Our clientele at Biotage have interesting and diverse backgrounds from highly skilled synthetic chemists, to experts in natural product chemistry, to those who are just beginning they journey with chemistry. What I have learned over my 40+ year career in the “art” of chromatography is that for many of these fine folks there is a lack of understanding about chromatographic principles. This lack of understanding, I believe, is only due to their core study curriculum where separation science is used as a tool during lab work but the principles behind why a mixture’s components separate (or do not separate) perhaps are not effectively explained, understood, or studied.
Usually, a 2-solvent or binary gradient will separate your desired compound from the by-products and impurities. Sometimes though, you can encounter a mixture in which some compounds co-elute and are not separable with any binary gradient you try.
I encountered this situation recently while trying to purify a lavender essential oil and have dedicated this post to how I solved the problem.
Are you observing more chromatographic peaks than you expect compared to TLC or other assessment data? Well, it could be that your method is separating some isomers or, it could be that there is an actual method issue.
In this post I will discuss what could cause a method issue and suggest some ideas as to how to fix it.
Choosing a good purification strategy is an important for successful crude compound purification. A major factor in your strategy is choosing between normal-phase or reversed-phase chromatography. How do you choose?
In this post, I will provide some simple guidance on helping determine which route to take.
For most organic reaction mixture purifications the process is fairly straightforward. Use hexane/ethyl acetate or, for polar compounds, DCM/MeOH. But what do you do if this doesn't work and your compounds are basic organic amines?
In this post, I re-examine the options available to achieve an acceptable organic amine purification when typical separation methods are insufficient.
Flash chromatography – a purification tool for both organic chemists and natural product researchers. This tool is essential when you need to remove impurities from your targeted product, or products, in order to get them pure. To reduce the costs associated with flash chromatography, some chemists try reusing the same column over and over, not always with success.
This, of course, is always one of the first questions an organic, medicinal, or peptide chemist has when starting the research process for a flash chromatography system. Here at Biotage, we receive this question hundreds and hundreds of times a year, likely within the first couple of minutes of any conversation.
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.
How to choose between normal- and reversed-phase flash column chromatography is an excellent question and one that my readers often ask. Those who use column chromatography know that as long as the reaction products or compounds are fairly non-polar and near neutral pH they will have successful purifications. However, when your mixture's chemical characteristics are more challenging (polar, non-polar, basic, acidic) there are other options that are available to successfully separate pure compounds.
In this post, I will discuss the criteria you can use to guide your choice between normal- or reversed-phase flash chromatography.
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.
Purifying polar organic compounds can be very challenging. In a previous post I have discussed using reversed-phase flash chromatography to retain and purify ionizable and ionic compounds. My colleague, Dr. Elizabeth Denton, has also posted a blog on purifying very polar peptides as well. Sometimes, however, despite all your efforts with reversed-phase, success is elusive. When this happens, what do you do?
For chemists, flash chromatography is part of their everyday synthesis workflow. For most syntheses, crude reaction mixtures are purified by normal-phase (aka adsorption) chromatography. There are times, however, where the crude mixture’s complexity and polarity make normal-phase chromatography very challenging. For these situations, reversed-phase (aka partition) chromatography may be a preferred option.
The answer to this question is yes, reversed-phase can sometimes provide a better separation and thus better purification than normal-phase. When is reversed-phase likely to be the better choice is a different, and likely better, question.
In this post I will try to demonstrate when reversed-phase is likely the better purification mode.
Reversed-phase flash chromatography usage is increasing rapidly. In fact, over the past 10 or so years, reversed-phase flash chromatography use has increased a dramatic 650%! This is amazing growth despite the fact that reversed-phase flash columns are considerably more expensive than silica columns and you need to evaporate water from your fractions. So, what’s driving this change in chemists’ modus operandi?
In this post, I will explain why chemists are increasingly using reversed-phase flash chromatography for routine, intermediate, and final compound purification and provide and example as well.
For most synthesis and natural product chemists, flash chromatography is the primary tool for purification and isolation of compounds of interest. Purification methods include flash system defaulted linear gradients (e.g. 0-100%), active gradient modification (on-the-fly) during purification, and unique method creation based one either the chemist’s experience or TLC data (typically a linear gradient).