Particle Size Analysis

3.2.7.1 Elzone Method

Particle size analysis above approximately 1.0 pm was carried out using an Elzone model 280 PC particle sizer (Particle Data Ltd., Cheltenham, U.K.) which uses the 'electrical sensing zone' method to determine particle size. The instrument consists o f a glass tube with a small orifice located in the tube wall (refer to Figure 3.1 overleaf for a schematic diagram). The tube is immersed in an electrolyte in which the particles to be analysed are suspended. A vacuum pump is used to apply negative pressure across the orifice, causing a flow o f electrolyte containing the suspended particles through it and the unbalancing o f a mercury column connected to the system. The vacuum source is then isolated and the flow o f electrolyte and particles continues through the orifice as the vacuum is slowly released and the mercury column re-balances. The advancing mercury column activates a counter by means o f start and stop probes so that a count is carried out while a known volume o f electrolyte passes through the orifice.

The instrument can also be operated so that the counter is activated for a given time interval at a constant vacuum, i.e. with a constant flow through the orifice. The electrical resistance across the orifice is measured by immersed electrodes on either side o f the tube wall. Any particles passing through the orifice cause a change in the resistance which is converted to a voltage pulse and then amplified. The volume o f each particle is proportional to the height o f the voltage pulse, while the number o f particles passing through the orifice is equal to the number o f pulses.

The electric sensing zone method has a lower size limit o f particle detection o f 0.6 pm. The upper size limit is reached when particles can no longer be kept in suspension (Allen, 1981). For all samples, a 30 pm orifice was used which was calibrated with latex particles o f sizes 2.02 pm, 5.00 pm and 9.86 pm (Particle Data Ltd., Cheltenham, U.K.). For whole yeast cells, the electrolyte used was 1% (w/v) sodium chloride in 100 mM potassium dihydrogen phosphate buffer pH 6.5. For homogenate, the electrolyte used was 5% (w/v) sodium chloride in 100 mM potassium dihydrogen phosphate buffer pH 6.5. The measured size range was from 0.93 to 14.55 pm divided into 128 channels.

To vacuum cc

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All electrolytes were filtered and degassed using 0.2 pm, 47 mm diameter cellulose nitrate filters (Whatman, Maidstone, U.K.) in a vacuum filter apparatus (Millipore, Whatford, U.K.). Adequate dilution (approximately 1 in 2000) was made to eliminate coincidence effects and all particle size distributions were corrected for electrolyte background noise. For each sample three replicate counts were made to ensure consistent results to within ±3.0%.

3.2.7.2 L aser Sizing

Particle size analysis below 1.0 pm was carried out using a Series 4700/ PCS 100 spectrometer laser sizer (Malvern, Worcestershire, U.K.) which uses the photon correlation spectroscopy (PCS) method to determine particle size. In the PCS method, a photon counting detector tracks rapid changes in laser light scattered by a sample containing particulate material. Measurements are taken over given times and fluctuations in intensity are characterised by a digital correlator in order to describe the diffusive movement o f the particles. For monosized particles the diffusivity, Df, is related to particle diameter using Stoke's law and the Einstein equation for Brownian motion (Berne and Percora, 1976) as:

(equation 3.5) where T is the temperature, kg is the Boltzmann constant, p is the solvent viscosity and d^ is the hydrodynamic particle diameter which is slightly larger than the geometric diameter due to solvation and interaction effects. In practice, the diffusion process is quantified by an auto-correlation function, which arises from the multiplication o f the intensity o f scattered light by itself, which is known as 'homodyne processing', i.e. successive signals are stored and every 'old signal' is multiplied by the current value o f the signal as it is measured. Various methods can then be used to relate to a distribution o f intensity over a range of particle sizes.

The main elements of the Series 4700/ PCS 100 spectrometer system are shown in Figure 3.2 on page 90. Monochromatic light emitted by an argon laser is focused onto the sample cell, which is held in a glass vat filled with a liquid. The 'waist o f focus', i.e. the point where the beam is narrowest, coincides with the axis o f rotation o f the photomultiplier.

The beam enters and leaves the vat through flat optical quality windows. An attenuator is mounted on the exit window to reduce back reflection o f the laser light. Liquid is placed in the vat to reduce flare at the vat/sample cell interface, and is controlled to a given temperature and filtered using the filter pump to remove dust. The sample is held in a 10 mm circular quart cell. Light scattered by the sample is collected by an optical system and sensed by the photomultiplier, which is sensitive enough to count individual photons. A narrow band filter between the optics and photomultiplier ensures that only light at the wavelength o f the laser is detected. The stepper motor allows the scattering angle to be changed from 10° to 150°. Digital signals coming from the photomultiplier are processed by the correlator and then passed to the computer for final analysis and display. A fixed angle o f 90° was used during analysis to restrict the laser from seeing the edge o f the cuvette. The liquid used in the vat was water and temperature o f the vat was set to 20°C.

The intensity distribution is determined by the Series 4700/ PCS 100 spectrometer using both cumulative and multimodal analysis methods:

i) Cumulative method: A polynomial is fitted to the log o f the normalised correlation function. The 1 st moment is used to derive a mean size (known as the 'Z Average') and the 2nd to give a measure o f the width o f the distribution (known as the 'polydispersity').

ii) Multimodal Analysis: The overall solution is formed by combining cumulative method solutions for subsets of the overall distribution. The resolution is altered by adjusting the width of the subsets, with the overall number fixed at 24. The solution found is used to generate a 'fit error' by back calculating a correlation function corresponding to the set o f size classes in the solution. An iterative process is then carried out in which the width o f subsets is reduced until the error no longer improves.

Specifying the refractive index o f the particles and sample liquid enabled the intensity distribution to be transformed into a number distribution by dividing the intensity for each size class by a scattering efficiency factor calculated by the instrument. The volume distribution could then be found by assuming the particles were spherical.

TEMPERATURE