2013-2014 SPRING SEMESTERS

MARMARA UNIVERSITY
DEPARTMENT OF BIOENGINEERING

BIOE1011.1

GENERAL BIOLOGY FOR BIOENGINEERS

Experiment 4: Dilutions, Buffers and pH Measurement

EZGİ CİNAN

Course Instructor: Prof. Dr. Ahu Altınkut Uncuoğlu

Submitted to: Ceyhun Bereketoğlu

Date of Experiment: 17.04.2014

Date of Submission: 29.04.2014

Marmara University
Istanbul
INTRODUCTION

Acids and Bases

There are two common acid and base definitions that can explain efficiently some aspects of
the behaviour of acids and bases: Arrhenius and Brønsted-Lowry definitions. [1]

Arrhenius Definition of Acids and Bases:

Any substance that ionizes when it dissolves in water to release hydrogen ion (H+) is called
Arrhenius acid. Additionally, an Arrhenius base is any substance that releases hydroxide
ion (OH-) when it dissolves in water. HCl, HCN and H2SO4 are some examples of Arrhenius
acids that include hydrogen and give hydrogen ion when it ionizes in water. Arrhenius base
includes ionic compounds that comprise OH- ion such as Ca(OH)2, KOH and NaOH. [2]

HCl(g) → H+(aq) + Cl-(aq) NaOH(s) →Na+(aq) + OH-(aq) [2]

On the other hand, any substance that include H+ ion is not acid. As an example of this, CH4
is not an Arrhenius acid because of not giving H+ ion in water. Although some compounds
such as Na2CO3 contain OH- ion but they are not Arrhenius bases. [2]

Brønsted Definition of Acids and Bases:

According to Brønsted-Lowry theory, a Brønsted acid is a proton donor (a donor of H+ ions)

and a Brønsted base is a proton acceptor. [1]

NH3 + H2O → NH4+ + OH- [1]

In this reaction, H+ ion is transferred into NH3 (Ammonia). H2O acts as an acid because of
giving H+ ion. [1]

HCl is a strong acid. A strong acid is very good at transferring H+ ion to a water molecule in
contrast to weak acid. Moreover a strong base is completely ionized in solutions but a weak
base is not. [2]

pH Measurement

The pH concept is a measure of the molar concentration of hydrogen ions in a solution. It is a

measure of the acidity or basicity of the solution. The numerical value of the pH is defined as
the negative of the power of 10 of the molar concentration of H+ ions. [3]
 pH= -log[H+] [3]

The range of pH scale is between 0 and 14. 7 is the value for neutral pH. A pH less than 7 is
acidic; contrary to this a pH greater than 7 indicates alkalinity. [3]

Figure 1: pH scale. [3]

pH can be measured by many ways: [4]
 pH papers (give approximate pH values)
 A colorimeter (with indicators and series of standards)
 pH meter (gives the most precise value of pH)

