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3
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5
After interacting with this Learning Object, the learner
will be able to:
1.
Define buffer preparation and the mechanism involved
2.
Operate to optimize the pH of the buffer
3.
Assess the troubleshooting steps involved in the
experiments.
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Master Layout
Mechanism of buffer action (Slide: 5-10)
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3
Buffer Preparation (Slide: 11-12)
pH meter standarization (Slide: 13)
pH instrument working mechanism (Slide: 14-15)
T4: pH measurement (Slide: 16)
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5
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Definitions and Keywords
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
strong base
3) Basic buffer: solution mixture containing base and salt of the same
base with strong acid.
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4
5
4) pH meter: An instrument that measures the differences in ionic
strength between the sample and the standard and display it . Mostly
hydrogen ions are detected to calculate the pH
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Step 1:
T1:Mechanism of buffer action
2
Acidic
buffer
3
Hcl
1)CH3COONa (aq) ————-> CH3COO- (aq) + Na+ (aq)
2) CH3COOH (acetic acid)
B)After Hcl addition
4
CH3COO- (aq) + H+ (aq) ⇌ CH3COOH
Dissociated
sodium acetate
5
From Hcl
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Step 1:
T1: Mechanism of buffer action
Description of the action
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3
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5
Show a bottle labeled as “ acidic
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
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
towards the CH3COO- ion and
bind to it.
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
2
3
Step 2:
T1:Mechanism of buffer action
NaOH
Acidic buffer
1)CH3COONa (aq) ————-> CH3COO- (aq) + Na+ (aq)
2) CH3COOH (acetic acid)
B)After NaOH addition
4
5
1)CH3COOH (aq) ————-> CH3COO- (aq) + H+ (aq)
2) H+ + OH- --------------->H2O
From
acetic
acid
From NaOH
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Step 2:
T1:Mechanism of buffer action
Description of the action
2
3
4
5
Show a bottle labeled as “ acidic
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
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)
moving towards the H+ ion and
bind to it.
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.
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Step 2:
T1:Mechanism of buffer action
Calculating the pKa, [H+] and pH
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3
4
1) if we know the value for Ka , hydrogen ion concentration and pH of the buffer
can be calculated
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
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) To calculate ka value
Ka=[H+][A-]/[HA]
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Step 3:
T1: Mechanism of buffer action
Description of the action
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3
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5
Instruct the user to go through the
calculation and the formulas as in
the previous slide with the audio
narration accompanying it.
Animate like user performing the
calculation
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
methodology.
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Step 4:
T2: Buffer Preparation
Tween 20
2
Tris
base
Nacl
Kcl
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4
5
Description of the action
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
required amount with help of spatula on a paper over the balance.
Display a gradual increase in reading with quantity addition. if the
gram exceeds user should remove some quantity or if it less add to
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.
Audio Narration
Prepare
TBST buffer
can be used
as
distain
solution and
also can be
used
for
washing the
membrane.
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Step 5:
T2: Buffer Preparation
Tween 20
2
Tris
base
Nacl
Kcl
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4
5
Description of the action
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
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.
Audio Narration
The pH of the
TBST buffer need
to set to 7.6 .
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Step 6:
T3: pH meter standarization
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STD 1
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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.
Initially for the pH rod is dipped in water, when user
clicks on read button, display must show a reading “7”.
Now show like taking out the rod and washing it with
deionized-water, let user cleans the rod with tissue. Now
pick the STD 1 , uncap it, dip the cleaned rod into the
solution, user must click read button with display
showing “4”. now clean the rod and repeat the step to
note down the reading for STD 2 and now the display
should show “9”
STD 2
Audio Narration
Before the pH reading, pH
instrument need to be
calibrated with standards.
Once with STD 1 at pH 4
and with STD 2 at pH 9. the
pH meter senses the
hydrogen ion concentration
with the standard inside the
electrode and the differences
are displayed as pH
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Step :
T4:pH instrument mechanism working
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3
Glass
electrode
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5
Nernst equation
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Step :
T4:pH instrument mechanism working
Description of the action
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3
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5
Show the figures
one by one so that
the user can
understand the
principle of working
of pH electrode and
audio narration
should go
simultaneously.
Audio Narration
Glass electrode consists of internal reference
electrode (silver/silver chloride electrode) and the
glass bulb at the end which is sensitive to the
hydrogen ion concentration. This is actually a
galvanic cell Both sides of the bulb will be
protonated during the pH measurement which
causes the potential difference, this protonation
occurs till the equilibration is achieved. The
potential difference is governed by the nernst
equation and the difference the measure of pH .
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Step 7:
T4: pH measurement
2
NaOH
3
4
5
Description of the action
Instruct user to set the pH for TBST pH at 7.6. Now take the
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
allow the user to click on NaOH bottle so that drops of NaOH
should be added with filler, user need to mix the solution with
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.
HCl
Audio Narration
Prepare TBST
buffer of pH 7.6.
Slide 510
Tab 01
Slide
11-12
Tab 02
Slide 14-15
Slide 13
Tab 03
Tab 04
Slide 16
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
Instruction
Button 02
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