Flash chromatography has emerged as a valuable tool in peptide purification workflows, especially as a pre-purification or cleanup step. While it shares the same underlying principle as preparative HPLC, they differ in scale, efficiency, and application. In this blog, we discuss these points.
Peptides are synthesized by chemical or recombinant methods or extracted from natural sources. Isolating and purifying peptides from a complex mixture is a critical step in manufacturing various peptide-based products, including pharmaceuticals, diagnostics, and research reagents.
The growing use of peptides has increased production demands, requiring faster and more efficient purification processes to maintain high quality standards. Innovative purification strategies are essential to meet the demand for efficient workflows. Impurities in peptide preparations can significantly impact downstream processes. They can compete with the desired peptide in downstream reactions, leading to lower yields. Impurities can affect the peptide's biological activity and compromise the product quality, or they may interfere with downstream assays, leading to inaccurate results. This may warrant additional purification steps to remove impurities leading to increased production costs.
Sample cleaning is essential in peptide purification to eliminate impurities that can interfere with downstream processes. It involves removing contaminants such as salts, reagents, truncated peptides, and other byproducts generated during peptide synthesis. Reverse phase chromatography (RP-HPLC) is a popular choice for peptide purification. While other techniques, such as ion exchange chromatography and size exclusion chromatography, can also be used, RP-HPLC remains the preferred method due to its superior performance and versatility.
Preparative HPLC (High-Performance Liquid Chromatography) has been the standard method for peptide purification for many years. Its ability to provide high resolution and high purity levels has made it the preferred choice for both research and industrial settings. However, it also has cost, time consumption, and solvent usage limitations.
Cost: Preparative HPLC systems are generally more expensive to purchase and maintain due to the high-pressure pumps, detectors, and column technology involved.
Time Consumption: HPLC runs can be significantly longer, especially for larger sample volumes. This can impact throughput and productivity.
Solvent Consumption: Preparative HPLC often requires larger volumes of solvents, leading to higher costs and environmental concerns.
Column Life: Loading crude mixtures into HPLC columns can deteriorate column separation parameters (efficiency, resolution, plate numbers), and can reduce the lifespan of columns, increasing operational costs.
Sample Loading Capacity: Preparative HPLC columns are filled with smaller particle media (5-10 µm), which become overloaded quickly and thus lower loading capacities. As a result, multiple injections are needed for larger sample volumes. For this reason, it is preferable to use 10 µm media instead of 5 µm media for preparative chromatography. The typical loading capacities can range from 0.01% to 0.1% of the media weight.
These limitations have driven the exploration of alternative methods, such as flash chromatography, to address the challenges [1, 2]..
Some key benefits of flash chromatography compared to HPLC are:
Speed: The run time for flash chromatography can be significantly lower, allowing rapid processing.
Cost-effectiveness: The technique uses less solvent and requires simpler instrumentation, lowering overall operation and maintenance costs.
Scalability: Methods developed on HPLC can be easily adapted to flash chromatography, allowing for the purification of larger peptide quantities.
Versatility: It can be used as a standalone purification method for peptides if there is spacing between impurity and peptide peaks or as a pre-purification step before HPLC for peptides with closely eluting peaks.
Flash chromatography also offers several sustainability advantages over preparative HPLC for peptide purification:
Reduced Solvent Consumption: Compared to preparative HPLC, flash chromatography often requires less solvent. This directly translates to lower chemical waste generation and reduced environmental impact.
Energy Efficiency: Flash chromatography systems operate at lower pressures (50-200 psi) than HPLC (>1000 psi), leading to lower energy consumption.
Waste Reduction: Minimizing solvent waste, another positive environmental impact, is possible by lowering the amount of organic solvents per gram of media weight packed into columns and optimizing purification conditions.
Green Solvents: Flash chromatography is compatible with greener (non-chlorinated) solvents, further enhancing its sustainability profile.
Flash chromatography can be an efficient and cost-effective method for purifying peptides. However, it may not always be sufficient to achieve the desired purity levels, especially for complex peptide mixtures or when high resolution is required. For complex peptide mixtures with many components or closely related isomers, preparative HPLC's higher resolution and sensitivity are often necessary to obtain pure fractions. Flash chromatography often has a lower resolution than preparative HPLC, making it more challenging to separate closely related peptides.
Flash purification is a robust and efficient front-end purification step, optimizing the subsequent preparative HPLC process for peptide isolation. It allows for more efficient use of the preparative HPLC column by initially reducing sample complexity and removing significant impurities. A common strategy is to use flash chromatography as a workup step to remove most impurities and then employ RP HPLC for final purification. This approach can often optimize efficiency and cost-effectiveness, especially when dealing with complex mixtures or when high purity is a critical requirement.
In summary, the choice between flash chromatography and preparative HPLC depends on the specific purification goals such as the amount of material to be purified, the desired resolution, and the cost. Flash chromatography is ideal for initial sample cleanup or large-scale purification. Preparative HPLC is best suited for achieving high purity, isolating closely eluting peaks from complex mixtures and when low sample volumes are available. In many cases, both techniques can be employed to optimize the purification process. Flash chromatography can be used for initial sample enrichment, followed by preparative HPLC for final purification and isolation.
At Millennial Scientific, we develop next-generation products for separation and purification of biomolecules. Get in touch with us to discuss how we can support your reverse phase flash chromatography needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
References
[1] W.C. Stevens, D.C. Hill, General Methods for Flash Chromatography Using Disposable Columns, Molecular Diversity 13(2) (2009) 247-252.
[2] K.K. Sørensen, N.K. Mishra, M.P. Paprocki, A. Mehrotra, K.J. Jensen, High-Performance Reversed-Phase Flash Chromatography Purification of Peptides and Chemically Modified Insulins, ChemBioChem 22(10) (2021) 1818-1822.
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