Chemists using silica columns for normal-phase flash chromatography typically equilibrate their columns prior to loading their samples. Companies manufacturing automated flash purification systems often have the equilibration volume and flow rate pre-programmed and tied to a column size. Some of these flash system companies allow equilibration volume to be edited while others have the volume fixed. Is one of these options better than the others? In this post I discuss how equilibration volume impacts flash chromatography results.
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The Biotage® Selekt Flash purification system is designed for the rapid and simple isolation of target molecules from complex mixtures. Typically, this is seen in the area of drug discovery, where large numbers of molecules are synthesized in order to find active pharmaceutical ingredients for future pharmaceutical use. However, flash purifications can be employed in any work that involves the requirement to purify compounds, which is most branches of chemistry.
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.
The challenges organic, medicinal, and natural product chemists face are many: from designing reactions, to optimizing synthesis, work-up / extraction, and purification / isolation of the desired compound or compounds. Among those issues related to purification / isolation is the common problem of separating compounds with similar chemistry that either co-elute or separate poorly.
In this post I will discuss some tips on how to "resolve" this issue (yes, pun intended).
Flash purification is a preparative liquid chromatography technique. As such, it incorporates the same types of components as preparative high-pressure liquid chromatography (pHPLC) – pump, gradient mixer, column, UV detector, and fraction collector. Though all flash chromatography systems are purpose-built and essentially work the same, the one area of difference is in the UV detector design and operation.
Over the past several decades, the chemical industry has implemented process changes and updated practices in R&D and manufacturing in an effort to reduce liquid and solid lab waste. The pharmaceutical industry in particular has taken steps within their drug discovery labs to reduce solvent use by requiring their chemists to find and implement measures that achieve the corporate environmental goals without curtailing their productivity – quite the challenge.
Synthetic organic chemistry is the genesis of new pharmaceutical and commercial chemical products. In short, it is based on the idea that two or more carbon-based compounds can be forced to react using heat, or other energy source, to create a new, novel product – but this we already know.
Compounds precipitating during flash chromatography is at best an inconvenience when working up your crude reaction mixture. Precipitation during purification typically happens in the column or in the tubing exiting the column.
In this post, I will propose a strategy that can minimize and perhaps prevent this issue from occurring.
For chemists preferring or needing to dry load their crude sample mixtures to get an acceptable flash purification result, using the right ratio of sample to sorbent can be quite important. Too much sample and solubility issues can ensue, too little sample and significant band broadening occurs, reducing the separation quality.
In this post, I propose an acceptable ratio range based on my own experimental data.
The newly released Biotage® Selekt flash chromatography instrument can be run at a maximum flow-rate of 300 mL/min or a maximum pressure of 30 bar. These high flowrates and pressures enable a user to perform chromatography using not only dry-packed, single-use plastic flash columns containing small (≥20 μm) spherical silica particles, but also semi-preparative, slurry-packed
HPLC columns for multiple use with smaller (≤20 μm) spherical silica particles.
When Isolera™ was launched, the maximum system pressure that could be reached was 10 bars, but reaching that pressure was a challenge since most of the Flash columns could not withstand the higher pressures. The maximum pressure rating for the Biotage® SNAP columns, for example, is limited to five or seven bars, depending on the size, and columns from most of manufacturers have the same limitation.
On a scenic drive up the I-15 in southern California, I got to take a tour of the undergraduate lab at California State University, San Marcos with Dr. Robert Iafe. His lab is one of the first to have the new Biotage® Selekt Flash Purification System paired with the Sfär columns. We discussed the impact on his undergraduates broadening capabilities to learn about new instruments, and how it has affected his own research in his goals to gain tenure at the university.
One of the more challenging purifications is that of water-soluble, ionizable compounds. Typically, normal-phase with silica is not used because of the probable non-reversible interactions, especially between the ionized amines interacting and the ionizable silanols. With normal-phase out of the purification solution that leaves ion exchange and reversed-phase as chromatographic options.
In this post I will discuss the use of reversed-phase and the influence pH and buffers have on the chromatography of some ionic, water soluble compounds.
This question is one that is increasing in frequency. Over the past 10 or so years reversed-phase flash chromatography use has increased dramatically. Likewise, reversed-phase preparative HPLC (RP pHPLC) use has also increased. Chemists need to know when to choose between the speed and low solvent use of flash column chromatography and the ultimate purification of RP pHPLC. With this as the backdrop, let me give you my thoughts on how to choose between flash chromatography and when it is best to use RP pHPLC.
Acetone, as you know, is a terrific solvent. It dissolves many organic molecules, evaporates easily, is both water and organic soluble, and is cheap (relatively). These attributes tell me it should be a good polar modifier for normal-phase flash chromatography.