1. EyeTech Particle Size & Shape Analyzer

2. What is the size of a particle? Size Related Properties : V = Volume W = Weight S = Surface Area A = Projected Area R = Sedimentation Rate Conventional techniques derive particle size from a (physical) property. Usually Particle Size is expressed as the diameter of a sphere with similar property -> equivalent sphere D min D max AMBIVALUE BV

3. Equivalent Sphere These diameters are fundamentally different for the same particle. The equivalent diameters differ for any non-spherical material. Thus, every PSA technique yields different results for non-spherical materials AMBIVALUE BV

4. Conventional PSA Techniques The measurement of particle properties has several drawbacks: - Sphere is an extreme particle shape, not reflecting real particles - Significant errors for non-spherical particles - Particle size results dependent on used technique - Instrument response requires calibration - Particle size analysis limited to the measured property ‘Laser Obscuration Time’ measurement solves these drawbacks AMBIVALUE BV

5. Laser Channel: Particle Size & Concentration EyeTech - Particles Analyzer AMBIVALUE BV

6. EyeTech Measurement Principle Single Instrument – Dual Measurement Channels AMBIVALUE BV

7. EyeTech Measurement Principle The EyeTech Laser uses the proven optical bench AMBIVALUE BV

8. EyeTech Measurement Principle The rotating laser beam scans single particles within its focus. The diameter of the particle is directly correlated to the duration of the obscuration. The principle measurement is a time interval, not an intensity measurement. The time domain is independent of particles optical or physical properties A B C D E F T a T b T c T d T e T f Time V Scanning Laser AMBIVALUE BV

9. EyeTech Measurement Principle From the duration of the obscuration (t) and the known rotation velocity of the laser beam (v) the particle diameter (D) can be calculated: D = v. t In relation to the high speed of the rotating laser, the particles are stationary. So, particle movement does not effect particle size measurement. No requirement for pre-knowledge of: - Refractive index -Temperature -Viscosity variation -Electrical conductivity AMBIVALUE BV

10. EyeTech Pulse Analysis; Off-Center rejections Pulse Profile Off-Center Pulse Transition Derivative Profile Pulse Transition The Derivative Pulse Width of an On-Center pulse transition corresponds to the laser beam width On-Center

11. EyeTech Pulse Analysis; Derivative Pulse Signal Minimum width and maximum amplitude indicate on-center interactions. Amplitud e For accepted particle crossings, the angle between the particle boundaries and the laser beam trajectory approaches 90°.

12. EyeTech Pulse Analysis; Out-of-Focus rejections Pulse Profile Derivative Pulse Focused Laser Beam Derivative Pulses too wide; Out-Of-Focus pulses are rejected

14. EyeTech Pulse Analysis Concept; Transparent Particles Opaque ParticlesTranslucent Particles Transparent Particles Pulse Profile Particle Size Measurement is independent of particle’s transparency

15. EyeTech Measurement Principle Advantages: - Data is collected on single particles - Detection of minor fractions - Direct measurement of correct particle size, not influenced by physical or optical properties - Operation in liquid, dry or slide - No calibration required - Excellent traceability to (BCR/NIST) standards - Robust measurement, no alignment required by user - Concentration measurement AMBIVALUE BV

16. FDA SCIENCE POSTER FEB 2001 COMPARATIVE ANALYSIS OF COMMON PARTICLE SIZING TECHNIQUES FOR PHARMACEUTICAL POWDERS H.R. Prasanna, E.H. Jefferson, J.S. Taylor, A.S. Hussain, R.F. Karuhn, R.C. Lyon In the FDA poster presented in Feb 2001 they concluded that: “The large population of fine particles was only detected by the particle counting techniques (image analysis and light obscuration) and not by the ensemble technique (laser diffraction)” In the FDA poster presented in Feb 2001 they concluded that: “The large population of fine particles was only detected by the particle counting techniques (image analysis and light obscuration) and not by the ensemble technique (laser diffraction)” AMBIVALUE BV

