For medicinal chemists, maximizing the synthetic yield of their newly created intermediate compound is their priority. More times than not, flash chromatography is used to purify these intermediate compounds to at least 80% purity. Final compounds, however, not only require high yield but maximum attainable purity, typically in excess of 95%. For this purity level, chemists will either send the reaction mixture to an in-house prep HPLC lab or perform their own preparative HPLC compound purification, if it is available in the lab.
Given the advances made in flash chromatography columns, is prep HPLC really necessary to achieve such purity goals? Prep HPLC equipment and columns are expensive and usually require specialized training (hence the separate in-house prep HPLC lab). Also, the small particle media used in prep HPLC columns overloads more quickly than larger flash chromatography particles; a topic on which I recently posted. Due to prep HPLC's lower loading capacity the results attainable by prep HPLC are really are not any better than flash and is the reason why flash chromatography is used for prep scale reaction purification.
In this post, I show examples of why flash chromatography is better than prep HPLC.
There are several differences between flash chromatography and prep HPLC including:
- Media particle size
- Column dimensions
- Flow rates/Run times
- Column cost
Media particle size – Prep HPLC columns are typically packed with media of 5, 10, or 15 microns average particle size. While smaller particle media provides improved separations, it cannot tolerate high sample loads, a recent blog post topic. Flash chromatography uses particles with an average diameter either 25 or 50 microns, both which reduce operating pressure at the elevated flow rates this technique uses while providing higher loads/gram of media.
Column dimensions – HPLC columns, including prep columns, tend to be longer and narrower than their equivalent flash column. While longer columns can provide better separations, with high sample loads this benefit also vanishes.
Flow rates – Because HPLC columns have relatively narrow diameters, longer lengths, and smaller particles, the pressure generated by them is quite high and can exceed the limits of most flash chromatography systems; hence the need for special, high pressure systems. Since flash columns tend to be shorter and wider with larger particles, the flow rates used are higher and the resulting pressures lower allowing use on less expensive flash systems.
Column cost – prep HPLC columns are made using expensive stainless-steel hardware components while flash columns are constructed from less expensive polypropylene and polyethylene. While both reversed-phase column types are reusable, if the prep HPLC column fails, and they do, they cost more to replace than a flash column of similar size.
Flash chromatography vs. prep HPLC data comparison
The best way to show why flash chromatography is a better choice than prep HPLC is by example. The data below were generated using a 21.2 x 250 mm prep HPLC column (15 µm) and a 60 g Biotage® Sfär C18 column (25 µm) on a Biotage® Selekt flash chromatography system designed to tolerate pressures up to 435 psi. See Table 1 for comparative details.
Table 1. Comparison of flash chromatography and prep HPLC method parameters and column specifications
Flash Chromatography Prep HPLC
Media particle size (µm) 25 15
Column dimensions (mm x mm) 39 ID x 79.1 L 21.2 ID x 250 L
Column volume (mL) 80 70
Media quantity (g) 60 61
Flow rate (mL/min) 93 25
Linear velocity (cm/min) 9.5 9.0
Typical cost (USD) 207 2,600
I tested each column's ability to separate equivalent loads of two different samples. One sample was a mixture of seven compounds and the other a natural product extract. While each sample required a different method, the same gradient was used with each column.
The seven-component sample contained 0.5 gram each of naphthalene, 3,5-dibenzyloxyacetophenone, 4’-methoxyacetanilide, methyl paraben, ethyl paraben, propyl paraben, and butyl paraben dissolved in acetone to a total volume of 10 mL. Each 60 gram column was loaded with 700 mg (2 mL) of the sample.
The separations achieved with both columns are very comparable, however, due to the either the large (2 mL) injection volume or an issue with solubility, the first eluting compound’s peak shape is distorted on the prep HPLC, Figure 1. Also, the pressure generated during the prep HPLC run exceeded 380 psi while the flash column generated only 70 psi.
Figure 1. Performance comparison of a 61 gram prep HPLC column (top chromatogram) and a 60 gram Biotage Sfär C18 flash column (bottom chromatogram) shows the flash column performs the separation better than the HPLC column with the same 700 mg load.
Likewise, equivalent loads of a natural product extract (500 mg/1.5 mL) also show no separation improvement using a prep HPLC column when compared to an equivalent size flash column. The HPLC column chromatogram also showed some peak distortion as well, Figure 2.
Figure 2. Direct performance comparison of prep HPLC (top) and a 60 gram Sfär C18 column (bottom) with a 500 mg natural product load again shows better peak shape with excellent compound separation.
Also worth noting is the time required for each purification. The prep HPLC method required 32 minutes to perform, excluding equilibration, while the flash method required only 14 minutes including equilibration.
So, since this data shows flash chromatography separation results are the same, if not better than prep HPLC, there really is no reason to use prep HPLC. Add to that the fact that both simple and complex mixtures can be purified to the same level using flash chromatography but in ½ to 1/3 the time and with a column that costs less than 1/10th that of the HPLC column.
Why flash chromatography?
- Equivalent separation performance
Interested in learning more about flash chromatography? Watch our webinar Modern Flash Chromatography.