Unit D3.3. EMULSION DROPLET SIZE DETERMINATION
Quality attributes of food emulsions, such as appearance, stability and rheology, are strongly influenced by the size of the droplets that they contain (Friberg and Larsson 1997, McClements 1999). For example, the creaming stability of an emulsion decreases as the droplet size increases. Analytical techniques that provide quantitative information about droplet size are therefore required to aid in the development and production of high quality emulsion-based food products. A variety of analytical techniques have been developed to measure droplet size, e.g., laser diffraction, electrical pulse counting, sedimentation techniques and ultrasonic spectrometry (McClements 1999). These techniques are used for fundamental research, product development and quality assurance. This unit focuses on the two most commonly used techniques in the food industry: laser diffraction and electrical pulse counting.
Basic Protocol 1. Laser diffraction determination of droplet size distribution
This protocol describes a laser diffraction technique used to measure the droplet size distribution of emulsions. A monochromatic beam of laser light is transmitted through a dilute emulsion and the resulting diffraction pattern is measured using a series of light sensitive silicon detectors (Figure 1). The diffraction pattern is the result of scattering of the laser beam by the droplets in the emulsion being analyzed, and its precise form depends on the droplet characteristics (i.e. size, concentration, and complex refractive index). Information about droplet size distribution and concentration is determined by finding the values that give the best agreement between the measured diffraction pattern and that predicted by light scattering theory. This theory assumes that each photon of light is only scattered by a single droplet, which means that the emulsion must be dilute (< 0.05% droplets) to avoid multiple scattering. Most commercially available instruments can measure droplet diameters between 0.1 and 1000 mm, although some have special features that enable them to analyze smaller droplets. The precise operating procedure for a particular laser diffraction instrument depends on its design, which varies between manufacturers. The protocol given below is therefore fairly general, and the laboratory manuals of specific instrument manufacturers should be consulted for more a detailed description. In the near future, an ISO standard for the installation and validation of Laser Diffraction units will be published (ISO 13320: part 1).
Materials
Sample emulsion
Distilled water or buffer solution
Laser diffraction instrument designed for particle size analysis
Calibration standard containing particles of known diameter
1. Turn on the laser diffraction instrument and allow it to warm up for 30 min before taking measurements. This gives the laser enough time to reach a consistent and reliable output.
2. Zero the instrument by measuring the BACKGROUND diffraction pattern of distilled water or buffer solution. Most commercial instruments display the variation of light intensity with scattering angle. This should be fairly flat across the range of scattering angles where the measurements are made (except at smaller angles where the laser beam directly passes through the sample). If it is not flat there may be problems with a dirty cuvette, insufficiently clean solution, air bubbles in the solution or incorrect alignment of the cuvette (see trouble shooting).
3a. If the sample emulsion has a droplet concentration within the measurement range of the instrument it can be analyzed directly. In this case, replace the distilled water or buffer solution with sample, and measure its diffraction pattern. The measurement range of an instrument depends on a number of instrumental and sample factors and must be determined for each system. Most commercial instruments have an indicator that shows when the droplet concentration is in the appropriate measurement range.
3b. If the sample emulsion is too concentrated to analyze directly then it must be diluted prior to analysis. If only the droplet size distribution is required, then dilution can be carried out by placing a sufficient number of drops of emulsion into the distilled water or buffer solution to reach the optimum transmittance range for the instrument (which is normally indicated on a computer screen). The sample should be stirred for about 1 min to ensure homogeneity and then the diffraction pattern should be measured. If both the droplet size distribution and concentration of the emulsion is required, and the instrument is capable of measuring both, then the emulsion must be diluted to a concentration that is within the optimum transmittance range of the instrument using a known amount of distilled water of buffer solution. The diluted emulsion is then placed in the instrument and the diffraction pattern is measured as in section 3a. Once the droplet concentration of the diluted emulsion has been measured, it is possible to determine the droplet concentration of the initial emulsion: fI = fDVI/VD, where f is the droplet volume fraction, V is emulsion volume, and the subscripts I and D refer to the initial and diluted emulsions, respectively.
