Expert Insight: Non-specific adsorption: Common causes, how it affects chromatographic results, and how to prevent it

Watch this on-demand webinar to learn how to prevent non-specific adsorption during your chromatography workflow

15 Apr 2021


How to prevent non-specific adsorption
Kim Haynes, principal product marketing manager at Waters

Did you know that non-specific adsorption (NSA) is a common cause of poor reproducibility, broad peak shapes, and limited sensitivity? Have you ever had to include long column conditioning or passivation steps in your workflow to achieve your analytical results?

In this on-demand webinar, Kim Haynes, principal product marketing manager at Waters, discusses the common causes of analyte loss, the approaches that have been used to solve them, and the consequences of those choices. Haynes also introduces a new family of technologies and columns that were developed specifically to solve these challenges by reducing analyte-surface interactions to give chromatographers more control over their separations.

Read on for highlights from the live event’s Q&A discussion or register to watch the full webinar on demand.

Q: Are the surfaces suitable for size-exclusion or ion-exchange chromatography?

KH: Typically, we know that molecules in those separation mechanisms could suffer from absorption problems. Specific high-performance surfaces, PREMIER and MaxPeak High Performance surfaces, are designed and tested for reverse-phase separations, and we have applications for HILIC mechanisms and retention mechanisms as well. However, we are still exploring and looking at processes like size-exclusion and ion-exchange. As I mentioned in the presentation, MaxPeak High Performance services is a family of technologies for these processes. These are separations that we plan to look for in the future to explore, but right now the focus is on reverse-phase separations primarily.

Q: What about absorption of positively charged analytes?

KH: We talked about the mechanism to prevent chelation. We've looked at things like phosphate-containing compounds and we've looked at acidic compounds. Those are typically the ones that we've seen chelation issues with. Whether that would help in your specific scenario, I think is something that you'd have to test. If there is an issue with metal interactions with stainless steel surfaces, then this is potentially something that could help. With regards to testing those specific compounds, I'm uncertain, but I would look at the mechanism of loss to explore whether this would be a help. 

Q: When using the PREMIER columns, do you have to use a biocompatible metal-free flow path?

KH: When we were exploring the challenges with metal interactions and looking at nonspecific absorption, we started with the piece that had the most metal and comes into the most contact with the analyte. That's the column, the frit, and the stainless-steel body of the column itself. Depending on the sensitivity of your analyte to nonspecific absorption interactions, you may need to look at other pieces of that flow path as well. That's where processes like system passivation help. If you've got something that's really sensitive to nonspecific absorption and metal chelation, you may still need to look at that. I also encourage you to keep in touch with Waters and keep your eyes open. We know that there are a lot of pieces that contribute to nonspecific absorption losses.

Q: How durable is non-specific binding? How and when during LC are bound macromolecules interactions with the metal surface broken?

KH: It's not steady necessarily, if you have non-specific absorption you will have those binding losses to a certain point of saturation. That binding can be disrupted with different amounts of organic substances that you may be running in a reverse space separation. That's where variability comes in, and you see large error bars in those types of situations. The best approach is to prevent that type of loss to begin with, so you don't have to worry about the fact that at different times you may get some analyte back-off that leads to variability. It also leads to the need for repeat conditioning, which isn't a steady state.

Q: How do I know that I'm having a problem with non-specific absorption? Are some applications more recognized for having this problem?

KH: Some of the symptoms of the problem are variability in your analysis. You can look at the structure. If you have things that tend to chelate with metal surfaces, that's a good indicator. It can be phosphate-containing compounds as well, but I think a good indicator is if you have peak tailing, for example. Could that be caused by interaction with the metal surfaces? Do you have reproducibility challenges? Do you find that you need to condition your column or your system extensively in order to get repeatable results? There are a number of different things that can indicate to you that you might be having this issue. I would encourage you to explore the technical white paper that we have published about the issue of non-specific absorption and that may give you some indications as well.

Find out more about the common causes of non-specific adsorption and how to avoid it>>