17. Laserdiffraction Fast & repeatable…. But good? Assembling technology…general approach. Assumptions…. Spherical particles, calibration Restrictions….. Concentration, minor fraction, T pre-knowledge of sample required… RI (mixture?, unknown sample?), AI (color), Transparency, mirror like (scattering, metals)

18. Video Channel: Dynamic Shape Analysis AMBIVALUE BV

19. Why Dynamic Shape Analysis Conventional particle sizing techniques will always give an equivalent spherical diameter in Number or Volume. The only information given is a SIZE information expressed in Number or Volume. Sometimes, these techniques are not sensitive enough to discriminate subtle variations. AMBIVALUE BV

20. Reasons for Dynamic Shape Analysis 1) Seeing is believing, See your particles. 2) Better characterization of your materials. 3) High resolution data. 4) Detection and quantification of shape influences. 5) Validation technique for the laser channel or other particle size equipment. 6) Number distribution, more sensitive for detection of minor fractions of fines or agglomerates. 7) Identification of the homogenity of the material 8) Characterisation of shape differences. AMBIVALUE BV

21. EyeTech Measurement Principle Single Instrument – Dual Measurement Channels AMBIVALUE BV

22. EyeTech Video Channel Video Channel collects digital images used for: Visualization during laser measurement Dynamic Shape Analysis Grabbing Images and Recording Movie AMBIVALUE BV

23. Seeing is believing No difference in particle size distributions for different materials With Dynamic Image Analysis, the difference is evident AMBIVALUE BV

24. Quantifying of shape differences The difference in the shape factor and maximum diameter are obvious! Max. Ferret Diameter: Shape Factor: AMBIVALUE BV

25. Dynamic Shape Analysis Real time images of particles in motion are collected Images are converted into a grid containing a collection of picture elements (pixels) Each individual pixel has a value for brightness (grey level): 0 = Dark; 255 = White All images are analyzed according to a pre-defined set of Image Analysis characteristics Measurement is finished when the measurement end condition is met (# of particles/images, confidence level, time) 420 µm 18 µm Image Conversion AMBIVALUE BV

26. Image Analysis Terms Basic Image Analysis terms: Macro Size Descriptors Ferret Diameters Determination of distributions of: -Minimum Ferret -Maximum Ferret -Average Ferret Average Ferret diameter: F 1 + F 2 + F 3 + F 4 … F 36 36 For a sphere size = diameter

27. Shape Analysis Terms Basic Image Analysis terms: Macro Shape Descriptors [ min Ferret diam.] Aspect Ratio = ------------------------- - [ max Ferret diam.] [Area * 4  ] Shape Factor = ------------------ (Circularity) [Perimeter] 2

28. DSA Parameters Comparing Shape Factor to Aspect Ratio Shape FactorAspect Ratio1 <0,11 0,7850,707 0,6040,577 0,4360,250 0,1600,100

29. Object Data Base The complete shape information for every particle is saved in the object data base. Particles can be viewed individually

30. EyeTech Shape Analysis Features: Easy setup and measurement using software wizards Advanced pre-processing algorithms Size and Shape grouping Automated report generation Storage of real raw data (un- processed images) Re-processing of raw data Calibration and verification tools available Interface with 3 rd party hardware (e.g. microscopes) AMBIVALUE BV

31. Dynamic Shape Analysis For each individual particle, size and shape parameters are determined. The EyeTech Image Analysis software automates set-up and measurement. EyeTech software includes many IA procedures such as: Pre-processing procedures Image Filters Region of Interest Out-of-focus Rejection Morphology Operations Grouping according to size/shape (Re-) processing of stored images and movies Lens Calibration AMBIVALUE BV

32. Grouping / Shape Filters Shape Filters can be set up to analyse a specific group of particles based on size and/or shape Appications: Fibre Analysis Shape of Sieve Fraction Abrasives All: Shape Factor < 0,8: Shape Filter Average Size of non-spherical particles in sample AMBIVALUE BV

