PRACTICAL 4: PH AND BUFFERS LAB REPORT

SURNAME: MODISENYANE
INITIALS: MEE
STUDENT NUMBER: 221079825
MODULE: BIOCHEMISTRY
MODULE CODE: BIC12B1
DUE DATE: 30 AUGUST 2023
INTRODUCTION
pH is a measure of how basic or acidic a substance is. pH has a range of 0-14. A pH
greater than 7 means the substance is basic. A pH less than 7 means the substance is
acidic. When the pH is exactly 7 that indicates that the substance is neutral. The pH is
usually measured with a pH meter. pH meter is an instrument used to measure acidity
or alkalinity of a solution (Raven, 2014).
The quantitative information provided by the pH value expresses the degree of the
activity of an acid or base in terms of hydrogen ion activity. The pH value of a substance
is directly related to the ratio of the hydrogen ion [H+] and the hydroxyl ion [OH-]
concentrations. If the H+ concentration is greater than OH-, the material is acidic, i.e.,
the pH value is less than 7. If the OH- concentration is greater than H+, the material is
basic, with a pH value greater than 7. If equal amounts of H+ and OH- ions are present,
the material is neutral, with a pH of 7 (Harries, 2005). Acids and bases have free
hydrogen and hydroxyl ions, respectively. The relationship between hydrogen ions and
hydroxyl ions in a given solution is constant for a given set of conditions, either one can
be determined by knowing the other (Dannis, 2012).
A rough indication of pH can be obtained using pH papers or indicators, which change
color as the pH level varies. These indicators have limitations on their accuracy, and can
be difficult to interpret correctly in colored or murky samples. More accurate pH
measurements are obtained with a pH meter. A pH measurement system consists of
three parts: a pH measuring electrode, a reference electrode, and a high input
impedance meter. The pH electrode can be thought of as a battery, with a voltage that
varies with the pH of the measured solution (Reece, 2011). The pH measuring electrode
is a hydrogen ion sensitive glass bulb, with a millivolt output that varies with the
changes in the relative hydrogen ion concentration inside and outside of the bulb. The
reference electrode output does not vary with the activity of the hydrogen ion. The pH
electrode has very high internal resistance, making the voltage change with pH difficult
to measure. The input impedance of the pH meter and leakage resistances are
therefore important factors. The pH meter is basically a high impedance amplifier that
accurately measures the minute electrode voltages and displays the results directly in
pH units on either an analog or digital display. In some cases, voltages can also be read
for special applications or for use with ion-selective or Oxidation-Reduction Potential
(ORP) electrodes (Raven, 2005).
Buffers are solutions that have constant pH values and the ability to resist changes in
that pH level. They are used to calibrate the pH measurement system (electrode and
meter). There can be small differences between the output of one electrode and
another, as well as changes in the output of electrodes over time. Therefore, the system
must be periodically calibrated. Buffers are available with a wide range of pH values,
and they come in both premixed liquid form or as convenient dry powder capsules. Most
pH meters require calibration at several specific pH values. One calibration is usually
performed near the isopotential point (the signal produced by an electrode at pH 7 is 0
mV at 25°C), and a second is typically performed at either pH 4 or pH 10. It is best to
select a buffer as close as possible to the actual pH value of the sample to be measured
(Jackson, 2011).
The Henderson-Hasselbalch equation is a mathematical expression used to find out the
pH of buffer solutions. This equation relates pH, dissociation of a weak acid, and the
ratio of the concentrations of its salt of the conjugate base and the concentration of
acids. It shows the approximate pH value of a Buffer solution. This equation represents
the relationship between the pH or pOH of an aqueous solution and the acid
dissociation constant and the ratio of the concentrations of the dissociated chemical
species (Hasselbalch, 1917).
The Henderson-Hasselbalch equation fails to predict accurate values for the strong
acids and strong bases because it assumes that the concentration of the acid and its
conjugate base at chemical equilibrium will remain the same as the formal concentration
(the binding of protons to the base is neglected). Since the Henderson-Hasselbalch
equation does not consider the self-dissociation undergone by water, it fails to offer
accurate pH values for extremely dilute buffer solutions (Hasselbalch, 1917).
Aim of the experiment was to observe the pH of the solutions using the indicator paper
and to demonstrate the buffering capacity using the pH meter. The end goal of this
experiment is to see if the pH of the solution changes or be different when we use the
indicator paper compared to when we use the pH meter. And to also conclude wich
solutions are better buffer and determine the effectiveness of a buffer.

