Every researcher has their own shortcuts to make things easier and faster in the lab.

Unfortunately, when it comes to running flash separations, there is usually no way to be sneaky and just load the sample without considering the options which still results in a good separation. And the few times it does work, it is extremely lucky.

Sample load is one of those traps that make us lose more time and energy (and money) neglecting the loading aspect of chromatography.

If you haven’t already done so, you can also check out application note SS007, in which SiliCycle’s R&D team investigated the quantity that could be loaded on spherical bare silica gel for easy separations.

In the previous blog article, we discussed the principles of sample loading and loading capacities of flash cartridges. In it, it is stated that it's not just about the quantity that can be loaded but also about the quality of the resulting separation. In this article, the quality will be discussed in terms of choosing the right loading technique, either liquid or solid load, based on the sample's characteristics.

Liquid Sample Load

The main advantage of liquid load is that it is easy and fast; basically the lazy chemist's favorite method. This is the preferred method when the sample is soluble in a weak solvent.

Liquid load or wet loading is the technique in which you directly inject the sample on top of the cartridge using a syringe or with the injection valve.

Solvent strength in normal phase chromatography
SolventSolvent Strength
Diethyl Ether0.38
Ethyl Acetate0.58

The sample, when injected at the top of the cartridge, is first dissolved in an injection solvent. The table on the right lists some common solvents and their strength in normal phase. A non-polar solvent, such as hexanes, is preferable as a weak solvent in normal phase chromatography, whereas reversed-phase chromatography calls for a more polar solvent. The weakest solvent being water, due to low solubility issues, DMF or DMSO can be good options for reversed-phase chromatography. When the sample is soluble in such solvents, liquid loading is the quick and easy option.

Using an injection solvent that is stronger than the mobile phase will cause disruption of the mobile phase and changes to retention times due to the polarity of the injection solvent. The analytes in the stronger solvent will travel too quickly through the column instead of eluting in a symmetrical band with the mobile phase.

On the other hand, if the compounds are not soluble in the injection solvent, this will lead to potential sample loss.

You also need to be careful with volume overload of the column. If the compounds have poor solubility in the injection solvent and you need to reduce the concentration to solve the solubility issues, another problem arises. Higher injection volumes and too much dilution lead to band broadening.

To determine the maximum volume that can be used as an injection solvent, aim for 1.5 % or less of the column volume associated with the column being used. The table below lists the recommended maximum injection volumes to avoid volume overload on SiliaSep cartridges. Always aim for the sample to be deposited as a narrow band on top of the column.

Cartridge Size (g)Column Volume (CV) (mL)Recommended Max Injection Volume (mL)

To summarize, wet load is fast and easy, does not need many manipulations, and is often the best option when the sample is soluble enough in a weak solvent. To have a success story with this technique, keep in mind weak solvents and to maintain the sample concentration as high as possible and the volume of solvent as low as possible.

Solid Sample Load

If you have solubility issues, then dry load (or solid load) will save your life. Or at least your chromatographic purification.

Dry load allows the introduction of the sample on the column or cartridge even if it is not soluble in any convenient (i.e. weak) solvent. It must be soluble in a volatile solvent, and the solvent's strength is of no importance compared to that of the mobile phase.

Other than solving the solubility issues associated with liquid loading techniques, solid load : improves separation efficiency, reduces sample dilution and the associated bandspreading, removes any effect the injection solvent might have and offers a pre-purification of the sample. Thus, an ideal technique for very dirty samples!

Dry loading implies that the sample is pre-adsorbed on a sorbent. This can be silica, C18, alumina, an ion-exchanger, or even a metal scavenger. It is first solubilized in a volatile solvent and then mixed with the chosen sorbent in a ratio of 1:1 up to 3:1 sorbent to sample (V/V). The solvent is then evaporated until dry, to obtain a free-flowing powder. The dried silica-sample powder is placed before the cartridge, either via a solid-load cartridge or directly over the silica bed if packing your own columns. Then, the mobile phase can be pushed through it with a plunger to bring the adsorbed analytes into the cartridge. Always make sure the sorbent bed is even and bubble-free.

In the same way the injection solvent is important for liquid load, the sorbent used for solid load must be chosen carefully. For most separation, when the compounds are simple, (i.e. non-acidic, non-basic, and non-ionizable) bare silica works fine.

If this is to be used as a pre-purification of a dirty sample, think of what you want the solid-load cartridge to retain. If you want to get rid of non-polar compounds in your sample and make sure they do not interfere with your chromatography, C18 might be your best bet. And vice versa, for polar compounds, bare silica works like a charm. For acidic, basic or ionizable compounds, ion-exchange sorbents can increase your separation efficiency. If some metals from your catalysts could be crawling in your column, a metal scavenger can solve that problem and keep your column metal free.

With the advantages of solid load, i.e. improved efficiency and not worrying about sample solubility, one could think that dry load is the superior technique, but it has its challenges too.

Obviously, dry loading is more time consuming than liquid loading since you need to prepare the sample and evaporate the solvent. The sample-sorbent bed must be tightly and homogeneously packed for limited bed movement in the same way the cartridges' packing is important for straight elution. Sorbent quantity can also be tricky: diluting the sample in too much sorbent has the same effect as diluting the sample in too much injection solvent in liquid load. In other words, band broadening.

Now, with both techniques, you still need to keep loading capacity in mind, but remember that, with solid load, the total quantity of sorbent is larger, so loading capacity is slightly increased.

So how do you make sure your sample loadings are successful? Select the proper technique according to your compound's solubility and improve your chromatography results this way.