1. December 2012 Analysis of Drinking Water Using LIBS Applied Research Associates, Inc. Proprietary Information

2. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 2 Applied Research Associates, Inc. Proprietary Information To Be Covered  LIBS Overview  Advantages  Methods of Analysis  Direct Analysis  Pre-concentration Prior to LIBS Analysis  PMACS 1000 (for pre-concentration)  Summary

3. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 3 Applied Research Associates, Inc. Proprietary Information How does LIBS work? LIBS is a form of atomic emission spectroscopy Laser pulse creates a spark on the sample material Light emissions are collected and input into a spectrometer Processor is used to compare the emissions with our database Analysis results listed in seconds to minutes depending on sample complexity Laser spark on soil 3-5 mm laser receiver plasma on sample The Laser Plasma  Hotter than the sun’s surface  Plasma lasts ~20 µs

4. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 4 Applied Research Associates, Inc. Proprietary Information LIBS Instrument Configurations are Application Specific SPECTROGRAPH LASER MIRROR LENS SAMPLE DETECTOR FIBER OPTIC COMPUTER General LIBS Set-up Table top ARA built food prototype: distinguishes E. Coli from Salmonella LIBS Instruments Customized for the application Lab and production line deployment Size, cost, complexity determined by the application LIBS spectrum analyzed to ID pathogens

5. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 5 Applied Research Associates, Inc. Proprietary Information Advantages of LIBS for Water Analysis  Rapid analysis for targeted species (<5 min for comprehensive analysis).  Simultaneous detection of all targeted species (eliminates separate tests for individual targets)  No chemical agents or consumables required in contrast to current reagent based methods.  Field deployable LIBS instrument through the use of compact and rugged components available commercially-off-the-shelf.  Automated analysis of all targets through instrument control and analysis software.

6. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 6 Applied Research Associates, Inc. Proprietary Information Methods of Analysis  Without Pre-concentration of sample using LIBS Useful when contaminant concentrations are high enough (10 3 – 10 9 cells/mL) Samples could be dried on slides or a direct sampling method could be developed.  Pre-concentration of samples prior to LIBS testing Useful when contaminant concentration are very low Without Pre-concentration With Pre-concentration

7. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 7 Applied Research Associates, Inc. Proprietary Information Direct Analysis  Pathogens and other contaminants can be detected in a variety of water matrices by creating chemometric differentiation models for analysis of LIBS spectra and deploying models in an analysis algorithm specifically designed for the detection application.  Recent study of LIBS detection capability in tap water indicates detection is possible in many types of water for concentrations ranging ≥ 10 3 cells/mL

8. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 8 Applied Research Associates, Inc. Proprietary Information Example of Analysis Algorithm for E. coli, Salmonella, and E. faecalis in Tap water Blank slide or Other? Blank Slide Tap Water Flow Tap water or Other? Tap water E. coli or Other? E. coli Salmonella or E. faecalis? Salmonella E. faecalis

9. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 9 Applied Research Associates, Inc. Proprietary Information Differentiation of uncontaminated Tap Water

10. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 10 Applied Research Associates, Inc. Proprietary Information Differentiation of E. coli in Tap Water

11. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 11 Applied Research Associates, Inc. Proprietary Information Differentiation of Salmonella from E. faecalis in Tap Water

12. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 12 Applied Research Associates, Inc. Proprietary Information Pre-concentration prior to LIBS Analysis of Sample  Collect and pre-concentrate particles and organisms in 200 mL volume of aqueous suspension using the PMACS 1000 IntelliSense Design TM flow system  Collect particles and organisms in the aqueous suspension on a filter substrate  Collect metal ions from the aqueous suspension on ion exchange membrane filters (e.g. 3M Empore® Extraction Disks; 3M Corp. St. Paul, MN).  Interrogate particles, organisms, and metals ions collected on filters using laser spark. Spark scanned along surface R.C. Chinni, D.A. Cremers, and R.A. Multari, “Analysis of material collected on swipes using Laser-Induced Breakdown Spectroscopy (LIBS),” Applied Optics, Vol. 49, Issue 13, pp. C143-C152 (2010).

13. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 13 Applied Research Associates, Inc. Proprietary Information The unit as currently configured has dimensions 36x12x12 inches and a flow rate of 4 L/min. A typical concentration factor is x1000. PMACS 1000 The device is shown in use at the Dunedin, FL water utility for groundwater collection of indicator organisms. Kearns, E.A., S. Magana, and D.V. Lim. 2008. “Automated concentration and recovery of microorganisms from drinking water using dead-end ultrafiltration,” J. Appl. Microbiol. 105:432-442. Leskinen, S.D. and D.V. Lim. 2008. “Rapid ultrafiltration concentration and biosensor detection of enterococci from large volumes of Florida recreational water,” Appl. Environ. Microbiol. 74:4792-4798. Could be used for pre-concentration if necessary.

14. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 14 Applied Research Associates, Inc. Proprietary Information Collection using the PMACS 1000 (1) Filtration of several liters of drinking water supply (4 L/min. flow rate) (2) Backflush to collect targeted materials on filters Diagram showing flow from a water supply through the PMACS unit to concentrate particles and organisms (metal ions collected on filter ). Diagram showing flow during the Backflush to collect particles and organisms on a filter ( ).

15. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 15 Applied Research Associates, Inc. Proprietary Information Differentiation of E. coli post pre-concentration

16. Copyright 2009. All rights reserved. Applied Research Associates, Inc. 16 Applied Research Associates, Inc. Proprietary Information Summary  ARA has demonstrated the ability to differentiate contaminates in water matrices using LIBS Multiple studies using samples provided by the University of South Florida  Basic Detection Blind Study (100%)  Detection Limit Study (direct detection limit ~10 3 cells/mL)  Study of LIBS post pre-concentration of sample (10 cells/mL demonstrated)  LIBS for water analysis could be used in real time to Differentiate “good” from bad water Monitor for specific contaminations in water Flag changes in water composition  Advantages of using LIBS: Fast Simultaneous analysis of multiple parameters is easily possible Results can be obtained on-site, in real time