How small can you go?

The other day I had an interesting request about measuring molecules with DLS. And coincidentally we just released a relevant application note on a similar topic on small molecule detection with SEC. Let’s see how this all started.

“While measuring water, I am observing a correlation that lasts for >1 second and has an intercept >0.1 . I use 1 µm PTFE filter to filter prior to all measurements but I am wondering if this could be due to some residual dust. If so, are there any alternative methods that I could use to eliminate the contribution from dust particles? I work with particles that are expected to be <1 nm. Could I use the correlation function to show that my samples are <1 nm by comparing them with that of pure solvent. Or would it be better to compare the derived count rate of my samples to that of pure water?”

Small molecule : just water – how to get it clean?

In principle, water should only show a flat, yet noisy, correlation function. There should be no size distribution. (Well, the software may display something, however, with several peaks, and respective warning indicators in the data quality guidance). Yes, in the data you describe there appear to be some particles present, since you see repeatable correlation functions with a significant intercept. How can we make sure the water is clean? Is the water suitable for our light scattering experiment?  

Water can be difficult to keep clean, since it is such an excellent solvent. It can also happen that the filters may shed some particles or produce air bubbles in your sample. My recommendation: Try simple distilled water, to get an idea of clean water and what it can look like. If you do not have distilled water in the lab, try distilled water from the supermarket. The type in a sealed plastic bottle. That is usually surpringly clean.

Measuring small molecules – what to consider

You expect a size of your nanoparticles lower than 1nm (i.e. the molecules). Since these are small scattering objects they will not scatter much light. So you probably require enough concentration in order to achieve a good correlation function. And yes, observe the derived count rate to make sure there is enough signal.
The project could work, as it is similar to measuring sucrose, which does work in your Zetasizer.
Please let me know how it goes!

See molecules: Bradykinin – 1060 Dalton – with SEC

In the application note “How small can we go: Analysis of Peptides” the molecule Bradykinin, a peptide (sequence of amino acids Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg or short RPPGFSPFR) of about 1060Da shows a resolvable peak in size exclusion chromatography or SEC. The image below shows a 2D representation, and we can roughly estimate the image to be about 0.5 by 1nm. (A typical C-C bond is about 0.15nm.) The experimentally determined hydrodynamic size of the monomer in this peak is between 0.7 and 0.8 nm.

Since the molar mass is almost three times that of sucrose, we expect to be able to measure this with DLS as well. And the Zetasizer software calculator can predict an estimate of the hydrodynamic size. For a peptide we would expect a size to fall somewhere between a linear or a branched polysaccharide structure, i.e. we predict 0.7nm to 0.8nm – and that is in excellent agreement with the result obtained in the application note.

PS: if you really want to dive into it, we have previously measured even lower molecular weight with chromatography. Even just 300 Daltons could be resolved, detailed observations in an earlier low molecular weight with OMNISEC blog.

Previously on the topic of small molecules, plus related info

• Blog on the opposite problem: “Solvent Peak – how to avoid the buffer or salt or additive contribution in a DLS result?”
• Application Note on “Molecular weight determination of polymers and polysaccharides” down to 980 Dalton
• Blog on low molecular weight analysis with OMNISEC – down to just 300 Dalton !
• Blog: “Why does DLS say my particles are too small?”
• Application Note on “Using Dynamic Light Scattering for Sub Nanometer Sizes” from a disaccharide
• For a simple animation video on dynamic light scattering, how it works and what the hydrodynamic size is watch the popular “Introduction on Dynamic Light Scattering Analysis“
• If you are interested in binding of small molecules, there is a different technique “Sensitive kinetic analysis of small molecules binding to large drug targets“