Measuring Lysozyme Monomer at 0.1 mg/ml Concentration. Equipment used : Sample Preparation and Measurement :

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1 Application Report #001 Measuring Lysozyme Monomer at 0.1 mg/ml Concentration Equipment used : ALV-NIBS / HPPS High Sensitivity Version, Lysozyme (MERCK), 0.1 molar Sodium-Acetate buffer (ph 4.25), syringe / 0.2 µm syringe filter, glass cuvette 12,5 mm square (2 mm x 10 mm inner dimensions, ~150 µl sample volume required). 100 mg Lysozyme was solved in 1000 ml Sodium-Acetate buffer and 0.2 ml filtered via a 0.2 µm syringe filter into the clean and dust free glass cuvette. The concentration of the Lysozyme monomer was thus 0.1 mg/ml. The glass cuvette was inserted into the ALV-NIBS / HPPS High Sensitivity Version and a waiting time of 3 minutes was introduced to ensure thermal equilibration of the sample to the 25 C control temperature. After the 3 minutes waiting time, a measurement of 300 second duration was taken and analyzed. Results ( Measurement Time 300 s, Count Rate 29 khz ) : Despite the low concentration and rather short measurement time, the monomer radius of Lysozyme (~ 2 nm) was clearly resolved to remarkable precision. In addition, the (unavoidable) aggregate contribution was measurable as well, although the mass contribution of these aggregates was measured to be less then 0.3 % only. The sample concentration of 0.1 mg/ml could be confirmed by re-computing it via the scattering intensity of the monomer (8.9 khz) versus the scattering intensity of a standard with known Rayleigh Ratio (toluene, 220 khz). Assuming a dn/dc for Lysozyme of nm and a monomer Mw of Dalton, the concentration was re-computed to be mg/ml, which was well within the weighing accuracy of +/-5 mg (and this weighing accuracy was the major reason of using up 100 mg Lysozyme, whereas 15 µg would have been sufficient). The ALV-NIBS / HPPS High Sensitivity Version is perfectly suited for DLS measurements on small particles even at very low concentration, as demonstrated here. Despite of the small concentration and thus count rate situation, the full particle size range from the nm to the µm regime was accessible, which ensures the instrument being a fast and reliable tool for monomer control and aggregation screening.

2 Application Report #002 Estimating the Mw of a Sample with known dn/dc For particles much smaller than 100 nm diameter, the angular dependency of the scattering intensity is not very significant (if at all visible) and Mw estimation can be safely performed at virtually any scattering angle via a series of measurements at different sample concentration or, if a rough estimate of the Mw is sufficient, via a single measurement at low enough sample concentration. Typically, many bio-molecules, but as well smaller polymers, are much smaller in size than 100 nm, and for these particle classes, the measurement of the scattering intensity in relation to the scattering of a standard with known Rayleigh Ratio allows fast and easy Mw estimation. For applications which require discrimination on monomer / dimer... conformation of the sample, the Mw estimation has the particular advantage of being very sensitive in contrast to a hydrodynamic radius measurement, which is fairly insensitive to Mw changes (a Mw change of a factor of 8 results in a hydrodynamic radius change of just a factor of 2). Requirements : One parameter required to perform this estimation is the refractive index change per concentration change of the sample, also known as dn/dc parameter. For many sample material / solvent combinations, the dn/dc value is tabulated - alternatively, the dn/dc can be measured on a differential refractometer, such as the ALV- DR 1. In terms of instrument, a very stable optical set-up and a monitorization of the primary laser intensity is required, both requirements are fulfilled with the ALV-NIBS / HPPS instrument. Example and Results : A standard was prepared using clean and dust free toluene, filtered through a 0.2 µm filter into a glass cuvette. The count rate of this standard was measured for 60 s. After this measurement, a 0.1 mg/ml Lysozyme sample (Sodium-Acetate buffer, ph 4.25) was measured in the same cuvette for 60 s and the Sodium-Acetate buffer was measured as in the same cuvette as well. All count rates were recorded. Since the Lysozyme concentration was as low as 0.1 mg/ml, the viral coefficient, which usually must be accounted for by taking a series of measurements at different concentration, should not play a pronounced role and the Mw estimated from this single concentration measurement should be quite precise already. Applying the classical formulae to obtain Kc/R and using in there 0.2 l/mg as dn/dc for 632,8 nm laser wavelength and 1.32x10-5 1/cm for the Rayleigh Ratio of toluene, the estimation of the Mw yielded Dalton +/- 5%, which is very near to the expected Mw of for the Lysozyme monomer and clearly precise enough to discriminate between monomer or dimer conformation.

