When it comes to polar organic compound purification, many chemists turn to normal-phase flash chromatography often utilizing dichloromethane and methanol as the eluting solvents. While this can work, it often can be challenging to optimize due to methanol’s high polarity and protic chemistry.
Improvements in solid phase peptide synthesis strategies and development of resin linkages susceptible to low acid cleavage conditions has enabled synthesis of long peptides while keeping the protecting groups intact. This strategy is now used for the preparation of chemically synthesized proteins, wherein shorter peptide fragments are ligated together. They are also found in the synthesis of peptide macrocycles that utilize head-to-tail cyclization strategies. Although linear synthesis of protected peptides is generally straightforward, purification of these compounds using traditional reversed phase methods is quite challenging. Herein we describe the use of normal phase chromatography for purification of fully protected peptides.
Biotage FLASH 400™ Production-Scale System The FLASH 400 is a complete skid-mounted system designed for large-scale flash chromatography and adsorption purification. The FLASH 400 uses prepacked cartridges and radial compression for performance and reliability. Built with industrial-grade components that are appropriate for operations under FDA regulations and cGMP standards, the FLASH 400 is rapidly becoming the first choice of pharmaceutical and contract manufacturing companies around the world for critical purification applications.
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.
Purify up to 400 grams of compound at 1 L/min with the Biotage Flash 150™ system, up to 2x faster than traditional glass columns. This robust stainless steel system safely operates at 100 psi enabling high flow rates and the use of higher viscosity solvents. A variety of cartridge media provides chemists with selectivity choices for optimal purification conditions. Simple and reliable, this system contains everything needed to begin your separations.
As reversed-phase flash chromatography gains traction in medicinal chemistry labs the need to monitor its cost and safety are becoming more important. Commonly used reversed-phase solvents typically include water with an organic solvent such as methanol or acetonitrile – each have advantages and disadvantages.
Reversed-phase chromatography is typically used when you need to separate several milligrams of relatively polar compounds that either are not soluble in normal-phase solvents or are not compatible with bare silica because they react, stick, or both. If you are currently using reversed-phase at preparative scale, such as flash chromatography, you know the mobile phase limitations – water with either methanol, acetonitrile, or THF. As with normal-phase flash chromatography, when it comes time to purify you want your crude sample fully solubilized in the weakest possible solvent at the highest possible concentration. ACS 2016
Although capable of very high resolution, RP-HPLC is often limited by low column loading capacity, therefore demanding a significant time investment for peptide purification. As an alternative strategy, reversed-phase flash chromatography can also be used to purify synthetic peptides. The larger particle size used in flash column chromatography enables much larger loading capacity, thereby significantly reducing the time required for peptide purification.
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Natural product chemistry deals with discovering the previously unknown in nature. Compounds found in nature are typically found in low quantity and thus extractions are needed to isolate certain compounds classes or at least compounds with similar solubility.
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
For those chemists performing organic synthesis, reaction mixture purification by flash column chromatography is an integral and necessary part of the synthesis process. However, flash chromatography consumes large volumes of solvent which either needs to be recycled or disposed. ACS 2016.