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Chinhoyi University of

Technology
NAME:

LEE DARYL TSVANGIRAI

REG NUMBER:

C14124160O

COURSE:

ANALYTICAL BIOCHEMISTRY

COURSE LECTURER:

DR K. CHITINDINGU

PROGRAM:

BSC. HON BIOTECHNOLOGY

PRACTICAL 1:

Use of pH meter.

LEVEL:

2.1

Lee Daryl Tsvangirai

TITLE
Use of pH meter.

OBJECTIVES
1. Calibration of pH meter.
2. Titrations for acetic acid and sodium hydroxide, glycine and sodium hydroxide &
glycine and hydrochloric acid using a pH meter to capture pH.

ABSTRACT
An experiment where calibration of pH meter using potassium hydrogen phthalate
and borax sodium of known pH in water at room temperature, was done. After
successfully calibrating the meter, it should give accurate results, provided that some
basic guidelines are followed. Titrations for acetic acid and sodium hydroxide where
performed by adding 1.00ml of 0.2M sodium hydroxide from a burette into 20.00ml 0f
0.1M acetic acid in a beaker and recording the pH with a pH meter until 11.00ml of
sodium hydroxide have been added. A solution of 20.00ml of 0.1M glycine was
prepared and titrations for glycine and sodium hydroxide where performed by adding
1.00ml of 0.2M sodium hydroxide from a burette into 20.00ml 0f 0.1N glycine in a
beaker and recording the pH with a pH meter until 20.00ml of sodium hydroxide have
been added. Another solution of 20.00ml of 0.1M glycine was prepared and titrations
for glycine and hydrochloric acid where performed by adding 1.00ml of 0.2M
hydrochloric acid into 20.00ml 0f 0.1N glycine in a beaker and recording the pH with
a pH meter until 11.00ml of hydrochloric acid have been added. Results where
plotted on the same sheet of graph paper. The whole experiment is completed and
successfully conducted.

INTRODUCTION
A pH Meter is a device used for potentiometrically measuring the pH, which is the
concentration of hydrogen ions, of an aqueous solution. It usually has a glass
electrode plus a calomel reference electrode, or a combination electrode. pH meters
are usually used to measure the pH of liquids.
Lee Daryl Tsvangirai

pH meters range from simple and inexpensive pen-like devices to complex and
expensive laboratory instruments with computer interfaces and several inputs for
indicator and temperature measurements to be entered to adjust for the variation in
pH caused by temperature. Specialty meters and probes are available for use in
special applications, harsh environments, etc. There are also holographic pH
sensors, which allow pH measurement colorimetrically (C. R Lowe 2013).
For very precise work the pH meter should be calibrated before each measurement.
For normal use calibration should be performed at the beginning of each day. The
reason for this is that the glass electrode does not give a reproducible e.m.f. over
longer periods of time.
Calibration should be performed with at least two standard buffer solutions that span
the range of pH values to be measured. For general purposes buffers at pH 4.01 and
pH 10.00 are acceptable. The pH meter has one control to set the meter reading
equal to the value of the first standard buffer and a second control which is used to
adjust the meter reading to the value of the second buffer. A third control allows the
temperature to be set (C. R Lowe 2013).
After each single measurement, the probe is rinsed with distilled water to remove
any traces of the solution being measured, blotted with a scientific wipe to absorb
any remaining water which could dilute the sample and thus alter the reading, and
then quickly immersed in a solution suitable for storage of the particular probe type.
Probes need to be kept clean of contamination as far as possible, and not touched
by hand. Probes are best kept moist with a medium appropriate for the particular
probe when not in use. If the bulb becomes contaminated with use it can be cleaned
in the manner recommended by the manufacturer; a quick rinse in distilled water
immediately after use, blotted off may be sufficient.
A titration is an analytical procedure used to determine the concentration of a sample
by reacting it with a standard solution.
In a titration, the standard solution, which appears to be NaOH solution, goes in a
burette, which is a piece of glassware used to measure the volume of solvent to
approximately 0.1 milliliters of accuracy. The solution that undergoes titration goes in
Lee Daryl Tsvangirai

an Erlenmeyer flask, which should be large enough to accommodate both the


sample and the standard solution that is being added.
Amino acids are compounds containing an amine group, and a carboxylic acid
group. In addition there is an R group that differs for each amino acid. The symbol
R is used here to represent a generalized abbreviation for an organic group.
H
H 2N