MATERIALS AND METHODS

Sodim acetate (0,1 mol/litre), Acetic acid (0,1 mol/litre), Hydrochloric acid (0,1mol/litre),
pH meter and calibration buffer, pH-indicator paper strips and various household
solutions such as distilled water, Vinegar, Bleach, Sprite Zero, Coke and soap are the
materials that were used to perform the experiment.
PART A
The PH of each solutions provided were predicted. The PH indicator strips were used,
dipped the end with the coloured squares into the solution briefly because it only
necessary for the squares to come into contact with the liquid, not the entire strip. The
strip was then removed and tapped the excess liquid off gently. Placed the wet strip on
absorbent towel/tissue and waited for about 15 seconds before reading the results. The
strip was then compared to the PH chart that was provided and wrote down the
measured PH range. This procedure was performed for all solutions using new strips.
PART B
The solution of sodium acetate and acetic acid were mixed. For test tube 1, 1ml of
sodium acetate mixed with 8ml of acetic acid; test tube 2 ,3ml with 6ml; test tube 2, 5ml
with 4ml; test tube 4, 7ml with 2ml; test tube 5 was only 9ml of sodium acetate and no
acetic acid then test tube 6 was 9ml of sodium acetate and 1ml of H2O. The PH was
then measured using the PH meter. The Henderson-Hasselbach equation was used to
calculate what the PH should be. The 1ml of 0,1mol/litre HCL was added further to each
mixture then measured the PH again and compared the values with that obtained by
calculation.

RESULTS
TABLE 1: THE RESULTS OF THE ESTIMATION OF PH OF
DIFFERENT SAMPLES USING THE INDICATOR PAPER.
SAMPLES ESTIMATED pH MEASURED PH RANGE

Distilled water 5 4-6

Vinegar 2 1-3

Bleach Between 11 and 12 10-13

Sprite zero 4 3-5

Coke 3 2-4

Soap 5 4-6

The table above shows the estimated pH of the different samples by using the indicator
paper. To obtain this results, the indicator strip end with the coloured squares was
dipped into all samples/ solutions briefly so that they both come into contact. It was
observed that the distilled water is sightly acidic with PH of 5 and it ranges between 4
and 6, vinegar is acidic with the PH of 2 that ranges between 1 and 3, bleach is basic
with the PH between 11 and 12 and it ranges between 10 and 13, Sprite zero is acidic
with the PH of 4 and ranges between 3 and 5, Coke is acidic with the PH of 3 that
ranges between 2 and 4 and lastly, soap is acidic with the PH of 5 that ranges between
4 and 6.

TABLE 2: RESULTS OF DETERMINING PH USING PH METER

AND HANDERSON-HASSELBALCH EQUATION.
TUBE NUMBER 1 2 3 4 5 6

Sodium acetate 1 3 5 7 9 9
(ml)
Acetic acid (ml) 8 6 4 2 0 1ml of
H2O
Calculated pH 3,85 4,45 4,85 5,29 13,00 4,75
(equation)
Measured PH 3,65 4,23 4,59 5,01 5,24 6,92
(value x)
HCL (0 1 1 1 1 1 1
,1 mol/ litre)
Measured PH 2,69 4,11 4,38 4,93 4,94 5,45
(value y)
Measured PH 0,96 0,34 0,21 0,08 0,30 1,47
difference (x-y)

The table above shows the buffering capacity of each solutions, This results were
obtained after using the pH meter and the Henderson-Hasselbach equation for the
determination of PH. The pH was calculated using the equation first then it was
measured using the PH meter and we observed that they differ sightly from each other
as indicated on the table above. We then went further and added HCl into the solutions
were observed that the PH of the solution decreases when we measured it again using
the same PH meter.

