The column loading depends on what sample is, how clean it is or how dirty/impurities it is, how soluble it is insolvent and the mobile phase. When we are performing a separation of analytes in HPLC, the column should not be overloaded because the response will be affected. As a common rule, do not load more than 5% of the column volume. Volume overload and mass overload are the types of overload in chromatography.
In mass overload, excess amount of analyte is injected and in volume overload excess amount of liquid is injected onto the column. The peak symmetry of analyte in high-performance liquid chromatography depends on the mobile phase composition, the column or stationary phase and the sample volume used. If the column is overloaded it will affect peak shape and peak height by increasing the peak broadening, tailing, and width.
The maximum injection volume in a column depends on several parameters such as.
- The type of detector has been used in it as it will affect the sensitivity issue.
- Column dimension i.e. particle size, internal diameter, and length.
- Type of the column used to separate the components.
- Once the analytical method is optimized, to determine the capacity of the particular packing material a loading study is performed on the analytical column.
Find out how much mass we need to separate the components. Once the required mass is recognized, several simple equations can be used to calculate the size of the column required for purification. As well, the preparative HPLC system needs to consider the maximum flow rate and backpressure and may limit the column size.
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