journal of Clinical Pharmacy and Therapeutics (1992)17,233-239

Determination of dissociation constants of some pharmaceutical compounds using derivative spectrophotometry I. I. Hewala, F. A. El-Yazbi, A. Awad" and A. M. Wahbi Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, University of Alexandria, Alexandria 2 152 I ; 'Alexandria Co. of Pharmaceutical and Chemical Industries,Alexandria, Egypt SUMMARY

Derivative spectrophotometry is applied to the determination of the dissociation constants (pKa)of some weakly dissociating compounds. The pKa value(s) can be calculated by measuring the amplitude of derivative maxima (0)at the selected wavelengths for the dissociated, undissociated and partially dissociated forms of the compound. A graphical technique, based on plotting the derivative spectrophotometric titration curves and interpolating pKa at Dt. is also presented. Both methods are applied to pharmaceutical compounds with single and double dissociation constants. The results are in good agreement with the reportedpK, values of the investigated compounds. INTRODUCTION

The absorption spectra of organic compounds with acidic or basic functional groups are dependent on the pH of the medium, which affects their maxima and intensity. If the absorption spectra of the species involved in the reaction are sufficiently different, an absorption spectrophotometric method can be used for the determination of the dissociation constant of these compounds provided that Beer's law is obeyed. The correct selection of a wavelength at which the absorbances of the dissociated and undissociated forms are significantly different results in the calculation of the pKa values of weakly acidic and weakly basic drugs using equations (I) and (2),respectively.

&-At,) pKa= pH log (Ab- A,)

+

pKa =pH

-A,)

+log(Ad- Ab) (Ab

Correspondence:Professor Abdel-Mi M. Wahbi.

where A,, Ad and A, are the absorbances of the buffered, dissociated and undissociated forms of the drug solution, respectively. The dissociated and undissociated forms of a compound can be obtained by using a solution of high acidity or alkalinity. By using a series of buffer solutions of different pH values, a mixture of the two forms can be obtained. The absorbance method was used to determine the pK, values of different acidic and basic compounds (1-4). The application of the absorbance method is limited by the fact that the absorption spectra of the dissociated and undissociated forms must be sufficiently different. If these two spectra overlap or have no significant difference, the absorbance method may give erroneous results. The application of a derivative technique to spectrophotometry is very useful in resolving spectral overlap or interference (5).The literature reveals many applications of derivative spectrophotometry in pharmaceutical analysis of single or multicomponent dosage forms (6,7) and in stability studies of pharmaceutical products for the determination of intact drug in the presence of its degradation products (8). We present a general scheme for the application of derivative spectrophotometry for the determination of dissociation constants of some weakly dissociating drugs. Thus, to determine the pKa of weakly acidic or basic drugs, equations (3) and (4)can be applied. (3)

pKa =pH (1)

(2)

+log((Db-- 0,) Dd

(4)

Db)

where D,,,Dd and D, are the derivative (first, second or higher) amplitude of the buffered, dissociated and undissociated drug solutions, respectively. The method is applied for the determination of pKa value(s) of phenol,

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I. I. Hewala et al.

Compound

Concentration (mg %)

Phenol Propylparaben sodium Chlorocresol Benzoic acid Paracetamol Trimethoprim Phenobarbitone

2-5 0125 2.5 1 1 04 1

Table 1. Buffer solution used to dilute drug solutions

Buffer pH values* 8 8-15 8-8

4-14 9.29 71 6 10.5

10 8.3 90 4.24 9.49 7.2 7 11.5

12 8.5 9.25 4.34 9.68 73 8 12.5 'Reference no. (9).

Table 2. Assay parameters for the determination of dissociation constants of certain drugs Compound Phenol Propylparaben sodium

Method

Selected A (nm)

Dl D2 D,

293 297 300 305 300 305 270 or 227 275 or 232 290 295 288 300 265 268 248 253

0 2

Benzoic acid

Dl D2 Dl

Paracetamol

Dl

Chlorocresol

0 2

0 2

Trimethoprim Phenobarbitone

Dl D2 Dl 0 2

D, 0 2

pH meter model S.A. 520 Orion was used to measure PHMaterials and reagents

All the reagents used were of analytical grade. Phosphate and acetate buffer (9)solutions (double strength) were used. Reference solutions

