Reversed-phase flash chromatography is a very popular purification technique using a non-polar stationary phase. Main application areas include separation of polar, ionizable and highly lipophilic compounds which cannot easily be separated by normal-phase techniques.
Flash purification is a separation technique developed in 1978 by Professor W.C. Still that uses a stationary phase (a column or cartridge filled with an insoluble solid support) and a mobile phase (elution solvent mixture) to separate and purify a mixture of organic compounds.
Normal-phase flash purification is commonly used by organic chemists in pharmaceutical drug discovery and process development labs. However, for many synthesized products (e.g. peptides, nucleotides and basic drug candidates) purification on standard flash silica is not an option due to irreversible adsorption, chemical interaction and/or solubility issues. Reversed-phase flash purification is an excellent solution for these applications. Yet, this technique has been used sparingly because of perceived lower loading capacity, higher operating pressures and a scarcity of publications addressing reversed-phase flash chromatography.
Peptide purification using standard reversed phase HPLC methods are hampered by low loading capacity, resulting in purifications that demand significant time investment. Recently, the use of reversed phase flash chromatography has increased in popularity for peptide purification due to the significant reduction of purification time, enabled by the increased loading levels of the larger stationary phase particles. Resolution, though, is somewhat diminished with the larger particle size, demanding creative techniques to retain a highly pure peptide product.
Flash purification involves a simple liquid chromatography technique » Method development uses TLC as a way of deciding the parameters for the separation » Isocratic separations are easiest to develop, but gradient separations are more powerful » Software in the Isolera helps with conversion of an isocratic separation to a gradient » It is possible with the Spektra software to run step gradients » Loading options are dependent on the column type » SNAP offers the most flexibility » Care must be taken to choose the best loading option to get good purifications
Pure fractions are in high demand – impurities mean more work after purification. With new technology, fraction purity can be digitally analyzed directly during chromatography to reveal any problems on the fly. In this application we will show how the Isolera Spektra is used to determine fraction purity, eliminating the need for other post-purification analysis.
Reversed-phase chromatography facilitates the isolation of milligram to multi-gram quantities of polar compounds from naturally occurring materials or from synthetic reaction products. Biotage reversed phase KP-C18-HS SNAP cartridges have been shown in a number of applications to be effective for the purification of natural products. The methodology is robust and may be applied generically to new extraction purification runs. Keywords: natural, products, reversed, phase, normal, extraction, spinach.
Normal-phase flash chromatography1 has been widely adopted as the method of choice for separation of product mixtures and reaction by-products. One of the most significant developments in this area concerns the practical separation of polar molecules. Reversed-phase purification is a modification of normalphase chromatography that provides an efficient mechanism for the separation of polar compounds.
Modelle haben gezeigt, dass sich die zur Herstellung eines Zielmoleküls benötigte Zeit mit dem automatisierten Workflow von Biotage um bis zu achtzig Prozent verkürzen lässt, sodass auch die Projektlaufzeiten entsprechend verkürzt werden können.
To save money on consumables, many chemists choose to reuse silica flash cartridges. This is true but risks purification results because chromatographic separation performance will change from run to run which reduces purification quality, especially in normal phase systems. Regardless of the cartridge brand used, repeated use of silica flash cartridges results in loss of compound resolution and therefore fraction purity.