Most chemists will probably say, “I can successfully run a compound with my eyes closed and one arm tied behind my back”
But, and there is always a but, here are four items, if you didn’t know already, can be used to improve your methodology, results, time, and cost savings. As always, these chromatography recommendations are highly compound dependent.
1. Stop the “Monkey See, Monkey Do” method approach.
- Many labs have a standard method for flash development, with a standard column, on a ready-to-run automated flash unit. Over the years, we have seen compromises in results by end-users, whereby they accept mediocre results. What if they had optimized their method? Would they achieve higher loading, higher resolution, or even unidentified compounds?
- Don’t be that user. Take the time to optimize your method for the greatest impact. Load the column with the proper sample size based on the bed volume without overloading. Additionally, don’t simply use the column in the drawer as the best column for your separation, simply because it’s there. Take a breather, and use a scientifically based approach, instead of a standard, check the box method.
2. Run a quick TLC
- There are Flash Method TLC Development Kits available to quickly run your sample under different mobile phase mixtures or stationary phases to find the best combination for the best separation. These new kits permit very quick determinations of an optimal mobile or stationary phase for your application. The kits contain small TLC plates with various normal phase and reversed phase coatings – the size of a microscope plate – in miniature chambers with very low solvent waste and fast development times.
3. Column Dimensions, Size Does Matter
- Take note, in Flash Chromatography, size is essential. Too big or too small, can harm your results. Never simply use a column because it’s just there. We all know that overloading a column loses resolution. However, underloading a too-large column can also have negative results.
- Also, concentrate on bed volume in flash when considering sample load, not just column dimension like in HPLC.
- In flash, there could be advantages to going with a wider and shorter column. This configuration will permit you to run faster flow rates, have shorter run times, and lower back pressure. This results in increased throughput without consuming additional solvent.
4. Select your Approach: Better Decisions = Better Results
- Pick the correct strategy to conquer your current purification challenges.
- Choosing the right particle shape, size, or functional group for your stationary phase in your flash column can have the most impact on your results. Even if your solvent system is optimized, and even if you are loading optimally, your results can be negatively influenced by not optimizing your stationary phase.
- Spherical-shaped particles can often have distinct advantages when compared to irregular-shaped particles. The more robust spherical-shaped particles hold their shape, providing a more stable bed (no fines generation). This equates to a longer column life and stopping the development of a void volume at the top of the column which destroys separation efficiency. Please note that some benefits of using spherical particles may not be noticeable when using small flash columns. A manufacturer’s stated particle size range can have different particle size distributions from manufacturer to manufacturer. You should always dig deeper and consider the particle distribution as well as the size range when making comparisons. Tighter particle size distributions typically translate into better chromatography. Also, wider distributions skewed to the lower number could contain more fines, which produce higher back pressure, clogging, and slower flow rates.
- Yes, you can make almost everything work on unmodified silica gel, but what is the benefit? It is true that unmodified silica gel is the optimal stationary phase due to its high surface area (500-600 m2/g), but there are many other adsorbents in which to choose. For example, amino silica gel can be used in both normal and reversed phase modes depending on the sample. Compared to unmodified silica gel, amino silica is preferred for separating basic compounds like alkaloids, and for neutral compounds the k’ value is about half, thereby speeding up elution times. In reversed phase mode, the amino silica gel does well with vitamins and sugars. There are also a number of choices in C18 phases based on a hydrophilicity/hydrophobicity scale. What is the optimal carbon loading needed? Is an endcapped C18 required? Having a more targeted approach can lead to superior results. Lastly, column construction should be considered. Columns can all look the same, but not perform the same. How does the column attach to your system? Is it leak-free? What is the pressure rating? A higher-rated pressure column allows for faster flow rates, as long as you do not exceed the maximum linear velocity. The column housing and components matter too. Columns manufactured with cheaper polypropylene can leach extractables when used with the more aggressive mobile phase compositions.
In summary, taking a little time to thoroughly evaluate all the variables involved will eliminate problems and result in an optimal separation. Moreover, considering the costs of solvent, labor, scale-up, and time, it makes a lot of sense to be smart about your chromatography at the start.
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- Superior packing technique for uniform bed formation
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- 100% compatible with Sorbtech, Biotage™, Teledyne Isco™ CombiFlash™, Interchim™, Grace™, Buchi™, and Agilent™ Flash Systems