MT 5003 50904989

Practical Report on the “Measurement of pKa value of

Sulphadimidine”

Aim:

To determine the pKa of a drug (Sulphadimidine) using spectroscopy.

Introduction:

Most of the drugs are either weak acids or weak bases. Weak acids or
base has a pKa value.

In general chemistry, the dissociation constant of an acid is called as

pKa, whereas the dissociation constant of a base is called as pKb. from
the pka values we can conclude that lower the pKa values, higher the ka
and stronger the acids is, or the lower the pKb values, the weaker the
bases.
(http://www.pharmacy.wsu.edu/courses/PharS532/Oral/Phy.html)

pKa value of a drug is very important because it affects the drug

molecules that are ionized and that which are in unionized forms. The
ratio of ionized over unionized form affects drug's absorption,
distribution and excretion at a given pH.
(http://www.pharmacy.wsu.edu/courses/PharS532/Oral/Phy.html)

For weak acid, the dissociation can be represented by-

HA↔ H+ +A- and the dissociation constant is Ka

Ka= [H+] [A-]

[HA]

[H+] = Ka [HA]

[A-]

Taking - log both the sides

-log [H+] = -log Ka -log [HA][A-]

We know -log [H+]=pH and -logKa =pka

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pH = pka +log [A-][HA] Henderson-Hasselbalch equation for

acids

similarly for weak basic, pH = pkb+ log [B+][HB+] or pH = pka+ log

[proton donor][proton acceptor]

rearranging the Henderson-hasselbalch equation

we get, pka = pH+ log [HA][A-]

(Nelson et al,2005)

For a majority of the drugs, dissociation constants are denoted as pKa,

irrespective of whether the drug is a weak acid or a weak base.

For acids, Ka denotes the ability of an acid to donate the proton. Higher
the tendency of an acid to loose the proton, the stronger the acid is,
whereas for bases, they have a tendency to accept the electron.
Strong base has high pKa value.

pka value can be determined by knowing the proportions of ionized and

unionized forms. There are various methods for the determination of
pka and spectroscopy is also one of the available methods which
depend on the change in absorbance shown with the changing pH.

Sulphadimidine is a weak acid which partly ionizes by loss of proton.

Sulphadimidine has 2 ionize able groups namely, NH2 and NHSO2 which
contributes to the pka .

Materials Required:

Sulphadimidine 50 μg/ml in water

Potassium dihydrogen phosphate (KH2PO4) 0.1 M (Solution A)

Disodium hydrogen phosphate (Na2HPO4) 0.1 M (Solution B)

Sodium hydroxide (NaOH) 0.1 M

Procedure:

1. pH meter is standardized using the provided standard buffer

solutions of pH 4, 7 and 9.
2. Switch on the Spectrophotometer.

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3. Eight buffers with pH ranging from 5.6 to 9.0 are prepared using
Solution A (0.1 M KH2PO4) and Solution B (0.1 M Na2HPO4) provided.
The pH of all buffers prepared is checked and recorded.
4. From the information available in Geigy table we can prepare buffer
of the below mentioned pH.

pH Solution A (0.1 M Solution B (0.1 M

KH2PO4) Na2PO4)

5.6 95.5ml 4.5ml

6.6 65.3ml 34.7ml

6.8 53.4ml 46.6ml

7.0 41.3ml 58.7ml

7.2 29.6ml 70.4ml

7.6 12.8ml 87.2ml

7.8 7.4ml 92.6ml

8.0 3.7ml 96.3ml

5. 9 test tubes are prepared, to each of the 9 tests tubes contains

Sulphadimidine drug with buffer solution in only 8 test tubes. In one
of test tube instead of buffer take 0.1 M NaOH solution.
6. Pipette 3.0 ml of Sulphadimidine solution and 3.0 ml of the
appropriate buffer in 8 test tubes and in one test tube 3.0 ml of
NaOH solution instead of buffer. Stopper the test tube and invert to
mix.
7. 9 test tubes of reference samples are prepared for the above
solutions. Each of the 9 test tubes contains, 3.0 ml of distilled water
and 3.0 ml of appropriate buffer and for one of the test tube 3.0 ml
of NaOH. Stopper them and invert to mix.
8. Using pH meter measure and note the pH of each combined
solution.(refer table 1)
9. Spectrophotometer is calibrated using the distilled water as blank.
10. Using the buffer solutions of pH ranging from 5.6 to 8.0 are taken in
quartz cuvettes and a absorbance spectrum is recorded for each
solution between 200nm and 400nm wavelengths.(refer graph 1)

