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ContentsLoading the ULM, 6-64ULM Software, 6-65Suspension Fluids, 6-73Cleaning the ULM, 6-75Replacing the Diffraction Cell Window, 6-78ULM Troubleshooting, 6-82ULM Specifications, 6-82APPENDIX A:Specifications, A-1APPENDIX B:Sample Handling, B-1Material Handling, B-1Liquid Sampling, B-1Solid Sampling, B-1Liquid Sample Dispersion, B-4Liquid Sample Dispersion, B-5Physical and Chemical Methods of Liquid Dispersion, B-7Special cases, B-7A Practical Recipe For Liquid Sample Dispersion, B-7Solid Sample Dispersion, B-8Diluent Selection, B-8Consider Solvation Effects, B-8Consider Other Effects, B-8Consider the Effects the Diluent Has on the Sample's SuspensionFluid, B-8Consider Characteristics of the Diluent, B-9APPENDIX C:Optical Models, C-1Introduction, C-1Refractive Index, C-1Extended Optical Model, C-2Creating an Optical Model, C-2Creating an Optical Model from the RunFile Menu, C-3Statistics, C-5User-Defined Mean, C-5Median and Mode, C-6Characterizing the Distribution, C-6APPENDIX D:Troubleshooting, D-1Background, D-1Troubleshooting High Backgrounds, D-3Reference Background Set-Up, D-4Part 1 - Setting Up a Reference Background, D-5xvi

ContentsPart 2 - Using the Reference Background, D-9APPENDIX E:Maintenance, E-1Cleaning Procedures, E-1Cleaning Fluids, E-1Cleaning the Lens, E-1Fourier Lenses, E-2Projection Lens, E-3xvii

Contentsxviii

LS 13 320 IntroductionThe Beckman Coulter LS 13 320, Figure 1, measures the size distribution of particles suspendedeither in a liquid or in dry powder form by using the principles of light scattering. This particle sizeanalyzer provides reliable and reproducible results for researchers, quality control laboratories,product and process control departments, or anyone with the need to measure particle sizedistributions.Figure 1 LS 13 320 With The Tornado DPSThe LS 13 320 consists of an optical bench and five different sample handling modules: Universal Liquid Module (ULM) Aqueous Liquid Module (ALM) Tornado Dry Powder System (DPS) Micro Liquid Module (MLM)In addition an AutoPrep Station can be used in conjunction with the ALM.The LS 13 320 incorporates Beckman Coulter's patented PIDS (Polarization Intensity DifferentialScattering) technology to provide a dynamic range of 0.017 μm to 2000 μm. Figure 2 shows the PIDSdetectors.xix

LS 13 320 IntroductionFourier OpticsFigure 2 PIDS DetectorsThe LS 13 320 is designed from conception to be fully compliant with the ISO standard coveringparticle sizing by the laser scattering method (ISO 13320-1 Particle size analysis - Laser scatteringmethods - Part 1: General principles).The LS 13 320 includes a sophisticated software package with a multi-component StandardOperating Procedure (SOP). This multi-component Standard Operating Procedure (SOP) will assistoperators by ensuring that analyses are the same run-after-run.Every element of the analysis from method set-up to the final printout can be locked into a userdefinable SOP. The SOP routine is divided into two distinct components; an SOM (StandardOperating Method) and a Preference (.prf) file. The SOM includes all aspects of the analysis relatingto the instrument settings. The Preference file (.prf) includes choices relating to data presentationand output formats.Fourier OpticsMeasuring Moving ParticlesThe LS 13 320 measures particle size distributions by measuring the pattern of light scattered by theparticles in the sample. This pattern of scattered light is often called a scattering pattern orscattering function. More specifically, a scattering pattern is formed by light intensity as a functionof scattering angle. Each particle's scattering pattern is characteristic of its size. The patternmeasured by the LS 13 320 is the sum of the patterns scattered by each constituent particle in thesample.An important component of making this measurement in an LS 13 320 instrument is the Fourierlens (Figure 3). A Fourier lens serves two functions: it focuses the incident beam so it will notinterfere with the scattered light, and it also transforms the angularly scattered light into a functionof location on the detection plane. The most important feature of Fourier optics is that the scatteredlight of any particle at a specific angle will be refracted by the lens so as to fall onto a particulardetector, regardless of the particle's position in the beam.xx

