The following represents the procedure for measuring the NTA Zeta Potential standard NTA4096, derived from Malvern Zeta Potential transfer standard (DTS1235 ZP Transfer Standard) and is applicable to all NanoSight NS500 zeta potential-enabled instruments using NTA software (version 2.3 or later). It is recommended that the zeta potential of a standard material be checked at least every month. This protocol outlines how to measure the NTA Zeta Potential standard NTA4096 to check that your instrument is performing within specification.
- Ensure that the sample chamber is fully clean before use.
- Prepare a 4-fold dilution of the zeta potential standard using the supplied diluent, and mix by gently inverting.
- Run the NS500 flush cycle with particle-free water as described in the user manual.
- Load the sample using the Load sample function in the NTA software
- Select the zero position (laser thumb print) and adjust to center and focus if required. Check the five pre-calibrated zeta measurement positions to confirm that the particles at each position are in focus and have an appropriate brightness. Adjust the camera level so the particles are clearly visible, in focus and with under 30% particles being colored (indicates saturated pixels) at all positions. Examples of poor and good particle light scatter images are shown in Figure 1.
Figure 1a: Visual of a good image, and 1b a poor image of light scatter from particles visualized with the NanoSight instrument.
- Ensure that the electrodes are free of air bubbles and follow the protocol in the manual to remove bubbles if required.
- Check the voltage response by applying both positive and negative voltages at 24V confirming the particles change direction.
- Select and run the Zeta Measurement from the SOP drop-down menu and analyse with all default settings.
The use of Advanced Analysis settings is not required.
Select an appropriate Detect Threshold setting to process the captured videos. The setting will be correct when each white dot that should be included as a particle is highlighted with a red + (Figure 2a). If the detection threshold setting is too high, the white particle dot will not contain a red cross and is not included in the data profile (Figure2 b). If the detection threshold setting is too low, there is potential that noise will be included in the data profile which is indicated as a blue cross in the image (Figure 2c).
Figure 2: Impact of changing detection threshold setting on sample analysis with NanoSight instruments with the light scatter from each particle is seen as a white dot. Each particle should be highlighted with a red + to indicate that the particle will be in included in the data set (a). If the Detection threshold setting is too high not all particles will contain the red + (b). If the setting is too low noise may be tracked and included in the data set, indicated by blue +’s (c).
Following analysis, confirm that the electro-osmosis profile is symmetrical and parabolic. An example of this is shown in Figure 3.
Figure 3: Typical electro-osmosis profile measured on the NanoSight NS500 instrument using NTA3.0 software.
NanoSight instruments produce particle-by-particle data distributions. The modal zeta potential should have a modal zeta potential value within +/- 10% of the value supplied with the sample and coefficient of variance of under 5%. A typical data profile for NTA4096 zeta potential standards is shown in Figure 4.
Figure 4: Typical expected zeta potential distribution profile for NTA4096 measured on the NanoSight NS500 zeta instrument
Possible Reasons for Errors
All NanoSight instruments are factory set for accurate data output. Provided the instrument is used as stated in the user manuals errors in size or zeta potential data should not occur. However, excessive shock or changes in any physical condition might affect the accuracy of the instrument. If this is suspected contact the Malvern helpdesk at firstname.lastname@example.org or for US users, email@example.com for assistance. It will be helpful to include a data report file and associated compressed sample video.
Single particle populations, such as zeta potential standards are often only described in terms of an average or modal zeta potential value. In reality however, sample particles will always have a range of values and knowledge of the full zeta potential distribution through Z-NTA measurements can provide much more information. This is especially critical where small changes in the zeta potential distribution have a large effect on the behavior of a nanoparticle product, or for the detailed comparison of samples which may be close to the stability threshold. It is recommended that the zeta potential of a standard sample is measured by zeta-NTA on a monthly basis.
For a wide range of monodisperse and complex polydisperse sample populations, the Z-NTA particle-by-particle measurement technique provides high resolution zeta potential distributions, giving you further insight into the characteristics of your sample.