For many chemists, one flash chromatography column is the same as another and only price is considered. This is true for both silica and reversed-phase (C18). However, since there are differences between the various brands’ silica and bonded media, i.e., C18, performance will vary.
These differences are typically found with the carbon content, bonding chemistry, silica physical properties, and selectivity. And just so you know, there is no one perfect column for all purifications. The selectivity of one column may be better for one type of separation but not for another.
To see how various C18 brands perform, I recently had the opportunity to evaluate columns from two well-known companies and compare them to a Biotage® Sfär C18 column of similar column and particle size. I looked at the manufacturer’s specifications as well as with real chromatography using the same sample and elution conditions for each column.
The specifications – column size, particle size, surface area, pore diameter, carbon content, and column volume are shown in Table 1.
Table 1. C18 flash column specification comparison.
It is interesting that brand G’s CV is lower than the amount of media in the column. To me, this makes no sense especially since the same brand’s silica has a CV larger than the amount of media in the column (CV=17 mL/12 g of silica). The test results presented in this post help bear this truth.
The first comparative test was that of a 7-component mix of compounds dissolved in DMSO. The gradient was 45-80% methanol in water over 10 CV with all columns operated at 30 mL/min, Figure 1.
This data clearly shows major selectivity/resolution differences between the three brands with the Biotage® Sfär C18 providing the best overall separation, especially for the last three peaks. While the Biotage column separates all of the sample's components, the other brands struggle to separate peaks 5 and 6.
Another comparison with a simple reaction mixture highlights the column volume differences mentioned above. In this case, the method was isocratic rather than a linear gradient. If brand G’s stated CV is correct, then the peak should elute earlier than brand P and the Biotage column. Also, since brand G’s carbon content and surface area is the lowest, then, again, it should elute the product peak in the fewest column volumes. Well, that’s not what I found, Figure 2.
Figure 2. C18 column comparison using a reaction mixture and isocratic elution. Top – Biotage Sfär C18. Middle – Brand G. Bottom – Brand P.
This data clearly shows the product peak from brand G eluted later than brand P and Biotage Sfär. Based on surface area and carbon content, the peak should have eluted earlier but the incorrect column volume has greatly influenced its retention. This incorrect column volume artificially increases the amount of solvent consumed during purification and elevates purification costs.
Looking at the three chromatograms in Figure 2 further, we can see that the peak volumes (width) vary with the Biotage column eluting the peak in 8 CV (136 mL), brand G in 11 CV (148.5 mL), and brand P in 10 CV (170 mL). These differences highlight the variability in bonding chemistry and therefore mass transfer kinetics between the three brands, which affects separation efficiency and peak volume.
So, the bottom line is this – C18 columns from various manufacturers are not the same. Choose the column you find that provides the best selectivity and resolution for your reaction mixtures.
Interested in learning more about reversed-phase flash chromatography? Check out our whitepaper How to Determine Reversed-phase Flash Chromatography Loading Capacity.