HPLC column loading capacity

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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Injection volume in HPLC

Injecting samples into the HPLC injector is very simple; this is done by manual syringe or auto-sampler injection.  The Rheodyne sample loops and auto-sampler have fixed or variable volumes (0.1 – 100 μL) that can inject into the HPLC column. The peak area and height increase in proportion to the injection volume of the sample solution. When the injection volume becomes too high the result in the peak is broadened and broadening this peak may reduce the resolution between adjacent peaks. The injection volume must be chosen in accord with the column geometry. We can use a volume between 1 and 5% of the column dead volume as the maximum injection load per column; however, we have to determine this. The best injection volumes are related to the volume of the HPLC column and, as a result, are dependent on the column length and cross-sectional area.

Here some approximate volume range of the sample component is mentioned if it is prepared in the same mobile phase as used in the system.

 

Column ID	Length of Column	Injection Volume
2.1 mm	30 to 100 mm	1 to 3 µL
3.0 to 3.2 mm	50 to 150 mm	2 to12 µL
4.6 mm	50 to 150 mm	8 to 40 µL
10 mm	50 to 250 mm	40 to 100 µL
21.2 mm	50 to 250 mm	150 to 300 µL
30 mm	50 to 250 mm	300 to 700 µL
50 mm	50 to 250 mm	1000 to 2000 µL

 

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