LS 13 320 IntroductionDetermining PSDFigure 3 Fourier OpticsThe result is that the Fourier lens forms an image of the composite scattering pattern of all particles,the pattern being centered at a fixed point in the Fourier plane. This pattern is centered at the samefixed point regardless of the position or velocity of the particle in the sensing zone.The individual scattering patterns from the many moving particles in the sample cell aresuperimposed, creating a single composite scattering pattern that represents the contributionsfrom all the particles in the sample cell. Detectors placed in the Fourier plane record this compositescattering pattern. Over the course of a measurement, a running average is created from thechanging flux patterns. When the duration of the measurement is long enough that the flux patternaccurately represents the contributions from all particles, an analysis of the resulting pattern willyield the true particle size distribution of the sample.Determining PSDThe composite scattering pattern is measured by 126 detectors placed at angles up to approximately35 degrees from the optical axis. When you view intensity in flux units (light intensity per unitarea), you are looking at the scattering pattern.In order to compute the size distribution, the composite scattering pattern is deconvolved into a setof individual number, one for each size classification, and the relative amplitude of each number isa measure of the relative volume of equivalent spherical particles of that size. This deconvolution isbased on either the Fraunhofer or Mie theories of light scattering.xxi

LS 13 320 IntroductionSystem ComponentsSystem ComponentsThe LS 13 320 Optical Bench OverviewThe LS 13 320 optical system is comprised of a source of illumination, a sample chamber in whichthe sample interacts with the illuminating beam, a Fourier lens system used to focus the scatteredlight, and an array of photodetectors that record the scattered light intensity patterns.The laser's radiation passes through a spatial filter and projection lens to form a beam of light.The beam passes through the sample cell where particles suspended in liquid or air scatter theincident light in characteristic patterns according to their size. Fourier optics collect the diffractedlight and focuses it onto three sets of detectors, one for the low-angle scattering, the second formid-angle scattering, and the third for high angle scattering. A block diagram of the LS 13 320optical system is presented in Figure 4.Figure 4 Optical System of the LS 13 320The sample modules are attached to the optical bench by an automatic docking system that can beactivated by the push of a button (Figure 5) or via software commands.xxii

LS 13 320 IntroductionSystem ComponentsFigure 5 Module Eject and Door Open ButtonsBefore a module can be docked the sample cell chamber door must be retracted. This is easily doneby pressing the Open button found on the left side of the optical bench (Figure 5).Light SourceThe LS 13 320 uses a 5 mW laser diode with a wavelength of 750 nm (or 780 nm) as the mainillumination source. It also has a secondary tungsten-halogen light source for the PIDS system. Thelight from the tungsten-halogen lamp is projected through a set of filters which transmit threewavelengths (450 nm, 600 nm, and 900 nm) through two orthogonally oriented polarizers at eachwavelength.Light from a laser diode is monochromatic- a requirement of the theoretical models that describe aparticle's scattering function. However, as opposed to gas or liquid lasers, the light from a laserdiode is not “focused”. The monochromatic light from a laser diode must also be “treated” toproduce a “clean” beam. The device that is most often used to condition an illuminating source iscommonly known as a spatial filter. Most spatial filters consist of a set of optical elements components such as lenses, pinholes, apertures, etc. - designed to refine the beam to the desiredquality. Figure 6 depicts a mechanical spatial filter. One feature of the LS 13 320 is that itincorporates a (patented) fiber optic spatial filter.xxiii

LS 13 320 IntroductionSystem ComponentsFigure 6 Mechanical Spatial FilterSample ModulesThe main function of the sample-handling module is to deliver particles in the sample, withoutdiscrimination to their sizes, to the sensing zone while avoiding the introduction of any undesirableeffects such as air bubbles and/or thermal turbulence. The sample module is typically composed ofthe sample cell and a delivery system. The delivery system may include certain features such as acirculation pump, an ultrasonic probe, or stirring bars to help better disperse and circulate theparticles. The LS 13 320 operates with sample cells designed for particles suspended in liquids or indry powder form. These modules are: Tornado Dry Powder System (DPS) Universal Liquid Module (ULM) Aqueous Liquid Module (ALM) Micro Liquid Module (MLM)In addition the ALM can be used in conjunction with the AutoPrep Station.Computer SystemThe operation of the LS 13 320 requires a personal computer (PC) and the PC-based control andanalysis software. If you are not using a Beckman Coulter-supplied PC, you must provide one thatmeets the minimum configuration requirements shown in Table 1, and preferably one that meetsthe recommended configuration requirements (shown in the same table).Table 1 Minimum and Recommended Computer ConfigurationsItemxxivMinimum ConfigurationRecommended ConfigurationMicroprocessor800 MHz1 GHZ Intel or equivalentRAM512 Mb1 Gb or betterHard Drive20 Gb35 Gb or better