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11. The spectrum obtained is carefully examined to identify where the

maximum changes in spectral lines of solutions are observed. The
spot where there is maximum diffraction between the lines is the
wavelength suitable for measuring the absorbance of all solutions.
(refer graph 1)
12.Now wavelength is set and absorbance for each solution containing
drug is recorded along with the appropriate buffer solution as
reference. (refer table 1)

Graph 1. Showing the spectral lines obtained for different pH solution

between 200nm and 400nm

Tabular column:

Table 1. showing the pH obtained and absorbance of different

solutions

Given pH pH of the pH of the Absorbance Absorbance

buffer prepared of the of the
prepared reference buffer at reference
samples 310nm samples at
310 nm

5.6 5.44 5.62 0.421 0

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6.6 6.76 6.61 0.398 0

6.8 6.68 6.66 0.399 0

7.0 6.88 6.9 0.383 0

7.2 7.18 7.13 0.340 0

7.6 7.54 7.51 0.259 0

7.8 7.77 7.74 0.205 0

8.0 7.96 7.92 0.168 0

NaOH - - 0.072 0

Result:

The pka for the Sulphadimidine was found to be 7.66 and the %
ionization achieved at 7.4 pH is 35.58%. (refer Q1 and Q2)

Discussion:

The result obtained clearly shows that sulphadimidine has a low

ionization which means that less number of molecules are available to bind
to the receptor hence efficacy can further be increased if greater ionization
of Sulphadimidine is achieved. As we know Sulphadimidine is a weak base
changing its various groups to make it a strong acid will increase its ka and
hence more ionization.

It is also important to note that the accuracy of the spectrophotometer is

not uniform in all the transmission range. Maximum accuracy is found at
36.8% transmission while between 20% -80% transmission there is an
relative error of ± 2%. Despite the short comings, the spectrophotometric
techniques are widely used for qualitative analysis of biological mixtures and
compound in their pure states.

Q1. Calculate the pKa of Sulphadimidine.

We have the pH and from pH we can find the pKa of the Sulphadimidine, by
the formula-

pka= pH ± log d-di(dm-d)

where, dm= Absorbance of the lowest pH

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di = Absorbance of the NaOH

d=Absorbance of each pH

1. Calculating the pka for the solution with pH=5.44

pka= pH ± log 0.421-0.072(0.421-0.0421)

=5.6± ∞

=0

2. Calculating the pKa for the solution for the pH=6.76,

pka= 6.76 ± log 0.398-0.072(0.421-0.398)

=6.76± log0.326(0.023)

= 6.76± log 14.17

=6.76 + 1.15

pka2 = 7.91

3. Calculating the pKa for the solution for the pH=6.68,

pka= 6.68 ± log 0.399-0.072(0.421-0.399)

= 6.68 ± log 14.86

=6.68 +1.17

pka3=7.85

4. Calculating the pKa for the solution for the pH=6.88,

pka= 6.88 ± log 0.383-0.072(0.421-0.383)
=6.88 ± log 8.18
=6.88+0.91
pka4= 7.79
5. Calculating the pKa for the solution for the pH=7.18,
pka= 7.18 ± log 0.340-0.072(0.421-0.340)
= 7.18 ± log 3.30
= 7.18 + 0.51
pka5=7.69

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6. Calculating the pKa for the solution for the pH=7.54,

pka= 7.54 ± log 0.259-0.072(0.421-0.259)
= 7.54 ± log 1.15
= 7.54 +0.06
pka6=7.60
7. Calculating the pKa for the solution for the pH=7.77,
pka= 7.77 ± log 0.205-0.072(0.421-0.205)
= 7.77 ± log 0.61
= 7.77 -0.21
pka7=7.56

8. Calculating the pKa for the solution for the pH=7.96,

pka= 7.96 ± log 0.168-0.072(0.421-0.168)
= 7.96 ± log 0.37
= 7.96 -0.42
pka8=7.54

To find the mean we antilog all the values and then find the mean of all
the pka’s and then again log it to get the pka of Sulphadimidine

