Dispersion analysis by analytical centrifugation

A common goal in the formation of stable emulsions during a homogenization process is to obtain the smallest possible particle size distribution of the dispersed phase.

The emulsification process and process parameters (type of homogenizer, temperature, energy input, time, and homogenization cycles) used to achieve this goal depend on the desired characteristics of the emulsion.

The use of a Microfluidizer Processor has shown to be both a flexible and proven method to achieve optimum product quality.

In the search for the most effective emulsification parameters, a method for fast comparative emulsion stability measurements is required.

The multisample analytical centrifugation (Step technology) allows the accelerated characterization of any demixing processes (creaming, sedimentation, phase separation), in addition to the quantification of time dependent structural alterations (e.g flocculation, coalescence) without dilution of the samples.

This paper demonstrates the effectiveness of analytical centrifugation as a technique for fast emulsion stability measurements in addition to homogenization parameter optimization.

Materials and methods.

Preparation of the pre-emulsion.

A rotor-stator mixer (IKA) was used to form the pre-emulsion consisting of 88.5% water and 10% sunflower oil.

Emulsifiers used were Tween-80 (1%) and Span-80 (0.5%).

The ingredients were mixed at 10,000 rpm for a total 120 seconds during which sample aliquots were taken at 30, 60 and 120 seconds.

The pre-emulsion formed at the end of the 120 second mixing period was the basis for the final emulsions formed using the Microfluidizer processor.

Preparation of the final emulsions In the Microfluidizer processor, the starting material is pumped under high pressures (up to 40,000psi/2750bar) through a patented interaction chamber in which the material is accelerated at high velocities through a channel of a fixed geometry.

The velocity inside the interaction chamber reaches several hundred meters per second.

The resulting very high shear rates and impact effects created inside of the interaction chambers lead to a highly efficient droplet or particle size reduction.

Various interaction chamber types and sizes are available for different applications: Y-type chambers in which the product stream is divided into two streams which impinge upon another and Z-type chambers in which the product stream zigzags through the interaction chamber are the two general types of interaction chambers which are available with different cross-sectional areas for different shear requirements.

For the described experiments, an M-110EH Microfluidizer processor equipped with an F20Y interaction chamber (75um minimum dimension) upstream and a H30Z interaction chamber (200um minimum dimension) downstream was used.

The effect of operating pressures (5000, 10,000, 20,000psi/345, 690, 1380bar) and system passes (1, 3, 5) on both emulsions stability and formation efficiency were investigated.

Analytical centrifugation.

Analytical centrifugation allows for the accelerated measurement of the separation process, and therefore gives the user a fast and accurate means of evaluating emulsion dispersion stabilities.

The stability analyzer Lumifuge measures the intensity of the transmitted light over the full sample length instantaneously as function of time using the Step technology - space and time extinction profiles.

All data are stored and displayed in real time as a function of time and radial position, allowing for micron-scale accuracy and precise analysis of any changes in the dispersion characteristics.

Up to eight samples can be analyzed simultaneously.

The separation behavior of the individual samples can be compared and measured in detail by analyzing the changes in the transmission at any part of the sample or by tracing the movement of any phase boundary.

Based on these changes, the rate of clarification and the sedimentation/creaming velocity can be easily calculated.

With the Lumifuge, both of the calculations are performed semi-automatically.

Due to differences in stabilities, the respective emulsions were analyzed at 20 minutes (running at 1500rpm) or 30 minutes (running at 3000rpm), resulting in a corresponding acceleration of 290g and 1100g.

Results and discussion.

The top of the sample and thus the air-liquid interface is at the 92.5mm position (measured from the center of rotation).

The bottom of the sample is at 114mm.

The first profile taken is shown in red.

All subsequent measurements are depicted as lines having a red-green gradient with full green indicating the last measurement taken.

At the beginning of the analysis, the emulsion was well dispersed and most of the incident light was absorbed.

Therefore, the first transmission reading taken is very low (around 7%).

Thanks to the density difference between the dispersed oil droplets and the continuous water phase, creaming will occur.

This results in increasing light transmission percentages near the bottom of the sample as a function of time.

The green transmission profile line indicates that complete clarification (100% transmission) occurred after 30 minutes for the lowest 1 mm part of the samples.

Since the clarification of the samples occurs at the bottom of the sample first, it becomes clear that we are dealing with an oil-in-water emulsion (o/w).

The Step technology helps determine whether the emulsion is oil-in-water or water-in-oil without having this knowledge a priori.

For a water-in-oil emulsion, one would observe sedimentation of the water droplets resulting in a clarification of the top of the sample.

The SepView software allows for a detailed and quantitative analysis of phase separation kinetics of water and oil and coalescence.

In the following emulsion stability, ie, their separation tendency was quantified by the creaming velocity and the ratio of total liquid phase volume separated after a given time of centrifugation to the volume of the whole sample.

Since the oil-water interface for this emulsion was not distinct during the creaming process, its position was measured along the 30% transmission line.

Thus after 30 minutes the phase boundary moved from the bottom of the sample up to the position of 110.5mm.

Pre-emulsion stability as function of mixing time Since the raw emulsion was extremely unstable, a low acceleration setting (290xg) was chosen for the Lumifuge.

The stability of the pre-emulsion is highly dependent on the length of mixing time using the rotor-stator mixer.

While the pre-emulsion showed great instability for the 30s mixing time, mixing the emulsion for two minutes does produce a stable enough emulsion to ensure consistent and reproducible behavior for the additional homogenization using the Microfluidizer processor.

Effect of process pressure and cycles on final emulsion stability Even low homogenization pressures have a noticeably positive effect on the stability of the pre-emulsion, as depicted using only the first and last measured transmission profiles.

The stability improves with increasing homogenization pressure, ie, the amount of water separated is reduced.

The application of higher shear forces on the emulsion by increasing the homogenization pressure led to a remarkable increase in emulsion stability.

At lower homogenization pressures (5000psi/345bar), increasing the number of cycles resulted in progressively more stable emulsions.

At 10,000psi (690bar) only the first two cycles showed meaningful increases in emulsion stability, with any additional cycles having only a marginal effect.

At the highest applied pressure of 20,000psi (1380bar), the emulsion stability actually decreased slightly with an increasing number of homogenization cycles (over-emulsification).

For this pre-emulsion then, it is more efficient to increase the homogenization pressure as opposed to cycles in order to achieve the highest stability towards creaming.

As an alternative to measuring emulsion stability on the final phase separation volumes, one can measure the rate of the phase separation process in real time.

The differences in the slopes indicate different separation velocities.

These separation velocities are also calculated semi-automatically by the software and can be used to predict the stability or shelf-life of the respective emulsion.

Conclusion.

Using a water-oil emulsion, it was shown that homogenization using a Microfluidizer processor can dramatically improve the stability of an emulsion.

The optimal process parameters can be readily and accurately determined using multisample analytical centrifugation.

The on-site use of such a technique then allows the operator in charge of quality control to immediately adjust and optimize the emulsification and homogenization process.