LS 13 320 IntroductionSystem ComponentsTable 1 Minimum and Recommended Computer Configurations (Continued)ItemMonitorKeyboardMouseMS Windows VersionMinimum ConfigurationRecommended Configuration800X6001024X768Enhanced 101/102Enhanced 101/1022 Button3 ButtonWin 98 or betterWin VistaSoftwareThe Microsoft Windows-based LS 13 320 control program provides both hardware control and datamanagement. Among other functions, the program allows you to: Display, print, store, and export data. Customize (user interface) on-screen and printed reports. Define analysis profiles to automate your most frequently used analysis protocols. Use the built-in security features (supervisor modes, automatic 21 CFR Part 11 compliancefunction use with the compliant version of software only).xxv

LS 13 320 IntroductionSystem Componentsxxvi

CHAPTER 1TheoryThe scattering of light is one of the most widely used techniques for measuring the size distributionof particles. In practice, the technique is fast and flexible, offering precise measurements that canbe easily adapted to samples presented to the analyzer in various forms. The method involves theanalysis (deconvolution) of the patterns of scattered light produced when particles of different sizesare exposed to a beam of light. The LS 13 320 series of instruments takes advantage of theseprinciple to rapidly provide precise and reproducible particle size distributions.Theoretical BackgroundLight ScatteringWhen light illuminates a particle having a dielectric constant different from that of the medium,depending on the wavelength of the light and the optical properties of the particle, light will bescattered in a unique way. We commonly describe scattering phenomena in terms of diffraction,reflection, refraction, and absorption. When light interacts with the electrons bound in the materialthat re-radiate light, scattering is observed. Because most materials exhibit strong absorption in theinfrared and ultraviolet regions which greatly reduces scattering intensity, most light scatteringmeasurements are performed using visible light of wavelengths from 350 nm to 900 nm. Thescattering intensity from a unit volume that is illuminated by a unit flux of light is a function of thecomplex refractive index ratio between the material and its surrounding medium. This intensityfalls within the regime of Rayleigh scattering and is inversely proportional to the fourth order ofthe light wavelength, i.e., the shorter the wavelength, the stronger the scattering. The reason thatthe sky is blue at midday and red at sunrise or sunset is that one sees the scattered sunlight duringdaytime and sees the transmitted sunlight during dawn and dusk. Utilizing this wavelengthdependence, we use red as the color for the stoplight and for all traffic control warning signsbecause red light has the least scattering power in the visible light spectrum. This allows thetransmitted light to go through fog, rain, and dust particles and reach the intended detector: in thiscase the human eye.Several technologies make use of light scattering to obtain information about materials. Amongthese technologies elastic light scattering (ELS) is the main method for the characterization ofparticles of sizes ranging from microns to millimeters. In ELS the scattered light has the samefrequency as the incident light, and the intensity of scattered light is a function of the particle'soptical properties and dimensions. In general, the scattered light intensity of a particle is a functionof the following variables: particle dimension, particle refractive index, medium refractive index,1-1

