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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
Level(UG/PG): UG
*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)
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.
5
1 Step 1: T1:Mechanism of buffer action
2
Acidic
buffer Hcl
Dissociated
sodium acetate From Hcl
5
1 Step 1: T1: Mechanism of buffer action
3
and the user should click on the acetic acid will be in non
bottle open the lid and take a filler dissociated form, when an strong
and add drops of the Hcl to the acid like Hcl was added the H+
acidic buffer bottle . Then the user ions from the acid will react with
should see the reactions as in the the dissociated form acetate ion
figure simultaneously while adding and form the acetic acid thereby
the HCl to the solution. Show like constantly maintaining the pH of
H+ ion in reaction (B) moving the buffer.
4 towards the CH3COO- ion and
bind to it.
5
1 Step 2: T1:Mechanism of buffer action
2 Acidic buffer
NaOH
From
acetic From NaOH
5 acid
1 Step 2: T1:Mechanism of buffer action
3
NaOH and the user should click on acetic acid will be in non
the bottle open the lid and take a dissociated form, when an strong
filler and add drops of the NaOH to acid like NaOH was added the H+
the acidic buffer bottle. Then the ions from the acetic acid
user should see the reactions as in dissociate and will react with the
the figure simultaneously while hydoxide ion from the sodium
adding the NaOH to the solution. hydroxide and form water, thereby
Show like OH- ion in reaction (B) constantly maintaining the pH of
4 moving towards the H+ ion and the buffer.
bind to it.
5
1 Step 2: T1:Mechanism of buffer action
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
3
calculation 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
4 methodology.
5
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 .
5
1 Step 6: T3: pH meter standarization
2
STD 1
STD 2
2
Glass
electrode
3
4
Nernst equation
5
1 Step : T4:pH instrument mechanism working
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
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
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:
Question 3:
Question 5:
pH is measured using
a)Ammeter
b)Voltmeter
c)Litmus paper
d) electrodes
APPENDIX 2
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