Zeta Potential Performance Verification

May 13, 2022
Applications: Sample PrepZeta
Instruments: NanoBrook Series


The purpose of this guide is to demonstrate the proper method for preparing a zeta potential validation sample used for checking the performance of the NanoBrook ZetaPlus, ZetaPALS, 90PlusPALS, and Omni instrumentation. The BI-ZR5 validation material is a dark blue, inorganic powder that is insoluble in water. It has a mean particle size of approximately 300 nm. 

Included in this guide are two filtration methods to ensure that there are no dust particles in the solution used for the performance verification.



· 1 L volumetric flask·  KCl (CAS#: 7447-40-7)
· (2) BI-DFAQ filter (0.2 µm 12 mm for syringe,
0.1 µm 32 mm for pump)
·  BI-ZR5 Zeta potential validation
material (ζ = -44 ± 8 mV)
·  Weighing paper/ weighing boat
·  Spatula(s)
·  Deionized (DI) water, squirt bottle
·  BI-RC27 20 mL sample vials
·  20 mL syringe or filtration pump
· 1 L solution container
·  Kimwipes or equivalent
·  Paper towels

Solution Preparation

1 mM KCl solution preparation

  1. Measure 1 L of DI water into a clean volumetric flask
  2. Weigh out 0.075 g of KCl
  3. Add solid KCl to the flask
  4. Stir the solution until dissolved with a magnetic stir bar or place on a roller-mixer
  5. Pour your solution into a clean container and cap it; label the container with “1 mM KCl” and the date that the solution was made

BI-ZR5 reference solution preparation – 2 Method Options for filtering KCl solution

With the Brookhaven Sample Filtration System (BI-SFS)

  1. Rinse a 20 mL scintillation vial and its cap with DI
  2. Take two 0.1 µm filters (32 mm diameter) and assemble them on the online filter holder included with the BI-SFS
  3. Place the inlet tubing into the bottle containing the 1 mM KCl; if necessary, the tubing can be secured with tape
  4. Place the dispensing tubing into a beaker and turn on the pump system
  5. Let the system run for 1 minute, and observe if there are any leaks
  6. Take the sample vial and fill it with 20 mL of the newly filtered solution
  7. Cap and label the vial

With a 20 mL syringe

  1. Pre-rinse a 20 mL vial with DI water including cap
  2. Pour approximately 30 mL of 1 mM KCl into a beaker
  3. Use a 20 mL syringe and aspirate solution until filled
  4. Take two 0.2 µm filters (13 mm diameter) and assemble them on Luer lockable syringe 
  5. Discard the first few drops into a waste container, then start dispensing the filtrated solution into the vial
  6. Cap and label the vial

Measuring BI-ZR5

  1. Rinse and wipe clean a spatula; make sure it is completely dry
  2. Open the vial that contains 20 ml of filtered KCl solution and rest the cap (threaded side face down) on a Kim wipe
  3. Open the bottle containing solid BI-ZR5 powder, use the spatula to pick up a tiny flake (~0.5 mg) of the reference material and add it to the vial
  4. Close the cap tightly and place in an ultrasonic bath to sonicate or alternatively gently turn the vial back and forth to mix the reference material with the KCl solution
  5. The ZR5 reference solution should be prepared the day of use
Properly prepared BI-ZR5 reference solution in a range of acceptable color intensities.

Performance Verification

The Particle Solutions software comes pre-loaded with Standard Operating Procedures (SOPs) for performance qualification. Open the Particle Solutions software and select either ELS or PALS measurement. In the resulting measurement window, click on “SOP.” Click “Load” and select “Performance Qualification ELS” or “Performance Qualification PALS” respectively. Click “Open.”

Go to “Instrument Parameters.” Confirm your Cell Type and Electrode Assembly, using the dropdown menus to make any necessary changes. All other parameters should be left as their defaults.

The zeta potential electrode can be placed in the BI-ZR5 solution and inserted into your NanoBrook instrument. Start your measurement using the Performance Qualification SOP and allow the sample to stabilize according to the default timer. This will allow the sample to acclimate to the appropriate temperature defined by the SOP.


The ZR5 reference solution should result in a -44 +/- 8 mV reading. The conductance of the sample should be 320 µS ± 10%.

Electrodes will discolor slowly in the presence of dilute chloride solutions including the 1 mM KCl solution; this is normal. The discoloration is not an issue as long as the conductance is within range.

If the conductance of a 1 mM, filtered Kcl solution is significantly outside the range of 270-370 µS range, the two inner electrode surfaces can be cleaned with an ethanol-soaked cotton swab or paper towel and then rinsed with some of the standards before testing. It is recommended that the electrodes are cleaned regularly between measurements. Keep the electronics/connector dry at all times. The electrode can be placed upright in a BI-RC27 scintillation vial for sonication to keep the connecter dry.

If rubbing with alcohol does not clean the contaminant from the surface, then use the BI-ELECCK electrode cleaning kit. Use it sparingly as excessive removal of the metal from the surface can damage the electrode leading to poor results.

If you have any questions, please contact us at info@brookhaveninstruments.com and we will have one of our specialists contact you for help.