I am currently doing some initial tests on the size of carboxylate-modified polystyrene beads in deionised water to determine their interaction with BSA with and without the addition of PLL-g-PEG polymer. I have performed some size measurements on 500 nm carboxylate modified beads to determine their initial size (linked below). However, I am running into a lot of errors, which upon changing various parameters, do not seem to disappear.
My uncoated beads samples was prepared at a concentration of 0.1% v/v via sonication for 15 mins to ensure the beads were well distributed, and then after an equilibration time of 20 minutes, size measurements were taken using non-invasive back scattering at 25 degrees Celcius, at an optimised position found by the DLS. Errors were given for the cumulant fit, correlation function, multimodal fit, polydispersity index, and the count rate, with comments on the sample fluorescence and absorbance, sedimentation, aggregation, and concentration. The Size peak was below 500 nm and so 0.01% and 0.001% v/v concentrations were also performed with no improvement.
I am unsure as to whether the azo dye molecules within the beads are interfering with the size measurements (Ex/Em 505/515) and if this needs to be accounted for in the SOP. Please can someone advise?
Beads - FluoSpheres™ Carboxylate-Modified Microspheres (thermofisher.com)
Naomi Crabbe At this size, laser diffraction would be the preferred light scattering technique. At a density of ~ 1.02 g/cm3 you’ll still have significant settling/sedimentation. Carry out a Stokes’ Law calculation to confirm.
At 500 nm (0.5 micron) size, both DLS and laser diffraction are options. What wavelength does your DLS use? That will answer your fluorescence question. Laser diffraction has an advantage that it is performed with mixing to combat sedimentation.
1) First of all, 0.1% v/v concentration for 500 nm beads is far-far too much. As the scattering intensity is increasing with radius according to power law, large particles should be measured roughly at ppm level, depending on particles material and liquid phase.
If you're using automated SOP, ZetaSizer is trying to compensate your error by introducing the maximum possible attenuator and placing the measurement position as close to cuvette wall as possible, but these measures did not help.
> The Size peak was below 500 nm
This is exactly was is expected in high concentration due to multiple scattering effect.
So, try to dilute your sample until the average count rate of 300-400 kcps at reasonable attenuator position of 4-9 (out of 11).
2) Second, pure water is not the best diluent when a precise size measurement is required. In pure water the thickness of electrical double layer (EDL) on the particle's surface may vary. So try to use filtered 1 or 5 mM of KCl as diluent to create some constant ionic strength that keeps the thickness of EDL constant. Keep this ionic strength constant in all measurements.
You may need the narrow band filter (optional item that needs to be installed in the instrument) to prevent the excitation emission from interfering at the detector.
The mean free path for 0.1% v/v of 500nm polysty in water is ~400micrometer (for a 630nm instrument wavelength), therefore the measurement position should be well below that to obtain single scattering signal.
Using a Spatially Resolved DLS instrument, a 0.1% v/v of 500nm can be measured directly, since depths down to
Some additional, fundamental reasons for deviations between good quality DLS-Z-av size and specified-size of larger, even fairly monodisperse particles are discussed in:Poster Instrument dependence of DLS particle size data: implication...
Naomi Crabbe
When measuring gels, surfactant solutions, titanium dioxide dispersions and your polystyrene particles, the environment matters. To accurately measure size, it is necessary to add an inorganic electrolyte such as potassium chloride. to reduce the thickness of the electrical double layer, destroy water and water-polymer associates.
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