Welcome to the Biotage Flash Purification Blogs.

    Is reversed-phase flash chromatography less expensive than normal-phase?

    April 26, 2022 at 2:41 PM / by Bob Bickler posted in Reversed-phase, Cost, Normal Phase

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    For many labs on a tight budget, keeping expenses at a minimum is crucial. Many of these same labs are also required to become more sustainable, or greener, by reducing the amount of organic solvent they use. Reducing solvent also reduces expenses, but what is the impact on lab productivity?

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    How many times can I reuse my reversed-phase flash column?

    February 22, 2022 at 2:41 PM / by Bob Bickler posted in Reversed-phase, Reuse, flash chromatography

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    Reversed-phase flash chromatography use continues to increase for a variety of reasons. Unlike silica normal-phase flash columns, which typically are used only once, reversed-phase flash columns can be cleaned, stored, and reused. How many times can a column be reused is a frequent question I receive. In this post, I will do my best to answer this question.

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    When Should I Replace My Flash C18 Column?

    January 25, 2022 at 3:36 PM / by Bob Bickler posted in Reversed-phase, replace

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    Knowing when it is time to replace your reversed-phase flash column is a question I am asked frequently along with…

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    How does reaction time impact synthetic product purity and yield?

    November 30, 2021 at 4:59 PM / by Bob Bickler posted in Reversed-phase, Normal Phase, microwave

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    For my purification blog I often will synthesize compounds so I can show representative, real-world reaction product purification. In doing so, I decided I would also post on the impact of various synthesis variable. This post looks at the impact of reaction temperature time on an amide synthesis.

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    What is Orthogonal Flash Chromatography and Why Should I do it?

    September 7, 2021 at 3:15 PM / by Bob Bickler posted in Reversed-phase, Normal Phase, orthogonal

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    Think of orthogonal flash chromatography as 2D-chromatography where a reaction mixture or natural product extract is purified first with one methodology or solvent gradient then re-purified with a different method or solvent pair in order to remove co-eluting impurities. This is a technique practiced in medicinal chemistry, especially for final compound purification, when the final product is purified first with normal-phase flash followed by reversed-phase prep HPLC.


    There are two general flash chromatography techniques...

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    How does an acid pH affect reversed-phase chromatography separations?

    August 10, 2021 at 6:00 PM / by Bob Bickler posted in Reversed-phase, acid, pH

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    Chromatography is as much an art as it is a science. Between synthetic reaction products and natural products, the range of compounds requiring separation, purification, and isolation is broad and diverse creating challenges from time to time. Because of this diversity, not all chromatographic separations can be performed with a “neutral” solvent system – one without added pH modifiers or buffers.

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    Does dry load media choice impact reversed-phase flash purification results?

    June 15, 2021 at 4:07 PM / by Bob Bickler posted in Reversed-phase, dry load, media

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    Flash chromatography is a purification technique used by chemists to isolate their targeted compound from by-products and impurities. Because the reaction mixture (or natural product extract) may have its best solubility in a solvent that is chromatographically “stronger” than the mobile phase, liquid sample loading can be problematic causing early eluting and/or broad peaks with poor purity. In those cases, a technique called dry loading is frequently used.

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    What is the maximum volume I can load on my reversed-phase flash column?

    May 19, 2021 at 2:11 PM / by Bob Bickler posted in Reversed-phase, Solvents, loading capacity

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    With all forms of chromatography there are limitations relating to sample load – both mass and volume. These are independent variables which, for the best results, should be investigated separately. In this post, I will address the impact of increasing solvent volume on flash chromatographic separations.

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    What is my C18 flash column's loading capacity?

    May 5, 2021 at 2:27 PM / by Bob Bickler posted in Reversed-phase, loading capacity

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    A good question I get asked frequently to which there is no specific value. When asked this question I answer, “it depends on your sample and how good your separation is”; not a satisfying response, but it is the truth.

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    How does gradient slope impact flash chromatography loading capacity?

    February 9, 2021 at 1:40 PM / by Bob Bickler posted in Chromatography Fundamentals, Reversed-phase, Troubleshooting and Optimization, Scale-Up

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    For many synthetic chemists the primary purification goal is to isolate as much synthetic product as possible with a minimum of 80% purity. The go-to technique for product isolation is flash purification (flash chromatography), especially for intermediates.

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    Using pH to optimize reversed-phase flash chromatography separations

    December 8, 2020 at 8:51 PM / by Bob Bickler posted in Chromatography Fundamentals, Reversed-phase, Solvents, Media and Resin, Detector

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    I have previously posted on the topic of normal-phase optimization by evaluating different solvent blends or mixtures. I have also touched on reversed-phase method development as well suggesting chemists use HPLC to optimize their purification.

    In this post, I will look at the impact modifying mobile phase pH can have on reversed-phase separations.

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    Can reversed-phase flash column chromatography be greener?

    December 3, 2020 at 7:24 PM / by Bob Bickler posted in Reversed-phase, Green

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    In previous posts I offered some suggestions to improve the “greenness” of normal-phase flash purification.  As the use of reversed-phase flash purification has increased the past few years I thought I would explore how to potentially make it greener too.

    So, with that in mind, let's take a look at the use of acetone in place of acetonitrile as a reversed-phase flash chromatography solvent.

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    When should I add acid to my detector make-up solvent when using mass-directed flash chromatography?

    November 20, 2020 at 2:57 PM / by Bob Bickler posted in Amine, Chromatography Fundamentals, Reversed-phase, Solvents

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    Increasingly, organic and medicinal chemistry labs use mass-directed flash chromatography to isolate synthesized compounds. Mass-directed flash chromatography benefits are many, including collecting only the targeted molecule(s) in the reaction mixture. This approach simplifies compound purification since you know what you have made and it's associated mass.

    However, there are mass detection nuances that can be challenging. One of these is to know when an acid should be added to the mass detector’s make-up solvent to protonate targeted molecules. In this post, I will provide some insight on this topic.

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    Can reversed-phase chromatography compound elution volume/time be predicted?

    November 18, 2020 at 9:26 PM / by Bob Bickler posted in Chromatography Fundamentals, Reversed-phase, Method development

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    When developing reversed-phase flash chromatography methods it is important to understand the impact that a change in solvent ratio has on compound retention and, therefore, separation performance. Unlike normal-phase chromatography where you can optimize separations using TLC and a wide variety of solvents and solvent ratios, reversed-phase limits you to 3 to 4 solvents, including water, using either HPLC or small flash columns for method development. Those solvents include:

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

    October 23, 2020 at 5:25 PM / by Bob Bickler posted in Chromatography Fundamentals, Reversed-phase, purification

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    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.

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    How do I convert my HPLC method to flash chromatography?

    July 14, 2020 at 2:00 PM / by Bob Bickler posted in Chromatography Fundamentals, Reversed-phase, HPLC

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    As synthetic chemistry has evolved, so has flash chromatography. Target molecule synthesis is becoming more complicated and the synthetic products more polar. This shift in compound polarity has changed purification strategy from almost entirely normal-phase flash chromatography using silica to a significant percentage of flash chromatography now being reversed-phase during the past 9 or so years.

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