Why would my new C18 column show longer retention for a strong acid (pH 3.7) than an old column of the same part number when running the same isocratic method but show no retention difference for a neutral compound? - WKB94543
ENVIRONMENT
- Reversed-phase chromatographyRetention
- C18 columns
- Selectivity shift
- Retention shift
ANSWER
New C18 columns tend to have more C18 ligand and endcap groups compared with old (used) C18 columns, which can affect retention and selectivity.
ADDITIONAL INFORMATION
Older C18 columns tend to lose both endcap groups and C18 ligand through a process called hydrolysis.
As hydrolysis occurs, more and more endcap groups and C18 ligands are cleaved off of the particle surface.
Each time an endcap group or ligand cleaves off of the particle surface, a silanol is exposed (Si-OH).
In a pH of 3.7, a strong acid is negatively charged. In addition, exposed silanols are mostly negatively charged.
If an old column has more exposed silanols, the packing will exhibit many more negative surface charges on the packing, compard with a new column of the same part number.
These negatively charged (deprotonated) silanols will repel the negatively charged acid more quickly as the number of exposed silanols increases.
On a new column of the same part number, there are far fewer exposed silanols, and, as a result, the strong acid will not be repelled as much by negative surface charges. It will, therefore, show a longer retention time on the new column.
In this scenario, because neutral compounds show no difference in retention on the old column vs. the new column, the amount of C18 ligand on the two columns should still be similar.
Also see: Should I develop a new LC method on a column that is used or old?
id94543,