APPENDIX
TEST TUBE 1
C= N/V C=n/v
0,1M= n/1x10^-3 L 0,1M= n/8x10^-3L
N= 1x10^-4mols of CH3COONa n= 8x10^-4mols of CH3COOH

PH= pka + log ([A]/ [HA])

PH= 4.75 + log ((1x10^-4)/(8x10^-4))
PH= 3,85
TEST TUBE 2
C= N/V C=n/v
0,1M= n/3x10^-3 L 0,1M= n/6x10^-3L
N= 3x10^-4mols of CH3COONa n= 6x10^-4mols of CH3COOH

PH= pka + log ([A]/ [HA])

PH= 4.75 + log ((3x10^-4)/(6x10^-4))
PH= 4,45

TEST TUBE 3
C= N/V C=n/v
0,1M= n/5x10^-3 L 0,1M= n/4x10^-3L
N= 5x10^-4mols of CH3COONa n= 4x10^-4mols of CH3COOH

PH= pka + log ([A]/ [HA])

PH= 4.75 + log ((5x10^-4)/(4x10^-4))
PH= 4,85

TEST TUBE 4
C= N/V C=n/v
0,1M= n/7x10^-3 L 0,1M= n/2x10^-3L
N= 7x10^-4mols of CH3COONa n= 2x10^-4mols of CH3COOH

PH= pka + log ([A]/ [HA])

PH= 4.75 + log ((7x10^-4)/(2x10^-4))
PH= 5,29

TEST TUBE 5
Ka= 10^-pka Ka x Kb= 1,0x 10^-14
Ka= 10 ^-4,75 (1,78x10^-5) x Kb= 1,0x10^-14
Ka= 1,78x10^-5 Kb= 5,62x10^-10

CH3COONa = CH3COO^- + Na^+

C= N/V
0,1M= n/9x10^-3 L
N= 9x10^-4mols of CH3COONa
CH3C00^- : CH3COONa Na : CH3COONa
1 : 1 1 : 1
N= 9x10^-4 mols of CH3COO n= 9x10^-4 mols of Na
[H^+] = ((Kb) x ([CH3COO])/ [Na]
[H^+] = ((5,62X10^-10) X (9X10^-4))/ (9X10^-4)
[H^+] = 5,62X10^-10 M

PH= -log [H^+]

PH= -log (5,62x10^-10)
PH= 9,25
TEST TUBE 6
C1V1=C2V2
(0,1)(0,009)= C2(0,001)
C2=0,9M of H20

C= N/V C= N/V
0,1 M= N/0,009L 0,9M= n/0,001l
N= 0,0009 mols of CH3COONa n=0,0009 mols of H2O

PH= pka + log ([A]/ [HA])

