Calcium Induced Flocculation of Intralipid

Introduction

In a previous application note, the measurement of Intralipid at high concentrations using the high concentration zeta potential cell (ZEN1010) accessory was discussed. In the study, zeta potential measurements were made at sample concentrations up to 4% w/v [1]. However, a concentration dependence in the zeta potential results obtained was observed which was due to increasing sample turbidity (figure 1) [1-4]. For Intralipid concentrations of 0.5% w/v and below, the zeta potential values were consistent (-50.7mV ± 0.83mV). At concentrations of 0.75% w/v and above, there is a gradual decrease in the zeta potential mean values obtained. This is due to scattered light being detected from particles close to the cell wall which will have reduced mobility due to the electric field being lower than assumed.

Figure 1: A plot of the zeta potential values obtained for various concentrations of Intralipid

Even though the zeta potential values at high concentrations are influenced by the turbidity of the samples, for certain applications the values obtained can be used in a relative sense.

In this application note, calcium induced flocculation of Intralipid was studied at two different sample concentrations to look at whether the isoelectric points (the point at which the zeta potential is zero) values obtained were consistent.

Experimental

The Intralipid used in this study was kindly provided by Fresenius Kabi at a neat concentration of 20% w/v. This was diluted to 1 and 0.1% w/v respectively in 2.25% w/v glycerol with the pH adjusted to 7.9 with NaOH. This was to ensure that any change in the measured zeta potential was not due to changes in the dispersant conditions upon sample dilution. The 1% w/v concentration had previously shown an influence of turbidity on the mean zeta potential values obtained whereas the 0.1% w/v concentration did not (see figure 1). These two concentrations allowed for comparison of the influence of sample concentration on the position of the isoelectric point of Intralipid.

Various aliquots of a calcium chloride solution were added to the Intralipid samples to give the appropriate concentration of Ca²⁺. These were incubated at room temperature for 30 minutes prior to measurement.

All samples were measured on a Zetasizer Nano ZS at 25°C using the high concentration zeta potential cell ZEN1010. A field of 40V was applied across the electrode spacing of 16mm (field strength of 25V/cm) and five repeat measurements were made on each sample to check the repeatability of the results obtained.

Results and Discussion

The high concentration zeta potential cell ZEN1010 accessory allows measurements to be made of concentrated samples. This is due to the reduced optical path length of the cell compared to the default folded capillary cell supplied with the Zetasizer Nano.

Figure 2 shows the zeta potential mean values obtained as a function of Ca²⁺ concentration for 1% and 0.1% w/v concentrations of Intralipid. In the absence of Ca²⁺ (0mM), the mean zeta potential values show a difference (-42.6mV for the 1% w/v concentration and -56.7mV for the 0.1% w/v concentration). This difference in mean values is due to the increased turbidity associated with the more concentrated 1% w/v sample.

Figure 2: Zeta potential values (in mV) as a function of Ca²⁺ concentration (in mM) obtained for 1% and 0.1% w/v concentrations of Intralipid

The addition of Ca²⁺ to both Intralipid concentrations results in a rapid reduction in the mean zeta potential value. The isoelectric points obtained for both concentrations are very consistent at 4.5mM Ca²⁺. The Intralipid droplets then become positively charged at Ca²⁺ concentrations above this. This reduction in the zeta potential of the Intralipid and consequent reversal of charge is due to the specific binding of the Ca²⁺ ions to the surface of the emulsion droplets. These results are in excellent agreement with other studies of the effect of Ca²⁺ on Intralipid [5, 6].

These results show that even though the zeta potential mean values obtained for the higher Intralipid concentration are influenced by the turbidity of the sample, the position of the isoelectric point is consistent with the lower concentration. This confirms that even though absolute zeta potential values may be influenced by sample concentration, the relative values obtained show consistent effects.

Conclusions

The results obtained in this study show that zeta potential measurements can be successfully made on turbid samples using the high concentration zeta potential ZEN1010 cell.

The isoelectric point of the Intralipid samples is consistent with previous studies.

References

[1] Zeta Potential Characterization of Concentrated Lipid Emulsions, Malvern Instruments Application Note MRK1383-01.

[2] Zeta Potential Measurements of Skimmed and Semi-Skimmed Milk Using the ZEN1010 High Concentration Cell, Malvern Instruments Application Note MRK 1301.

[3] Zeta Potential Characterization of Concentrated Titanium Dioxide Slurries with the ZEN1010 High Concentration Cell, Malvern Instruments Application Note MRK 1326.

[4] Surface Coating Polyurethane Dispersions: Measuring Zeta Potential at Concentrations Up To 40% w/v, Malvern Instruments Application Note MRK 1358.

[5] C. Washington (1990) Int. J. Pharmaceut. 66, 1-21.

[6] C. Washington (1996) Advanced Drug Delivery Reviews 20, 131-145.