Liquid chromatography (LC) is used for two different purposes – analytical work and purification – distinct applications which place differing requirements on the equipment and columns used. It is difficult to imagine a single system and columns perfectly designed to work perfectly in both roles. However, it is clear that recent technological developments have allowed flash purification to learn a few tricks from HPLC, to some extent blurring the distinction between the two. This kind of cross-over can only go so far, but commonality in the principles of operation means that cross-fertilisation is inevitable in purification applications – and as can be seen by the advantages of commercial products like Biotage® SNAP Ultra, Isolera™ Spektra and Isolera™ Dalton – invaluable.
Liquid chromatography (LC) involves the separation of a mixture into components based on partitioning between a stationary phase (typically a solid material such as silica or a polymer) and a flowing liquid mobile phase. With wide ranging application, LC is one of the most ubiquitous techniques of the analytical laboratory. However, analytical chromatography is not the only environment where LC flourishes, and for many years purification by LC has been a mainstay of organic synthesis laboratories, such as those in pharmaceutical and agrochemical companies. Traditionally, analysis and purification by LC have been kept entirely separate, with dedicated systems designed for each, however in recent times there has been a blurring of this distinction between the two forms of LC.
In liquid chromatography, the technique most chemists are familiar with is high-performance liquid chromatography (HPLC). This powerful analytical technique is ubiquitous in laboratories around the world, and a basic understanding of the mechanisms of separation is taught to undergraduates. Systems designed for this application involve high performance pumps, sensitive detectors and an often complex and powerful software suite to tie the components together. Separations are typically performed on expensive, reusable columns. Anyone familiar with a typical chemical QC or analytical laboratory will have seen HPLC systems in abundance.
In a purification context, however, LC is used somewhat differently. Although high-performance preparative LC systems are available, they are expensive and relatively niche. Instead it is simpler and cheaper techniques such as flash purification that are more commonly seen. This technique involves a dedicated system where ease-of-use is imparted by a dedicated software focus on purification. Typically a simple pump is employed with a relatively low sensitivity UV, and disposable, one-shot pre-packed plastic columns are used.
To most chemists, flash purification is a simple tool to convert raw reactor products into cleaned up samples ready for a further synthesis step or analytical study. To analytical chemists, flash is seen as a relatively quick and dirty technique compared to HPLC. In the past, these two very divergent forms of LC would rarely see cross-over, but with new developments the line between HPLC and purification is blurring.
This article discusses two recent developments in flash purification that have increased cross-over with traditional HPLC techniques
The use of expensive HPLC-type small particle size, high pressure stainless steel columns is not suitable for flash purification. Loadings on such columns tend to be relatively small, and to achieve high flow rates and allow purification of material on the gram scale, very large columns are needed with extremely high flow rates at pressure and costs that are prohibitive to most laboratories. Instead, cheaper columns with relatively high particle size packings are desirable, which can be disposed of after a single use to reduce contamination risk and solvent-intensive washing. As a result, flash purification has traditionally been performed using irregular silica particles that are large (around 50 μm) in size compared to HPLC packings.
Recently LC has seen a move towards spherical particles, smaller in size and to some extent mimicking the evolution of HPLC. These spherical media have many advantages in purification, giving greater resolution than irregular counterparts. Interestingly enough, this is not the major benefit of these columns. Although resolution is often the criterion of performance in HPLC, the desired goal in flash purification is high column loading. Innovative spherical media like Biotage’s SNAP Ultra 25 μm packings have almost twice the surface area of older column formats, leading to a vastly increased loading. This allows smaller columns to be used for the same purification, resulting in reduced solvent consumption, faster run times and less solvent evaporating after the purification. Also, despite the smaller column format, SNAP Ultra gives lower pressures for comparable column sizes than irregular materials due to the way in which spherical particles pack.
For details of Biotage’s column offerings, see the Biotage Flash Cartridge User Guide - The Definitive Guide to Flash Chromatography
A traditional flash purification set up employs a UV detector, a simple concentration detector revealing when a compound containing a chromophore is eluting from the column, but providing no further information about the actual nature of the compound. This is in stark contrast to HPLC, where dedicated information-rich detector techniques such as mass spectrometry reveal information about the composition and chemical nature of the eluting species. In flash purification, being seen as a low cost, simple technique, the use of such devices has previously been limited by their relative cost and complexity. As technological developments unfold and the cost and complexity of information rich detectors decreases, they have now become viable as tools in flash purification as well.
The first such advance was the inclusion of a photo-diode array (PDA) within a flash system. For example, the Isolera™ product from Biotage with the Spektra upgrade includes PDA functionality along with a host of software features. This allows post-run analysis of the separated components across the spectral range, with the ability to assess component purity after collection. Recently, flash purification has been taken to a new level with the inclusion of mass-detection. The Isolera Dalton flash instrument is capable of mass-directed flash, with the collection of fractions triggered by the detection of the m/z values of target compounds either entered manually or identified after a direct injection of the braw reaction product.
For more information of the Isolera series of instruments, Spektra and the Isolera Dalton, download the brochure Biotage Flash Purification Systems - Pure Compounds in a Flash
Ten Years with Environmental SPE Workflow - a User's Perspective
How St. John's University Accelerated their Cancer Research
Application Video: Extraction of Polyfluorinated Compounds in Water
Yes, You Can Add Biotage® Sfär Columns to Your Isolera™ System
Two New Applications Notes - Sample Preparation of a Drugs of Abuse Panel in Human Hair