Objectives:

1. To determine the analytical wavelength of furosemide by using different pH.

2. To determine the ionization constant of furosemide using UV spectrophotometer.

Introduction:
Ionisation constant, which is also known as dissociation constant is quantitative
measure used to determine the strength of acid and base based on their degree of ionization.1
Ionization constant can be used to determine the percentage of ions formed from a molecular
species. A strong acid will have high ionization constant and higher percentage of the
molecular species will dissociate in ionic species when compared to weak acid. However, the
percentage of ionization of a molecular species is greatly influenced by the pH of the
medium. When there is a small change in pH, this will lead to large change of the percentage
of ionization of molecular species. The value of ionization constant is normally expressed in
term of pKa. Henderson-Hasselbach defined the equilibrium expression in mathematical
equation.

pH= p K a +log
The pKa value is important in pharmaceutical industry. The pKa of a drug is an important
characteristic used to determine its pharmacokinetic action like solubility, degree of protein
binding and rate of absorption.2
Different ionic species have different ultraviolet absorbance spectra. Therefore,
spectrophotometer can be used to assess the substance. The direct determination of the ration
of the neutral molecular species against a series of ionized species in buffer solution of
known pH is used to determine the pKa of the species.
In this experiment, a pure unionized species is exposed to different wavelength and
the absorbances in different wavelength are recorded. The same step is repeated for pure
ionosied species. A wavelength is selected at which the greatest difference between the
absorbances of the two species is determined. This wavelength is known as analytical
wavelength. This analytical wavelength is important to calculate the ratio of ionized species
to molecular species at various pH values.
1

Results:

pH of the buffer solution

pH of the mixture after addition of

furosemide

2.00

2.29

3.00

3.10

4.00

4.04

5.00

5.03

6.00

6.26

Table 1: pH of the mixture containing the buffer solution and furosemide

pH

d-dI

dM-d

2.0
3.0
3.2
3.4
3.6
3.8
4.0
4.2
5.0
6.0

0.936
0.839
0.837
0.795
0.735
0.672
0.814
0.609
0.666
0.690

0.258
0.161
0.159
0.117
0.057
-0.006
0.136
-0.069
-0.012
0.012

-0.0485
0.0485
0.505
0.0925
0.1525
0.2155
0.0735
0.2785
0.2215
0.1975

Log |(d-dI)|
|(dM-d)|
0.726
0.521
-0.502
0.102
-0.427
0.00278
0.267
0.248
0.00542
0.00608

pKa
2.726
3.521
2.698
3.502
3.173
3.802
4.267
4.448
5.005
6.006

Table 2: Absorbance values for analytical wavelength of 230 nm and pKa values for furosemide

Room temperature =24C

Concentration of furosemide mixture = 1mL in 10mL (10%v/v)
Analytical wavelength = 230nm
Calculations:
Averaged absorbance value at pH 2 and 3 = dM

0.936 +0.839
2

= 0.8875

Averaged absorbance values at pH 5 and 6 = dI

0.666 +0.690
2

= 0.678

To find pKa, use the formula:

pKa= pH + Log |(d-dI)|
|(dM-d)|

Average pKa

2.276+ 3.251+ 2.698+ 3.502+ 3.173+ 3.802+ 4.267+ 4.448+5.005+6.00 6

10

3.843
pKa

= -log Ka

3.843 = -log Ka
Ka = 1.435 x 10-4

Discussion:
Furosemide is a loop diuretic drug which acts on the thick ascending limb of the
loop of Henle. It inhibits the Na+/K+/2Cl- carrier in the luminal membrane by combining with
the Cl- binding site.3 Thus it decreases the reabsorption of Na+ and Cl- by the ascending loop
of Henle.4 It is known as anthranilic acid or o-aminobenzoic acid derivatives. It possesses a
free carboxyl group which in fact is a weak acid.5 structure of furosemide is shown below:

Figure 1: Structure of furosemide

It contains three ionisable groups with different pKa values. However the pKa value of acidic
group (carboxylic group) is always be the focus. The pKa of carboxylic group of furosemide
is ranges from 3.9 to 4.2. The molecular species will predominate in acidic pH while the
ionized species will predominate at alkaline pH. UV spectrophotometer can be used to
determine the pKa value of furosemide. A pure unionized species is exposed to different
wavelength and the absorbances in different wavelength are recorded. The same step is
3

repeated for pure ionized species. Hence, a wavelength which is known as analytical
wavelength can be selected when the absorbance shows the greatest difference between the
two species. Based on the result obtained in this practical, the analytical wavelength of
furosemide is 230nm.
Table 1 shows the changes in pH on addition of furosemide to different buffer. Buffer
system of pH 2.0, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 5.0 and 6.0 has been prepared. After
addition of 1ml of furosemide to the buffer solutions, the change in pH is maintained within
0.05, where the addition of furosemide decreases pH up to 0.15. Buffers are solutions that
tend to resist changes in their pH as acid or base is added. Typically, buffer solution is
composed of a weak acid and its conjugate base.6 To resist the change in pH, the weak base in
the buffer solution accepts the hydrogen produced from the carboxyl group of the
furosemide. As a result, concentration of H+ ion in the solution remains fairly constant, pH
did not change much even after adding acidic furosemide.
From the results, the average absorbance values at pH 2 and 3, (d M) and average
absorbance values at pH 5 and 6 (dI) are calculated. The dM and dI values are 0.8875 and
0.678 respectively. Since furosemide is an acidic drug and d M value is greater than dI value,
the pKa value of furosemide is calculated using the following equation.
pKa = pH + log(d - dI) / (dM d)
The average pKa value of furosemide calculated is 3.843 which is slightly deviated from its
theoretical value which is between 3.9 to 4.2. This slight deviation of calculated pKa from its
theoretical value may due to experimental errors when conducting this practical. The
inaccurate absorbance value of spectrophotometer may due to the presence of impurities in
the prepared furosemide solution. Presence of impurities in furosemide solution increase the
overall concentration of solution and affect the scattering of UV wavelength in
spectrophotometer, thus an inaccurate absorbance value is obtained. This leads to inaccurate
result. Improper handling and operation of spectrophotometer can also cause inaccurate
absorbance values to be measured and recorded.

Conclusion:
As a conclusion, the ionization constant of a compound can be determined by using
UV spectrophotometer. The analytical wavelength of furosemide determined in this practical
is 230nm while the pKa value of furosemide calculated in this practical is 3.843. It is slightly
4

deviated from the theoretical value which the pKa ranges from 3.90 to 4.2. The slight
deviation may due to experimental errors when conducting this practical.
References:
1. Rang HP, Dale MM, Ritter JM, Flower RJ. Rang and Dales Pharmacology. 6th ed.
London: Churchill Livingstone; 2007.
2. Garrett RH, Grisham C. Biochemistry. Maryland (MA): Brooks/Cole Cengage
Learning; 2010. p. 41.
3. Troy DB. Reminton: the science and practice of pharmacy. 21st ed. Philadelphia (PA):
Lippincott Williams & Wilkins; 2006. p. 239.
4. Sinko PJ. Martins Physical Pharmacy And Pharmaceutical Sciences: Physical
Pharmacy and Biopharmaceutical Principles in the Pharmaceutical Sciences. 5th Ed..
Philadelphia: Lippincott Williams & Wilkins.
5. Ives HE. Diuretic Agents. In: Katzung B. G. Basic & Clinical Pharmacology. 8th ed.
United States: Mc-Graw Hill; 2001.
6. Marieb EN, Hoehn K. Human Anatomy & Physiology. 7th ed. San Francisco: Pearson
Benjamin Cumming; 2007. p. 1050.

Faculty of Pharmaceutical Sciences

PP 264/3 Pulmonary, Renal and Pharmacotherapy
Practical III: Determination of ionization constant by UV
spectrophotometer

Practical No.

Practical 3

Date of Laboratory

11th March 2015

Dickson Hng Zhi

Practical
Students Name & ID
No.
Group Members & ID
No.

1001335646

Wei
:

Cheng Wai Kit

1001334813

Eng Wan Yun

1001334907

Group No.

Group A9

Lecturer

Dr Anand

Absorbance
210
220
230
240
250
260
270
280

2
0.397
0.646
0.936
0.381
0.159
0.243
0.431
0.351

3
0.44
0.566
0.839
0.321
0.155
0.185
0.401
0.394

pH
4
0.431
0.619
0.814
0.348
0.151
0.221
0.391
0.425

5
0.526
0.646
0.666
0.297
0.133
0.213
0.389
0.43

6
0.477
0.606
0.69
0.245
0.089
0.21
0.384
0.408

Absorption spectrum of Furosemide

1
0.9
0.8
0.7
0.6
pH2
Absorbance

0.5

pH3

pH4

pH5

pH6

0.4
0.3
0.2
0.1
0
200

210

220

230

240

250

260

270

280

290

Wavelength