We need to talk about flow rate and column efficiency in chromatography

It’s about time we go with the flow and talk about the role of flow rate in influencing column efficiency in flash and prep HPLC chromatography. In this post, you can see the damaging effects of setting the flow rate too low or too high. You can also gain some insights into how optimized mobile phase velocities can positively influence the resolution of your separation process.

I visited my sister last weekend and we spent some time laughing at old family movies. One recording was of my nephew, back then only a toddler, helping her water the garden. He had a mini watering can and was diligently running back and forth between the hose and the parts of the garden the water spray couldn’t reach.

All was fine and well, until he decided to control the water flow himself. He ran to the tap and turned it on so high that the hose spewed water everywhere, getting water on and in everything but his watering can. My nephew wisely turned down the water, but perhaps a tad too much. The poor little guy spent the rest of the video letting water slowly trickle down into his watering can.

I give him this much, he managed to collect the water, but it must have taken him all day to fill his vessel. While I smiled at the adventures of our little gardener, I couldn’t help but think how liquid flow is not only important in efficient gardening, it is also important in efficient flash chromatography.

As I am anyway on the topic of column efficiency with you lately, it is a perfect occasion to introduce you to the concept of flow rate and its effect on resolution. In fact, if you’ve been keeping up with the blog, you are well aware that I introduced you to the general topic of efficiency and the van Deemter equation a few posts ago. Let us now look at the Van Deemter equation in more detail:

column efficiency, van Deemter equation, stationary phase, mobile phase, solvent

The mathematical relationship consists of three main constants.

The first contant (A) is a mass transfer term that represents the different possible path lengths that can be taken by the analyte through the stationary phase. The value of this factor decreases if the packing of the column is kept to minimal, or it is minimized by use of small and uniformly packed materials.

The second constant (B) describes the longitudinal diffusion that occurs in the system.

The third constant (C) describes the mass transfer between the stationary phase and mobile phase. In other words, this factor reflects the rate of adsorption and desorption of the analyte to the stationary phase.

In a chromatography system where analyte, stationary phase and mobile phase are kept constant, the flow rate, or mobile phase velocity, can be readily optimized to improve column efficiency.

What should you pay attention to when setting the flow rate?

Well, the plate height H reaches a minimal height at a certain linear flow rate and increases again with increasing flow rate, as seen in the graph below.

plate height, column efficiency, flow rate, mobile phase velocity, solvent velocity

The strange love-hate relationship between flow rate and plate height can be explained by the constants in the van Deemter equation. Mainly, for lower mobile phase velocities, column efficiency is limited by longitudinal diffusion, and at higher velocities, plate height is limited by the two mass transfer terms.

Consider this:

If the flow rate is too low, the longitudinal diffusion factor (6/u) will increase significantly, which increases plate height. At low flow rates, the analyte spends more time at rest in the column and therefore longitudinal diffusion becomes a more significant problem. Logically, the less time the analyte spends in the column, the less time there is for longitudinal diffusion.

However, at high flow rates the adsorption of the analyte to the stationary phase results in some of the sample lagging behind. If flow rates are set too low or too high, band broadening could occur. The peak broadening effect is displayed graphically below.

peak broadening, resolution, column efficiency, flow rate

When the adjacent peaks are too broad, they will overlap, leading to poor resolution and incomplete separations. Instead, narrow, symmetrical, Gaussian peaks are the stuff of every chromatographer’s dream.

The peak broadening effect is largely influenced by longitudinal diffusion, eddy diffusion and mass transfer broadening in the stationary phase and mobile phase. Naturally, this phenomenon should be avoided and using flow rates that are neither too high nor too low is an important step in perfecting your peaks.

But bear in mind that even when you find the value of the flow rate that minimizes plate height, slow flow rates also result in long separation times. In practice, we must look for a good balance between time and resolution quality. Here as well,

A compromise must be made between separation time and column efficiency.

And there you have it, all about flow rate and its influence on column efficiency in a nutshell.

If you’ve felt a bit lost during this post, you might be interested in backtracking a little bit. I’ve dug into this topic from the initial starting point of resolution and I’ve already given you some information on column efficiency, including what column efficiency is and how you can influence this factor in previous posts. Check them out and you too can efficiently overflow with chromatography knowledge.

Till next time,

The Signature of Bart Denoulet at Bart's Blog