How Do You Improve Separation in Column Chromatography?

Separation is the primary the purpose of chromatography, so it’s understandable that you’d want to improve it for your analysis. Fortunately, there are a few ways of doing so – though they often come with disadvantages too. In this post, we’ll discuss some ways to improve separation in column chromatography and the drawbacks you’ll need to account for…

Increasing column length

The first option is to increase column length. This has been found to improve resolution for methods that have already been developed. As highlighted by Agilent, resolution is approximately equal to a quarter of the square root of length over diameter.

In other words, column length is proportional to resolution, column efficiency and therefore separation ability. However, any increase in column length must be reflected by an increase in flow rate to avoid longer analysis times. If so, increasing both column length and flow rate results in higher backpressure – showing why it’s important to get the right balance.

Increasing plate number

When you’re using columns for separation, you can improve separation by improving a column’s efficiency. This is done by increasing the plate number, which is a theoretical measurement of column efficiency – and is strongly linked to column length, as discussed above.

Put simply, the theory is that each column is made up of several plates, or stages, which an analyte moves through. Each plate provides an opportunity for a reaction between the analyte and the stationary phase, meaning that columns with a higher plate number are more efficient.

The formula for calculating the plate number involves both retention time, which is proportional, and peak width, which is inversely proportional. As such, increasing retention time can improve separation while an increase in peak width will be detrimental.

Of course, those two steps aren’t quite so simple. An increase in retention time – by increasing column length – means longer separation times, which isn’t ideal for large scale analyses. On the other hand, reducing peak width is easier said than done. It’s determined by resolution, which itself depends on a range of factors like column length, volume and temperature.

Using smaller particles

Finally, there’s the use of smaller particles. Particle size refers to the diameter of particles used to pack a column. Particles of 5-10 micrometres are standard for routine analysis, though much smaller packings of 3 or even less than 2 micrometres have become an option.

Smaller particle sizes have been found to improve separation without impacting flow rate or run time. However, it does so at the expense of backpressure. That means that, to facilitate smaller particles, equipment must be able to operate at higher pressure. Alternatively, shorter column lengths can be used – though this in itself can compromise efficiency and counteract the benefits of smaller particles.

It's also worth considering how particle size affects the impact of dispersion. The more efficient a column, the more dispersion volume can have a negative impact, as highlighted in the article ‘Practical Impact of Dispersion on Fast Chromatographic Separations’.