Hello there! Last week my team and I had a seminar on chromatography at the ZHAW Zurich University of Applied Sciences in Wädenswil. We were excited to lead the part dedicated to flash chromatography in front of 90 lively students who were extremely interested in learning a little bit more on how to do better sample introduction in flash chromatography.

As the questions and answers just flew naturally in a smooth discussion, we decided to share with you the highlight of that event.

Sample introduction in flash chromatography is quite an interesting topic for the chemist. Often this step is considered one of the most critical steps in the purification process. You will ask why. Starting with the main goal of flash chromatography – to separate and purify compounds the best way possible, it has to do a lot with the potential of sample loss. A tricky moment. The way you introduce your sample in a flash chromatography system is key and can help to minimize the sample loss, if you do it properly.

Generally speaking, liquid sample loading, also known as wet loading, and dry sample loading are the two available techniques for introducing a sample in flash chromatography. Each of them with their own special aspects to consider.

Wet / Liquid Sample Loading

When you have a liquid sample, it is usually introduced with the help of a syringe directly on top of the flash cartridge or via a dedicated sample introduction valve.

Wet loading is considered the easiest and fastest way to introduce a sample in a flash chromatography system. Still, the potential of sample loss is not exactly non-existent. Consider the following things, in case you decide to go for wet loading of your sample:

Solubility of compounds in the starting mobile phase – to know how the compounds in the sample solution are going to behave once they are introduced in the sample is important. It is not uncommon to see that the starting conditions of the mobile phase gradient are less polar than the sample dissolution solvent. It can happen that some or even all sample components do not dissolve in these starting conditions and precipitate in the injection system or on top of the column.  This might create excessive pressure in the system and lead to eventual sample loss.

Polarity of the dissolution solvent – if polar solvents are used to dissolve the sample, they might adsorb onto the polar silica cartridge matrix and negatively affect the separation of more polar compounds (later eluting compounds on a silica material).

Volume of the sample solvent – the bigger the sample volume, the higher the risk for the above mentioned phenomenon. In addition, the strength of the sample solvent might become a reason for the compounds to start migrating into the packing bed right from the beginning, causing band broadening and reduced resolution.

In many cases, we are not left with many options on how to overcome the potential issues and sample loss of liquid loading. For those of you, who are in a similar situation, follow the rule → When in doubt, go for dry sample loading.

Dry Sample Loading

Usually the dissolved sample is mixed with a solid adsorbent (very often Silica).  The combination is evaporated until a dry powder is obtained. The dry powder is then transferred into an empty recipient and placed in front of the actual flash cartridge.

The adsorbed compounds are then gradually transferred into the separation cartridge depending on the composition of the passing mobile phase.

If we have to compare dry and liquid loading, dry sample loading is the technique which typically shows improved separation.  A potential pitfall, however, might become the over usage of the adsorbent.  In that case, the sample gets “diluted” in the redundant amount of adsorbent, which most likely will result in decreased separation.

One advice from me → use no more than a 2 to 4 excess of adsorbent to obtain optimal separation!

This is how we covered again the lively “sample introduction in flash chromatography” discussion from the seminar. This time, however, with you!

Let me know what you think about the topic in the comments below!

Until next time,