What is the purpose of adding a small volume, e.g 1ml of glacial acetic acid in the mobile phase during HPLC analysis? Thanks.
The purpose of adding a small volume of glacial acetic acid in the mobile phase during High Performance Liquid Chromatography (HPLC) analysis is to control the pH of the mobile phase and enhance the separation of certain compounds.
To understand why glacial acetic acid is added, let's first discuss the concept of pH. pH is a measure of acidity or alkalinity of a solution and ranges from 0 (acidic) to 14 (basic), with 7 being neutral. In HPLC analysis, the pH of the mobile phase can directly affect the separation and retention of analytes.
Glacial acetic acid is a weak acid with a pKa value of 4.76. By adding a small volume of glacial acetic acid to the mobile phase, the pH of the mobile phase can be adjusted slightly to be more acidic. This acidic pH helps in several ways:
1. Ionization of analytes: Some compounds may exist as charged species (ions) at specific pH values. By adjusting the pH, these compounds can be converted into their desired ionic form, making them more easily separated.
2. Enhanced separation: Changing the pH can alter the interactions between the analyte molecules and the stationary phase (column material). This can lead to improved separation between different compounds in the mixture, as well as increased selectivity and resolution.
3. Prevention of analyte degradation: Some compounds may be sensitive to pH changes and can undergo degradation or hydrolysis under certain conditions. By controlling the pH, the stability of these compounds can be preserved during the analysis.
It is important to note that the addition of glacial acetic acid should be done carefully, and the amount added should be optimized for each specific analysis. The concentration and gradient of glacial acetic acid can vary depending on the nature of the analytes, the column used, and the desired separation conditions. Consultation of method development guides or expert advice is recommended to ensure the appropriate use of glacial acetic acid in HPLC analysis.