COOH

In physiological systems where the pH is near neutrality, the amino group of an


amino acid will be protonated and the carboxylic acid group will be deprotonated.
This is called the zwitterion form.
H
H 3N

COO

In strongly acidic solutions the carboxylic acid group will also be protonated, while in
strongly basic solutions both the carboxylic acid group and the amino group will both
be unprotonated.
H
H 3N

H
COOH

Acidic

H 2N

COO

Basic

The acid-base behavior of amino acids is best described by the Brnsted-Lowry


theory of acids and bases. A simple amino acid that does not have an acid or base
group in the R group is a diprotic acid and in fully protonated form it can donate two
protons during its complete titration with a base. The titration with NaOH is a twostage titration represented by the reactions below.
+

NH3CH(R)COOH + OH- +NH3CH(R)COO- + H2O


+

NH3CH(R)COO- + OH- NH2CH(R)COO- + H2O

The titration curve will be biphasic. There will be two separate flat portions on the
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titration curve. The midpoint of the first leg is where the amino acid is half in the
acidic form and half in the zwitterion form. The point of inflection occurs when all of
the original amino acid is in the zwitterions form (assuming the R group has no
charge). The actual pH at which this occurs is called the isoelectric pH (or isoelectric
point), and is given the symbol pI. During the pH titration of an amino acid with a
non-ionizable R group, the equivalence point occurs at the pI of the amino acid. At
the midpoint of the second leg, half the amino acid is in the zwitterion form and half
is in the basic form. The apparent pK values for the two dissociation steps may be
extrapolated from the midpoints of each step. This can be shown by the HendersonHasselbach equation:
pH = pKa + log([Base]/[Acid])

MATERIALS AND METHODS


Refer to Practical Handbook 2.1, CUBT213 pg25.

RESULTS
Table for change in pH for titration of acetic acid and sodium hydroxide (Initial
pH 3.5).
Volume of
NaOH

4.5 4.7

4.9

5.1

5.2

5.5

5.9

10

11

10.4 10.9

11.1

added/ml
pH

4.3

Table for change in pH for titration of glycine and hydrochloric acid (Initial pH
6.9).
Volume of
HCl

3.7

3.5

3.4

3.3

3.2

3.1

3.0

2.9

10

11

added/ml
pH

4.0

2.9

2.8

Lee Daryl Tsvangirai

Table for change in pH for titration of glycine and sodium hydroxide (Initial pH
6.9).
Volume of
HCl

10

8.4

8.7

8.9

9.0

9.1

9.3

9.4

9.5

9.7

9.8

11

12

13

14

15

16

17

18

19

20

10.1

10.4

11.1

11.2

11.3

11.3

11.3

11.3

11.4

11.4

added/ml
pH
Volume of
HCl
added/ml
pH

DISCUSSION
A pH meter is a more accurate means of measuring pH value because it can be
calibrated to measure one tenth of a pH unit, whereas the indicator solution may only
measures to one pH unit. Thus, pH meter is used to conduct this experiment instead
of phenolphthalein as indicator solution. However, the errors when using the pH
meter should be considered too. The correct ways to calibrate the pH meter before it
can be used to measure any pH are as below:
1 The electrode must be removed from the distilled water and placed in pH 4 buffer
solution.
2 The pH meter is set to pH 4 and the electrode is rinsed with distilled water to
remove any excess solution.
3 The electrode is then placed in pH 10 buffer solution.
4 The pH meter is set to pH 10 and the electrode is rinsed, and placed in distilled
water.
5 The pH meter should now be calibrated and is ready to be used to measure any
pH accurately.
Lee Daryl Tsvangirai