pH meter:
pH meter is used to measure the hydrogen ion
concentration (pH) of solutions. A pH meter
consists of two electrodes that are connected to a
voltmeter: glass (or indicator) and reference (or
calomel) electrode. The pH is analysed by a glass
electrode. It benefited from the development of
voltage difference between two electrodes. One of
them is reference and the other (indicator
electrode) is sensitive to changes in pH. [4, 5]
Figure 2: A pH meter. [6]
Important Parameters of pH Measurement:
1. The pH meter should be warmed up 15-30 minutes. Generally the temperature should
be 25 oC.
2. Before you begin the measurement, rinse the probe with deionized water and pat the
tip dry with a soft tissue. [5]
3. The meter must be calibrated prior to making pH measurements. [5]
4. pH calibration can be affected by temperature and carbon dioxide absorption. [7]
5. Temperature changes in the sample cause the shift in hydrogen-ion activity. If you
allow the electrodes, buffers and container to equilibrate to the same temperature, the
changes will be minimized. [8]
6. A clean, undamaged glass membrane is necessary for performing an accurate
measurement of pH. [8]
7. Prevent the electrode from coming into contact with the sides of bottom of your
beaker or other measurement vessels. [8]
8. Get rid of any mineral precipitate off the electrode with DIW. [8]
9. Inspect the pH electrode. [8]
Buffers
A buffer solution is that which resists changes in pH when adding small quantities of acid or
alkali. Buffers consist of a weak acid and a salt of a strong base; or a weak base and a salt of a
strong acid. In other words buffers are solutions that have conjugated pair of acid-base. [4]
The ability of a solution to resist changes in pH is quantified by buffer capacity. Buffer
capacity is described as the moles of an acid or base required to change the pH of a solution
by 1. [9]
Biochemical reactions are responsive to pH. Most biological molecules contain functional
groups that have a significant effect on the biological activity of the molecule. Two important
biological buffer systems are the dihydrogen phosphate system and the carbonic acid system.
In mammal, cellular fluid has a pH in the range 6.9 to 7.4. The phosphate buffer, which
operates in the internal fluid of all cells, maintains this pH range. The carbonic acid which is a
biological buffer has an important role in maintaining pH of blood plasma. [10]
Important parameters for biological buffer: [11]
1. pKa: Ideal buffers should be in the range of pH 6 to 8 that provides maximum buffer
capacity.
2. Solubility: High solubility in water is necessary and low solubility in nonpolar
solvents is considered useful.
 3. Permeability: Permeability should be low because of reducing accumulation of buffer
compound within cells.
4. Dissociation: The effects of buffer concentration, temperature of the system and ionic
composition of the medium about buffer dissociation should be minimal.
5. Optical Absorbance: Buffers should not absorb any light at wave-lengths longer than
230 nm.
6. Costs: Buffers should be prepared from inexpensive materials and easily purified.
The technique of Serial Dilution:
As a first step, take a known volume (1 mL in general) of stock and transfer it into a known
volume of distilled water (9 mL in general). In this way, 10 mL of dilute solution is obtained.
This dilute solution has 1 mL of extract / 10 mL, producing a 10-fold dilution. The technique
which is used for making a single dilution is repeated sequentially to make serial dilution.
[12]

Figure 3: Successive Serial Dilution. [12]

The aim of this experiment is describing the terms of acid and base, pH, dilution and buffer.
Additionally, to learn about how pH and dilutions are measured. On the other hand, to
practise about the usage of pH meter.

MATERIALS

 Beakers
 Graduated Cylinder
 Scientific Balance
  Weighing dish
 Spatula
 Wash bottles
 NaOH
 1M Tris-HCl buffer (pH 8.02)
 Distilled Water
 Magnetic Stirrer Hotplates
 Pipettes
 pH meter

METHODS

1- 20 ml 5M NaOH solution is prepared by Group 1.

2- Dilute the prepared solution into 20 ml 1M NaOH solution. (Group 2)

3- Dilute the former one into 20 ml 0.5M NaOH solution. (Group 3)

4- Dilute the former one into 20 ml 0.1M NaOH solution. (Group 4)

5- The prepared 50 ml 1M Tris buffer is placed on magnetic stirrer.

6- Later, the pH of tris buffer is measured by pH meter. (pH 8.02)

7- Add 250 µl liquid from 20 ml 5M NaOH solution into the tris buffer solution by
micropipette and note the pH value.

8- This process is repeated until tris buffer loses its resistance across the shift in pH and
values of pH are noted.

9- Before measuring the pH of pure water, rinse and wipe the electrode.

10- After the pH value of pure water is noted, 250 µl 5M NaOH solution is added and results
are noted.

RESULTS

We made some calculations for preparing and diluting the solutions.

Group 1: This group prepared 20 mL 5M NaOH solution.

MW NaOH=40 g/mol

20 mL= 20.10-3 L

M=n/V → n=5x20.10-3= 10-1 mol

n=m/MW → m=10-1x40= 4 g

4 g NaOH is required for preparing 20 ml 5M solution.

Group 2: This group found the volume that will be taken from the main solution and diluted
prepared solution into 20 ml 1M NaOH solution.

M1: The concentration of main solution.

V1: The volume that will be taken from the main solution for dilution.

