54 February / March 2019


Chromatography Today Help Desk Column Overloading - An issue of some sensitivity


Injecting too much onto a chromatographic system can distort the chromatography, diminishing efficiency and reducing assay sensitivity. Reducing column diameters will reduce sample consumption, solvent consumption and also improve sensitivity, however this needs to be done with care as column overloading can result. For columns that are equivalent in the type of stationary phase that is being used, and in length, a simple squared relationship exists between the different diameter columns and the injection volume. Thus reducing the diameter of the column by a factor of two will require a reduction in the injection volume of a factor of 4. Where different stationary phases and column lengths are being used the relationship is more complicated. The issue with injecting too much onto any form of chromatographic system is that of overloading the column. This article will highlight the symptoms, and identify the sources of this situation.


There are two types of overloading that can occur: mass overloading of the analyte as well as volume overloading. In addition to these two types of overload, care must be excercised with the selection of injection solvent as one with excessive elutropic strength can have deleterious effects which mimic volume overload.


Mass loading considerations


The peak shape associated with mass overloading is often referred to as a ‘shark fin’. This abnormal peak shape results from the bulk of the analyte eluting earlier than expected as a results of the stationary phase not having the capacity to retain the analyte.


Equation 1 Ap Vp εi


phase and will elute earlier than the analytes which were retained prior to reaching the overload state. This effectively shifts the centre of mass for the eluting peak further down the column, resulting in a reduced retention time. It is important to note that the surface area per pore volume plays a role in this loading capacity and it is not dcitated by surface area alone. Packing possessing a low pore volume results in more mass being packed into a column, and the amount of surface thereby increases with the mass of packing in a column. Thus, the loading capability of a column is dependent on the structure of the stationary phase media and the surface area. The surface area on typical packing materials can vary from 4 m2/g for non-porous beads up to 400 m2/g for highly porous media. It is evident from these surface area numbers, coupled with the array of pore structures, that the amount of material that can be retained will vary substantially and will result in a very large, typically a hundred-fold difference in the loading capacity between columns of the same dimension.


It is possible to estimate the amount that can be injected on different columns by using the following formula, which gives the phase ratio, a measure of the loading capacity of a column [1];


– surface area of packing (m2/g) – specific pore volume (mL/g) – interstitial fraction


ρ - density of substrate material (g/mm3)


Table 1 provides an idea of what can be loaded onto a column before overloading occurs, however it should be noted that these are very approximate and the loading capacity for each analyte / column configuration should be investigated experimentally.


Figure 1. Schematic example of mass overloading on a HPLC column, showing a characteristic shark fin.


When considering the acceptable mass loading of a column, it is important to appreciate that within any chromatographic system there are only so many active sites where retention can occur. Once all of these sites are consumed, any subsequent analyte molecules travelling through the column will not be retained by the stationary


Table 1. A guide to how much can be loaded onto a column before overloading occurs.


Column Dimension (length x i.d. / mm)


150 x 4.6 50 x 4.6 150 x 2.1 50 x 2.1


Loading estimate (mg)


15 5


0.3 0.1


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