Determination of Dissociation Constant (PKA) of Enalapril maleate by Electromagnetic Instrument Based Method

 

Amrutkar R. D.1*, Shahare H. V.2, Rakibe V. D.1

1Mahatma Gandhi Vidyamandir’s Samajshri Prashantdada Hiray College of Pharmacy,

Loknete Vyanktrao Hiray Marg, Malegaon Camp, Malegaon-423105, Dist: Nasik, Maharashtra, India.

2SSDJ SNJB College of Pharmacy, Chandwad Dist: Nasik, Maharashtra, India.

*Corresponding Author E-mail: rakesh_2504@yahoo.co.in

 

ABSTRACT:

A simple, rapid and precise Electromagnetic Instrument Based Method (Conductometry and Potentiometry) has been developed for the determination of Dissociation constant. Dissociation constant is playing a great role in the drug absorption in vivo, especially in case of oral administration. So, in drug designing, pKa i.e. the negative logarithm of dissociation constant determination is very important. In this regards we report electromagnetic types of instrument-based procedures with principles elaborated to determine the Pka of Enalapril Maleate, an ACE inhibitor type of antihypertensive drug. The Dissociation Constant (PKa) of Enalapril Maleate was found to be 2.7 and 2.6 by the Potentiometric titration and Conductometry methods respectively.

 

KEYWORDS: Potentiometer, Conductometer, ACE inhibitor, pKa (Dissociation constant).

 

 

 


INTRODUCTION:

The negative logarithm of the acid dissociation constant (Ka).  Just like the PH, the Pka tell us the acid or basic properties of a substance. Pka < 2 means strong acid, pKa > 2 but < 7 means weak acid, > 7 but < 10 means weak base, pKa >10 means strong base.1 The Ka value is a value used to describe the tendency of compounds or ions to dissociate. The Ka value is also called the dissociation constant, the ionization constant and the acid constant.

 

 

 

 

 

 

The dissociation constant and lipid solubility of the drug as well as the PH at absorption site often dictate the absorption characteristics of a drug from solution the fraction of drug in solution that exist in Non-ionized form is a function of both the dissociation constant of the drug and the PH of solution. The dissociation constant is often expressed for both acids and bases as Pka (Negative logarithm of dissociation constant). The Pka value is related to the Ka value in a logic way. Pka values are easier to remember than Ka values and pKa values are in many cases easier to use than Ka values for fast approximations of concentrations of compounds and ions in equilibriums.2

 

AIM OF THE PRESENT WORK:

The present research work compiled electromagnetic types of instrument-based procedures with principles elaborated to determine the Pka of Enalapril Maleate, an ACE inhibitor type of antihypertensive drug. The electromagnetic methods used were Potentiometric and Conductometric method.

 

APPARATUS AND INSTRUMENTS:

 

1.     PH-METER /POTENTIOMETER (MAKE - CHEMOLAB)

 

 

2.     CONDUCTOMETER (MAKE – TOSHCON)

 

CHEMICALS:

0.1N KOH solution, 0.1N HCl solution, BUFFER solution (pH- 4, pH-7 and pH- 10).( 0.01N KCl solution., 0.01M, 0.02M, 0.001M, 0.002M and 0.005M solution of Enalapril Maleate.

 

ENALAPRIL MALEATE:

Enalapril maleate is chemically described as 1-[N-[(S) - 1 –carboxy- 3-phenylpropyl] L - proline 1’- ethyl ester, maleate (1:1). Its molecular weight is 492.53.1 It is the active angiotensin converting enzyme inhibitor. It is an ideal drug for hypersensitive patients who are intolerant to beta-blockers. The drug and their tablets are official in IP 1996.1 It is of off white crystalline powder. The Enalapril Maleate is freely soluble in methanol and in Dimethylformamide, also soluble in ethanol (95%), sparingly soluble in water, and slightly soluble in semi polar organic solvents, practically insoluble in non-polar organic solvents. It stores in well closed containers. It contains not less than 98% and not more than 102 % of Enalapril maleate, calculated with reference to dried substance. The Specific optical rotation of Enalapril Maleate between 410 and -43.5 0, determine in a 1% w/v solution in methanol. Melting point: 1440C.5

 

 

APPLICATIONS AND SIGNIFICANCE:

