Validation report: Testing and validation of NanoCuvette™ S, SpectroWorks™ together with UV-Vis spectrophotometer

 

 

Report written by Wenbo Wang, QC, and approved by Søren Lund Kristensen, QA at Particle Analytical ApS - Agern Alle 3, 2970 Hørsholm, Denmark. 

Date: 30-jul-2021

 

Table of contents

 

Abstract

The present report evaluates the particle size distribution and concentration of different polystyrene latex beads using NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer The report includes:

• Evaluate mean particle size and concentration of polystyrene latex beads analyzed in SpectroWorks™ by using UV-Vis measurements with NanoCuvette™ S.
• Compare the result of concentration of polystyrene from NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer and diluted sample from stock provided by suppliers. Samples were prepared by diluting the stock provided by supplier (Sigma Aldrich).
• Compare the result of mean particle size of polystyrene latex beads from NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer and dynamic light scattering (DLS).

The performance of Nanocuvette™ S and Spectroworks™ is assessed by comparing the results of particle size and concentration of polystyrene latex beads done with DLS. Optical filter inserted cuvette, Nanocuvette™ S and software together with spectrophotometer provided reliable results where the concentrations of all samples (except for polystyrene latex beads 1100 nm) have deviation less than 30% compared to the concentration of  sample dilutions. Furthermore, the particle size results obtained from analysis of the UV-Vis measurements with NanoCuvette™ S by SpectroWorks™ have less than 25% deviation from the standard for all the size range.

 

1        Aim

The present report documents the results of mean particle size and concentration of polystyrene latex beads analyzed by SpectroWorks™ using UV-Vis measurements with NanoCuvette™ S. To evaluate the reliability of the results obtained from the cphnano products, the results will be compared with DLS results and product specification obtained from sample dilutions and product specification sheets provided by suppliers (Sigma).

2        Materials

The material used for measurement with NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotomter are listed in Table 1.

 

Table 1: Sample received for UV-Vis measurement with NanoCuvetteTM S and SpectroWorksTM modeling

Sample name	Concentration (Vol.) ( diluted samples)
Polystyrene Latex beads 100 nm	0.01%
Polystyrene Latex beads 460 nm	0.003%
Polystyrene Latex beads 600 nm	0.003%
Polystyrene Latex beads 800 nm	0.003%
Polystyrene Latex beads 1100nm	0.001%
Polystyrene Latex beads 3000 nm	0.001%

 

The material used for DLS measurement are listed in Table 2.

 

Table 2: Samples received for testing and validation of NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer

Sample name	Batch number	Internal number
Polystyrene Latex beads 100 nm	2021/07/23	32027
Polystyrene Latex beads 460 nm	2021/07/23	32028
Polystyrene Latex beads 600 nm	2021/07/23	32029
Polystyrene Latex beads 800 nm	2021/07/23	32030
Polystyrene Latex beads 1100nm	2021/07/23	32031
Polystyrene Latex beads 3000 nm	2021/07/23	32032

3        Analytical equipment

Analytical equipment is listed in Table 3.

Table 3. Analytical equipment included in the project.

UV-Vis spectrophotometer	VWR UV-6300 Double beam spectrophotometer
DLS unit	Malvern Zetasizer Nano ZS

 

4        Method background

4.1      UV-Vis measurements with NanoCuvette™ S and SpectroWorks™ modeling

UV-Vis measurements using NanoCuvette™ S was done from 200nm-1100nm. Firstly, reference measurement was done by pipetting 100 µL of reference solution, DI water in the NanoCuvette™ S. B, A and D side measurements (see figure 1) were done respectively, and the spectra were saved. Secondly, the reference sample was removed and 100 µL of diluted samples were added in the NanoCuvette™ S and B, A and D side spectra are recorded and saved for further analysis. Each measurement was replicated. Reference spectra A, B, D sides and sample spectra A, B and D sides were respectively dragged and dropped in the SpectroWorks™ to obtain the results.

 

Figure 1. Measurement protocol including the demonstration of side A, side B and side D configuration.

 

4.2      DLS measurement

The measurement conditions for dynamic light scattering are described in Table 4. 

 

Table 4. Measurement conditions of samples.

