Chromatography is one of the most versatile and widespread techniques used in modern science. It plays a vital role in the advancement of chemistry, biology, and medicine. The significance lies in its ability to separate, identify, and purify components within a mixture for qualitative and quantitative analysis.
This biophysical method is instrumental in protein purification, using characteristics such as size, shape, total charge, surface hydrophobicity, and binding capacity to isolate target molecules. Because of the straightforward protocol, chromatography has been used for many years to successfully characterize and purify products for a range of applications.
Getting the most out of chromatography isn’t always easy since the number of variables can quickly add up to make the technique feel complex. With the right knowledge and technical expertise you can boost the efficiency and effectiveness of the technique.
In this blog we explore our ten best tips to optimize the performance of your chromatography resins.
1. Choose the right resin for your application
Resin describes the fine, solid beads that make up the stationary matrices in chromatography columns. They are used to capture and polish molecules, from DNA to antibodies.
Resin particles can be physically or chemically modified to provide specificity in capturing or repelling molecules within mixtures. This enables the separation of molecules based on size, net charge, hydrophobicity, specific binding affinities, and more.
Choosing the right resin for your application directly influences the level of purity and recovery of your target molecule. There are several parameters to consider for optimal performance of your chromatography process such as the most suitable separation technique for your target, bead and pore size, and compatibility with your target molecule.
Capto™ Core resins offer a diverse selection of sizes and properties that greatly facilitate the separation process. A recent study demonstrated the successful separation of extracellular vesicles (EVs) and viral particles using Capto Core 700 beads [1]. These beads effectively removed impurities like proteins and DNA fragments due to their pore size, hydrophobic and positively charged nature.
This removal step was crucial in the subsequent purification of viral particles from EVs through affinity chromatography. This type of innovation helps researchers gain deeper insights into the intricacies of viral diseases.
For help finding the best resin for your application, use our app or selection guide
2. Optimize mobile phase composition
The mobile phase composition plays an integral role in chromatographic separations. Try experimenting with different solvent systems, buffers, and pH conditions to optimize your elution profile and achieve better resolution and peak shape.
In situations where the molecules to be separated have a wide range of polarities or the mixture contains numerous components, consider a gradient elution. The mobile phase composition transitions gradually from "weak" to "strong" during the chromatographic run. This approach enhances the quality of the separation and can reduce the overall run time.
3. Precondition the resin
It is essential to precondition the chromatography resin to remove any impurities or contaminants. If the resin is not pre-treated in the initial stage of use organic contaminants can gradually dissolve and release which affects the quality of the effluent or product. Follow the manufacturer's guidelines for resin equilibration, washing, and regeneration for optimal resin performance.
4. Pack the column properly
Proper packing of the chromatography column is vital to achieve good resolution and efficient separations, especially when using gradient elution. Pack the resin uniformly and avoid voids or channeling that lead to uneven flow and poor performance. Use appropriate packing techniques such as slurry packing or compression packing depending on the resin type.
5. Optimize flow rate
The flow rate during protein purification impacts performance, influences the protein structure, biological activity, and recovery yield. A flow rate that’s too high can result in poor resolution and separation, and a flow rate that’s too low leads to longer cycle times.
Though lower flow rates result in longer run times, sometimes the increased recovery yield, resolution, and column efficiency can be worth the wait.
Tired of the wait? Modern Capto resin technology provides increased rigidity and cross-linking which leads to high recovery levels even at high flow rates and significantly reduced retention times.
6. Automate your protein purification
The future of chromatography is automation. As sample preparation techniques become more complex and systems need more optimization, automation is crucial for improving timeframes and attaining optimal reproducibility.
7. Minimize sample load
Overloading the chromatography resin can decrease retention time, reduce column efficiency, and negatively affect resolution and peak shape (Fig 1). It’s important to optimize the sample load and ensure that the amount of analyte is within the resin's capacity.
A general rule is to never apply more than 30% of the total binding capacity of the chromatography column to maintain optimal resolution using gradient elution.
Fig 1. Decreased sample load typically improves the resolution. By decreasing the load from (A) 10 mg to (B) 1 mg, the resolution between the first two peaks increased, as indicated by the green circles.
8. Employ proper cleaning and maintenance
Regular cleaning and maintenance of your chromatography system is crucial for optimal resin performance and longevity (Fig 2). Cleaning columns routinely removes precipitated proteins or other contaminants that may have built up on the column. Also, consider ad hoc cleaning when you see colored bands in the top of the column or if the backpressure has substantially increased.
Fig 2. Microscope pictures of the resin beads. Comparison between foul MabSelect™ particles after inefficient cleaning with 0.1 M phosphoric acid (A), cleaning with 0.1 M NaOH for (B) 40 mins and (C) up to 3h.
9. Stay updated on advances in resin technology
The field of chromatography is continuously evolving and new resin technologies are regularly introduced. Modern resins are shown to be mechanically more robust, have a higher pore count, higher apparent porosity, and an increased distribution of pore sizes compared to legacy resins. These advancements mean modern resins are more effective, long-lasting, and capable of purifying a wider variety of molecules.
Other recent advancements include fiber-based chromatography, which provides a another way to increase the specificity and speed of chromatography. By offering a more open pore structure than resin beads, fiber-based chromatography can easily isolate larger molecules and complete cycles in minutes instead of hours.
Read about the latest innovations in resin technology
10. Don't pinch pennies that cost dollars
The most expensive element in any lab is you, so prioritize your time and resources. Implement efficient systems that minimize your time and effort while maximizing your overall output.
When thinking about purchasing new equipment, it is important to consider more than just the initial cost. Higher sample loads, flow rates, and binding capacity will reduce your time and labor — the most significant expense.
If your chromatography resins don’t give you optimal performance or require more frequent cleaning, it may be time to invest in a modern resin column. The advances in modern resins save you significant time and costs in the long run, and allow you to quickly move from purification to analysis and characterization.
By following and implementing these chromatography tips, you’re sure to boost the performance of any chromatography resin that you choose. For further support in any aspect of your workflow, contact our Scientific Support team.
- Reiter K, Aguilar PP, Wetter V, Steppert P, Tover A, Jungbauer A. Separation of virus-like particles and extracellular vesicles by flow-through and heparin affinity chromatography. Journal of Chromatography A. 2019;1588:77-84. doi:10.1016/j.chroma.2018.12.035
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