One of the pleasures of working in the Applications Lab is that I get to analyze a variety of GPC/SEC sample types. I can be finishing up a set of biodegradable polyesters, currently running low molecular weight oligomers, while getting an OMNISEC ready to study polysaccharides from wine. It never gets boring!
That range of sample types leads to a lot of interesting chromatograms and peak shapes. But, regardless of sample type, one question I frequently receive is: What is this peak in the light scattering signal?
And often, after a quick review I can reply: That peak has all the characteristics of an aggregate!
What is an aggregate?
An aggregate is a cluster of macromolecules that are entangled or clumped together through supramolecular interactions. These species typically exhibit high molecular weight and are larger in size than the main sample component – sometimes significantly larger.
How can I tell it’s an aggregate?
The profile of an aggregate peak will have certain features that reveal its identity. These include:
- Early elution volume – aggregates are large and thus tend to elute prior to the sample peak
- Strong light scattering response – due to the aggregate’s high molecular weight
- Refractive index &/or UV response – low or non-existent due to aggregate’s low concentration
- Viscometer response – sometimes visible, but usually weak due to high density of aggregate
When you add those up, you get a large material that exists in low concentration relative to the rest of your sample. This large material has an extremely high molecular weight, as evidenced by the strong light scattering response in combination with the low refractive index/UV response (meaning that the strong light scattering response is not because of the high concentration of the species). Even though they’re large and exhibit high molecular weight, aggregates tend to be very dense and often produce a minimal viscometer response.
Does it have to be a separate peak?
No! If they’re not significantly larger than the main sample, aggregates can show up as a shoulder of the sample peak. This shoulder will be on the earlier eluting, higher molecular weight (larger) side of the peak and will be most notable in the light scattering signal.
A frequent question users ask in this situation is whether to include the aggregate within the integration limits. I recommend placing the upper limit according to the refractive index signal, which will most likely exclude much of the aggregate peak. This prevents the aggregate from distorting your calculated molecular weight and keeps your results from being extrapolated beyond the existing refractive index signal.
Why does it matter?
The effect of aggregates can range from harmless to dangerous, depending on the sample. With a synthetic polymer sample, an aggregate may indicate the dissolution solvent and procedure are not optimized. Or maybe that the sample is too old and has begun to degrade.
However, in protein samples where there is a strong relationship between structure and function, an aggregate can be catastrophic. In therapeutic proteins aggregates can reduce efficacy and, more significantly, stimulate an immune response. Because it is critical to know if aggregates are present, many users in the biopharmaceutical industry use light scattering detectors primarily to identify aggregates, with absolute molecular weight determination a secondary function.
Can I prevent aggregates from forming?
Sometimes you can minimize the presence of aggregates in your sample by adjusting the dissolution or storage procedure. Other times you may have to physically remove the aggregate via filtration. Things to try include:
- Sample purification, including filtration
- Finding a better solvent for dissolution
- Apply the least amount of heat (temperature & time) required for dissolution
- Adjusting identity and concentration of additives in the dissolution solvent
- Preparing sample as close to injection time as you can (while ensuring complete dissolution)
- Test your sample with DLS before injection for a quick, low-resolution aggregation check
In conclusion, I hope I’ve been able to shed a little light on that mysterious peak in your chromatogram! Furthermore, if you have any questions please don’t hesitate to contact us or email me directly at firstname.lastname@example.org.