4. After the diffraction pattern from the sample emulsion is measured it is automatically corrected by the instrument using a BACKGROUND subtraction routine to account for any extraneous scattering from the solvent or imperfections in the optical system. The instrument automatically calculates the droplet size distribution (and sometimes the droplet concentration) that give the best agreement between the measured diffraction pattern and that predicted by light scattering theory. Finally, the results are presented as a table and/or graph. Be sure to use the correct values for the refractive index and absorptivity of the droplets and continuous phase in the mathematical analysis. These can usually be found in the literature, obtained from the instrument manufacturer or measured in the laboratory.
5. Periodically, it is advisable to ensure the instrument is working correctly by measuring the particle size distribution of a calibration standard and checking that it agrees with the known particle size. Again it is important to be use the correct refractive index and absorptivity of the particles and continuous phase in the mathematically analysis.
Alternate Protocol 1. Electrical pulse counting determination of droplet size distribution
This protocol describes an electrical pulse counting (also called “electrozone sensing”) technique used to measure the droplet size distribution of emulsions. These instruments have been commercially available for many years and are widely used in the food industry (McClements 1999). The emulsion to be analyzed is diluted in a weak electrolyte solution, which is placed in a beaker that has two electrodes dipping into it (Figure 2). One of the electrodes is contained in a glass tube that has a small aperture in it, through which the emulsion is drawn (Hunter 1986, Mikula 1992, Lines 1994). When an oil droplet passes through the aperture it causes a decrease in the current between the electrodes because oil has a much lower electrical conductivity than water. Each time a droplet passes through the aperture, the instrument records a decrease in current that it converts into an electrical pulse. The instrument controls the volume of liquid that passes through the aperture and so the droplet concentration can be determined by counting the number of electrical pulses in a known volume. When the droplets are small compared to the diameter of the aperture, the droplet size is simply related to the height of the pulses: d3 » kP, where, d is the droplet diameter, P is the pulse height and k is an instrument constant. The instrument constant is determined by recording the pulse height of a suspension of monodisperse particles of known diameter. Most commercially available instruments can measure droplet diameters between 0.4 and 1200 mm, although a number of tubes with different sized apertures are required to cover the whole of this range (Lines 1994). The measured droplet diameter is precise to better than 1%. The operating procedure for a particular instrument depends on its design, which varies between manufacturers. The protocol given below is therefore fairly general, and the laboratory manuals of specific instrument manufacturers should be consulted for more a detailed description.
Materials
Sample emulsion
Electrolyte solution
Electrical pulse counting instrument
Tube with appropriate aperture for sample being analyzed
Calibration standard containing particles of known diameter
1. Turn on the instrument and allow it to warm up for 30 min before taking measurements.
2. The instrument must be calibrated using spherical particles of known diameter, which should be about 5-20% of the diameter of the aperture in the glass tube. Monodisperse latex particles are commercially available, which can be used for this purpose. The measuring cell is filled with electrolyte solution containing an appropriate concentration of suspended particles. A stirrer is switched-on to ensure homogenous distribution of the particles within the measurement cell. The instrument measures the pulse height of a large number of particles and then calculates the calibration factor, k (= d3/P). The instrument only needs to be calibrated periodically, but it is useful to always analyze a calibration standard before making a set of measurements to ensure that the instrument is performing correctly
3. The emulsion to be analyzed is dispersed in electrolyte solution to obtain a final droplet concentration less than about 105 droplets per ml. The instrument automatically pulls the particles through the aperture and measures the pulse height (P) and number of pulses per unit volume of a large number of particles (» 10,000). It then uses this information to calculate the particle size distribution (d3 = kP) and number of particles per unit volume. This information is reported to the user in the form of a table or a plot.
4. After the analysis has been completed it is necessary to remove the emulsion and thoroughly clean the measurement cell with electrolyte solution.
REAGENTS AND SOLUTIONS
Use deionized, distilled water in all recipes and protocol steps. If necessary filter the water before use to remove any particulate matter e.g., using a 0.22 or 0.44 mm filter.
