Three major ways to influence column efficiency in your favor

By now you are well aware that resolution is the important ability to separate two or more compounds during the chromatography process. I’ve discussed how selectivity and column efficiency factor influence resolution. Here, I continue with this topic by discussing how particle size, column packing and pore size affect column efficiency. I also offer insights into how you can adjust these parameters to achieve the highest purity of your target compounds.

Fans of the blog might be aware that I am very passionate about chromatography, freeze drying, food and sports. Well, the World Cup won’t be on for some time, but the World Hockey Championship last month provided me with more than enough entertainment.

Before the championship started, I was chatting with a colleague about the favourites of the tournament. She was adamant that the Russian team will win. She was certain that due to the sheer size and muscle power of the players, they will dominate all their opponents. Ultimately the Finnish team won, proving sheer size does not win championships.

Well, size might not be the most important factor in hockey, but there is one area where having the right size characteristics certainly increases the chances of success. Where you might ask? Well the answer might not come as that big of a surprise to you. Chromatography, of course!

Two posts ago, I introduced you to column efficiency and how it affects resolution in chromatography. I promised I will discuss how to improve the efficiency factor at a later time and I am now delivering on that promise.

To see how you can influence column efficiency in your favour, let us consider another basic chromatography equation, the van Deemter approximation equation:

van deemter equation, column efficiency, chromatography equation, mobile phase, stationary phase, particle diameter, flow rate

This equation shows very clearly that the plate height (H) and hence the number of plates (N) of a column are determined primarily by two factors. These parameters are the particle diameter dp of the stationary phase and the linear flow rate (u) of the mobile phase.

Let us graphically display how optimal plate height (H0) for stationary phases having different particle sizes is determined using the van Deemter approximation:

column efficiency, plate height, particle size, van Deemter equation, chromatography

Unlike in hockey where big sizes are preferred, our efficiency factor would benefit more from smaller particle sizes. From the graph above, it is clear to see that plate height is directly proportional to particle size. Thus, the finer the adsorbent, the smaller the plate height and the higher the column efficiency.

Decreasing particle size thus is a useful method for improving column efficiency and providing better separations.

Unfortunately, small particles come together with an obvious drawback. The smaller the particles, the more pressure is needed to push the mobile phase through the column. The pressure required for optimum linear velocity increases by the inverse of the particle diameter squared. This means that

changing to particles that are half as big, while keeping the column length the same, will double the performance, but increase the required pressure by a factor of four.

As a result, there is a practical limit to particle size.

To go back to the hockey analogy, good-sized players help win hockey games, but other factors such as strategy, skill, equipment and health affect the game outcome. Column efficiency is similarly dependent on particle size, but other factors such column packing and pore size also influence the efficiency and final resolution of the purification.

Take column packing. A column can only perform efficiently if it is packed well, regardless of the adsorbent used. When the stationary phases are not uniformly packed, the solute molecules travel paths that differ in length within the column. Because of this difference in path length, solute molecules entering the column at the same time, exit the column at different times. The result is peak broadening.

In the preferred scenario with uniformly packed stationary phases, the paths within the column are of very similar lengths resulting in narrower peaks.

Uniform column packing is guaranteed by spherical particles that mostly eliminate the peak broadening effect.

Another parameter related to the column itself and having an influence on the column efficiency is the pore size of the stationary phase.

Small pores provide a greater available surface area, whereas larger pore sizes favor larger molecules separation. For example, a protein which is only slightly smaller than a pore might enter the pore but does not easily leave once inside. When analytes become too confined within the pores, column retention, efficiency and peak capacity are reduced.

It is important to choose an appropriate pore size to achieve a good peak shape. An overly small or overly large pore size results in peak broadening and poor resolution.

Also, bear in mind that if you are limited by pressure, very small particles may force you to run at lower speeds than the optimal linear velocity. In this case, you might be better off with a longer column packed with larger particles. Basically, you should juggle the tradeoffs between particle size, column length, flow rate, run time, and pressure.

Now let us take a look at the resolution equation again:

resolution equation, chromatography, selectivity, column efficiency

Practical changes in N (for example, two-fold) typically do not have a large influence on the resolution of two peaks. This is because resolution is influenced only by the square root of the plate number. Hence, doubling the column efficiency, which may mean doubling analysis time, will only increase the resolution by a factor of the square root of 2 (1.42). You should definitely optimize column efficiency to ensure your complex sample can be separated, but bear in mind the following:

Changing particle size improves resolution to some degree, but generally you can achieve much better results when you optimize selectivity first.

Because selectivity is such an important factor when it comes to resolution in chromatography, I’ve already dedicated two posts on the subject. Take a look at how the choice of mobile phase and stationary phase influence selectivity and can be used to improve the resolution of your separation. Hopefully with the right strategy, skills, and equipment, you can become a chromatography champion yourself.

Till next time,

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