TheoryTheoretical Backgroundlight wavelength, polarization, and scattering angle. The scattered intensity from a particulatesample is, in addition to the above variables, a function of particle concentration and particleparticle interaction. Some of the variables are constants in a particular experimental setup, such aslight wavelength and the particle refractive index. In characterizing particle size using lightscattering, one optimizes sample concentration to a proper range so that the sample will scatterenough intensity to enable the measurement to be completed with a desired signal to noise ratio,but not to scatter so much as to saturate the detecting system. Sample concentration is alsooptimized for minimal particle-particle interaction and minimal multiple scattering so that themeasurement is performed based on elastic single particle scattering. In addition, in a lightscattering measurement one has to assume that the refractive index and density of particles in thesample are uniform, which is true for most particulate systems. Thus, the scattered intensity is onlya function of scattering angle, particle shape, and particle size. If the relations between scatteringintensity, scattering angle, particle shape and particle size are known, one is able to resolve sizedistribution for particles of a particular shape from the measured angular scattering intensitypattern. Theories have been developed to aid in the extraction of the information needed for thedetermination of particle size from light scattering measurements.Figure 1.1 Scattering Patterns for SpheresMie TheoryThe Mie theory describes the interaction of light with a particle of arbitrary size as a function ofangle, given that the wavelength and polarization of the light are known and that the particle issmooth, spherical, homogeneous, and of known refractive index. This theory is more complex thanthe theory set forth by Fraunhofer, in that it accounts for all possible interactions between particlesand light, yet it's only applicable to spheres.Spheres produce light scattering patterns that are characterized by the presence of scatteringminima and maxima at different locations (Figure 1.1) depending on the properties of the particles.At small angles (typically smaller than 10 degrees) the scattering pattern for spheres is centrallysymmetric instead of axially symmetric, i.e., it displays concentric rings in the direction of theincident light. Therefore, large particles produce scattering intensities that are concentrated atsmall angles and due mainly to diffraction effects from the edge of the particle.1-2

TheoryPIDS3.1.2 Fraunhofer TheoryWhen the particle size is much larger than the wavelength of light or the materials are highlyabsorptive, the edge effect of the particles contributes more to the total scattered intensity.Interference effects are now due to the bending of light at the particle's boundary (diffraction). Ina light scattering measurement, because the light source is far away from scatterers and the opticsare usually designed so that the incident beam illuminating the scatterers is homogeneouslyparallel, only Fraunhofer diffraction will take place. For these spheres, the Fraunhofer diffraction isjust a simplified form of the Mie theory with the limiting condition that d l.Fraunhofer theory can only be used for particles that are 1) much larger than the wavelength of thelight (typically 30 mm) and non-transparent, i.e., the particles have different refractive indexvalues than that of the medium (typically with the relative refractive index being larger than 1.2),or 2) highly absorptive (typically with absorption coefficients higher than 0.5). In Fraunhofertheory the refractive index of the material is irrelevant because for large particles the scatteringintensity is concentrated in the forward direction -- typically at angles smaller than 10 degrees. Forthis reason Fraunhofer diffraction is also known as forward scattering. The angle for the firstminimum of scattering intensity is simply related to the size by EQ 1.EQ 1Most of the scattering intensity is concentrated in a very sharp central lobe, which provides a muchsimpler solution applicable to sizing large particles in a light scattering measurement.Light scattering is an absolute measurement technology only in the sense that once theexperimental setup is correct, calibration or scaling are not necessary in order to obtain the volume(or weight) percentage of each component. In addition, choosing a correct optical model is often thekey step in obtaining the correct results.PIDSMany samples have particle sizes that extend into the submicron range creating a wider sizedistribution range. However, as a particle size gets smaller, the ratio of particle dimension to lightwavelength (d/l) is reduced. Interference effects are thus reduced and the scattering patternbecomes smoother and less angular dependent. At this smaller size range, the sensitivity of particlesize to scattering intensity pattern is greatly reduced causing it to be more and more difficult toobtain correct size values. Obviously, if light of a short wavelength is used, the ratio will be great,and so the lower size limit will be effectively extended. Combining the polarization effects of lightscattering with the wavelength dependence at high angles, we can extend the lower size limit to aslow as 40 nm, almost reaching the theoretical limit. This is the patented Polarization IntensityDifferential Scattering (PIDS) technology.The origin of the polarization effect can be understood in the following way. If a small particle, muchsmaller than the light wavelength (d l), is located in a light beam, the oscillating electric field ofthe light induces an oscillating dipole moment in the particle, i.e., the electrons in the atomscomprising the particle move back

listed in this manual. Proper handling procedures for diluents and reagents used in particle analysis should be adhered to at all times. Consult appropriate safety manuals and Material Safety Data Sheets for all samples, diluents and reagents used. Care should be taken when m