Accuracy and reproducibility in automated dynamic light scattering measurements

Introduction

The Zetasizer Nano range delivers excellent accuracy and reproducibility in determination of particle or molecular size, providing the highest level of confidence in results. Sample throughput can now be increased without compromising performance with the Zetasizer NanoSampler.

The Zetasizer Nano utilizes light scattering to derive results from first principles and therefore requires no calibration. For GLP performance verification, Polystyrene (PS) latex standards of known size are commonly employed to validate performance. In this application note, PS latex standards are used to demonstrate the accuracy, reliability and reproducibility of the NanoSampler accessory for the Zetasizer Nano series of instruments.

Materials and methods

Sample Preparation

A buffer was prepared by diluting 7mL of a 0.92M NaCl solution with 100ml of ultrapure deionised water prior to filtration through a 0.2µm membrane filter.

60nm polystyrene latex

4 drops of a 60nm Latex Sample (LTX3060A from Thermo Scientific) was added to 20mL of buffer.

200nm polystyrene latex

7 drops of a 200nm Latex Sample (LTX3200A from Thermo Scientific) was added to 2mL of buffer.

Experimental

A Zetasizer NanoSampler (ZEN2000) with the supplied quartz flow cell was used in combination with a Zetasizer Nano ZSP (ZEN5600) to measure the size of two different polystyrene latex samples. The Zetasizer software (v 7.10) was used to assess the reproducibility of the results. For batch measurements the disposable plastic cuvette (DTS0012) was used.

Results and discussion

The simplicity of running batch measurements on the Zetasizer Nano remains achievable even with the NanoSampler attached. Prior to automated measurements, the samples were analyzed by the Zetasizer in batch mode. Figure 1 shows an overlay of 30 measurements, comprising of 10 repeat measurements of 3 aliquots of 60nm PS. The size distribution profile of the entire data set overlay with a standard deviation of 0.287, Table 1. Also included in the table is the data from 200nm PS samples, and an overlay of every size distribution profile is shown in Figure 2.

Figure 1: Overlay of 30 measurements of a 60nm PS latex, three aliquots with 10 measurements each

Figure 2: Overlay of 30 measurements of a 200nm PS latex, three aliquots with 10 measurements each

Batch to automated transferability

For each of the two different samples, aliquots were pipetted into 10 separate vials, placed in one of the two 48 vial capacity well plates and place inside the NanoSampler. Up to three loadings were delivered from each vial and repeat measurements were taken to ensure statistical accuracy.

The measurements using the NanoSampler are comparable with those from the batch measurements, as demonstrated with representative overlays in Figure 3. Three measurements per loading have been displayed for visual clarity however; Table 2 has been populated from 30 measurements for a robust calculation of averages and standard deviation.

Figure 3: Overlay of distributions from representative batch & NanoSampler repeat measurements, 60nm

Figure 4: Overlay of distributions from representative batch and NanoSampler repeat measurements, 200nm

NanoSampler reproducibility

The data presented so far establishes the transferability and the reproducibility of the NanoSampler. To highlight both the aliquot to aliquot and the vial to vial reproducibility, the data is split further below.

Aliquot to aliquot reproducibility

Figure 5: Overlay of distributions from 5 representative measurements of 3 aliquots from a single vial loaded using the NanoSampler, 60nm

Figure 6: Overlay of distributions from 5 representative measurements of 3 aliquots from a single vial loaded using the NanoSampler, 200nm

Vial to vial reproducibility

Figure 7: Overlay of distributions from a representative measurement of single aliquots from 10 vials loaded using the NanoSampler, 60nm

Figure 8: Overlay of distributions from a representative measurement of single aliquots from 10 vials loaded using the NanoSampler, 200nm

Multiple aliquots from multiple vials

Figure 9: Overlay of distributions from representative measurements of 3 aliquots from 3 vials loaded using the NanoSampler, 60nm

Figure 10: Overlay of distributions from representative measurement of 3 aliquots from 3 vials loaded using the NanoSampler, 200nm

Large data sets

Figure 11 shows the z-average diameter of every measurement that was made on the two sizes of polystyrene, from all aliquots and all sample vials, 1260 runs. This volume of data would take 10 times longer to measure using only batch mode. The average sizes were 65.2nm and 202.0nm which are both within the expected tolerance of the latex standards.

Figure 11: All data points measured using the NanoSampler (60nm & 200nm PS latex Z-average diameter)

Carryover

Confidence in knowing that the results presented are a genuine product of the current sample, and not an artefact of the previous sample is extremely important. The NanoSampler's flushing schedule virtually eliminates sample carryover, reducing it to less than 0.05%. Figure 12 demonstrates this with an overlay of the final loading of a 200nm PS latex following a run of 280 measurements, with the subsequent 60nm. The zoom to baseline shows no evidence of carryover, Figure 13.

Figure 12: The lack of any sample carry-over using the NanoSampler is demonstrated by this overlay of adjacent 60 and 200nm sample runs

Figure 13: The lack of any sample carry-over using the NanoSampler is demonstrated by this zoom to baseline overlay of adjacent 60 and 200nm sample runs"

Discussion

The Zetasizer NanoSampler is a compact sample delivery system for the highly precise and reproducible automated loading of samples into the Zetasizer Nano instruments. The data described here demonstrates that this versatile accessory can be trusted to make measurements of samples unattended, reducing operator time and maximizing sample throughput.

Polystyrene latex standards were successfully analyzed using the Zetasizer NanoSampler to automate 30 repeat measurements of each sample. Of the two data sets, the measurements of the 60nm samples were the most reproducible; however the standard deviation for both data sets were below 1.50.

The Zetasizer NanoSampler gives excellent reproducibility from aliquot to aliquot, and from vial to vial, and shows no carryover of sample between injections.

By automating the measurements of samples in the Zetasizer, which allow the instrument to be run overnight, the NanoSampler maximizes sample throughput while delivering high quality, reproducible data.