DETERMINATION OF PKA OF ACTIVE PHARMACEUTICAL INGREDIENT BY SPECTROMETRY

About Authors:
Kalpesh Ashara
Registered Pharmacist S.K.R.I.Medicines Centre, Rajkot, Gujarat, India,
M.Pharm Semester-I Student of B.K.Mody Govt.Pharmacy College Rajkot,
Department of Pharmaceutics GTU, Gujarat, India.
kalpeshashara@yahoo.com, kalpeshshr5@gmail.com*

Abstract:
Spectrophotometry is an attractive method for PKa determination in very diluted aqueous solution about 10^-5 to 10^-6M provided that the compound possesses P^H dependent light absorption due to the presence of a chromospheres in proximity to the ionization centers. Traditionally 6 to 8 aliquot solutions of samples in identical concentrations but with different P^H values are prepared & their absorption spectra are registered at single wavelength. This series of solutions can be generated by either preparing the sample in buffer solutions of known P^H or titrating the sample solution e.g. alkalimetry. Then half the absorbance of maximum plotted on graph & interpolated on x-axis that will give value of P^H is Pka & negative Antilog of that value at base 10 give value of ka i.e. Dissociation Constant (Pka=-logka). This exercise is carrying out in below assignment.

Reference Id: PHARMATUTOR-ART-1514

Introduction:
Very dilute solution of Aspirin i.e.10ug/ml in different P^H solutions gives Pka value directly from graph by use of UV absorption spectrometry & Digital P^H meter by means of titrimetry, but UV-visible spectrophotometry technique is most unique because of less time consuming, reproducible than other techniques.

Materials:
API (Aspirin), 0.2MKH2PO4, 0.2MNaOH

Instrument: UV-visible spectrophotometer, two matched quartz cuvettes with 1 cm path length, Digital P^H meter.

Method:
Prepare 6-8 aliquots of solutions of buffer. For preparations of buffer as IP 1996,firest take 27.18gm KH₂PO₄in 100ml of volumetric flask ,dissolve then make volume up to 100ml_.It will give 0.2M 100ml KH₂PO₄ & similar pattern freshly prepare 100ml 0.2M NaOH. Now place 50ml 0.02MKH₂PO₄ in 200ml volumetric flask add sufficient volume of 0.2MNaOH then make volume 200ml,so it will give 200ml specific P^Hbuffer solution:.

P^H	.ml of NaOH
6	5.6
8	46.1
4.2	3

For preparation of acid phosphate buffer 0.02MKH₂PO₄ in 200ml volumetric flask add sufficient volume of 0.2M HCl freshly prepared then add water to make volume 200ml which gives 200ml specific P^Hbuffer solution:

P^H	.ml of NaOH
2.2	49.5
4.0	0.1

Now with help of Digital P^Hmeter measure the P^Hof prepared buffer which is found that are as follows:

P^H of buffer	Actual P^H of Prepared Buffer by Digital P^H
2.2	2
4	3.5
4.2	6.6
6	7.7
8	9.4

And prepared 2:8 Aspirin: Buffer solution in which solution of aspirin is 100ug/ml, now measure the absorbance of each buffer aspirin preparation solution with help of UV-visible spectrophotometer at 226nm wavelength in which each buffer is considered as blank in each time.226nm λ_max is achieved first by taking spectrum of Aspirin solution in water by UV-visible spectrophotometer then 226 nm wavelength should remain constant even by diluting the solution of solution i.e. λ_max is that irrespective of concentration of substance. Here concentration of API should between in the range of 10^-5to 10^-6M so this method is applicable so concentration of aspirin is taking at 100µ/ml.

The absorbance of different prepared solutions is as follows:

2:8 Aspirin :Buffer Solution of Different P^H	Absorbance
2	0.658
3.5	0.615
6.6	0.283
7.7	0.111
9.4	0.016

The maximum Absorbance is 0.658 at 2 P^H. Plot a graph of Absorbance vs. P^H as follow:

Figure: Pka determination by Spectrometry

Now from half of max absorption if plotting from graph the value of P^Hon x-axis directly gives value of Pka, so from equation of chart i.e. y=0.094x+0.888 the value of y=0.658/2=0.329 is taken
.y=0.094x+0.888

0.329=0.094x+0.888

0.329-0.888/0.094=x=5.95

Pka=5.95

Now Pka=-logka

5.95=-logka

Ka=-Antilog of 5.95

.ka=1 X10^-6

Conclusion:
Pka determination by P^H metry is time consuming process, whereas by means of Spectrophotometry is easy & reproducible method.

Acknowledgement:
I am very much thankful to Analysis Department of B.K.Mody Govt.Pharmacy College Rajkot-3, Gujarat, India especially Dr.Nilesh K.Patel, Mr.Amit J.Vyas, Mr.Ajay I. Patel, Assistant Professors B.K.Mody Govt.Pharmacy College Rajkot, Gujarat, India & Mr.Vishal Rupapara & Mr.Nalin Rabadiya M.Pharm Semester-I Student of B.K.Mody Govt.Pharmacy College Rajkot, Department of Pharmaceutics GTU, and Gujarat, India for co-operation to me in this Assignment.

References:
1.   CHEM 335: Physical Biochemistry Lab pKa of a dye: UV-VIS Spectroscopy Flory Wong and Roxanne Cheung
2.   “UV-Visible Spectroscopy” URL: en.wikipedia.org/wiki/UV/VIS_spectroscopy> (Retrieved Apr 19 2007)
3.   MERCURY’S HELP DESK | UV-VIS SPECTROMETRY jr.stryker.tripod.com/physchem/pka.html> (Retrieved Apr 19 2007)
4.   Keiichiro Fuwa, B. L. Valle. “The Physical Basis of Analytical Atomic Absorption Spectrometry. The Pertinence of the Beer-Lambert Law.” Anal. Chem.; 1963; 35(8); 942-946.
5.   Mukerjee, Pasupati and Bannered, Kalyan. “A Study of the Surface pH of Micelles
6.   Using Solubilized Indicator Dyes” J. Phys. Chem., 68, 12, 3567 - 3574, 1964
7.   Indian Pharmacopoeia1996.

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