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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 ]¿¿

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

pH = pka +log ¿¿ Henderson-Hasselbalch equation for acids

similarly for weak basic, pH = pkb+ log ¿¿ or pH = pka+ log

[ proton donor ][ protonacceptor ]

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rearranging the Henderson-hasselbalch equation

we get, pka = pH+ log [HA ]¿¿

(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.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.

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pH Solution A (0.1 M KH2PO4) Solution B (0.1 M Na2PO4)5.6 95.5ml 4.5ml6.6 65.3ml 34.7ml6.8 53.4ml 46.6ml7.0 41.3ml 58.7ml7.2 29.6ml 70.4ml7.6 12.8ml 87.2ml7.8 7.4ml 92.6ml8.0 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)

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)

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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 buffer prepared

pH of the prepared reference samples

Absorbance of the buffer at 310nm

Absorbance of the reference samples at 310 nm

5.6 5.44 5.62 0.421 06.6 6.76 6.61 0.398 06.8 6.68 6.66 0.399 07.0 6.88 6.9 0.383 07.2 7.18 7.13 0.340 07.6 7.54 7.51 0.259 07.8 7.77 7.74 0.205 08.0 7.96 7.92 0.168 0NaOH - - 0.072 0

Result:

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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

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))

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=6.76± log( ¿0.326¿ (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

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

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

8

=373507219.8 =46688402.47 8

= log 46688402.47

pka= 7.66

Q2. Calculate the percentage ionization of Sulphadimidine at pH 7.4

Given, pH=7.4 and we know pka=7.66

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Using formula, pka=pH + log (non ionised¿ ionised )7.66= 7.4 + log (non ionised¿ ionised )7.66-7.4 = log (non ionised¿ ionised )0.26= log (non ionised¿ ionised )

We know that log10100= 2 and 102=100

Using the above law of logarithms,

100.26 = (non ionised¿ ionised )1.81 = (non ionised¿ ionised ) 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.

0 1 2 3 4 5 6 7 8 9

Series2

pH→

Abso

rban

ce a

t 310

nm →

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

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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

4. Capillary electrophoresis5. 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

aHA 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 groups2. 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.

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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.

- 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)

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- 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. (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 analogs: metabolism by CYP1A2. Drug Metab. Dispos.Vol 29.1377-1388

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