Laser Diffraction
Electrolyte solution. The droplets being analyzed must normally be suspended in an electrolyte solution to increase the flow of current between the electrodes. Commonly used electrolyte solutions are 0.85 wt% NaCl or 5wt% trisodium orthophosphate. The electrolyte solution should be free from any particulate matter and be chemically compatible with the sample.
Particle size calibration standard. Particle size calibration standards can be obtained from a number of chemical suppliers or from the National Institute of Science and Technology. Lines (1994) lists a number of standards that are appropriate for this purpose.
Electrical Pulse Counting
Buffer solution. The composition of the buffer solution used to dilute an emulsion depends on the characteristics of the emulsion. To avoid changes in the aggregation of emulsion droplets it is normally best to use a buffer solution with the same pH and composition as the continuous phase of the emulsion. To prevent droplet aggregation it may be necessary to incorporate a small amount of surfactant in the buffer solution.
Particle size calibration standard. Particle size calibration standards can be obtained from a number of chemical suppliers or from the National Institute of Standards (e.g., NBS 1003b).
COMMENTARY
Background Information
Laser Diffraction
Laser diffraction is the most commonly used instrumental method for determining the droplet size distribution of emulsions. The possibility of using laser diffraction for this purposed was realized many years ago (van der Hulst 1957, Kerker 1969, Bohren and Huffman 1983). Nevertheless, it is only with the rapid advances in electronic components and computers that have occurred during the past decade or so that has led to the development of commercial analytical instruments that are specifically designed for particle size characterization. These instruments are simple to use, generate precise data and rapidly provide full particle size distributions. It is for this reason that they have largely replaced the more time-consuming and laborious optical and electron microscopy techniques.
The technique is most suitable for analyzing dilute emulsions that are fluid, and therefore competes directly with electrical pulse counting methods, which are applicable to similar systems (see Alternate Protocol 1). Most laser diffraction instruments (0.01 to 1000 mm) can cover a wider range of particle sizes than electrical pulse counting instruments (0.4 to 1000 mm using a number of different aperture sizes), and do not require the presence of electrolyte in the aqueous phase (which could destabilize some electrostatically stabilized emulsions). Nevertheless, electrical pulse counting techniques are considered to have a greater resolution.
The accuracy that the droplet size distribution of an emulsion can be determined by a properly functioning and correctly operated laser diffraction instrument depends on two major factors. First, it depends on the design of the optical system used to measure the diffraction pattern resulting from the transmission of a laser beam through the cuvette. Second, it depends on the sophistication of the mathematical model used to convert the measured diffraction pattern into a droplet size distribution. The number, position and quality of the detectors used to measure the angular dependency of the laser beam determine the accuracy to which the diffraction pattern can be measured. The greater the number, the wider the range of angles covered and the greater the sensitivity of the detectors the more accurately can the diffraction pattern be determined. More sophisticated instruments, which are normally only used in research laboratories, have a detector that can be positioned at any angle to the laser beam.
The droplet size distribution and concentration are determined by finding the values that give the best agreement between the measured diffraction pattern and that predicted by light scattering theory. The most rigorous light scattering theory, called the Mie theory, is applicable to suspensions containing any droplet size and refractive index (Kerker 1969, Farinato and Rowell 1983). On the older particle sizing instruments it used to take a considerable amount of time to determine the droplet size distribution because a great deal of computation was required. Consequently, a number of instrument manufacturers used simpler approximations to the Mie theory that were limited to certain particle size ranges (Kerker 1969). The use of these theories reduced the required computation time, however, it also meant that the accuracy of the results was sacrificed. Another method of reducing the computation time was to assume that the particle size distribution followed a certain mathematical form (e.g., normal or log-normal), however, this also led to a reduction in accuracy. Recent advances in electronics and computers have led to the availability of modern instruments that can rapidly solve the Mie theory without requiring any simplifying assumptions. Consequently, these instruments have a greater accuracy than their older counterparts.