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BUFFERS

Experiment 1
Ebru AKHARMAN, Gebze Technical University, Turkey

AIM:
You should be aware that buffers play a critical role in almost all biochemical systems. Biochemical
experiments routinely require a buffer. In this laboratory you will cover the basics of buffer
preparation and test the buffering capacity of the resulting solution. This buffer will be used in the
other experiments later in the term.

INTRODUCTION:
Equation 1:
A buffer is an aqueous solution that has a
highly stable . If you add acid or base to a
= + log
buffered solution, its will not change

significantly. Similarly, adding water to a


Equation 2:
buffer or allowing water to evaporate will not
change the of a buffer. When hydrogen + = []
ions are added to a buffer, they will be
neutralized by the base in the buffer. What are the characteristics of a good
Hydroxide ions will be neutralized by the
buffer?
acid. These neutralization reactions will not
have much effect on the overall of the A pKa between 6 and 8. Most
buffer solution. biochemical experiments have an
optimal in the range of 68. The
When you select an acid for a buffer solution, optimal buffering range for a buffer is
try to choose an acid that has a close to the dissociation constant of the weak
your desired . This will give your buffer acid component of the buffer (pKa)
nearly equivalent amounts of acid plus or minus unit.
and conjugate base so it will be able to Solubility in water. Biological
neutralize as much H+ and OH- as possible. reactions, for the most part, occur in
aqueous environments, and the buffer
Buffer solutions are one of the most should be water-soluble for this
important types of chemical reagent used in reason.
chemical research, biological research and Exclusion by biological
industry. membranes. This is not important for
all biochemical reactions. However, if
A buffer contains a weak Brnsted base. The this is an important criterion for your
pH of any given buffer can be related to the particular experiment, it is helpful to
remember that zwitterionic buffers
of the weak acid involved and the
(positive and negative charges on
logarithm of the molar ratio of conjugate base different atoms within the molecule)
to acid by means of the Henderson- do not pass through biological
Hasselbalch equation: membranes. Examples of zwitterionic
buffers include MOPS and HEPES; Tris
and phosphate buffers do not Experimental Application:
isomerize into zwitterions.
Minimal salt effects. In other words, 0,1 M and 5,0 acetic acid sodium acetate
the buffer components should not buffer is prepared. According to Henderson
interact or affect ions involved in the Hasselbach equation, amounts of acetic acid
biochemical reactions being explored. and acetate ion contained in 100 ml of such a
Minimal effects on the dissociation
buffer are calculated.
from changes in temperature and
concentration. 1 M acetic acid and 1 M sodium acetate
Minimal interactions between buffer
solutions are mixed with 90 ml 2 for
components and critical reaction
components. prepairing 100 ml sample of this buffer. Thus,
Chemical stability. equation 1 and equation 2 are used for
Light absorption. The buffer should amounts of acetic acid and acetate ion.
not absorb UV light at wavelengths

that may be used for readouts in Equation 1: = + log

photometric experiments.
Ease of Use. The buffer components Equation 2: + = []
should be easy to obtain and prepare.
NOT: Sodium acetate solutions have of
Buffer Capacity: The purpose of a buffer is to 4,99. is 4,76.
minimize changes in when either acids or
According to the equation 1, values are
bases are added to a solution. The ability of a
placed.
buffer to resist changes depends both on
the absolute concentration of the buffer [ ] []:conjugatedBrnsted Base
4,99 = 4,76 + log [] []: Brnsted acid
components and the ratio of these to each
other. [ ] [ ]
4,99 4,76 = log [] 0,23 = = log []
Molarity and Ionic Strength of A Buffer:
[ ]
100,23 = [] . Thus,
The molarity of a buffer is defined as the sum
of the molarities of the Brnsted acid and 100,23 * [] = [ ]
conjugate contained in the buffer. The
concept of ionic strength differs from molarity According to the equation 2, values are
in two ways. First, it considers only the ionic placed.
species present in solution. Secondly, it
+ = [0,1] (100,23 * [] = []). Thus,
considers both the concentrations and the
charges of these ionic species. Mathematically + 100,23 [] = [0,1]
the ionic strength( ) of a solution is defined
0,1
as follows: * ( 1+ 100,23 ) = [0,1] =
1+ 10 0,23

Equation 3: is found as 0,037 M in solution.