In biochemistry; the pKa values of proteins and amino acid side chains are of major importance for the activity of enzymes and the stability of proteins. Protein pKa values cannot always be measured directly, it may be calculated using theoretical methods. Buffer solutions are used extensively to provide solutions at or near the physiological pH for the study of biochemical reactions.3 The isoelectric point of a given molecule is a function of its pKa values, so different molecules have different isoelectric points. This permits a technique called isoelectric focusing , which is used for separation of proteins by 2-D gel polyacrylamide gel electrophoresis.3 In acid-base extraction, the efficiency of extraction of a compound into an organic phase, such as an ether, can be optimized by adjusting the pH of the aqueous phase using an appropriate buffer. At the optimum pH, the concentration of the electrically neutral species is maximised; such a species is more soluble in organic solvents having a low dielectric constant than it is in water. This technique is used for the purification of weak acids and bases. A pH indicator is a weak acid or weak base that changes colour in the transition pH range, which is approximately pKa ± 1. The design of a universal indicator requires a mixture of indicators whose adjacent pKa values differ by about two, so that their transition pH ranges just overlap. In pharmacology ionization of a compound alters its physical behavior and macro properties such as solubility and lipophilicity (log p). For example, ionization of any compound will increase the solubility in water but decrease the lipophilicity. This is exploited in drug development to increase the concentration of a compound in the blood by adjusting the pKa of an ionizable group. Knowledge of pKa values is important for the understanding of coordination complexes, which are formed by the interaction of a metal ion, Mm+, acting as a Lewis acid, with a ligand, L, acting as a Lewis base. However, the ligand may also undergo protonation reactions, so the formation of a complex in aqueous solution could be represented symbolically by the reaction

 

[M(H2O)n]m+ +LH [M(H2O)n−1L](m−1)+ + H3O+

 

To determine the equilibrium constant for this reaction, in which the ligand loses a proton, the pKa of the protonated ligand must be known. For example, hydrogen cyanide is a very toxic gas, because the cyanide ion inhibits the iron-containing enzyme cytochrome c oxidase. Hydrogen cyanide is a weak acid in aqueous solution with a pKa of about 9. In strongly alkaline solutions, above pH 11, say, it follows that sodium cyanide is "fully dissociated" so the hazard due to the hydrogen cyanide gas is much reduced. An acidic solution, on the other hand, is very hazardous because all the cyanide is in its acid form. Ingestion of cyanide by mouth is potentially fatal, independently of pH, because of the reaction with cytochrome-C oxidase. In environmental science acid–base equilibria are important for lakes and rivers; for example, humic acids are important components of natural waters. Another example occurs in chemical oceanography  in order to quantify the solubility of iron(III) in seawater at various salinities, the pKa values for the formation of the iron(III) hydrolysis products Fe(OH)2+, Fe(OH)2+ and Fe(OH)3 were determined, along with the solubility product of iron hydroxide.4 Stomach has a pH range of 1 to 3. Beyond the pylorus, in the duodenum of the small intestine, the pH changes sharply and range between 5 to 7. The pH of the intestinal fluid increases gradually until a maximum pH of 8 is reached in the colon. In the stomach the absorption of the weak organic acids is more, whereas absorption of weak organic base will be minimal. The reverse will be true in the lower intestine. Studied revealed that weak acid and strong bases have a larg pKa value, whereas weak bases and strong acids have a small pKa value. It is also noted that, a small change in pH can make a large alteration of the percentage ionized. The presence of least ionized substances describes the maximum isolation, which is important in preparative chemistry.3 A significant ultraviolet spectrophotometry can be done only when the pH is so chosen that only one ionic species is present. Ionization constants, too, can help to discover the structure of newly isolated substances. Tautomerism can also be identified, as the structure with the weaklier acidic proton is favored because it must have the mobile hydrogen more firmly bonded. The presence of covalently bound water can be detected in a heterocyclic amine by the high pKa values that it endangers, by strengthening bases and weakening acids. To distinguish between a zwitter ion and an ordinary ampholyte, the pKa determination is very important. For the easily applied test for zwitter ions, the followings are the best.3

 

I. If one of the pKa values is markedly different from that of a partly blocked derivative, such as an ester or an O-ether, the substance is a zwitter- ion.

II. If the pKa, found by titration with acid, rises or is stationary instead of falling, when the titration is repeated in 50-70% ethanol, the substance is a zwitter- ion.

III. If the substance absorbs in the ultraviolet and one group is a carboxylic acid (the spectrum of which should not change appreciably on ionization), and the other group is an aromatic amino group, then a large shift, in the long wave band of the spectrum, to shorter wavelengths on adding alkali indicates that the substance is a zwitter-on.

 

Geometrical isomers can be identified by determination of pKa e.g. axial carboxy groups are weaker than their equatorial analogues. Differences in pKa values from the basis for separating many chemically similar substances e.g. penicillins.3 It is observed that the rate of decrease of pKa found as the temperature is raised. The temperature effect is much greater with stronger than with weaker substances (e.g. bases). It is also reported that nitrogenous bases are highly sensitive to temperature and become weaker as the temperature is increased. Generally, the pKa values express its positive value, but sometime it is also found that some organic substances show its pKa value with negative sign, e.g. Hydrazinium ion in water at 250C is -0.88, for 2-nitro aniline it is 0.26.3

 

EXPERIMENTAL WORK:

The Dissociation Constant (PKa) of Enalapril Maleate is 2.97 determined by the Potentiometric titration is reported.6 Several methods for determination of dissociation constant is reported methods including Potentiometric method are Spectroscopic method, Solubility method, Conductometric method etc3, the present work reveals the Potentiometric and Conductometric (Electromagnetic) method for determination of Dissociation constant.