Therefore, the calibration of the pH meter may not be done thoroughly before it is
used to measure the pH value of the titrated solutions. In addition, the pH electrode
may have come into contact with the magnetic stirrer inside the Erlenmeyer flask,
which may also interrupt the pH readings.
Besides that, the whole experiments are conducted only once, which means that no
average value will be obtained in order to get more accurate results.
Glycine, has two dissociation steps: firstly the loss of H+ from the acidic carboxyl
group at low pH; and the loss of H+ from the more basic amino group at high pH.
The pKa value for each dissociable group of an amino acid is determined from
titration curve by extrapolating the midpoint of each buffering region within the curve.
The isoelectric point (pI), is defined as the pH where the amino acid is predominantly
a zwitterion. The pI can be approximated as halfway between the two points of
strongest buffering capacity and can be estimated by:

Where pK1 and pK2 are the dissociation constants for the deprotonation of glycines
carboxylic acid and amino groups.
The titration between acetic acid and sodium hydroxide is a titration of a weak acid
and a strong base were acetic acid id the weak acid and sodium hydroxide is the
strong base. The initial ph is 3.5 which one of the things that show us that the acetic
acid is a weak acid. Acetic acid is the analyte while the sodium hydroxide is the
titrant. There is a shape increase in pH at the beginning of the titration because the
is the rapid reduction of the hydrogen ions to water due to the addition of the
hydroxide ion present in the sodium hydroxide. After the shape increase there is a
gradual change in the curve of the titration, the gradual change is because the
solution is acting as buffer. This will continue until the sodium hydroxide overcomes
the buffer capacity. On the graph of tis titration the equivalent point is high than pH 7
because all the acetic acid has been neutralized and the ph increases until it become
the pH of sodium hydroxide.
Lee Daryl Tsvangirai

QUESTIONS
1. What are the pK values of acetic and glycine?
2. Indicate on your plot the regions of efficient buffering action for each
compound.
3. Explain the action of amino group in affecting the pH of the neighbouring

carboxyl group.
SOLUTIONS
1. pK values of acetic acid= 4.75
pK values of glycine= 2.34 and 9.60
2. On graph paper.
3. The amino group affects the pH of the neighbouring carboxyl group by
donating hydrogen ions to the carboxyl group.

CONCLUSIONS
A pH meter needs to be calibrated against a known set of values before it will yield
accurate readings.

RECOMMENDATIONS
There are a few recommendations, and precautions that have to be considered
during the experiment in order to get an accurate value and readings of data.
Firstly, the standard solution that is used should be a hundred percent pure and
stable at room temperatures. Thus, it is more preferable to use a dried standard
material before weighing and diluting.
Secondly, in order to reduce experimental error when conducting an experiment, the
standard solution that is chosen must have a high molecular weight. In this
experiment, KHP solution is used as a standard solution and since it has a large
molecular weight, which is 204.22 g/mol, therefore a large mole can be obtained to
perform the titration. The relative error in weighing a greater amount of material is
smaller than for a small amount.
Next, the neutralization reaction should be quantitative which means that the
reaction should proceed to completion. A marked change in some property of the
solution indicates that the reaction has come to completion.

Lee Daryl Tsvangirai

REFERENCES
AK Yetisen, H Butt, CR Lowe (2013). "Light-Directed Writing of Chemically Tunable
Narrow-Band Holographic Sensors." Advanced Optical Materials.

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Arnold Thackray and Minor Myers, Jr.; foreword by James D. Watson. (2000). Arnold
O. Beckman: one hundred years of excellence. Philadelphia, Pa.: Chemical Heritage
Foundation.
Douglas Skory, Donald West, Stanley Couch (2004) Fundamentals of Analytical
Chemistry 8th Edition
John E Murray, Robert C. Fay (2008) Chemistry 5th Edition
Steven L. Murov, (2004). Experiments in General Chemistry, 4 th Edition, United
States
Travis, Anthony S.; Schrter, H.G.; Homburg, E.; Morris, P.J.T. (1998). Determinants
in the evolution of the European chemical industry: 1900-1939: new technologies,
political frameworks, markets and companies. Dordrecht: Kluwer Acad. Publ. p. 332.
Verhappen I. (1993) Advanced Instrument Control. The effects of winterizing pH
buffers.
Y Montelongo, J Blyth, JB Carmody (2012). How to Care for Your pH Meter.

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