Sample cell	Disposable standard 1 cm cuvette, volume 3 ml
Sample preparation	The sample is diluted Ca. 50 times: 20 uL of sample is added to 1 mL of water.
Dispersant refractive index	1.330
Viscosity	0.8872 cP
Material refractive index	1.590
Material Absorption	0.010
Temperature	25.0 °C
Equilibration time	180 seconds
Evaluation model	General purpose
Display range	0.600 to 6000 nm
Multimodal – analysis resolution	Normal
Lower threshold	0.05
Upper threshold	0.01
Measurement position	Automatic
Attenuator	Automatic
Run duration	Automatic
Number of runs per measurement	Automatic
Measurements per sampling	3
Delay between measurements	0 seconds
Number of samplings	2

 

5        Analysis

5.1      Comparison of the diluted polystyrene latex beads concentration from stock provided by distributor with the concentration obtained from NanoCuvette™ S and SpectroWorks™

 

The results obtained from analyzed data using SpectroWorks™ are listed in table 5.

 

 

Table 5. Concentration of the diluted polystyrene latex beads obtained using NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer.

Sample name	Concentration (Vol.) Diluted from the stock	Concentration (Vol.) From NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer. Result (% deviation)
		Run 1		Run 2
Polystyrene Latex beads 100 nm	0.01%	0.0099% (1.0%)	0.0081% (19.0%)
Polystyrene Latex beads 460 nm	0.003%	0.0038% (26.7%)	0.0038% (26.7%)
Polystyrene Latex beads 600 nm	0.003%	0.0026% (13.3%)	0.0026% (13.3%)
Polystyrene Latex beads 800 nm	0.003%	0.0030% (0.0%)	0.0031% (3.3%)
Polystyrene Latex beads 1100nm	0.001%	0.0016% (60.0%)	0.0016% (60.0%)
Polystyrene Latex beads 3000 nm	0.001%	0.0008% (20.0%)	0.0007% (30.0%)

 

The results of concentration obtained using NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer are considered reliable. The concentrations of all samples (except for polystyrene latex beads 1100 nm) from SpectroWroks™ have deviation less than 30%, when compared to the concentration of the dilution made from stock. However, due to the low concentration of polystyrene latex beads 1100 nm, the deviation of 60% might be also acceptable.

 

5.2      Comparison of the mean particle size of polystyrene latex beads from DLS and NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer.

The results of the mean particle size of polystyrene latex beads from DLS and NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer are listed in table 6.

 

Table 6. Mean particle size (Vol.) of polystyrene latex beads measured by DLS and NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer

Size of Polystyrene latex beads	PDI DLS		Mean particle size (D50, nm) from DLS (% deviation from standard)		Mean particle size (nm) NanoCuvette™ S and SpectroWorks™ together with UV-vis spectrophotometer (% deviation from standard)
	Run 1	Run 2	Run 1	Run 2	Run 1	Run 2
100 nm	0.026	0.058	109 (9.0%)	107 (7.0%)	115.00 (15.0%)	148.90 (48.9%)
460 nm	0.025	0.048	442 (3.9%)	431 (6.3%)	414.10 (10.0%)	413.04 (10.2%)
600 nm	0.040	0.086	662 (10.3%)	633 (5.5%)	546.26 (9.0%)	549.85 (8.4%)
800 nm	0.070	0.102	704 (12.0%)	730 (8.8%)	633.44 (20.8%)	637.50 (20.3%)
1100nm	0.147	0.040	873 (20.6%)	953 (13.4%)	1097.66 (0.2%)	1083.85 (1.5%)
3000 nm	0.365	0.334	1690 (43.7%)	1480 (50.7%)	3637.24 (21.4%)	3050.95 (1.9%)

 

For the results from DLS measurements, the deviation from the standard increases with the increase of the particle size of the sample. This phenomenon is also in line with the increase of PDI with the increase in the particle size of the sample and might be due to more sedimentation of particles when the particle size became larger. The results from replicate measurements done using NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer are considerably consistent except for 100 nm (Table 6). Furthermore, particle size measurements done using NanoCuvette™ S with SpectroWorks™ gives the deviation less than 22% for all measurements except for replicate 100 nm measurements, whereas the deviation range from 3.9% to 50.7% for DLS measurements from the theoretical concentration. It should also be noted that the polystyrene latex beads provided by sigma has the standard deviation ranging from 5%-15%.

 

6        Conclusion

NanoCuvette™ S and SpectroWorks™ together with UV-Vis spectrophotometer is considered reliable to evaluate the concentration and particle size of the sample. The concentrations of all samples (except for polystyrene latex beads 1100 nm) from SpectroWorks™ have deviation less than 30% compared to the diluted samples from the stock provided by supplier. Furthermore, the particle size results obtained from SpectroWorks™ using measurements in UV-Vis spectrophotometer and NanoCuvette™ S have less than 22% deviation from the standard for all the size range.

 

7        Appendix

 

 

Report written by Particle Analytical ApS - Agern Alle 3, 2970 Hørsholm, Denmark.