Antilog of pka2=81283051.62
Antilog of pka3= 70794578.44
Antilog of pka4= 61659500.19
Antilog of pka5= 48977881.94
Antilog of pka6= 39810717.06
Antilog of pka7= 36307805.48
Antilog of pka8= 34673685.05

Mean pka= pka1+pka2 +pka3+pka4+pka5+pka6+pka7+pka88

Mean pka =
81283051.62+70794578.44+61659500.19+48977881.94+39810717.06+3630780
5.48+34673685.05

=373507219.8 =46688402.47
8

= log 46688402.47

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pka= 7.66

Q2. Calculate the percentage ionization of Sulphadimidine at pH 7.4

Given, pH=7.4 and we know pka=7.66

Using formula, pka=pH + log non ionisedionised

7.66= 7.4 + log non ionisedionised

7.66-7.4 = log non ionisedionised

0.26= log non ionisedionised

We know that log10100= 2 and 102=100

Using the above law of logarithms,

100.26 = non ionisedionised

1.81 = non ionisedionised equation 1.

Non ionized + ionized = 100 equation 2.

Rearranging equation 1.

1.81 ionized = non ionized putting this in equation 2

1.81 ionized +ionized =100 %

2.81 ionized =100

Ionized =1002.81

Ionized = 35.58

Thus, Sulphadimidine is 35.58% ionized at a pH of 7.4

Q3.As an additional experiment, another pka value for

Sulphadimidine (approx 2.0) can be determined. How might this
arise?

It is absolutely possible to find another pka value for Sulphadimidine, because

Sulphadimidine has 2 ionize able groups that are NH2 and NHSO2 and each
group ionizes at a different pH. In general case, proton donor or acidic group
ionizes at basic pH and basic group or proton acceptor ionizes at acidic pH.

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e.g from the titration curves of glycine we came to know that the two
charged groups- carboxyl group and the amino
group have different pka. Pka of carboxyl group is
2.34 where as pka for NH3+ group is 9.60. (Nelson et
al,2005)

Figure 1. Titration curves of glycine (image

taken from Nelson et al, 2005)

Q4. Plot a graph of absorbance against pH. What information can

you obtain from the graph? Why do we choose to read absorbance
at 300-310nm rather than say, 250nm?

The graph obtained is a bell shaped graph and we can infer from this graph
that as the pH is increasing absorbance increasing till a point then the
absorbance starts decreasing gradually.

We choose to read absorbance only at 310nm because while scanning to find

the appropriate wavelength for the drug, maximum split in the spectral lines
was observed at 310nm. So 310nm is the most appropriate wavelength at
which solutions could be measured.

Scale: 1 cm on x-axis = 2 units of pH

1 cm on y-axis =0.05 units of absorbance

Graph 2. Showing relationship between pH and absorbance

Q5. Comment on other methods for the determination of pka.

pka could be determined by-

1. Potentiometeric titration,
2. measuring the electrical conductance,
3. surface plasmon resonance analysis- we can measure the dissociation
constant and we know pka = 1/ka

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4. Capillary electrophoresis
5. Solubility methods (Barro’n et al,2000)
6. pka can also be measured as y-intercept of the graph where pH is
plotted against log

a HA x AA-

aA- x AHA

where AHA is the absorbance of HA, AA- is the absorbance of A- , aHA is the
absorbtivity of HA, and aA- is the absorbtivity of In- .
Q6. What factors influence the determination of pka?

1. Ionic strength of the charge groups

2. Temperature- The dissociation of a compound dependent on the
temperature thus it affects the pKa and the pH.
3. Addition of alcohol produces a change in the activity and concentration
of H+ hence pKa is affected.
4. Isoelectric point (pI): pI is that pH at which the amino acid has a net
total charge of zero. At this point pH=pka. Any change in the pI will
surely affect the pka. (http://www.iitk.ac.in/chm/Lecture1.html )

Q7. Discuss :

– The clinical applications of pka

The pka value decides how a compound would be in distributed
plasma, urine (alkaline) and gastric juice (acidic) at equilibrium.
Acidic drugs are present in the body where there exists basic or
high pH this is called ion trapping.
pH portioning in different compartments of the body explains the
renal excretion and penetration of the Blood-Brain Barrier by the
drug molecules.

