Instruments:
Introduction
Colloidal systems, which include emulsions, foams and dispersions, form the basis of many manufactured food products. One of the chief quality issues concerning these products is the physical stability of the colloidal ingredients including proteins, glycerides, phospholipids and fatty acids. Accurate measurements of the properties of food colloids are needed to control the properties of bulk foods. Important properties include sedimentation, creaming, phase separation, flocculation, gelation and crystallisation.
The structure and dynamics of colloidal structures are governed by a set of forces often not directly perceived at the bulk scale and include the electrostatic forces between charged bodies. In general, a suspension of homogeneously charged colloidal particles is more stable than a similar, uncharged suspension because of inter-particle electrostatic repulsion and, according to classical DLVO theory, the magnitude of the stabilising force is proportional to the square of the surface charge. Electrostatic repulsion/attraction can influence the binding of charged materials, such as proteins, polysaccharides, surfactants and ions, which can themselves mask charges present on the colloid and diminish the repulsion that prevents gelation or precipitation. Chemical activity may also be altered if ions are sequestered. For example in an emulsion, the binding of iron ions in the protein layer close to an oil droplet could enhance lipid oxidation, but binding them into an aqueous polysaccharide (away from the fat) could reduce it. The measurement of charge properties is, therefore, clearly important in any attempt to apply colloid science principles to food systems.
Electrophoresis
As surface charge is difficult to measure directly, it has to be estimated indirectly by electrophoresis. When a charged particle is subjected to an electric field it will move towards the oppositely charged electrode, attaining a constant velocity when the viscous drag force balances the electric force. This velocity, divided by the applied electric field, is the electrophoretic mobility and will depend on the size, shape and charge of the particle.
Presuming a uniform field can be established, the problem then becomes one of measuring the speed of small particles. The movement of relatively large (> 0.3 micrometer), fast particles can be followed using an optical microscope, a technique known as microelectrophoresis, but this is not usually suitable for the smaller, slower particles of real food colloids. The conventional method used in this type of work is laser Doppler electrophoresis (LDE).
LDE is based on the scattering of light from a moving particle, where the frequency of scattered light will be shifted by an amount dependent on the speed and direction of the movement. In the absence of a charge-induced particle flow, the observed frequency broadening is solely due to the Brownian motion of the particles and can be used to calculate the diffusion coefficient and hence size. When an electrical field is applied to a charged particle, its net motion becomes a sum of coherent (charge dependent) and incoherent (Brownian) motion. The electrical properties of the scattering particle can then be calculated if these terms can be distinguished.
However, the signal (coherent) to noise (incoherent) ratio becomes progressively smaller for low mobility particles. The charge-induced movement of the particles can be increased, and therefore more easily measured, by increasing the time they are exposed to the electric field or increasing the magnitude of that field. These long experiments often lead to an accumulation of particles at one of the electrodes or high voltages which can lead to overheating, both affecting measurements. Until recently, these limitations made measurement of the charge of low mobility food colloids extremely difficult.
Phase Analysis Light Scattering
The ZetaPALS system from Brookhaven Instruments has improved this situation by using phase analysis light scattering to determine the electrophoretic mobility of charged colloidal suspensions. Figure 1 shows a schematic diagram of the PALS apparatus. A fraction of a split beam is applied to a suspension of particles in an electric field and the resulting scattered light is then recombined with the remainder of the frequency modulated laser beam, which acts as a reference. Due to motion of the particles, the amplitude of the phase difference between the scattered and reference beams contains information from which both the sign and the magnitude of a particle’s electrophoretic mobility can be calculated. Unlike LDE the PALS technique does not require the application of large fields that can often result in thermal problems and that can cause irreversible chemical and physical changes in the samples. The PALS technique is more sensitive for suspended samples with very low mobilities.
The following experiment demonstrates how the PALS technique can be applied, with superior results, to a food colloid system.
Materials and methods
A 10% wt/wt whey protein isolate (WPI) solution was prepared with distilled water and stirred gently for two hours to ensure complete dissolution. The solution was then titrated against 0.1 molar HCl. Samples were extracted after each addition of acid and centrifuged to remove dust particles.
Samples of supernatant were transferred to a standard four-sided, 1cm polystyrene cuvette and a parallel plate electrode was inserted. The cuvette was placed in a temperature-controlled holder and allowed to come to thermal equilibrium before measurement. Electrophoretic mobility was measured using a NanoBrook ZetaPlus (conventional LDE) and a NanoBrook ZetaPALS (PALS) instrument, both from Brookhaven Instruments.
All scattering techniques depend on the accumulation of statistically valid data over time, so each measurement of these was made over 10 minute periods to allow meaningful comparison. Each was divided into 10 one minute periods and the standard deviation of these replicates was used to calculate the reproducibility of the measurement. Each experiment was conducted in duplicate.
Results
The electrophoretic mobility of WPI increased monotonically from –2.8 x 10-8 m2 s-1 V-1 at pH=6.3 to +1.75 x 10-8 m2 s-1 V-1 at pH=3.8 as measured by PALS. Both experiments gave nearly identical values indicating the good reproducibility of the PALS method.
The results obtained using conventional LDE showed a similar trend of increasing mobility with pH. However, the two replicates reported very dissimilar results. The mean standard error (within measurement variability) was less than 0.24 x 10-8 m2 s-1 V-1 for PALS and 0.84 x 10-8 m2 s-1 V-1 for LDE and showed no clear dependency on the absolute magnitude of the mobility.
The isoelectric point of WPI was estimated from the molar average of the values for the constituent proteins as pH~5. This value is close to the zero mobility point (pH~4.60-4.75) determined by PALS, indicating the technique is reliable for very low charge colloids. The large amount of random fluctuations in the LDE data meant no reliable estimate of the isoelectric point could be made.
Conclusions
The electrical properties of globular proteins in solution have not typically been characterised by light scattering as they are relatively weak scatters and have, at certain pH values, very low charge. These limitations make LDE unreliable for the task, but the PALS technique maintains good sensitivity, even close to the isoelectric point. The PALS system offers a new way to study the effect of ion binding and pH changes on the properties of the small, lowcharge, low-mobility colloids often found in food systems. Our most recent experiments, using the PALS system, on pH titrations of soy proteins modified with surfactant, and of complementary measurements of the surfactant titration at fixed pH, have enabled us to propose a mechanism for protein-surfactant interaction. In addition, we have been able to make similar measurements to those reported above on whey-stabilised emulsions, using titration with sodium and calcium chloride as an indicator of ion binding, something we would not have been able to achieve using LDE.