 

PH – METRY

 

Principle:

It Involves the measurement of the PH followed by the calculation of concentration of ionized and Non-ionized part of the test compound, which is a neutralization procedure by a standard acidic or basic solution.7,8,9

 

Procedure:

1.      0.01M solution of the Enalapril Maleate is prepared by dissolving in to 5 ml of methanol and diluted up to 50 ml with Distilled water.

2.      Resulting solution is titrated with 0.1 N aqueous potassium hydroxide solution (for acidic drug).

3.      The PH meter can be calibrated by the PH tablets before 30 minutes of the titration.

4.      The burette containing 0.1 N aqueous potassium hydroxide solution is now fitted.

5.      47.5 ml of 0.01M solution of the Enalapril Maleate in titrating vessel has to be taken and the pH is to be recorded.

6.      The titration has to be performed by addition of 0.5 ml portion of aqueous potassium hydroxide solution from burette and the pH after each addition to be measured.

7.      Addition of titrant volume is to be continued up to 5 ml. (10 nos. of addition.)

8.      The pKa is then calculated by addition of Henderson-Hassel batch equation for each addition of titrant and the mean value is generally reported.

 

For acidic drugs: pKa = pH + log[HA]/[A-]

 

For basic drugs: pKa = pH + log[BH+]/[B]

 

Where,

HA is the unionized form of acid,

A- is the ionized form of base.

 

CONDUCTOMETRY:

Principle involves the measurement of the electrical conductivity of solution of the test compound of known strength, which is entirely due the movement of ions. The cell constant of Conductometric cell can be determined first by determining the conductivity of 0.01 N KCl. The Specific Conductance of 0.01 N KCl at 18 0 C is 1221 micromhos in aqueous media. The cell constant (K) = specific conductance of KCl /Observed conductivity of KCl7,8,9

 

Procedure:

1.    The cell constant of conductometric cell can be determined first by determining the conductivity of 0-01 N KCl. The specific conductance of 0-01N KCl at 180C is 1221 micromhos in aqueous media. The cell constant (K) = specific conductance of KCl /Observed conductivity of KCl

2.    A series of known concentration of solution of drug sample is to be prepared in a suitable solvent. The concentration may be 0.1, 0.2, 0.5, 0.02, 0.05, 0.001, 0.002, 0.005M etc. At least four no. of concentrated solution has to be prepared and they are to be applied to observe the conductance.

3.    A plot is then to be drawn taking 1/conductance (1/A) in y-axis and molar concentration (—) in x-axis. A straight line, which will be obtained, to be extrapolated to y-axis and point of intercept to y-axis is to be found out and it indicates the conductance at infinite dilution.

4.    After the PKa can be calculated for each concentration according to the formula mentioned below and then mean value of PKa is generally reported.

 

(i)As=A.K (218) Ac= (As ×1000)/c (218) α=Ae/Ao (218v) Ka=cα2/ (1-α) (v) Pka= - log ka

 

Where A=Observed Conductance, A0= Conductance at infinite dilution, As=Specific Conductance, Ae=Equivalent Conductance, α= the change in the extent of Ionization (fraction ionized), Ka= Ionization or Dissociation constant, k is cell constant.

 

RESULT AND DISCUSSION:

Observations for pH-metry: Temperature – 27 0C

 

Observation Table:

SR. NO.

ML OF TITRANT ADDED (0.101 M KOH)

PH

1

0.0 ML

1.95

2

0.5 ML

2.06

3

1.0 ML

2.18

4

1.5 ML

2.30

5

2.0 ML

2.44

6

2.5 ML

2.60

7

3.0 ML

2.76

8

3.5 ML

2.99

9

4.0 ML

3.26

10

4.5 ML

3.58

11

5.0 ML

3.92


 

CALCULATIONS:

Volume of titrant (0.101 M KOH) ml

PH

[A-]=volume of titrant added×0.1

Total volume of solution

[HA]= 0.01-[A]

Log[HA]/[A]

Pka = pH+ Log[HA]/[A]

0.0 ML

1.95

-

-

-

-

0.5 ML

2.06

0.00104

0.00896

0.935

2.99

1.0 ML

2.18

0.00206

0.00794

0.585

2.76

1.5 ML

2.30

0.00306

0.00694

0.355

2.65

2.0 ML

2.44

0.00404

0.00596

0.168

2.6

2.5 ML

2.60

0.005

0.005

0

2.6

3.0 ML

2.76

0.00594

0.00406

- 0.16

2.6

3.5 ML

2.99

0.00686

0.00314

- 0.33

2.66

4.0 ML

3.26

0.00776

0.00224

- 0.53

2.73

4.5 ML

3.58

0.00865

0.00135

- 0.80

2.78

5.0 ML

3.92

0.00952

0.00048

- 1.29

2.63

 

TOTAL= 27.0

Mean value of pka was found to be 2.7.