Some of the effects that change in pH could produce-

– Urinary acidification speeds up excretion of weak bases
– Urinary alkalinisation speeds up excretion of weak acids
– Reducing the plasma pH causes weakly acidic drugs to become
concentrated in brain increasing the Neurotoxicity.

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– pH and pka influences the selectivity in liquid chromatography and

knowledge of pka is also essential to establish and optimize
analytical procedures for separation of compounds.
– The pka of E54 residue of c subunit of ATP synthase was found to be
7.7 which was higher than in alkaphiles. The high pka prevents the
loss of protons from c-rotor at high pH causing ATP synthesis defect.
(Torry et all,2004)
– pka values affect the binding of the drug to plasma proteins as well
as tissue protein and thereby affects the pharmacokinetics of the
drug.
– The variations in the pka of the carboxylic acid affects the binding
of human serum albumin and thereby affecting antibacterial
translational activity of antibiotic. (Stiff,2007)

– The pharmaceutical applications of pka.

– The majority of drugs are weak acids or weak bases, knowledge of

the dissociation constant helps in understanding the ionic form a
molecule. (Manallack,2007)

– The pKa of a drug influences lipophilicity, solubility, protein binding

and permeability which in also affects pharmacokinetic
characteristics such as absorption, distribution, metabolism and
excretion. (Manallack,2007)

– Formulation procedures for drug delivery and drug discovery

requires the knowledge of the pKa . (Manallack,2007)

– Knowledge of pka and its distribution in the body is very helpful

during screening purposes e.g. in combinatorial libraries.

– Molecular weight (MW), partition coefficient (log P), number of

hydrogen bond donors and acceptors, and polar surface area (PSA)
could be estimated from the pka value. (Manallack,2007)

– While dealing poor water solubility drugs adjusting the pka will
enhance the dissolution where dissolution is the rate-limiting step.

– Ionizable groups present on the drug affects its ability to interact

with a target. It has been shown that pKa influences the rate of
metabolism of drugs which are metabolized by CYP1A2.

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(Upathagrove,2001). From the study conducted on the compounds

targeting the human (hERG) potassium channel, it was found that
selectivity can be influenced by controlling pKa. (Lee et al,2008)

– Toxicity could also be related directly to a drug’s pKa at normal pH,

e.g. cardiovascular toxicity due to long duration between the start
of the Q wave and the end of the T wave or due to QT prolongation,
it is due to blockade of the hERG potassium channel. (Lee et
al,2008)

References:

– Annon. (1999).A Simplified Method for Finding the pKa of an Acid-

Base Indicator by Spectrophotometry. Journal of Chemical
Education. Vol. 76. p 395.
– Anon. Techniques and principles in Biophysics. p559-566
– Barro´n, D., Lozano, J.E., Irles, A., Barbosa, J., (2000). Influence of
pH and pKa values on electrophoretic behaviour of a quinolones in
aqueous and hydro-organic media. Journal of Chromatography .Vol
871.p 381–389
– http://www.iitk.ac.in/chm/Lecture1.html
(Indian Institute of Technology, Kanpur)
– http://www.pharmacy.wsu.edu/courses/PharS532/Oral/Phy.html
(Washington State University, College of Pharmacy)
– Lee,A.C., Yu, J.Y., Crippen,G.M (2008).pKa Prediction of Monoprotic
Small Molecules the SMARTS Way.J. Chem. Inf. Model. Vol 48 (10).p
2042–2053
– Manallack, T.D.,(2007). The pKa Distribution of Drugs: Application to
Drug Discovery Perspect Medicin Chem. 1: 25–38.
– Nelson, D.L.,Cox,M.M.(2005) Lehninger Principles of biochemistry.4th
edition
– Stiff,M.C.,(2007)Bioorganic & Medicinal Chemistry Letters 17 5479–
5482
– Torres,I.O.R., Koplin,R.D.K., Hicks,D.M., Cahill,S.M., Krulwich,T.A.,
Girvin,M.E.(2004). pKa of the essential Glu54 and backbone
conformation for subunit c from the H+-coupled F1F.FEBS Lett 575:
131-5.
– Upathagrove, A.L.,Nelson, W.L.(2001). Importance of Amine pKa and
distribution coefficient in the metabolism of fluorinated propanolol

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analogs: metabolism by CYP1A2. Drug Metab. Dispos.Vol 29.1377-

1388

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