Five hundred millilitres of aqueous solutions containing 25 rng of phenol, 25mg of propylparaben sodium, 25 mg of chlorocresol, 10 mg of benzoic acid, 10 mg of paracetamol, 80 mg of trimethoprim or 10 mg of phenobarbitone were prepared. The drugs were dissolved in about 10ml of ethanol, if necessary, followed by dilution with water to 500 ml. In the case of propylparaben sodium and trimethoprim, a 2 5 - d portion of the reference solution was diluted to 500 ml with water. Procedure

propylparaben, chlorocresol, benzoic acid, paracetamol, trimethoprim and phenobarbitone. MATERIALS A N D METHODS

Apparatus

A Perkin-Elmer model 551-S UV-VIS spectrophotometer and a Hitachi Model 561 recorder were used. The derivative spectra of test and reference solutions were recorded in I-cm quartz cuvettes over the range 350 to 200nm. Suitable settings are: scan speed 120 nm/min; chart speed 120 and 60 mm/min; mode D,and D,; response time 10 s; slit width 2 nm. A

Fifty-millilitre aliquots of the reference drug solution were transferred into 1OO-ml volumetric flasks and each flask was made up to volume with: (i) 0.2 N hydrochloric acid solution; (ii) 0.2 N sodium hydroxide solution; and (iii) buffer solutions, double strength, of different pH values (Table 1). The pH of each solution was measured. The absorbance, D,and D2values were measured at the selected wavelengths (Table 2) against a blank prepared similarly but using distilled water instead of the drug. RESULTS A N D DISCUSSION

Figures 1 and 2 show the absorption, D, and D2 spectra of the singly dissociated compounds: phenol,

Determination of dissociation constants

235

I-

I

I

250

I

270

l\-l

290

I

310

270

290

310

270

290

310

c

E

0

v

250

260 280 300 ?

260 280 300 3

X

330

\ I

!; I

l

l

260 280 300 20

(nm)

Fig. 1.Absorption, D,and D2spectra in 0.1N sodium hydroxide (-) 0-125mg%propylparaben sodium and (c) 2.5 mg%dorocresol.

propylparaben sodium, chlorocresol, benzoic acid, paracetamol and trimethoprim in 0.1N hydrochloric

290

and 0.1 N hydrochloricacid (- --)of: (a)2.5 mg%phenol, (b)

acid and 0.1N sodium hydroxide. The dissociation constant can be calculated by measuring the absorbance,

236

1.1. Hewalu et d.

J.

(0)

?\

I \

I:

I I

I

;

(-1

I

I I

I I

I

I

I

c:0

* I

I

I

I

I

L

210

I

250

290

250 270 310 35i0

X Fig. 2. Absorption, D,and D,spectra in 0 1 N sodium hydroxide (-) acid, (b) 1 mg%paracetamol and (c) 0.4 mg%trimethoprim.

260 280 300 320 (nm)

and 0 1 N hydrochloricacid (- - -) of: (a) I mg%benzoic

Determination of dissociafionconsfanfs 237 Table 3. Assay results for the determination of the dissociation constants of certain drugs

Mean 3- SDt Mathematical methods

Graphical method

Reported

D,

*PK,

Absorption

Phenol Propylparaben sodium Chlorocresol Benzoic acid Paracetamol Trimethoprim Phenobarbitone

10.0 8.4 92 4.2

9.91 9.93 8.26f 0.04 8.26 0.05 9-10f0005 9.20f0.03 4-69+0*11 4.16f 014 9.65f 0.03 9.63f 0.03 7.35k0.13 7-17 f0.03 736 f 0.23 7.25 f 0.07 11-98f020 11.95 f0-17

9.5 7.2 735 11.8

D,

DL

Compound

0,.

9.92 9-8 8.27& 005 8.26& 008 9-22f 002 9.17 f 006 4.26f 0007 9.52f 008 9-65 7.38& 008 7.1 7-30f 006 7.40f 0.22 11.85f031 12.04f 0.22

9.8 8.29f 0.05 9-2f 0.05 4-28f0-04 9.6 7-36f0.10 12.00f0-26

'For reported pK, values refer to reference (11). tMean of five experiments.

.1 i?

I

i \

(t 0 - (

(-

I

230

6

250

270

I

2

230

X (nm) Fig. 3.Absorption, D, and D2spectra of 1 mg% phenobarbitone in 0.1N sodium hydroxide (-), and buffer solution pH 10 (----).

D , and D , values of the full-dissociated, undissociated and buffered drug solutions at the selected wavelengths (Table 2 ) and substituting in the above mentioned equations 1-4. The results obtained are summarized in Table 3.