PH= 4.75 + log ((0,0009)/(0,0009))
PH= 4,75
QUESTIONS AND ANSWERS
1. Explain which of the solutions in the table above are better buffers than
others.
 Test tube 4 and 6 are the better buffers because the capacity of test tube 4 fall
within one to two PH units above or below the desired PH which is 7,00 and test
tube 6 has the same PH as the pka.
2. Which factors determine the effectiveness of a buffer?
 The actual concentrations of A^- and HA, the pka of the weak acid component
and the relative concentration of the weak acid and base component.
3. Explain how you would choose a buffer for a specific biological system.
 Before selecting a buffer it's necessary to figure out what pH level is right for the
experiment. If it is likely that the pH will decrease or increase during the
experimental process it's important to choose a buffer with pKa that is either
higher or lower than the optimum at the start of any experiment.
4. Why is it essential that near constant PH is maintained in biological
systems?
 Because all biological processes are dependent on pH, cells and organisms must
maintain a specific and constant pH in order to keep their enzymes in the
optimum state of protonation.
5. Why is the PH of distilled water not necessarily 7,0?
 The pH of distilled water is actually slightly acidic. This is because as soon as
distilled water comes into contact with the air, carbon dioxide gas dissolves in it
and creates a dilute solution of carbonic acid.
6. Explain the temperature dependency of pH (mention PH meters).
 The auto-dissociation activity increases as temperature increases. As such, the
temperature is very important in regards to pH measurements. As the pH value
changes with the change in temperature, the new measured pH value is
technically the true pH value.
7. What can you add to prevent the growth of bacteria/fungus in buffers?
 You can add a chemical preservative or add sufficient organic solvent to the
buffer so that it suppresses microbial growth.
DISCUSSION
A pH test strip is a strip of litmus paper with which you can measure the pH value of a
liquid. The substance in the paper causes the paper to show a different colour at
different acidities. The official pH scale is from 0 to 14, where 0 is very acidic and 14
very alkaline. Some pH test strips can measure a pH from 0 to 14, but there are also pH
test strips that can measure only acid or only alkaline substances (Helmenstine, 2019).
To determine the PH of the solution, you have to Dip the pH strip in the fluid which you
want to control for two seconds, then wait for the result for ten seconds. When the strip
contacts an acidic or alkaline substance, it discolours. The more acidic the solution, the
redder the strip becomes. In contrast, the more alkaline it is, the bluer the strip
becomes. Then the you have to use the indicator scale that comes with the strips to see
the colour chart that helps to determine the pH of the solution. When a pH strip is
dipped into a vial of an acidic solution, such as hydrochloric acid, it turns red. But when
it is dipped into an alkaline like sodium hydroxide, it turns into a blue or greenish colour.
Meanwhile, when the strip is in the presence of pure water or neutral, it turns purple
(Madura, 2011).
PH meter can also be used to determine the pH of the solution. More accurate pH
measurements are obtained with a pH meter. A pH measurement system consists of
three parts: a pH measuring electrode, a reference electrode, and a high input
impedance meter (Petrucci, 2011). A pH meter works through an electrochemical cell
mechanism, where the H + in the chemical reaction allow determining the concentration
of them in solution. When determining pH with a pH meter, a reference electrode and a
measurement electrode are used, which are sensitive to analytes. In other words, a pH
meter is a high impedance amplifier that accurately measures the minimum electrode
voltages and displays the results directly in pH units on an analog or digital display. In
some cases, the voltages can also be interpreted for special applications or use with ion
selective or oxidation / reduction potential electrodes (Ralph, 2011).
The most important difference between a pH test strip and pH meter is the accuracy of
the measurment. A pH meter usually contains a computer and generally gives results to
a hundredth of an inch. This is not the case with pH indicator strips, these are less
accurate. pH test strips are therefore very useful to quickly determine whether a solution
is acidic, neutral or alkaline, but are not suitable for accurate measurements (Kolb,
1979).
The Henderson-Hasselbalch equation tells us how the pH of the buffer solution changes
with the pKa of a weak acid. The best buffer solution is prepared when we take an equal
amount of salt and acid. The pH is controlled by the pKa of the acid. For example, for
the buffer of acetic acid and sodium acetate (Hasselbalch, 1917).
CONCLUSION
The experiment was successful as we were able observe the estimated PH of the
solutions using the indicator strips and also we were able to determine the PH using PH
meter to demonstrate the buffering capacity of the solutions and the Henderson-
Hasselbalch equation.

TITLE
Estimation of PH using indicator strips, PH meter for accuracy and demonstration of
buffering capacity using Henderson-Hasselbach equation.

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