Estimate Particle Concentration with the Nano

The new Zetasizer Advance Ultra (red) has a concentration feature that allows for convenient determination of particle concentration, for example for AAV capsid titer. A technical note “Particle Concentration measurements on the Zetasizer Ultra – how it works” explains the exact principles behind the mathematics.

While the classic Zetasizer Nano does not have this feature integrated directly, there is a method to utilize a similar estimate of particle concentration. The classic Concentration and Scattering calculator predicts the amount of light scattered for a sample with specified characteristics (like size, refractive index, concentration). We can then also turn this around and use a measured amount of light to estimate the concentration. Here are the steps with an example.

1 – Get a good DLS measurement of your sample

The Zetasizer Advance utilizes the unique MADLS concept to get a really good size distribution. In the classic Zetasizer we can “only” look for good data quality: You can observe this in the Intensity PSD as a statement Result Quality Good, where the word “Good” is green. You could also check the dedicated size quality report, if you want more details.

How you achieve good data quality is up to the sample. And some samples may just not be suitable for DLS, and then those would most certainly not be suitable for concentration estimate with this method.

2 – Record the size and the intensity from that sample

In this example, we will take the diameter 63.13 d.nm which is the same as 31.57 r.nm radius. The derived count rate for this sample was 41438.3 kcps. This is the normalized scattering intensity, taking the effects of an applied attenuator into account. It is a convenient number to express the theoretical count rate one would get if no attenuation was applied. Find more details on what that means and how to display this parameter in the blog.

3 – Enter these values in the Concentration Utilities Calculator

You can find the Concentration Utilities Calculator under Tools – Calculators. Set final volume to 10, Instrument to Zetasizer S, attenuator to 11. Now enter the radius, [31.57] and the refractive index of your material in the sample, in this example Polystyrene at 1.59 and imaginary or absorption 0.01. For other materials, select an appropriate refractive index for your sample. (Please note that this has a significant influence on the outcome for the particle concentration, you can test the effect by modifying the refractive index and checking the outcome.)

4 – Estimate the concentration by trial and error

You can now modify the Initial concentration until the Expected Derived Counts are as close to the observed derived count rate of 41438.3 as possible. After a few manual entries (press return after each trial) we find that 0.007717 is pretty close to the expected derived count rate. Yes, this is cumbersome, and that’s why the new generation makes it a lot easier – without the extra work.

5 – Read the particle concentration [#/mL] from the calculator

The result is in the upper right corner. In our example, we find 5.9*10^8 particles per microL which is the same as 5.9*10^11 particles per milliL or 5.9 E11 particles/mL.

 Limitations of this tool

The calculator is based on an average Zetasizer Nano, your instrument may be slightly more or less sensitive than the average. This will lead to additional uncertainty in the estimate. With additional peaks, the %intensity has to be applied to each peak. As an example if 16% of the intensity is for Peak 1 then 0.16 times the derived count rate of the sample has to be assigned to that contribution. And that number leads to the estimated particle concentration for Peak 1.

And just to reiterate, the latest generation has a Particle Concentration feature built into the software. With the additional advantage of achieving a higher resolution size distribution, it can provide results in samples, where the classic system would not. Plus you won’t have to manually trial values until you find a result for the estimate.

What else can the Particle Concentration in the Ultra do?

While we discussed the Nano above, let’s look at what is different in the Zetasizer Ultra (Red). Here, the software has all the intermediate steps built in, so there is no complicated trial and error. Plus there are four additional advantages:

• The higher resolution size distribution from MADLS provides a more precise size and thus a closer estimate on the concentration
• Concentration per peak when multiple peaks are present
• Cumulative concentration distribution to display versus size
• Method includes reference to toluene standard

MADLS takes the information from multiple angles into account to achieve a higher resolution size distribution. This improved size then improves the mathematical conversion to the concentration estimate. In fact, when there are for example 3 peaks present, the software can provide a particle concentration for each peak, in one measurement.

In addition, a complete cumulative concentration distribution can be selected for display, as well as easy exporting. An example of a cumulative particle concentration from a two-peak situation is shown here:

You can visually discern, how many particles there are present in the sample up to which size. And one reason why this is possible is that the method in the Zetasizer Ultra (Red) involves a “calibration” with a common light scattering standard, toluene, for each specific instrument (as opposed to the calculator for a generic, typical, average Zetasizer Nano). Pretty clever.