RESULT – The Pka Was Found to be 2.7 by Potentiometric Method.


 

OBSERVATIONS FOR CONDUCTOMETRY (Temperature – 26 0C)

CELL CONSTANT (K) = Specific conductance of KCl / Observed conductance of KCl

                                     =   13.10/ 14.11

                                       =   0.9                                     

 

 

 

OBSERVATION TABLE –

SR. NO.

CONCENTRATION OF SAMPLE (C)

OBSERVED CONDUCTANCE (A)

SPECIFIC CONDUCTANCE (AS)

1.

0.02 M

15.34

13.806

2.

0.01 M

11.1

9.99

3.

0.005 M

8.05

7.24

4.

0.002 M

4.74

4.26

5.

0.001 M

3.17

2.85


 

 

 

 

CALCULATIONS:

SR. NO.

C

A

AS = A × K

AC = (AS ×1000)/C

α =AC/A0

Ka =C α2/(1-α)

pKa = - Log Ka

1.

0.02 M

15.34

13.806

690300

0.242

0.00154

2.81

2.

0.01 M

11.1

9.99

999000

0.350

0.00187

2.72

3.

0.005 M

8.05

7.24

1448000

0.508

0.00262

2.58

4.

0.002 M

4.74

4.26

2130000

0.747

0.0044

2.35

5.

0.001 M

3.17

2.85

2850000

1.0

-

-

 

TOTAL = 10.46

Mean value of pka was found to be 2.61.

RESULT – The Pka was found to be 2.61 by Conductometric method.

 

 


CONCLUSION:

This work shows the application of pH-meter and conductometer other than assay. Students can be exposed to all practical aspects and thus, determination of pKa can be successfully implemented in routine practical classes of undergraduate laboratories. 

 

FUTURE PLAN OF WORK:

The Dissociation Constant (PKa) of Enalapril Maleate is 2.7 and 2.6 determined by the Potentiometric titration and Conductometry methods respectively. Several other methods for determination of dissociation constant which are reported methods are spectroscopic method solubility method etc. the present work reveals the Potentiometric and Conductometric method determination of Dissociation constant. However, in future the plan is to determine pKa of Enalapril maleate by10

 

1.    Spectrophotometric Method:

Involves the measurement of the absorbance of the test compound in different pH arranged gradually solution from acidic to basic range, which shows different absorbance due to increase or decrease of number of ionic species

2.    Involves the measurement of λ max in UV range in suitable pH solution thereby determination of intrinsic solubility and solubility at pH by applying the Beer’s plot, which demands the appropriate and correct dilution11-12.

 

REFERENCES:

1.       http://www.everythingbio.com/glos/definition.php?word=pKa

2.       http://www.phscale.net/pka-ka.html

3.       http://en.wikipedia.org/wiki/Acid_dissociation_constant

4.       William, O. F., Principles of medicinal Chemistry, 3rd edition, Varghese publishing House, 1989; 28.

5.       Indian pharmacopoeia 1996, published by the controller of publication, Delhi, Government of India ministry of health and family welfare, volume-1,4th edition, ; 281

6.       Analytical profiles of drug substances, edition 1st volume 16th, edited by Klaus Florey, published by Elsevier, ; 227

7.       Willard. H. et al., Instrumental Methods of Analysis, 6th edition. CBS, Delhi, 1999.

8.       Natarajan A, Sapre V., Hadkar U.B. and shorodkar p.y., Indian drugs, 1992; 29: 545.

9.       Albert, A. and sergeant, E.P., the determination ionization constants-a laboratory manual, 3rd edition, Chapman & hall, New York, 1984

10.     Dean, A.J., Analytical Chemistry, Handbook Mc-graw Hill, New Delhi,1955: 337

11.     Chatwal, G. and Anand, S., Instrumentals methods by chemical analysis, Himalaya, Bombay, 1995: 424.

12.     Das, R.C. and Behera, B., Experimental physical Chemistry, Tata Mc-graw Hill, New Delhi,1983: 201.

 

 

 

 

 

 

 

Received on 15.07.2018       Accepted on 16.10.2018     

© Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2018; 8(4): 215-219.

DOI: 10.5958/2231-5675.2018.00039.X