The method development in reversed-phase HPLC involves the optimization of different types of column and mobile phase parameters. The separation of acid and base molecules the pH plays a significant role in determining the selectivity, retention time, and in controlling the methods reproducibility and ruggedness. Below are some points on how the pH of the buffer/ mobile phase affects your columns and analysis of HPLC can help you quickly determine the best mobile phase conditions for an analyte.
Peak shape:
The pH of the buffer/mobile phase affects the peak area and retention time of the component as it affects the ionization state. The mobile phase pH can affect chromatography in many ways. Depending on the molecule being analyzed, pH can affect peak shape, retention, and selectivity. With a considerable non-polar analyte, the effect of pH will usually be unimportant for retention and resolution. In basic molecules, if the specific pH has not been determined based on the requirement of specific molecules, the silica surface silanols are protonated, reducing interactions with the basic molecules that cause the tailings.
Column stability and durability:
The pH of the buffer/mobile phase affects the life span and stability of the silica-based column. Most chromatographers use HPLC columns between the pH ranges from 3.00 – 08.00, however column degradation occurs within this pH range through two mechanisms.
Column degradation at higher pH:
The pH 07.00 or higher is the main cause of the dissolution of silica in the aqueous mobile phase. The dissolution rate will be influenced by numerous column parameters including bonding type and silica used in the column packing. The rate of dissolution of silica is also affected by many mobile phase parameters, such as organic composition, operating temperature and buffering strength.
Column degradation at lower pH:
At low pH (below 03), the acid hydrolysis of the bonded phase the surface of silica results in a change in the retention time of the component. Reducing pH increases the rate of hydrolysis.
Reproducibility:
The reproducible separation of the molecules is acquiring by choosing the pH of the mobile phase that gives good selectivity and retention, hence a column gives good durability and peak shape at the required pH.
Peak shape:
The pH of the buffer/mobile phase affects the peak area and retention time of the component as it affects the ionization state. The mobile phase pH can affect chromatography in many ways. Depending on the molecule being analyzed, pH can affect peak shape, retention, and selectivity. With a considerable non-polar analyte, the effect of pH will usually be unimportant for retention and resolution. In basic molecules, if the specific pH has not been determined based on the requirement of specific molecules, the silica surface silanols are protonated, reducing interactions with the basic molecules that cause the tailings.
Column stability and durability:
The pH of the buffer/mobile phase affects the life span and stability of the silica-based column. Most chromatographers use HPLC columns between the pH ranges from 3.00 – 08.00, however column degradation occurs within this pH range through two mechanisms.
Column degradation at higher pH:
The pH 07.00 or higher is the main cause of the dissolution of silica in the aqueous mobile phase. The dissolution rate will be influenced by numerous column parameters including bonding type and silica used in the column packing. The rate of dissolution of silica is also affected by many mobile phase parameters, such as organic composition, operating temperature and buffering strength.
Column degradation at lower pH:
At low pH (below 03), the acid hydrolysis of the bonded phase the surface of silica results in a change in the retention time of the component. Reducing pH increases the rate of hydrolysis.
Reproducibility:
The reproducible separation of the molecules is acquiring by choosing the pH of the mobile phase that gives good selectivity and retention, hence a column gives good durability and peak shape at the required pH.
You may also like this
No comments:
Post a Comment