M2: The desired concentration

V2: The amount of solution to be prepared at concentration of M2

M1xV1 = M2xV2

5x V1 = 1x20 → V1= 4 mL

Stock=Substance Content x Dilution Factor

5M= (1M) x DF → DF=5

Group 3: This group diluted the previous one into 20 ml 0.5M NaOH solution.

1x V1 = 0.5x20 → V1= 10 mL

5M= (0.5M) x DF → DF=10

Group 4: This group diluted the former one into 20 ml 0.1M NaOH solution.

M1xV1 = M2xV2

0.5x V1 = 0.1x20 → V1= 4 mL

5M= (0.1M) x DF → DF=50

After dilution, we measured the pH value of Tris-HCl buffer solution. (pH = 8.02) Then, the
capacity of tris-HCl buffer solution is observed by adding 250 µl 5M NaOH solution a couple
of times. The following shows how the pH of buffer solution changes:

Change of pH curve of buffer solution

14,00
pH value of Tris-HCl Buffer

12,00

10,00
pH value of Tris-HCl Buffer
8,00
Doğrusal (pH value of Tris-HCl
6,00
Buffer)
4,00

2,00

0,00
0 2000 4000 6000 8000
5M NaOH Solution (µl)

Figure 4: The graph of change in pH value of tris-HCl buffer solution.

The pH value of pure water was measured by pH meter. (pH=8,18) Next, the shift in pH value
was observed by adding 250 µl 5M NaOH solutions one time. The following graph shows
how the pH value of pure water changes:

change of pH curve of distilled water

14
pH Value of distilled water

12
10
8
pH value of distilled
6 water
4 Doğrusal (pH value of
distilled water)
2
0
0 50 100 150 200 250 300
5M NaOH Solution (µl)

Figure 5: The graph of change in pH value of pure water.

DISCUSSION

In this laboratory we learned the definition of acid-base. There are different kind of
description for acids and bases in chemistry: Arrhenius theory, Brønsted-Lowry theory and
Lewis theory. The definition of Brønsted is better than Arrhenius’ because Arrhenius theory is
restricted only with aqueous solutions. When moving from the Arrhenius theory, to Brønsted-
Lowry theory, to the Lewis theory; the definitions of acid and base become more general, less
limited.

There are many techniques to measure pH. The technique which is used depends upon how
accurately you want to know pH. pH indicators change colours according to the pH of
solution. We used pH meter for measuring, it is more professional than other techniques.

The temperature of the work environment has an important effect on pH. pH could vary
depend on the temperature fluctuation. Thus, the temperature of the system generally should
be 25 oC.

The process of preparing 20 ml 5M NaOH solution, we put 4 g NaOH into beaker and then
placed the beaker on magnetic stirrer. After a while, its temperature increases more. It is
because that NaOH solution is both as concentrated and as exothermic. To prevent any
accident because of increasing temperature of substance, the substance should be added
slowly.

The curves of Figure 4 and Figure 5 are very different from each other. When looking at
Figure 4, it is observed that the buffer solution tends to resist changes in pH when 250 µl
NaOH is added. Also the capacity of tris-HCl buffer solution has approximately pH range 7 to
9. Reduction in resistance is seen on pH values greater than 9. Otherwise, pure water did not
resist shift in pH. Its pH increased suddenly from 8.18 to 12.43. The pure water lost its
neutrality instantly.

Biological buffers have a great importance for living things. For instance, the carbonic acid
act as a biological buffer in blood. The pH of the medium must be specific and rapid changes
should be blocked. The carbonic acid system provides the blood to carry oxygen by make the
pH of medium stable. As is seen, the ability of resistance against changes in pH plays a
crucial role in human’s life as well as the life of other living things.
REFERENCES

1- Petrucci, R.H., Herring, F.G., Madura, J.D., Bissonnette, C., 2010. General Chemistry:
Principles and Modern Applications, 10th ed. Pearson Canada Inc., Toronto, pp.697-700

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11- Will, M.A. and Clark, N.A., Swain, J.E. (2011). Biological pH Buffers in IVF: help or
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