250

270

i 3

0.1N hydrochloric acid (-

- -)

Figure 3 shows the absorption, D , and D , spectra of phenobarbitone in 0.1 N hydrochloric acid, 0.1 N sodium hydroxide and phosphate buffer at pH 10.Two pKa values are present. Rosenberg i?z Jackson (10)used the D, method to determine only the first pKa value of

238

1.1. Hewala et al.

Fig. 4. Derivative spectrophotometrictitration curves of (a) 0-125mg%propylparaben sodium and (b)1 mg%phenobarbitone.

Determination of dissociation constants phenobarbitone using an in-the-cell (INC) titration method. The proposed method is used to determine the two pKa values of phenobarbitone. The first pKa value was calculated by measuring the derivative amplitude of the hydrochloric acid and the buffered (pH 10)drug solutions at the selected wavelengths (Table 2). Buffer pH 10 was chosen because the monodissociated form of phenabarbitone predominates. The calculation of the second pKa values, however, was based on measuring the derivative amplitude of the buffered (pH 10)and the sodium hydroxide drug solution at the nominated wavelengths (Table 2). Calculation of the pKa values of phenobarbitone were carried at two wavelengths (Table 2, Fig. 3).The results obtained are summarized in Table 3.

Graphical technique An alternative method is the graphical technique which is based on plotting the value of the derivative maxima, measured at the selected wavelengths, (Table 2 ) against the pH of the solution. The resulting curve is the derivative spectrophotometric titration curve. The dissociation constant is taken as the pH value when the concentration of the ionized and unionized forms of the compound are equal. Thus D+(at half neutralization) can be calculated by (D,+ Du)/2, where d and u are fully dissociated and undissociated forms of the compound. The pH which corresponds to the derivative value at Dt is taken as the pKa of the compound. Figure 4 shows the derivative spectrophotometric titration curves of single (4a)and double (4b)dissociated compounds. The single dissociated compounds have only one break. In the case of phenobarbitone, a compound with two pK, values, the titration curves are plotted at narrow pH intervals and show two breaks. Its shape differs according to the wavelengths used (Fig. 4b). The results obtained for the determination of pK, values of the above mentioned compounds by the mathematical and graphical methods are in good agreement with the reported pKa values (Table 3 ) (1 I).

239

REFERENCES 1. Flexser LA, Hammett LP, Ding Wall A. (1935) Determination of ionization by UV spectrophotometry: Its

validity and its application to the measurement of the strength of very weak bases. Journal of the American Chemical Society, 57,2103-2115. 2. King FT, Hirt RC. (1953) A continuous-flow method for determining dissociation constants by UV spectrophotometry. Applied Spectroscopy, 7, 164-167. 3. Brown HC, McDaniel DH. (1953) The base strengths and UV absorption spectra of the 2- and 3- monohalopyridines. Journal of the American Chemical Society, 77, 3752-3 755. 4. Mattco BN. (1956) Spectrophotometric determination

of dissociation constants of p-hydroxy benzoic acid. Transactions of the Faraday Society, 52, 1462-1465. 5. Wahbi AM, Ebel S. (1974) The use of the first-derivative curves of absorption spectra in quantitative analysis. Analytica Chimica Acta, 7 0 , s 7-63. 6. Fell AF. (1978) Analysis of Pharmaceutical dosage forms by second derivative UV. Spectrophotometry, Proceedings of the Analytical Division of the Chemical Society, 15, 260-267. 7. Korany MA. Wahbi AM, Mandour S, El-Sayed MA. (1985) Determination of certain drugs in mdticomponent formulations by first derivative UV spectrophotometry. Analytical Letters, 18(BI), 21-34. 8. Korany MA, Wahbi AM, Hewala 111. (1984) Determination of procaine hydrochloride and benzocaine in the

presence of 4-aminobenzoic acid using first derivative spectrophotometry.Archives of Pharmacy and Chemistry, Science Edition, 12, 26-30. 9. Lenter D. (ed.) (1984) Ceigy Scientific Tables, Vol. 3, 8th edn. Ceigy Pharmaceutical Company, Manchester. 10. Rosenberg LS, Jackson JL, (1989) Application of a second-order derivative spectroscopictechnique: Determination of the ionization constants of phenytoin and phenobarbital.Drug Development and Industrial Pharmacy, 15,373-386. 11. Moffat AC (ed.)(1986) Clark's Isolation and Identificationof

Drugs. The Pharmaceutical Press, London.

Determination of dissociation constants of some pharmaceutical compounds using derivative spectrophotometry.

Derivative spectrophotometry is applied to the determination of the dissociation constants (pKa) of some weakly dissociating compounds. The pKa value(...
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