Worksheet: EXPERIMENT 2.2: THE USE OF
BUFFERS IN BIOLOGICAL SCIENCES
14 October 2020
05:50
SESSION ORGANISATION
All timings are approximate
10.00 a. m. Make sure you arrive in good time. Download a copy of the
session worksheet from Minerva before or as soon as you arrive. You will
enter the breakout room with your group. Check your answers to the
questions with one another and with your demonstrator.
10.30 a.m. There will be a brief introduction by the Academic Lead together
with any course-related announcements, followed by a video demonstrating
the technique and experiment.
10.45 a.m. – 12.00 p.m. Complete worksheet in your groups with help from
your demonstrator (who will be supervising 3-4 groups): remember that
while you are working as a group, each of you must complete their own
worksheet, in your own words.
PREPARATION
Before you come to the session, you should:
Read the introduction for understanding and making buffers.
Using the information from these sources, write a 100-150 word
summary of the background material.
Answer the questions
Read through the session worksheet (experiment 2.2) so you
understand what you will be doing (but don’t start to complete it)
INTRODUCTION
In many instances it is necessary to use buffers in experiments involving
biological materials. This applies especially to systems isolated from the
normal (buffered) environment of the cell or organism. For example,
isolated mitochondria and chloroplasts will only remain biologically active
when suspended in a buffered, isotonic medium. When investigating
enzyme reactions using isolated enzyme systems it is essential to keep the
pH constant by the use of buffers. There are several reasons for this, the
main one being that enzyme activity depends on pH.
, There is a variety of buffers available, the one chosen depends primarily on
the pH needed, but also on other considerations such as whether the buffer
interferes with any of the assays or procedures to be performed.
The working pH ranges of some commonly used buffers are shown below.
In general the useful buffering range is pK a ± 1.0.
Summary:
Buffers resist PH changes even after the addition of acids or bases.
Buffering is necessary when assesting biological molecules, as they are
functionable at a specific PH.(protein activity is depended on PH)
The buffering capacity is maximal when PH=PK and a buffer is in a useful
buffer range when the PH is whithin one unit of its PK.
METHOD 1 :If you have the both the acidic and basic form of the buffering
compound available, you can make up solutions of both, then use the
Henderson-Hasselbach equation to determine the ratio of acidic and basic
forms that need to be mixed together, to get a buffer of the required pH.
METHOD 2: If only the acidic or basic form of the buffer is readily
available,then a solution of this compound is made, adjusted to the correct
pH with a strong base or acid,then the volume is adjusted to ensure the
correct concentration of the finished buffer. (eg.Tris)
Working pH range for some common buffer solutions
Acetate buffer pH 3.0-6.0
Citrate buffer pH 3.0-6.2
Phosphate buffer pH 5.7-8.0
Pipes buffer pH 5.8-7.8
Hepes buffer pH 6.6-8.6
Tris-HCl buffer pH 7.2-9.0
Glycine-NaOH buffer pH 8.6-10.6
BUFFERS IN BIOLOGICAL SCIENCES
14 October 2020
05:50
SESSION ORGANISATION
All timings are approximate
10.00 a. m. Make sure you arrive in good time. Download a copy of the
session worksheet from Minerva before or as soon as you arrive. You will
enter the breakout room with your group. Check your answers to the
questions with one another and with your demonstrator.
10.30 a.m. There will be a brief introduction by the Academic Lead together
with any course-related announcements, followed by a video demonstrating
the technique and experiment.
10.45 a.m. – 12.00 p.m. Complete worksheet in your groups with help from
your demonstrator (who will be supervising 3-4 groups): remember that
while you are working as a group, each of you must complete their own
worksheet, in your own words.
PREPARATION
Before you come to the session, you should:
Read the introduction for understanding and making buffers.
Using the information from these sources, write a 100-150 word
summary of the background material.
Answer the questions
Read through the session worksheet (experiment 2.2) so you
understand what you will be doing (but don’t start to complete it)
INTRODUCTION
In many instances it is necessary to use buffers in experiments involving
biological materials. This applies especially to systems isolated from the
normal (buffered) environment of the cell or organism. For example,
isolated mitochondria and chloroplasts will only remain biologically active
when suspended in a buffered, isotonic medium. When investigating
enzyme reactions using isolated enzyme systems it is essential to keep the
pH constant by the use of buffers. There are several reasons for this, the
main one being that enzyme activity depends on pH.
, There is a variety of buffers available, the one chosen depends primarily on
the pH needed, but also on other considerations such as whether the buffer
interferes with any of the assays or procedures to be performed.
The working pH ranges of some commonly used buffers are shown below.
In general the useful buffering range is pK a ± 1.0.
Summary:
Buffers resist PH changes even after the addition of acids or bases.
Buffering is necessary when assesting biological molecules, as they are
functionable at a specific PH.(protein activity is depended on PH)
The buffering capacity is maximal when PH=PK and a buffer is in a useful
buffer range when the PH is whithin one unit of its PK.
METHOD 1 :If you have the both the acidic and basic form of the buffering
compound available, you can make up solutions of both, then use the
Henderson-Hasselbach equation to determine the ratio of acidic and basic
forms that need to be mixed together, to get a buffer of the required pH.
METHOD 2: If only the acidic or basic form of the buffer is readily
available,then a solution of this compound is made, adjusted to the correct
pH with a strong base or acid,then the volume is adjusted to ensure the
correct concentration of the finished buffer. (eg.Tris)
Working pH range for some common buffer solutions
Acetate buffer pH 3.0-6.0
Citrate buffer pH 3.0-6.2
Phosphate buffer pH 5.7-8.0
Pipes buffer pH 5.8-7.8
Hepes buffer pH 6.6-8.6
Tris-HCl buffer pH 7.2-9.0
Glycine-NaOH buffer pH 8.6-10.6