3 Application Report #003 DLS measurements on sub-nm size particles - Cholesterol (Mw = 387 Dalton) Principally, any small particle size, even a particle size much smaller than 1 nm, for example, can be measured via the DLS method. All what is required is that the particles scatter enough light (has a good optical contrast to the solvent), that the particle freely diffuses and that the particle concentration in the solvent is not too low to ensure enough particles being present in the detection volume (which for sub 10 nm particles virtually never is a problem). However, for particles smaller than 10 nm, the light level of the scattered light usually is very small, even at larger concentrations, and only highly sensitive instruments are still capable of performing DLS measurements in this particle size regime. The ALV- NIBS/HPPS High Sensitivity Version for sure is one of the most sensitive particle sizers available on the market (probably it is the most sensitive particle sizer available today) and thus even sub-nm particle sizing is not at all impossible. We used Cholesterol at approximately 20 mg/ml concentration solved in 1,2-Butanone at 50 C for demonstrating sub-nm particle sizing with the ALV-NIBS / HPPS High Sensitivity Version. Equipment used : ALV-NIBS / HPPS High Sensitivity Version (+additional ALV-5000/FAST Tau Extension), Cholesterol, 1,2-Butanone, syringe / 0.2 µm syringe filter, glass cuvette 12,5 mm square (5 mm x 10 mm inner dimensions, ~375 µl sample volume required). 100 mg Cholesterol was solved in 5 ml de-gased 1,2-Butanone and filtered through a 0.2 µm syringe filter into the clean and dust free glass cuvette. The glass cuvette was inserted into the ALV-NIBS / HPPS High Sensitivity Version and a waiting time of 30 minutes was introduced to ensure thermal equilibration of the sample to the 50 C control temperature. After this waiting time, a measurement of 120 second duration was taken and analyzed. Example and Results : The measurement clearly resolves a very short fluctuation time component at less than 1 µs fluctuation time plus additional slower components in the order of 10 µs µs. While these slower components could be seen in a pure 1,2-Butanone sample as well, and thus are accounted for as being residuals in the solvent, the fast component is only present in the cholesterol sample. The hydrodynamic radius of this fast component is computed to be approximately 0.32 nm yielding a particle size of 0.64 nm. As expected, it is the dominating component in the mass weighted particle size distribution function as well. While we could not find literature values for the hydrodynamic radius of cholesterol in 1,2-Butanone, the particle size measured here still comes very near to the expectation using a (simple) Mw relation of a molecule with known Mw and hydrodynamic radius and the Mw for Cholesterol. The extremely high sensitivity of the ALV-NIBS / HPPS High Sensitivity Version allows even sub-nm particle sizing, as shown for Cholesterol at 20 mg/ml in 1,2-Butanone at 50 C. From the overall count rate obtained, even a concentration of as little as 10 mg/ml seems feasible for this particular sample material.

4 Application Report # nm & 300 nm particle size Silica samples at 40% mass concentration Silica samples at 40% mass concentration (and thus approx. 20% volume concentration, assuming a density of near to 2 g/cm³) are impossible to conduct particle size on using a standard 90 particle sizer. The reasons are mainly the fact that samples at such high sample concentrations are very opaque, and that multiple scattering of light is very pronounced and will strongly distort the DLS results yielding a much too small particle size. Another important factor is, that at such high sample concentrations, sample interactions can no longer be neglected. Hydrodynamic interactions, electrostatic interaction are to be expected and will have influence on the particle size results. While the later is difficult to account for without particular knowledge of several sample properties, multiple scattering effects can be avoided by the optical setup used and as will be shown herein, the ALV-NIBS / HPPS optical setup with 173 scattering angle performs excellent, even at such high sample concentrations. No special sample preparation was performed, but the samples were directly filled into disposable PS cuvettes. To ensure eventual aggregates to vanish, the samples were ultra-sonicated for approx. 3 minutes. Within this experiment, the possibility of changing the distance of the sample and the optics was used. All samples were measured at 8 different such positions, with the minimum position chosen such, that the detection volume was very near to the cuvette wall and the maximum position chosen such, that the detection volume was about 5.5 mm within the sample. While it can be expected, that the minimum position gives the maximum amount of single scattered light, the maximum position should be almost pure multiply scattered light and in fact a Diffusing Wave Spectroscopy (DWS) measurement was performed. At each position, a 60 s run was taken and analyzed using the Cumulant Analysis. Example and Results : For the 100 nm Silica sample, the first and second order Cumulant analysis gives constant and very precise results for any position smaller or equal the 0.0 mm position. Note however, that for any position trying to look into the sample, the computed diameters are way off, clearly caused by multiple scattering. In any case, the automatic search for optimum support in the ALV-NIBS / HPPS Software iterated to the -0.5 mm position, which clearly gives reliable results. For the 300 nm Silica sample, the results are a bit less obvious. The reason is, that with the increase in particle size, the multiple scattering effect quickly grows as well - for 40% mass and 300 nm, the multiple scattering is very high. Still, the computed diameters from the -1 mm and -1.5 mm position are not at all non-precise and again, the automatic search for optimum software support iterated to the -1 mm position which gives fairly reliable results as well. Diameter [nm] from 1. and 2. Order Cumulant Analysis 100 nm Silica Sample Order Cumulant Order Cumulant Cuvette Position in mm (0.0 mm is Original Position) Diameter [nm] from 1. and 2. Order Cumulant Analysis 300 nm Silica Sample Order Cumulant Order Cumulant Cuvette Position in mm (0.0 mm is Original Position) High concentration particle sizing is not a problem with the ALV-NIBS / HPPS. For particle size up to 300 nm diameter, even 40% mass concentration (20% volume) for a Silica sample was yielding excellent results in very good agreement with the sample measured in a dilute (0.1% volume) regime. Still, whenever possible, the > 5% volume concentration regime should be avoided whenever possible, simply because particle interactions take place and their influence on the DLS measurement result is usually unknown.

