The Malvern Zetasizer APS is used to measure the size of sucrose in a 96 well plate. Measurement of such a small molecule is considered a good test of the sensitivity and the lower size specification of a Dynamic Light Scattering system.
To explore the lower size limit of a dynamic light scattering (DLS) instrument, a suitable small, well defined particle or molecule that produces enough excess scattering needs to be used. Traceable latex standards are only available down to 20nm in diameter. Proteins such as lysozyme are often used as test samples for sizes below 10nm. However, for sizes less than a nanometer, there are no proteins available. Sucrose is an ideal candidate to test the performance of a light scattering system as it is small, easily obtained, available in a crystalline, pure form and readily dissolves in water. A sucrose molecule consists of a glucose joined to a fructose molecule via a linker and has a molecular weight of 342.3 Da.
A previous study measured the size of sucrose solutions using DLS [1]. These measurements were made on a Zetasizer Nano S instrument and proved that sub-nanometer sizes were possible by DLS. The diffusion coefficients and subsequent sizes obtained corresponded well with known structural data as well as diffusion coefficients of sucrose measured by other techniques [2,3]. In this application note, the measurements are repeated on a Zetasizer Automated Plate Sampler (APS) to demonstrate that its lower size limit is equivalent to the Zetasizer Nano S. The Zetasizer APS is a DLS instrument that automates the measurements of samples from industry standard 96 or 384 well plates.
Sucrose solutions of different concentrations (5% to 35% w/v) were prepared using de-ionized water. The samples were filtered through Whatman Anotop 20nm pore size filters to remove dust particles.
The technique of DLS measures the diffusion coefficients of molecules or particles undergoing Brownian motion. The measured diffusion coefficients are converted into hydrodynamic sizes through Stokes-Einstein's equation which requires the viscosity of the sample. As sucrose will change the viscosity of the samples, which in turn affects the measured size, it is important to compensate for the increased viscosity. The viscosities were determined by comparing the sizes obtained of a standard latex bead dispersed in each sucrose solution with the size obtained for the latex dispersed in a 10mM NaCl solution. This comparison allowed for the calculation of the viscosity of each sucrose solution.
The Zetasizer APS uses a 60mW 830nm laser and has built in temperature control from 2 to 90°C. All measurements for this application note were performed at 25°C. Figure 1 shows the plate navigator and summarizes how the samples were set up on the plate for measurement. The first column contained 60nm standard beads in 10mM NaCl, column 2: 35% sucrose in water, column 3: 30% sucrose, column 4: 25% sucrose, column 5: 20% sucrose, column 6: 15% sucrose, column 7: 10% sucrose and column 8 sucrose: 5%. Rows A to D were samples containing only sucrose whereas in rows E to H, a small aliquot of 60nm standard latex beads was added to the samples for the determination of viscosity.
The intensity particle size distributions (PSD) for the sucrose solutions show two populations, the first with a peak mean around 1nm and the second around 200nm in diameter (Figures 2A and 2B).
The 1nm population is interpreted as single sucrose molecules whereas the second population is thought to be collective diffusion of sucrose molecules. This interpretation is supported by the fact that the amplitude of the 200nm population increases with increasing sucrose concentration. If these intensity PSDs are converted into mass distributions using Mie theory, a single peak is obtained at less than 1 nanometer in size (Figure 2C)
The sucrose population of around 1nm shows a concentration dependence in the size obtained - a peak mean of 0.43nm at 35% w/v increases to 0.79nm at 5% w/v. A dynamic Debye plot (Figure 3) was therefore used to extrapolate the results to infinite dilution as suggested in the International Standard for DLS, ISO22412 [4]. The intercepts obtained at zero concentration give the hydrodynamic sizes with no molecule-molecule (or particle-particle interactions) which may occur in the sample. For the results obtained in this study, the intensity and volume mean sizes were 0.9nm and 0.86nm respectively.
The concentration dependence of the measured size has two possible explanations. Firstly, a concentration-dependent conformational change in the sucrose molecule, since water content has been reported to affect the mobility and reorganization of sucrose molecules, specifically the flexibility of the bond between the glucose and fructose increases with increasing sucrose concentration [5, 6]. Another plausible explanation for the concentration dependence of the size is that the sucrose molecules interact with each other in a way that cannot be compensated for by using the measured sample viscosity.
Is the size measured for sucrose realistic? If the result from the DLS measurements is compared with a structural model of sucrose, the dimensions of the three main axes are 1.06, 0.72 and 0.6 nm, respectively. The technique of DLS reports the size of the equivalent hard sphere that diffuses in the same way as the measured molecule. Therefore, the value of 1nm obtained from this study fits well with structural data.
Literature values of sucrose diffusion have previously been reported to be in the range of 5.0 to 5.5 x 10-6 cm2/s at 25°C, and if these values are used in the Stokes-Einstein equation which relates size to diffusion, the diameters obtained are 0.9 to 0.98nm. These values correspond well to those measured in this study.
Size measurements of sucrose have been successfully made on the Zetasizer APS. The values obtained correspond well with previously reported sizes. These results indicate that measurements of sub-nanometer sizes down to at least 0.6nm can be made with the Zetasizer APS if samples are carefully prepared. This application note has shown that both the backscatter detection in combination with fiber optics in the Zetasizer Nano, as well as the focused high power laser and 90 degree optics in the Zetasizer APS provides excellent sensitivity enabling measurements of samples at these small sizes.
[1] M. Kaszuba, D. McKnight, M.T. Connah, F.K. McNeil-Watson and U. Nobbmann (2008) J. Nanopart. Res. 10, 823-829.
[2] CRC Handbook of Chemistry and Physics: 87th Edition (2006-2007) CRC Press
[3] S.B. Engelsen and S. Pérez (1997) J. Mol. Graph Model. 15, 122-131.
[4] International Standard ISO22412:2008 Particle size analysis, Dynamic Light Scattering (DLS)
[5] M. Mathlouthi and P. Reiser (Editors) Sucrose: Properties and Applications (1995) Blackie Academic and Professional, London.
[6] S.G. Schultz and A.K. Solomon (1961) J. General Physiology 44, 1189-1199.
