How to perform zeta potential measurement with the diffusion barrier method?

Diffusion barrier figure 1 showing the plug of sample highlighted with blue dye, sitting at the lowest part of the capillary cell, DTS1070

The diffusion barrier method is particularly suitable for low sample volumes and high ionic strength buffers. The method consists of carefully injecting a “plug” of the sample into a folded capillary cell that already contains just the buffer. (The buffer has to be the same buffer of the protein preparation, same conductivity, same pH, same additives, in order to match the sample and the diffusion barrier as closely as possible). In this setup, primarily the zeta potential is of interest, however the size can be determined as well.

Practical details are discussed in “The Diffusion Barrier Technique, Practical Aspects and Data interpretation” , the web presentation recording “Protein zeta potential measurements using Malvern’s new diffusion barrier method” as well as the application note “The Diffusion Barrier Technique for Accurate and Reproducible Protein Mobility Measurement“.  Below is a brief summary of the practical overview of the key components of this method.

Why consider this diffusion barrier method ?

There are two main reasons you may want to look at this method. Both are due to the sample.

  1. Low sample availability. If you have only a very limited amount of sample, then this method allows a measurement even though only part of the cell contains sample.  (And the rest of the cell contains buffer).
  2. Protect sample from electrode interactions. In some conditions (e.g. high ionic strength, some phosphate salts, sulfur containing buffer components) electrode blackening (degradation) may occur. While blackening itself is not necessarily bad, it is an indication of potential sample-electrode interactions.

Other effects are Joule heating, sample degradation (aggregation), and electrode polarization. The use of the diffusion barrier method can help minimize these adverse effects.

How to introduce the sample ?

Diffusion barrier fig3 showing how to inject the sample plug at the bottom of a Malvern disposable capillary zeta cell, sample indicated by blue dye

The sample is gently injected into the buffer-filled DTS1070 capillary cell with a gel-loading tip. We have tested the Costar pipette tips (1-200µl, round, 0.5mm thick, Sigma cat:4853) and similar ones should work well.  For their use, gently push the gel-loading tip through one of the loading ports on the capillary cell until you feel a “natural” end then inject 70µl at first (you will never go wrong with slug location in that case). As you get more familiar with the method, it is is easy to reduce the injection volume to 50µl, then eventually even down to 20µl. You can practice this with either blue dextran solution, or by using the count rate monitor in the software (Tools – Count Rate Meter) to confirm that the scattering volume is within the sample slug.

Which settings in the software ?

You should select special instrument settings in order to be able to measure zeta potential of protein samples in buffers with relatively high ionic strengths. The automatic mode in the zeta analysis software will reduce the applied voltage. But for the most gentle handling of delicate sample, you can manually adjust the analysis settings according to the table below, taken from the application note.

To access the settings during the Manual or SOP setup go to:

  • Measurement -> Measurement Duration -> Automatic -> Maximum number of runs
    Leave the minimum at 10 and select maximum according to table
  • Measurement -> Measurements -> Delay between measurements (sec)
    When repeating measurements, it is a good idea to let the sample rest for 30 seconds
  • Measurement -> Advanced -> Voltage -> Automatic Voltage Selection -> No
    Enter the suggested voltage according to the table
Diffusion barrier table showing the recommended settings for voltage and number of subruns for select salt concentrations


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