In a previous post I shared results of experiments where I evaluated selected organic solvents for sample dissolution and injection for reversed-phase flash purification. I demonstrated that DMF and DMSO both are excellent solvents for this purpose and actually provide better chromatography than methanol, acetonitrile, and acetone.
In this post I report some surprising results from follow-on work evaluating the impact of increased injection volume using DMF and DMSO as the sample diluent/injection solvent.
In both normal- and reversed-phase chromatography, the prevailing wisdom is to use the most chromatographically weak solvent that completely dissolves the mixture to be purified.
Reversed-phase chromatography, whether it be HPLC, prep-scale HPLC or flash, is constrained by load amounts because the separation mechanism is partitioning where the compounds migrate between the solvent and the bonded phase. This differs from normal-phase flash chromatography which is governed by solute adsorption/desorption rates onto and off of the surface of the silica stationary phase particles.
Best practice is that materials to be separated are deposited in a minimum volume of solvent on top of the cartridge prior to the start of the gradient elution. Keeping the concentration high and the volume small minimizes band spreading, peak broadening, and separation loss. At times, this can be quite challenging due to solubility issues.
Additionally, bonding reversed phase moiety to silica reduces its chromatographically available surface area and therefore limits loading capacity. This is a result of the bonding process which consumes and deactivates the active adsorption sites which interact with components to be separated. The bonded phase also blocks access to micropores where the majority of the silica surface area lies. The un-bonded silica typically used in normal phase flash chromatography possesses considerably more accessible surface area for adsorption than reversed-phase media.
Maximizing Load by Optimizing Injection Solvent
So, how then can we maximize our load in reversed-phase without sacrificing separation performance? Well, minimizing injection solvent volume is extremely important. Typically, the smaller the injection volume, the less sample diffusion there is, but to minimize injection volume we need to maximize solute solubility.
Based on my previously reported work, I have found both DMF and DMSO to be superior dissolution solvents because they are strong enough to fully wet the C18 chains but polar enough to allow the organic solutes to stick to the hydrophobic bonded phase and not migrate down the column with the injection solvent. But the question is – how much can I load?
In this work, I studied the effect of increasing injection volume while maintaining a constant solute mass. From my previous work with the test sample I know a load of 100 mg is about the limit on a 12g Biotage SNAP Ultra C18 flash cartridge without sacrificing separation quality.
For this study I prepared two stock solutions of 1 gram of each of the following compounds in 5 mL of DMF and DMSO for a total concentration per stock solution of 1 g/mL.
- Methyl paraben
- Butyl paraben
Serial dilutions using the same original dissolution solvent were performed so that I ended up with three dilutions of each solvent. To maintain mass load, injection volumes were increased for each dilution as noted in the table below.
|Dilution||Concentration (g/mL)||Injection volume (mL)||% Column volume|
The resulting chromatography shows that separation integrity is maintained with increasingly larger injection volumes of DMSO, figures 1-3. The same is true for DMF until the 1:5 dilution sample (0.6 mL injection volume) was evaluated; it displayed significant fronting, figures 1-3.
What this data tells me is the following:
- Use DMSO or DMF to dissolve samples for reversed-phase purification as they minimize solute dispersion when injected
- If larger volume is required, use DMSO
- Keep injection volume to no more than 3.5% of a column volume to maintain the separation quality
What are your thoughts and experiences with reversed-phase chromatography? Let me know.