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.
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.
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.
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.
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.
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.