33. Dynamic Shape Analysis DSA provides a solution for characterization of non-spherical materials: Multiple size & shape parameters to adapt to any application. No assumptions about particle’s sphericity. Random orientation of particles. No preferred orientation as in microscopy. Grouping of particles based upon size or shape. Visualization for measurement validation/verification. AMBIVALUE BV

34. Dynamic Shape Analysis Summary - Advanced analysis of video images enables the determination of different shape parameters. - Feret Diameters, Shape Factor and Aspect Ratio for shape characterization. - Isolation of classified particles using strategic Shape Filter options. - Validation of particle size results: “SEEING IS BELIEVING” - More accurate characterization of non-spherical materials - Re-processing of stored data AMBIVALUE BV

35. S/W Features AMBIVALUE BV

36. Eyetech Analysis Package New Features: Easy setup and measurement using software wizards Automated Report Generator Results stored in SQL database Multiple User Levels Hardware Diagnostics Calibration and verification tools available 21 CFR Part 11 Compliant AMBIVALUE BV

37. EyeTech User Interface Laser and video in one program One click measurement Real time feedback Multiple data display of graphs, tables, statistics On-Line cycle compare On-Line help Wizard driven software AMBIVALUE BV

41. Setup Method Setup wizards for laser and video measurements Step-by-step guidance through the whole setup process All created methods are stored Methods can be linked to a sample, user or customer Load a method to start the measurement AMBIVALUE BV

44. Advanced Data Output Result Database supports: Data mining: search, sort… Image retrieval Networking: import \ export data, images… Archiving, back up Measurement cycles comparison Host mode: Remote data analysis 21 CFR compliant version includes: Secure log-in Electronic signatures Audit trails AMBIVALUE BV

47. Object Data Base The complete shape information for every particle is saved in the object data base. Particles can be viewed individually

48. EyeTech S/W Advantages Easy setup and measurement using software wizards Advanced pre-processing algorithmes Size and Shape grouping Automated report generator Storage of real raw data (un processed images) Re-processing of raw data (movie files) Interface with third party hardware ( e.g. microscope) Calibration and verification tools avaible Concentration measurement Integrated laser and video software Simultaneous laser and video results Pharmaceutical FDA 21 CFR Part 11 Compliant AMBIVALUE BV

49. One System = Variety of Applications “Adapting the technology to the application rather then adapting the application to the technology” Magnetic Stirring Fiber Cell Free Fall Liquid Flow (10mm) Slide Cell Liquid Flow (4mm) Heated Cell Mechanical Stirring Aerosol Flow Micro Flow AMBIVALUE BV

50. Unique & useful features… (1) True multi-tasking technology - multiple tasks enable during measurement Time based analysis - not depending on any optical properties of the sample Measurements applicable for a broad of concentration range - no dilution needed Possibility of kinetics measurements (Crystal growth, dissolution properties, etc) Investigation of flow ability - process conditions simulations Particle of interest in large matrix can be characterized selectively by using shape filters No alignment or calibration needed - not even when changing system compounds Advanced overflow protection in the automatic Liquid Flow Controller (EyeFlow) Advanced data base - single particle can be detect - clear & fast internal search engine AMBIVALUE BV

51. Unique & useful features… (2) High resolution & accuracy of dynamic measurement range for multi disperse samples No need for pre-knowledge of sample properties Ideal for non-spherical particles analysis Small volume samples - allowing sample volume down to 2 ml IQ, OQ, PQ validation support available 21 CFR part 11 compliant S/W Operation in harsh conditions - minimum maintenance requirements Easy access, maintenance and cleaning procedure to all system compounds Robust measurement - independent to laser intensity and no fluctuation adjustment Emergency measurement stop available at any time by the operator Particles size distribution based on measuring individual particles - not on ensembles Simple & easy to explain measurement principle Fast and easy preparation of samples and user controlled presentation of analysis data AMBIVALUE BV

52. Our Development ; Your Added Value AMBIVALUE BV