Column choice is a critical aspect for chromatography analysis. But it’s also one of the most difficult areas to advise on – given the breadth of options available. In this post, we’ll advise on some of the most important considerations when choosing a column.
The first question you’ll need to answer is which mode of separation you’ll be using. That includes things like normal-phase, reverse-phase and size-exclusion HPLC. This will determine the type of column you need.
Normal-phase columns contain a polar stationary phase, for instance, while reverse-phase columns are the opposite with a non-polar stationary phase. This in itself depends on the analytes in question, with reverse-phase chromatography better suited for separating organic compounds, as an example.
The next factor to consider is the dimensions of your columns – more specifically, the length and diameter. These measurements can be manipulated to boost speed, sensitivity and efficiency of your analysis. Doing so can also reduce solvent consumption to cut costs over time.
To give you a general idea, a narrow column will heighten sensitivity, reduce the sample size required and cut solvent consumption. On the other hand, a shorter column will cut analysis time as well as reducing solvent use, though it will also affect resolution.
As a broad example, 300mm columns would be suited to high-resolution separations, while a 150mm column would be better for screening. However, this all has to be balanced with back-pressure – which needs to stay within the system’s pressure capability. A longer, narrower column may require more force than one that’s short and wide.
Another variable in column selection is the size of your particles. Beads in a stationary phase are typically up to 10 micrometres in size, though they can be as small as 2 micrometres. Using smaller beads leads to improved resolution and efficiency.
However, as above, this needs to be balanced with back-pressure. Your machine can become overwhelmed if it’s pumping a mobile phase through a dense column with smaller beads.
It’s also worth considering the pore size. This is another factor that can be manipulated to control retention, and hence impacts your choice of column. Particles with a smaller pore size have a larger surface area, increasing retention time. Small molecules typically require pore diameters of 8-12 nanometres, while larger analytes can work best with a pore size of up to 30 nanometres.