5 Application Report #005 ph influence on the particle size of a Silica sample at 20% mass concentration The average hydrodynamic radius and distribution function of the particle size of a silica samples is very dependent on the actual ph of the solvent (buffer) used, specially at higher concentrations, were particle interactions quickly allow a change in these parameters. Depending on the ph, different electrostatic interaction is to be expected which will influence the particle size results. In this application report, a commercially available silica solution is used as sample, which was stated by the manufacturer to have an average particle size in the 10 nm nm diameter regime. The original silica solution was filtered through a 1 µm syringe filter to ensure larger aggregates to be removed from the sample. No dilution was used for the original sample, while for the two samples with changed ph a small dilution was introduced (however less than 10% additional volume). The ph of the original sample was 8.7, the low ph sample was prepared to have a ph of around 6.5 (CH3COOH), the high ph sample around 9,5 (NH 3 ). After ph change, a 4 hour waiting period was introduced to insure eventual sample restructuring was finished. Radius Distribution Silica 20% weight in different solvents 1.00E E+00 Rel. Mass Weight 7.50E E E-01 ph 8,7 ph 6,5 ph 9,5 7.50E E E-01 ph 8,7 ph 6.5 ph E E E E E E+03 Radius [nm] 0.00E E E E E E E+01 Lag Time [ms] Results : Quite obviously, the original sample is far from the stated particle size of 10 nm nm diameter, but much more in the 1 nm diameter regime. After a change in ph, the average particle size of the sample significantly changed - for both ph directions towards a larger average particle size. Lowering the ph lead to an average particle size very near to the manufacturers statement. An additional lowering of the ph to about 5 did not change to particle size distribution significantly. The ALV-NIBS/HPPS is perfectly suited for organic and inorganic nano-particles, even at very large concentration, as shown here for silica in different, water based solvents. Even for the very small size range of investigation here (1 nm nm radius), very precise distribution function results can be expected.

6 Application Report #006 Phenolphthalein (Mw = 318) in Water/Ammonia at ph 9,5 Absorbing samples are a real challenge for DLS instruments. For a standard 90 -DLS scattering setup, not too much absorption of a sample is allowed for two reasons - at first the overall scattering intensity quickly drops, at second, the fact that along the axis of propagation of the laser beam the intensity drops as well (in fact exponentially), difficult to correct for distortions of the correlation functions appear. In this application report, a highly absorbing sample, namely Phenolphthalein at ph 9,5 is measured. While the molecule itself is very small (Mw = 318 Dalton), this report will show, that even the small Phenolphthalein molecule itself was measurable. Equipment used : ALV-NIBS / HPPS High Sensitivity Version (+additional ALV-5000/FAST Tau Extension), PS cuvette. Phenolphthalein was solved to the saturation level in water/ammonia at ph 9,5. The sample was then filtered through a 0.2 µm syringe filter into a standard PS cuvette. A detection point position rather near to the inner cuvette wall was selected. Correlation data accumulation was started for a 120 s run and afterwards analyzed using the distribution function analysis. Results : The sample was highly absorbing. The 0 diode monitor of the ALV-NIBS/HPPS detected only 1% of the incoming laser beam, which means that 99% of the primary laser beam was absorbed along the 10 mm path length through the cuvette. Still, the measurement allowed both, the determination of the aggregate, which was surprisingly small in weight content (approx. 0,5 % at around 150 nm radius) and the determination of the Phenolphthalein molecule s hydrodynamic radius which was measured to be around 0.3 nm yielding a particle size of 0.6 nm, which is in very good agreement of the Cholesterol molecule result of APR#003. Particle Sizing on absorbing samples is not a problem for the ALV-NIBS/HPPS at all. The back-scattering angle combined with the extremely high sensitivity ensures optimum results, even if the sample is highly absorbing. This makes the ALV-NIBS/HPPS a perfect choice for measurements on dyes, pigments etc.