1. Mass Spectrometer Autosampler Jake Ahrens Stephen Pearson Kyle Sala Seth Yellin Project Mentor: Professor Jane Hill

2. Project Background Mass Spectrometer – Highly sensitive device that analyzes volatile organic compounds (VOC) Pathogen bacteria – Mass spectrometry is used to identify different pathogen bacteria Identification – Every pathogen has a specific VOC profile

3. Project Background Pathogen Library – Build a database of pathogen bacteria profile under various conditions. Unknown identification – Ultimately will be used to identify unknown bacteria in human lungs, by comparing the mass spectra of the humans breath, to the pathogen bacteria database

4. Project Motivation Currently, each sample must be connected by hand, tested, and then disconnected and cleaned. To build a large database, the sampling process needs to be automated The sampler will reduce bacterial aerosol & person contact Reduce sample contamination Increase data reproducibility

5. + = ? Design an autosampler to be integrated with a mass spectrometer. This device would allow the user to hook up multiple samples, and then test them all sequentially. Problem Objective

6. Project Goals Maintain temperature of samples at 37°C Sample bottles need be 100ml System needs to be air tight Flow rate needs to remain constant at 2 L/min System flushing and headspace replacement Stay within $5000 budget

7. Method of Sampling

8. Before the Autosampler…

9. Project Progression 1.Cartesian Robot 2.Valves, Needles, and Septa Bottle Tops 3.Manually Connect Bottles to Valves 4.Incubator  Cost  Fluid Dynamics in Needles  Familiar Set-up with Current Bottles and Tops  Acquired Instead of Built an Enclosure

10. Our Device

12. Circuit Board

13. Major Features Valve System Microcontrollers ▫Code Incubator

14. Valve System 3-Way Solenoid pinch valves Used to control the path of Air and CO2

15. Microcontrollers 2 Independent microcontrollers Push button switches used to open all valves to replace tubing for cleaning

16. Valve Code Written in C Uses subroutines for each phase ▫Flushing ▫Collect CO2 and medium spectra Constants for these times defined in beginning Easy for user to change time constants depending on the tests they are running

17. Incubator Temperature accurate within 1% of target temp Samples need to be at constant temperature for growth and testing

18. 2 Sample Schematic Valves

19. Initial Flush of Main Line

20. Collect CO2 Spectrum

21. Collect Medium Spectrum

22. Sample 1

23. Air Replacement

24. Flushing/Collect CO2 Spectrum

25. Collect Medium Spectrum

26. Sample 2

27. Sequence of Operation 1.Flush the main line with CO2 2.Collect CO2 spectrum 3.Collect medium spectrum 4.Collect sample 1 5.Replace headspace of sample 1 with air 6.Flush main line 7.Collect CO2 spectrum 8.Zero medium spectrum 9.Collect sample 2 10.Replace headspace of sample 2 with air 11.Flush main line Repeat…

28. Operation of device Attach sample bottles to the valve system (one inlet and one outlet per bottle) Turn on CO2 and Air supplies Turn the power switch on Save spectrum graphs for the samples

29. Operation Loading of sample bottles

30. Design Demonstration Three samples, all contain water ‘Bubbles’ depict the CO 2 flow into and out of the sample bottles Three subsequent tests ▫Testing with standard compounds that bacteria could produce in natural environment. The two we used are acetone and indole.

32. Test #1 Volumetric flow rate (2 L/min) Ran auto-sampler with all 9 sample bottles in place ▫Testing the code that controls the valves and subsequent gas flow Flushing and sampling sequences ▫Simulated in complete fashion Successful Test!

33. Test #2 Demonstration of TIC (total ion chromatograph) of sampling process Having the mass spectrometer effectively read samples from the autosampler Three sample test ▫One background control and two standard compounds ▫Water and two samples of acetone  Acetone – Commonly produced by bacteria Successful Test!

34. Test #2 Results Demonstration TIC (Total ion chromatograph) of sampling process of one background control and two standard compounds Step 3: Start collect sample 1 Step 5: Start collect sample 2 Step 4: CO2 flush Step 6: CO2 flush Step 1: Water background collection Step 2: CO2 flush Time (min) Intensity (%)

35. Mass spectrum of step 1 (signal response to bottle 1 – water) Intensity (%) Mass/charge

36. Mass spectrum of step 3 and 5 (signal response to bottles 2 and 3 – acetone)

37. Test #3 Second demonstration of TIC (total ion chromatograph) of sampling process Sampling the more volatile compound of indole in varying bottle positions ▫Indole – characteristic compound from E.Coli bacteria Able to get a momentary reading in the first bottle position and a successful reading in the last (ninth) bottle position Why is the mass spectrometer not reading this compound?

38. Test #3 Results Indole test at valve position 1 Short time response of Indole (peak 118) in position 1

39. Indole test at valve position 9 Stable response of Indole (peak 118) in position 9

40. Test Results Auto-sampler mechanically works and operates like it was designed too!! Effectively samples and flushes bottles Maintains volumetric flow rate (2 L/min) Able to send samples to mass spectrometer for identification But… Some compounds, as seen with indole, cannot be successfully read ▫Possible problem could be chemical interactions with the valves’ silicone tubing

41. Desirable Attributes Outcome Effectiveness ▫Temperature Control – Incubator (~37°C) ▫Constant CO 2 flow rate – 2 L/min ▫Automation – effectively samples nine bottles sequentially, with some user interaction Ease of Use ▫Setting up sampler – familiar process of manually connecting the tubing ▫Simplicity to operate – flipping a switch Economy ▫Cost of parts – under budget (< $5,000) ▫Replacement cheapness (cost to replace the parts that did not break because of the ones that did)– instructions from an annotated user manual, easy to replace parts

42. Project Outcome Sampling process automated, to build large library The sampler will reduce bacterial aerosol & person contact Reduce sample contamination Increase data reproducibility

43. Future Development Reduce dead space tubing for better results More samples to be taken Tube track to prevent any pinch points and allow for easier tube exchanges More chemically inert tubing Higher levels of automation ▫Computer automation

44. Future Development Computer automation: Develop a program to allow interface with sampler and mass spec Mass spec uses Analyst software that can communicate with other windows programs to create databases of samples

45. Beta

46. Acknowledgements Yin-Ming Kuo – Post Doctoral Fellow in The Hill Lab Jiangjiang Zhu – Ph.D student in The Hill Lab Professor Stephen Titcomb Paul Sala

47. Questions and Discussion