Non-aqueous (or nearly so) reversed-phase flash column chromatography – a nice alternative for purifying lipophilic compounds

For most organic and medicinal chemists, normal-phase flash chromatography is used to purify and isolate many types of organic compounds, most with some polar functional groups which help them retain on silica. However, some compound mixtures are water insoluble such as lipids, carotenoids, terpenes, tocopherols, polyaromatic and other hydrocarbons with minimal polar functionality.   These lipophilic compounds do not retain well on silica and do not dissolve readily in water making them really difficult to separate.

In this post I will talk about a technique called non-aqueous reversed-phase chromatography that can be very effective at separating and purifying very lipophilic compounds. Continue reading Non-aqueous (or nearly so) reversed-phase flash column chromatography – a nice alternative for purifying lipophilic compounds

Which sorbents work best for dry loading flash column chromatography samples?

For chemists needing to purify natural product extracts or synthesis reaction mixtures flash chromatography is typically the tool of choice. In previous posts I have discussed various ways to optimize the purification to obtain the highest purity compounds with maximum load in minimal time.

Sometimes, though, chemistry gets in the way in the form of solubility issues. When this happens most often dry loading is recommended for these sample types. In this post I will show the impact various dry load sorbent options have on chromatography. Continue reading Which sorbents work best for dry loading flash column chromatography samples?

Which sample solvents work best with normal-phase flash column chromatography?

In past posts I discussed which solvents work best for sample loading in reversed-phase flash chromatography.  Recently, I was asked to provide some insight as which solvents are best in normal-phase flash column chromatography.

Liquid loading of samples onto chromatography columns is not always straight-forward. If your sample is dissolved in the mobile phase, that can work but may not be the best choice, especially if running a gradient.

In this post I show you some work I have done that opens up quite a few options for solvents used for loading samples and some surprising results with DMF.

Continue reading Which sample solvents work best with normal-phase flash column chromatography?

Peptide purification with flash column chromatography – a beginner’s experience

With this post I would like to introduce a new colleague, Beth Denton, who I recently convinced to try flash chromatography for purification of peptides. She has kindly agreed to share her new flash column chromatography experience here.  So, now, here’s Beth.

As a peptide chemist, I was trained to purify my peptides with reversed-phase HPLC, just as many a peptide chemist before me. Despite the hundreds of hours I’ve logged in front of an HPLC, injecting samples and collecting peak fractions, I can’t imagine using any other method to purify my freshly synthesized and cleaved peptides.  In fact, you’d be hard pressed to convince me to try something else.  But here I am, trying something new.  Wish me luck!

In this post, I’ll describe my experiences using flash chromatography to purify a new peptide sample.

Continue reading Peptide purification with flash column chromatography – a beginner’s experience

Celebrating 1 year of the flash purification blog

It is hard for me to believe but the flash purification blog is a year old. This started as an idea within the technical group here at Biotage to share knowledge of flash column chromatography that we have learned over 30-plus years.  Though I was reticent at first, now after only a year its hard to imagine my world without this blog.

Those of you who read this blog regularly are growing in number and each time I post we set a new record for numbers of visitors, thank you.  There are certainly some posts that are popular.  I’ll spend the rest of this post sharing some details and at the end of the post, I ask that you take a couple of minutes to provide feedback in the form of an on-line survey.

Continue reading Celebrating 1 year of the flash purification blog

Detecting the undetectable in flash column chromatography using wavelength focusing

Sometimes it feels as if organic chemistry and chromatography are a mixture of art and science.  Maybe its because of the necessary creativity needed to address the variety of challenges that we face almost daily.  Frankly, its what I find most interesting about this world.

One of the bigger challenges facing chemists is the ability to detect and collect compounds with little or no UV absorption during flash purification.  In this post I will talk about a technique that I have found to be quite useful when trying to purify mixtures containing one or more poor absorbers.

Continue reading Detecting the undetectable in flash column chromatography using wavelength focusing

Acetone – a lower cost alternative to EtOAc in normal-phase flash column 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.

There is one attribute that keeps chemists from using acetone in chromatography – its strong UV absorbance above 250 nm which can mask compound detection and make UV-triggered peak fractionation a challenge, especially aromatic compounds. However, with some state-of-the-art flash systems, mobile phase UV absorption is zeroed in real time enabling the use of UV absorbing solvents such as acetone.

In this post I discuss some reasons to explore switching to acetone and how modern flash chromatography equipment deals the UV-absorbing mobile phase solvents. Continue reading Acetone – a lower cost alternative to EtOAc in normal-phase flash column chromatography

How and when to insert an isocratic hold in flash column chromatography

For many chemists using generic linear gradients and even gradients based on TLC the purification results often are not selective enough to separate all of the compounds in their mix.  This is especially true if your target has a closely eluting impurity. One method used to try and increase resolution is the use of an isocratic hold or gradient pause during purification.

In this post I examine the use of the isocratic hold to determine how well it works and when/if it should be inserted into a gradient method.

Continue reading How and when to insert an isocratic hold in flash column chromatography

Does methanol really dissolve silica during flash column chromatography?

This is an age-old question that has been around a long time, perhaps as long as me (and I have been around a while) –  “Does silica dissolve in methanol?”  For many years I have heard from organic and medicinal chemists as well as students of organic chemistry that silica is dissolved by methanol when running a DCM/MeOH gradient.

In this post I will share the results of some tests I performed that hopefully will help clarify what is happening to the silica when in contact with methanol.

Continue reading Does methanol really dissolve silica during flash column chromatography?

So, what exactly is a column volume in flash column chromatography and how is it determined?

In many of my previous posts I have used the term column volume, typically abbreviated as CV, as a value used to help determine separation quality and loading capacity.  However, I recently was asked a question about this topic from a chemist who understands the column volume concept but wanted to better understand its definition and how it is determined.

In this post I will explain what a column volume is and how it is determined empirically.

Continue reading So, what exactly is a column volume in flash column chromatography and how is it determined?