spectroscopy and chromatography in physiology

spectroscopy

1. Qn. You are presented with a stock solution containing a 50 µg mL−1 solution of a drug. Prepare 100 mL of solution to contain 5, 10, 20 and 30 µg mL−1 of drug. - calculate how much of the 50 µg mL−1 stock solution will be required for each dilution. -[Required]/ [Stock] × volume required. - where [ ] represent the concentrations of drug. -to remember easily [Want] [Got] × volume of flask

2. Remember -[Required]/ [Stock] × volume required. - where [ ] represent the concentrations of drug. Note T o remember easily [Want]/ [Got] × volume of flask

3. Questions Qn :Which of the following compounds would you expect to be the last to elute from a reverse phase liquid chromatography column? a) CH3OH b) CH3CH2OH c) CH3CH2CH2OH d) CH3CH2CH2CH2OH Answer: d) CH3CH2CH2CH2OH. Explanation: Compounds with low boiling points elute first, followed by compounds with higher boiling points. The boiling point rises with increasing molecular weight. As a result of its large molecular weight and long carbon chain, CH3CH2CH2CH2OH (Butanol) has the highest boiling point. Therefore, it will elute last from a reverse phase liquid chromatography column. 2) Qn : Explain the effect changing the mobile phase pH will have on the retention time of (a) a basic drug (b) an acidic drug when analysed by reversed phase HPLC

4. Solution: b) Acidic Drugs: -Effect of pH Change: Acidic drugs are proton donors and are more ionized (deprotonated) at higher pH levels. When the pH of the mobile phase increases, these drugs lose protons and become negatively charged, which increases their polarity and decreases their retention time. Example: Ibuprofen is an acidic drug that demonstrates this effect. At a higher pH, ibuprofen is more ionized, leading to a shorter retention time due to its increased polarity. Conversely, at a lower pH, it is less ionized (more protonated), resulting in a longer retention time as it interacts more strongly with the hydrophobic stationary phase Example: Tramadol is a basic drug that exhibits this behavior. At a lower pH, tramadol is more protonated and less hydrophobic, leading to a shorter retention time in the HPLC column. Conversely, at a higher pH, it remains unprotonated, increasing its hydrophobic character and resulting in a longer retention time.

5. Key point When a drug ionises , it becomes significantly more polar and will, therefore, be expected to elute more quickly in reversed phase. It follows that a basic drug (e.g. local anaesthetics , antihistamines, alkaloids) will elute more quickly with a mobile phase of low or acidic pH, while acidic drugs (e.g. NSAIDs, barbiturates, phenols) will elute more rapidly when the pH of the mobile phase is increased to alkaline levels.