Automation of pH-titrations using the BI-ZTU

Nov 25, 2020
Applications: TitrationsZeta
Instruments: BI-ZTUNanoBrook Series


The stability of dispersible nanomaterials often depends on having a high surface charge. These charges are often pH dependent, especially for surface chemistries with acidic or basic functionalities. A titration can be performed to rapidly profile a range of solution conditions to determine how stability might change with respect to formulation pH. This is especially useful for protein containing formulations, where the components often contain mixtures of positively and negatively charged sidechains.


The Brookhaven Instruments Autotitrator (BI-ZTU) provides researchers with a tool to study the effect of pH on zeta potential (ζ). The ZTU is an accessory and is used in conjunction with a NanoBrook Series instrument, to automate isoelectric point measurements for engineered and biologically sourced nanomaterials such as proteins, dendrimers, biopolymers, and nanoparticles. The ZTU uses multiple metering pumps for dispensing various concentrations of acids, bases, and potentially of other titrants. The titrator integrates directly with the NanoBrook for both zeta potential (ζ) and particle sizing experiments (dh). This system can be set up to titrate within a user-defined pH range, or additively, and produce aggregation data for particle size or zeta potential as a function of pH or additive concentration. In the following example, we will discuss the impact on zeta potential.

Many commercially relevant nanomaterials will aggregate under conditions where their surface charge, or zeta potential, is strongly diminished.  A well-defined variable for controlling surface charge is pH, which results in changes in the degree of ionization (α) of various surface groups.

Figure 1The degree of ionization, α, refers to the extent that a particular functional group will be ionized, or charged, at a given pH. By convention, α = 0 when a titratable group is uncharged, and α = 1 when the group holds a formal charge. This is independent of the sign of the charge.

Titratable sidechains are usually divided into two broad categories: acidic or basic. Acidic sidechains tend to have low pka values and hold formal charges at pH > pka. Basic sidechains, in contrast, vary from charged at pH < pka, to uncharged at high pH. In the context of pH titrations, we usually only consider protons that can be titrated between pH 2 and pH 12;  thus in the simple case where we only recognize carboxylic acids and amines, the relevant transitions going from low- to high-pH are R-CO2H to R-CO2-1, and R-NH3+1 to R-NH2.

As shown in figure 1, the degree of ionization for basic sidechains will be 1 at low pH, where the amine will contain a formal positive charge (Z = +1), and 0 at high pH where the proton is removed. The opposite is true for acidic sidechains, which are neutral when protonated at low pH, containing a formal Z = 0 charge, and only become charged at high pH when deprotonated, taking on a formal charge of Z = -1.

To understand how this impacts samples with mixtures of acidic and basic groups, such as is commonly the case with proteins and other biological samples, we need to look at the net charge (Znet) rather than the charges of individual sidechains. This concept is demonstrated below, in the simplified case where only a single sidechain of each type is present (Fig. 2).

Figure 2 – Simplified representation of a pH titration of a sample with mixtures of acidic and basic surface chemistries. The net charge, Znet, approaches zero when both oppositely charged sidechains are ionized, in this example near neutral pH. 

Synthetic nanoparticles tend to have a single dominant surface chemistry, meaning that these particles will either be charged, or uncharged depending on pH, with the sign of the charge being determined by surface chemistry. Biologically sourced materials, such as proteins or other biomacromolecules will typically have mixtures of acidic and basic groups. A pH titration can be an effective method for experimentally determining the isoelectric point of an unknown material.

As shown below, the exact pH dependence will depend on surface chemistry; for proteins the common titratable groups are the sidechains of various amino acids. Common acidic amino acids include glutamic- and aspartic-acid which both contain carboxylic acid sidechains, whereas basic amino acids such as histidine, lysine, and arginine all have amine-containing sidechains.


A commercial formulation whose major component is protein-based was prepared at low pH. An autotitrator was subsequently used to measure zeta potential as a function of pH (Fig. 1). The point where the titration curve crosses zero can be used to estimate the isoelectric point, as a zero-, or near-zero zeta potential would be expected when the net charge is zero.

Figure 3 – Autotitration of a protein-containing formulation using Phase Analysis Light Scattering (PALS) to measure zeta potential (ζ) as a function of pH. Titration is run from low- to -high pH. Corresponds to an isoelectric point (pI) around 6. Stability against aggregation would be expected to be lowest near the point of zero charge. 

As shown in figure 3, the ZTU can be used to automate a zeta potential titration of a protein-containing formulation.  Starting at low pH it is assumed that there must be an excess of positively charged groups present, leading to a positive zeta potential (approaching ζ = +25 mV). As neutral pH is approached, the negatively and positively charged sidechains are present in roughly equal numbers, dropping the net surface charge to near zero. As pH is raised further, the charge on the basic residues finally disappears, leaving only the acidic, or negatively charged groups to contribute to surface charge, resulting in a limiting negative zeta potential (ζ = -40 mV) as pH 12 is approached. From this titration experiment we can conclude that the isoelectric point of this protein is in the vicinity of pH 6, where ζ ~ 0 mV, and that the zeta potential is sufficiently high to prevent aggregation below pH 4, and above pH 9.  Additionally, the higher limiting zeta potential seen at basic pH, vs acidic pH, suggests that this sample contains an excess of negatively charged amino acids relative to positively charged groups.


  • An autotitrator can be used to determine protein stability across a wide pH range.
  • For materials with mixtures of acidic and basic sidechains the point of zero (net) charge frequently corresponds to a pH range where both groups will be ionized. 
  • Studying zeta-potential as a function of pH allows us to experimentally determine an isoelectric point.

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