=

[0,037] + [ ] = [0,1] [0,037] [0,1]=

[ ] is found as 0,063 M in solution.


C: concentration of ion i
: The charge of this ion
MATERIALS AND METHODS: 25 ml of diluted buffer are transfered in a
NaOH clean beaker and measured the . Then, 0,5
HCl ml of 0,1 N HCl are added to diluted buffer.
meter This mixture is mixed well and of mixture
Flask
is measured. Until is approximately 2
Beaker
below the for acetic acid is continued to
Acetic acid
Sodium acetate adding the HCl in 0,5 ml increments and
Acetic acid sodium acetate buffer is measured the of solutions.
prepared and measured of buffer with
meter. Thus, 100 ml undiluted buffer 25 ml of distilled water are transfered in a
solutions are obtained. clean beaker and measured the of water.
Then, 20 ml this buffer are transfered to a 1 ml of 0,1 N NaOH are added to distilled
clean 100 ml volumetric flask abd 80 ml of water. This mixture is mixed well and of
water are added to this buffer. This mixture is mixture is measured. Unbuffered systems are
mixed well. Thus, 100 ml dilted buffer compared with buffered systems.
solutions are obtained.
25 ml of diluted buffer are transfered in a 25 ml of distilled water are transfered in a
clean beaker and measured the . Then, 1
clean beaker and measured the of water.
ml of 0,1 N NaOH are added to undiluted
1 ml of 0,1 N HCl are added to distilled water.
buffer. This mixture is mixed well and of
mixture is measured. Until is This mixture is mixed well and of mixture
approximately 1 units above the for is measured. Unbuffered systems are
acetic acid is continued to adding NaOH in 1 compared with buffered systems.
ml increments. At this point, until is
approximately 2 units above the for RESULTS:
acetic acid is continued to adding 0,5 ml of
NaOH. stock(25 ddH2O(25
25 ml of diluted buffer are transfered in a ml) diluted(25 ml) ml) NaOH(ml)
clean beaker and measured the . Then, 0,5 5,02 5,08 8,75 0
ml of 0,1 N NaOH are added to diluted buffer.
This mixture is mixed well and of mixture 5,09 5,57 11,58 1
is measured. Until is approximately 2 5,16 11,09 2
units above the for acetic acid is 5,25 3
continued to adding NaOH in 0,5 ml
increments. 5,35 4
25 ml of undiluted buffer are transfered in a 5,49 5
clean beaker and measured the . Then, 1 5,65 6
ml of 0,1 N HCl are added to undiluted buffer. 5,88 7
This mixture is mixed well and of mixture
6,36 8
is measured. Until, is approximately 1
11,14 9
unit below the for acetic acid is
continued to adding the HCl in 1 ml
increments and measured the of This table shows the changes due to NaOH
solutions. At this point, until is according to the solution types.
approximately 2 units below the for
acetic acid is continued to adding 0,5 ml of If you want to show this data graphically:
HCl.
If you want to show this data graphically:
Farkl Deriimdeki zeltilerde
NaOH' a Bal pH Deiimleri
12
11,5 According to pH Change in Different
11 Concentration of Solutions
10,5
10 6
9,5 5,5
9 c
8,5 5
8 h
D 7,5 a 4,5
e 7 4
6,5 n
p 6 g 3,5

(
H e 5,5 e p
r 5 3
4,5 H
i 4 2,5

)
o

3,5 stok
f 2
3 diluted
2,5 seyreltil 1,5
2 mi ddH2O
1,5 p 1
1 ddH2O H stock
0,5 0,5
0 0
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 101112131415
NaOH miktar (ml)
HCl amounts (ml)
The of other solutions increase because of
NaOH is a basic solution. The of other solutions decrease because
of HCl is a acidic solution.
stock(25 diluted(25 ddH2O(2
ml) ml) 5 ml) HCl
5,01 5,05 5,37 0
4,92 4,69 2,82 1
4,84 4,29 2
4,78 3,52 3
4,7 2,79 4
4,62 5
4,54 6 Resources:
4,46 7
https://buffer.com/guides
4,36 8
4,26 9 https://www.degruyter.com/downloadpdf/bo
4,16 10 oks/9783110411331/9783110411331.11/9783
4,02 11 110411331.11.pdf
3,28 12
2,86 13 http://www.promegaconnections.com/what-
makes-a-good-buffer/

This table shows the changes due to HCl


according to the solution types.