Mechanism of buffer action and buffer preparation

1
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
• gt Prior Viewing IDD-1. Extraction of
  bacterial protein, IDD-17. SDS-PAGE
• gt 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-ShareAl
ike 2.5 India license
2
Learning objectives
1

• After interacting with this Learning Object, the
  learner will be able to
• Define buffer preparation and the mechanism
  involved
• Operate to optimize the pH of the buffer
• Assess the troubleshooting steps involved in the
  experiments.

2
3
4
5
3
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
4
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.
• 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

2
3
4
5
5
Step 1
1
T1Mechanism of buffer action
2
Acidic buffer
Hcl
3
1)CH3COONa (aq) -gt CH3COO- (aq) Na
(aq) 2) CH3COOH (acetic acid) B)After Hcl
addition CH3COO- (aq) H (aq) ? CH3COOH
4
Dissociated sodium acetate
From Hcl
5
6
Step 1
T1 Mechanism of buffer action?
1
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.
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.
2
3
4
5
7
Step 2
1
T1Mechanism of buffer action
NaOH
Acidic buffer
2
3
1)CH3COONa (aq) -gt CH3COO- (aq) Na
(aq) 2) CH3COOH (acetic acid) B)After NaOH
addition 1)CH3COOH (aq) -gt CH3COO- (aq) H
(aq) 2) H OH- ---------------gtH2O
4
From acetic acid
From NaOH
5
8
Step 2
T1Mechanism of buffer action
1
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.
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.
2
3
4
5
9
Step 2
T1Mechanism of buffer action
1
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 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 H6.9 10-6 mol dm-3. 3) To
calculate pH, substitute the H value in the
equation belowpH -log10 H pH- log10 6.9
10-6 mol dm-3 \ pH 5.1 4) To calculate ka
value KaHA-/HA
2
3
4
5
10
Step 3
T1 Mechanism of buffer action
1
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
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.
2
3
4
5
11
Step 4
1
T2 Buffer Preparation
Tween 20
Tris base
Nacl
2
Kcl
Audio Narration
Description of the action
3
Prepare TBST buffer can be used as distain
solution and also can be used for washing the
membrane.
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.
4
5
12
Step 5
1
T2 Buffer Preparation
Tween 20
Tris base
Nacl
2
Kcl
Audio Narration
Description of the action
3
The pH of the TBST buffer need to set to 7.6 .
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.
4
5
13
Step 6
1
T3 pH meter standarization
2
STD 1
STD 2
3
Audio Narration
Description of the action
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
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

4
5
14
Step
1
T4pH instrument mechanism working
2
Glass electrode
3

4
Nernst equation
5
15
Step
1
T4pH instrument mechanism working
Audio Narration
Description of the action
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 .
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.
2
3
4
5
16
Step 7
1
T4 pH measurement
2
NaOH
3
HCl
Description of the action
Audio Narration
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.
Prepare TBST buffer of pH 7.6.
4
5
17
Slide -14-15
Slide -16
Slide 5-10
Slide 11-12
Slide -13
Tab 02
Tab 03
Tab 04
Tab 05
Tab 06
Tab 07
Tab 01
Name of the section/stage

• Animation area

Interactivity area
Slide 16 Show like the user added excess NaOH
and the pH reaching to 9 instead of
7.6 Instruction 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
18
Questionnaire
APPENDIX 1

• Question1
• What is a buffer?
• Solution that maintains ionic strength
• Solution that helps to store two different sample
  together
• Solution that maintains pH
• Solution that has acid/base
• Question 2
• Acidic buffer contains
• Salt of strong base and acid along with the acid
• Acid with strong base
• Salt of the acid with water
• Water and acid
• Question 3

19
Questionnaire
APPENDIX 1

• Question 4
• Formula to find pH of the buffer
• logH
• logH
• 1/H
• 1/H-
• Question 5
• pH is measured using
• a)Ammeter
• b)Voltmeter
• c)Litmus paper
• d) electrodes

20
APPENDIX 2
Links for further reading

• Reference websites
• http//www.chemguide.co.uk/physical/acidbaseeqia/b
  uffers.html

21
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