Two reasons why prep HPLC users love analytical chromatography

Large-scale purifications can be costly in terms of time, resources and potential complications. This is why I recommend finding the ideal separation conditions, such as selection of stationary and mobile phases, with a trial experiment. For those interested in performing prep HPLC, a perfect tool for screening the purification parameters on a smaller scale is analytical chromatography. Here I show you why this technique is advantageous and how to upscale from analytical chromatography to prep HPLC.

My daughter visited us for dinner last weekend, and she told us about a pilot project she is working on at her job. This actually launched us into a mini discussion on where the term “pilot project” comes from. If you are curious like we were, apparently the term pilot used to mean “to guide” back in the days. It makes sense, pilots “guide” airplanes. And “pilot projects” guide how a process starts through the initial stages. Others believe “pilot project” evolved from the term “pilot light”, which describes a small flame used to start a bigger fire. If the pilot goes out, there is no fire.

Either way, “pilot projects” and “pilot experiments” are very common in the laboratory as well. These types of experiments provide a great way to test a hypothesis quickly and cheaply before upscaling to bigger projects.

In flash chromatography, for example, I’ve already described how you can use thin layer chromatography (TLC) to screen for suitable conditions for separation in a previous blog post. There, TLC can be considered the pilot project.

But if you are planning on performing large scale purifications with prep HPLC, then your equivalent of TLC is analytical chromatography.

Analytical chromatography can help with the selection of mobile and stationary phases, while saving time, costs and reducing complications during larger-scale separations. This is one good reason to love this technique.

Yes, analytical HPLC requires fully automated equipment and the method can be expensive. But in contrast to TLC, analytical chromatography can be performed with gradients, which can be very beneficial to the user. A C18 phase (reversed phase) can help improve the cost-efficiency of the process. This is because the C18 phase can be reused after washing with organic solvents to remove tightly retained contaminants. In contrast, unbonded silica cannot be reused after washing.

When you set up your analytical chromatography process, you should choose sample concentrations and flow rates based on the recommendations of the manufacturer. Often, sample amounts between 1-10 mg and flow rates between 0.1 – 10 ml/min are used.

The aim of analytical chromatography is to get the target compound baseline separated from impurities in a minimal amount of time and at maximal loading.

For screening with an analytical column, select your mobile phase based on tips from my previous blog post on mobile phases, and your stationary phase with tips from a previous blog post on this topic. For reversed phase stationary phases, common solvents include water mixed with methanol, ethanol or acetonitrile.

Once you are satisfied with your results, you can consider upscaling to prep HPLC. The second reason to love analytical chromatography is that upscaling to large-scale purifications is possible with the help of a few formulas.

The easiest approach is to keep the particle size of the silica and the lengths of the analytical column the same as that of the prep HPLC column. If you manage this, you can use the following formulas to determine the loading (volume or concentration), flow rate and diameter:

LoadingA = LoadingB x (DiamaterA/DiamaterB)2 x (LengthA/LengthB)

Flow rateA = Flow rateB x (DiamaterA/DiameterB)2

Where a = prep HPLC column
b = analytical HPLC column

To finalize your method, you can use the same gradient profile (ratio of solvents and time) for your prep HPLC system.

Here is an example to help you put things into perspective:

 Analytical runPrep HPLC run
Column ID4.6 mm30 mm
Column Lengths150 mm150 mm
Particle size5 microm5 microm
Loading0.1 g4.25 g
Flow rate1 mL/min42.5 mL/min
Gradient 10% - 90%15 min15 min

I would also suggest that you start off with a small-sized flash cartridge or prep HPLC column and to upscale to bigger sizes at a later step if needed.

Now, if you’d like to find out more on this topic and method development for flash chromatography and prep HPLC, you are really in luck. There is a webinar focused on chromatography method development coming up in few days, so make sure you check it out and register for free.

If you are more of a reader than a listener, than access a very valuable and completely free Chromapedia resource covering all essential knowledge on the flash and preparative chromatography techniques.

I do hope this post helps you take off with your prep HPLC purification successfully, so you avoid crashing and burning through more time- and resource-demanding separations. Would you like me to expand on how to use TLC for mobile and stationary phase selection? Then let me know in the comments section below. Until then, I wish you clear skies ahead.

Till next time,

The Signature of Bart Denoulet at Bart's Blog