Particle size analysis

Particle size measurements of retentate and selected permeate samples were performed using three different particle sizers depending on the size range of the substance to be measured.

3.3.2.1 Electrical Sensing Zone Method

Particle size analysis of polyethyleneimine flocculated yeast homogenate was carried out using an Elzone (Model 280 PC, Particle Data Ltd., Cheltenham, U.K.). The Elzone consists of a tube with an orifice in its wall which is immersed in an electrolyte solution containing the material to be analysed. A vacuum pump applies a negative pressure across the orifice which causes material and electrolyte solution to flow through it and unbalance a mercury column connected to the system. The system is then isolated from the vacuum source and flow through the orifice continues due to the balancing action of the mercury column. This activates a counter which performs a count on a known volume of material passing through the orifice. The change in electrical resistance across the orifice is monitored by means of immersed electrodes on either side of the tube wall. The resistance is converted to a voltage pulse. The number of particles passing through the orifice is proportional to the number of pulses and the height of a voltage pulse is proportional to the volume of particle.

An 18 |im orifice was used and calibrated using latex particles of sizes 2 |im and 5 |xm (Particle Data Ltd., Cheltenham, U.K.). Sodium chloride, 10 % w/v, in phosphate buffer (100 mM KH2PO4, pH 6.5) was used as electrolyte solution. The solution was filtered using 0.2 |Lim

cellulose nitrate filters (Whatman, Maidstone, U.K.). Samples, - 1 0 |liL , were dispensed into the

electrolyte solution and measurements were conducted as described above. Three replicate counts per sample were performed.

3.3.2.2 Laser Diffraction Method

Particle size analysis of whole yeast cells and polyethyleneimine flocculated yeast homogenate was carried out using a Malvern 3600E particle sizer (Malvern Instruments, Malvern, Worcestershire, U.K.). The instrument uses the principle of diffraction as a means of measurement. A low power monochromatic beam of light, incident on particles in a sample cell, is diffracted by the illuminated particles to give a stationary diffraction pattern. The diffraction pattern represents the instantaneous size distribution of particles in the illuminated area. This pattern changes as particles enter and leave the illuminated area. A lens focuses the diffraction pattern onto a photo-electric detector which produces an analogue signal proportional to the incident light intensity. The final measured diffraction pattern, which is representative of the bulk sample, is obtained by integration over a suitable period using a computer directly interfaced to the detector. Having obtained the diffraction pattern, the computer uses non-linear least squares analysis to find the size distribution which gives the most closely fitting diffraction pattern.

A 63 mm lens capable of detecting particle sizes in the range 1.2 p.m up to 118 |im was selected and details entered into the computer programme. The sample cell was cleaned with de­ ionised water and positioned within the range of the lens. The cell was then filled with 0.2 pm filtered phosphate buffer (100 mM KH2PO4, pH 6.5). The tilt (vertical angle) and twist (horizontal angle) adjustment of the cell was performed to ensure that the laser beam reflections from the cell windows were properly directed out of the measurement area. Alignment of the receiver unit was achieved using the X (horizontal adjustment) and Y (vertical adjustment) adjusters until the focused laser spot passed through the main detector, hitting the central detector. Measurements were carried out in two stages. The initial measurement was performed on diluent to eliminate the contribution of non-sample scattering sources. Sample, a pinhead amount, was then added to the cell contents and mixed using a magnetic stirrer. The sample concentration was checked prior to measuring the particle size distribution. Measurements were performed in duplicate for analytical purposes.

Particle size analysis of yeast homogenate, selected permeate samples and Escherichia coli whole cells and lysed cells were carried out using a Malvem Series 4700/PCS 100 spectrometer laser sizer (Malvem Instmments, Malvem, Worcestershire, U.K.). The

spectrometer, which operates by photon correlation spectroscopy, consists of a laser transmitter mounted on a turntable which sends a light beam into the sample cell at the centre of the turntable. Scattered light from the sample is recorded in a photon counting device and its intensity and fluctuations in intensity as a function of time are recorded using a digital correlator. The fluctuations in intensity describe the diffusive movement of particles, which is related to the size of the equivalent sphere using Stoke's law. The information from the correlator is passed to a computer for analysis and display. Typical measurements last 5-150 s, during which the sample must remain stable and representative.

The sample viscosity and an assumption of the refractive index of the sample liquid were inputted in the computer programme. The sample cell was cleaned with de-ionised water and filled with 0.2 pm filtered phosphate buffer (100 mM KH2PO4, pH 6.5). Sample, ~ 10 pL, was added to the contents in the cell and mixed gently by inversion. The sample cell was held in a glass vat filled with de-ionised water. Temperature of the water in the vat was controlled at 25°C and filtered to remove dust particles using a filter pump. The angle of measurement was set at 90 °. The concentration of sample in the cell was altered until an acceptable count rate of 50,000 to

100,000 counts per s was achieved. Measurements were then conducted in duplicate.