The total time spent for a compound to separate from the column
after an injection is a Retention time. In chromatography analysis, several
components need to be separated and each peak has the specific retention time,
Because of its affinity towards the stationary phase, the analytes will spend a
different amounts of time on the column.
The retention time (tR) has a major economic significance in
each type of chromatography. The ideal situation would be to have appropriate
separation of all components with the proper symmetric peak shape in a minimum
period. RT depends on several factors e.g. Type of column used, length of the
column, analysis conditions, flow rate, and system temperature. Here are
mentioned some factors that affect the retention time in chromatography.
The flow rate of the system:
The flow rate is the major influence on the retention time of
the component.
If the flow rate is high, then retention time will decrease and
if the flow rate is low then RT will increase. High flow rates are reduced the
retention times but result in poor separation and high back-pressure.
Column length:
The retention time of the molecule also depends on the column
length. The long column will give longer retention, while the short column will
give short retention, but the longer column gives better separation than the
short column.
Mobile phase composition:
The mobile phase or solvent composition can have a significant
effect on the RT of the component. In HPLC the retention time is controlled by
adjusting the properties of the mobile phase.
Column temperature:
If the column temperature is high, the retention time is reduced
and the separation of the analytes is poor. This is because the analytes don’t
get appropriate time to interact with the stationary phase. Due to the
volatility of the solvent the temperature can affect the separation in the thin
layer chromatography (TLC), paper chromatography, and column chromatography.
Polarity and boiling point of the sample:
The boiling temperature is often related to the polarity of a
compound. The more polar analytes the higher its boiling point and taking more
retention time to separate from the column. The molecule's polarity is relative
to the polarity of the stationary phase in the column. Hence, the molecules
elute as per their affinity toward the stationary phase.
The volatility of the component:
The volatile analytes travel faster through a column than
non-volatile components in gas chromatography (GC). The volatility is related
to the size of the analyte and the boiling point.
The pH of the mobile phase and sample
High-performance liquid chromatography (HPLC) the pH of the
sample solution and mobile phase can affect the peak shape and the retention
time of the analyte, as it affects the ionization state of the component, and
hence the chemistry of interactions happening inside the chromatographic
column.
Stationary phase:
The affinity of the molecule is significant for the stationary
phase, not just the stationary phase. The stronger the affinity betters the
interaction, resulting in longer retention time.
The polarity of the stationary phase:
The retention time is increased when the polarity of the
stationary phase and compound are the same. This happens, as the molecule has
more interaction with
the stationary phase.
Consequently, the stationary phase selection will affect the retention time of the molecule.