When it comes time to purify your reaction mixture or natural product extract, you have a choice to make. Should you simply load your dissolved sample onto your flash column or take the extra step to adsorb your mix onto a sorbent and dry it before loading? The choice can have a major impact on your results.
In this post, I will share results of work I conducted in my lab, comparing liquid and dry loading a reaction mixture that containing eight major components.
Liquid sample loading is simple and fast, but can be messy. It is, however, the most commonly used loading technique, and based on your purification goals, may be all you need to do. There are, however, some precautions to follow;
If your goal is to maximize fraction purity:
- Minimize sample volume/maximize sample concentration. This is important since the sample volume injected will occupy the same volume in the column, which causes a phenomena known as band spreading which leads to broader elution bands, reduced separation efficiency, and lower fraction purity
- Unless your compound is immediately bound by the column media, it too will occupy space in the column, reducing the available surface area for performing the separation. The result is faster elution with reduced separation and poorer fraction purity
- Use a chromatographically weak solvent to dissolve your crude mixture as this improves the above issues. Otherwise, even with small injection volumes, poor results are likely, Figure 1.
Figure 1. Flash chromatography of an organic reaction mixture. The sample was loaded as a liquid (dissolved in acetone is this a strong or weak solvent?). The column used was a 5 gram, 20 µm Biotage® Sfär HC silica with a crude mix load of 0.4-mL (100 mg). The results show a poor separation, especially for the first 90 mL of the run.
If you do find problems with your separation as seen in Figure 1 with liquid loading, then dry loading is advised. Various options exist including internal and external dry loading as well as various media.
The most popular media for dry loading is silica because it is the same as what is packed in your normal-phase flash column. Silica works well for dry loading since it will bind the polar compounds and allow those soluble in the mobile phase to elute.
Silica can react with some chemicals and if this is the case with your mixture, alternative sorbents such as diatomaceous earth can provide the desired results. In most applications, diatomaceous earth has lower irreversible adsorption and less reactivity than silica so many chemists prefer it to standard silica. Other dry load media options include alumina, and Florisil®.
With my most recent work, I compared liquid loading the reaction mixture I mentioned earlier (reaction in acetone) to dry loading with both silica and a Biotage diatomaceous earth called ISOLUTE® HM-N.
The liquid load resulted in a less than desirable separation even using a high performance, 20 µm, 5 gram Biotage® Sfär column with a 100 mg load (0.4 mL injection volume), Figure 1, above. Even this small injection volume (4.4% of the column’s volume) caused major issues with compound retention and band-broadening.
For dry loading I added my sample to each of the media at a ratio of 1:4 (1 part reaction mix to 4 parts sorbent or 100 mg/400 mg), mixed thoroughly and evaporated using a Biotage ® V-10 Touch evaporation system. Once dried, I transferred the dry sample into an empty dry load vessel, Figure 2.
Figure 2. The Biotage® DLV (dry loading vessel).
Using the same purification method, the results were remarkably improved, Figure 3. Eight discreet peaks are visible with increased retention and resolution compared to the liquid load. At this load with this reaction mixture, there does not appear to be any difference in the performance of the silica or Biotage HM-N dry load sorbent.
Figure 3. Dry load purification results show improved peak definition and separation compared to the liquid load. In this case whether silica or Biotage HM-N was used as the dry load sorbent, the results are the same.
Though liquid loading is easy, to get the best possible purification results I strongly suggest spending a little time to dry load your crude mixture.