Introduction
Ultraviolet-visible (UV-Vis) spectroscopy can be used to determine the pKa of an ionisable compound, especially if a change of absorbance is observed. The pKa in this instance can be determined from pKa = pH + log (Ai - A*)/(A* - Au), where A* is the measured absorbance in a buffer of known pH at a particular wavelength, Ai is the absorbance of the fully ionised species, and Au is the absorbance of the unionised species.Here the wavelength used is one where there is the largest difference between the ionisedand the non-ionised species. Generally a suitable pH value is chosen where it is within ±1of the pKa. Experimentally, several attempts may be needed to locate the appropriate pH value.
Objectives
To determine the pKa and the partition coefficient of several pharmaceutical compounds through the use of ultraviolet and visible spectroscopy
Procedure
As stated in the Pharmaceutical Analysis 1 lab manual page 10-12
Result
Table 1.1 Absorbance and pKa of paracetamol, procaine and phenolphthalein in different conditions
| | Paracetamol | Procaine | Phenolphthalein |
| Maximum wavelength | 268nm | 265nm | 555nm |
| Hydrochloric acid | 0.492 | 0.086 | 0.001 |
| Sodium hydroxide | 1.530 | 1.234 | 1.620 |
| Carbonate bicarbonate buffer pH 9.2 | 0.839 | | |
| 0.1 M potassium phosphate buffer pH 2.5 | | 0.806 | |
| 0.1 M potassium phosphate buffer pH 9.0 | | 0.264 | |
| pKa | 9.4991 | 2.2741 | 9.712 |
Calculation
pKa of paracetamol =
= 9.2 -log0.5022
= 9.4991
pKa of procaine hydrochloride =pH-log((0.086-0.806)/(0.806-1.234))
= 2.5 -log1.6822
= 2.2741
pKa of phenolphthalein =pH-log((0.001-0.264)/(0.264-1.620))
= 9.0-log0.1940
= 9.712
Discussion
The chromophores of paracetamol, procaine and phenolphthalein function to provide colour to their respective molecules. It did this by absorbing visible light of certain wavelength (this allows the excited electrons jump from ground state to excited state) and reflecting the other wavelength (this resulting in colour of the molecules). In the presence of auxochrome, which is a group of atoms attached to chromophore also helps to modify the chromophore’s potential to absorb light, examples of auxochromes are hydroxyl group, amine group and aldehydes. An auxochrome which is directly conjugated or attached system of chromophore will be able to alter the wavelength and intensity of absorption of its’ chromophore. Auxophores is defined as a group of compound with at least one non-bonding electron pairs example nitrogen or oxygen-containing compounds which can interact with the extended chromophores.
Structure 1.1 Chemical structure of paracetamol
The paracetamol’s structure is shown above, it has a hydroxyl group and an amide group in para position of the benzene ring. These two functional groups are known as its auxochrome and thus highly conjugated molecules. Under basic condition (NaOH), the phenolic group is ionized via deprotonation; therefore, the oxygen atom will have extra lone pairs of electrons, a total of 3 lone pairs of electrons. These extra lone pairs of electrons will delocalize into the π system, hence contributing to greater conjugation effect. As a result, paracetamol will show higher absorbance (hyperchromic) in basic condition. However, under acidic condition (HCl), the phenolic group is not ionized/deprotonated, hence the lone pairs of electrons interacting with the π system is less as compared to that under basic condition. Besides that ,the nitrogen atom is protonated and losses its' As a result, the absorbance of paracetamol under acidic condition is reduced (hypochromic effect) due to the lesser conjugation effect. The theoretical pKa of paracetamol is 9.50 while the pKa of paracetamol obtained from this experiment is 9.4491. The slight difference in experimental pKa and theoretical pKa might due to random error such as the surface of the cuvette is not clear, the cuvette might be contaminated.
Structure 1.2 Chemical structure of procaine hydrochloride
In procaine hydrochloride case, it has amino group as its auxochrome. Under acidic condition, protonation takes place in amino group so it can no longer function as auxochrome due to absence of lone pair of electrons to react with benzene ring. As a result, the conjugation effect of procaine hydrochloride in acidic condition is decrease which in turn contribute to a decreased in absorbance value in acidic condition (hypochromic). Conversely, in basic condition (NaOH), the lone pairs of electrons at nitrogen are readily available to delocalize into the π system which in turn increases the conjugation effect. Thus, the absorbance of procaine hydrochloride in basic condition increased (hyperchromic). The experimental pKa value of procaine hydrochloride is 2.2741 while the theoretical pKa value is 2.5.
Structure 1.3 Chemical structure of phenolphthalein
Phenolphthalein, meanwhile, has two hydroxyl groups which can take part in the protonation or deprotonation when undergoes pH changes. Under basic condition (NaOH), the two phenolic groups will undergo ionization, thus increases the number of lone pairs of electrons that are available to delocalize into the π system. Therefore, the phenolphthalein in basic condition will have greater conjugation effect. Consequently, the absorbance of phenolphthalein in basic condition increases (hyperchromic). But, the phenolic groups are not ionized under acidic condition (HCl), thus the lone pairs of electrons interacting with π system is lesser compared to basic condition. The conjugation effect will also be lesser which causes absorbance of phenolphathalein in acidic condition to be reduced (hypochromic).
The electron delocalization usually does occur around the ring when in the acidic environment. ,but because of the presence of a carbon (joined with 4 single bonds) at the centre of the phenolphthalein molecule , the interaction between the rings is inhibited and resulting incomplete delocalization throughout entire molecules when under acidic condition, this further decreases the absorbance. Basic environment on the other hand will allow the molecule to rearrange and allow the delocalized extend over the entire molecules. The carbonyl group also contributes its lone pairs to delocalization under basic condition
The theoretical pKa of phenolphthalein is 9.30 while experimental pKa of phenolphthalein is 9.712, both are basic pKa. Since the colour intensity of phenolphthalein fade away upon expose to the air it is said to be unstable in the basic condition .This is because it reacts quickly with sodium hydroxide and effectiveness reducing corresponding to time. Hence, the absorbance got to be obtained before 10 minutes passed, or else the value would be lowered.
Conclusion
pKa of paracetamol , procaine hydrochloride and phenolphthalein are 9.4491 (alkaline), 2.2741 (acidic) and 9.712 (alkaline) respectively.
References
1. wikipedia.org.com
2. www.drugs.com/mmx/procaine-hydrochloride.html
3. http://www.mhra.gov.uk/home/groups/pl-a/documents/websiteresources/con015073.pdf
4. http://ibchem.com/IB/ibnotes/full/aab_htm/18.6.htm
5. http://www.chemistry.ccsu.edu/glagovich/teaching/316/uvvis/uvbasics.html
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