Robert E. Synovec CPAC Department of Chemistry University of Washington

1. Robert E. Synovec CPAC Department of Chemistry University of Washington

2. LC-related Technologies and NeSSI Compatibility • On-line, real-time analysis of mixtures: > Drugs and Pharmaceuticals > Environmental Samples > Petroleum Industries > Clinical Chemistry > Pesticides and Their Residues > Foods • Development of analytical instrumentation: > Small-volume and microfabricated LC > Micro-scale Molecular Weight Sensor (m-MWS) > Sampling

3. NeSSI Compatibility • Requirements and Challenges • What should we strive for in instrument development in order to provide lab-based LC performance in a process LC analyzer? • Low – pressure, flow rate (low waste), maintenance • High separation efficiency (optimize peaks/time) • Broad range of chemical selectivity • Robust and quantitative sampling (readily calibrated) • FINALLY: Scaled & implemented so readily interfaced • to NeSSI hardware WHILE minimizing dead volumes

4. We need to consider: New products and new implementation New research level systems (that show promise) IDEAS: > Monolithic columns (perform well and low pressure drop is good, but need smaller id columns are needed) > Capillary columns (everything is good, need more work here), LC on-a-chip > m-MWS (size information without Chromatography!) > H-filter sampling

5. High speed LC of anions using a monolithic column: Very low pressure, but at ~ 5 mL/min (due to 4.6 mm id column) Reference P. Hatsis, C. A. Lucy, Anal. Chem. 2003, 75, 995 – 1001.

6. LC vs. Micro-fabricated LC Lab ScaleMicrofabricated Column Diameter 4.6 mm 10 mm x 100 mm Flow Rate 1 mL/min 10 nL/min Waste Generated 500 L/year 5 mL/year Sample Used 10 mL 1 nL or less Pump Pressure 1000 psi 5 psi Cost per column $400 and up $10 “A disposable, plug and play LC device.” “Easy to swap out chips for maintenance or to change the type of analysis.”

7. Microfabricated Liquid Analyzer Prototypes: Channels in PDMS We use microfabrication techniques such as soft lithography.

8. An integrated LC device: sampling, separation and detection Sample Inlet Sample By-pass Mobile Phase Inlet Typical channel dimensions 10mm Separation Channel 100mm References P. G. Vahey, S.H. Park, B. J. Marquardt, Y. Xia, L. W. Burgess and R.E. Synovec Talanta, 2000, 51, 1205 - 1212. P.G. Vahey, S. A. Smith, C. D. Costin, Y. Xia, A. Brodsky, L.W. Burgess and R. E. Synovec Analytical Chemistry, 2002, 74, 177 - 184. Detection Region Outlet

9. On-Chip Automation Provides Reproducible Injection 3 injections, 2 nL each 8 mM Bromocresol Green 5 mM phosphate mobile phase, pH 7, 40 nL/min PDMS channel 100 mm x 10 mm x 23 cm Valcor Solenoid Absorbance Detection Ocean Optics SD2000 80 40 Absorbance, mAU 0 4 6 8 10 Time, min

10. Micro-fabricated LC with Absorbance Detection Separated temporally and spectrally FD&C Blue #1 Water m.p., 6 nL/min Injected Volume 1 nL Separation Channel in PDMS 100 mm x 10 mm x 6.6 cm Absorbance Detection using Equitech Spectrophotometer FD&C Red #3

11. Micro-fabricated LC Separation of Two Dyes Temporal and Spectral Selectivity are Both Provided 800 Water m.p., 6 nL/min Injected Volume 1 nL Separation Channel in PDMS 100 mm x 10 mm x 6.6 cm Absorbance detection using Equitech spectrophotometer FD&C Red #3 750 700 Time, sec FD&C Blue #1 650 600 400 500 600 700 Wavelength, nm

12. Microfluidic Chip and Instrument Setup for m-MWS References C. D. Costin, R. E. Synovec, Talanta, 2002, 58, 551-560 and Analytical Chemistry, 2002, 74, 4558-4565.

13. Sample Inlet Mobile Phase Inlet Upstream  Deflected Beams Downstream  Laser Beams Position Sensitive Detectors (PSDs) Outlet m-MWS: Measure m-RIG Signals at Two PositionsReal-Time, Dual-Beam Detection Configuration

14. 180 Upstream 140 100 Downstream Signal (mrads) 60 0 400 800 1200 20 PEG 106 PEG 11840 Time (Seconds) Upstream and Downstream -MWS signals of PEG 106 and PEG 11840 • 20 L off chip sample injection • Signals were aligned in time • Diffusion and MW information

15. 1.0 0.8 PEG 106 PEG 11840 0.6 Ratio 0.4 0.2 0 0 200 400 0 200 400 600 Time (Seconds) Ratio of the Upstream and Downstream Position Signals forPEG 106 and PEG 11840 Independent of Concentration • Ratio signal as a function of time is independent of concentration • Diffusion and molar mass information

16. 0.9 0.8 0.7 0.6 Ratio 0.5 0.4 0.3 100 1000 10000 Molar Mass (g/mol) Molecular Weight Calibration for Linear Poly(ethylene)glycols (PEGs)Excellent Molar Mass Resolution!!Imagine application as on-line sensor of polymerization, etc. • Ratio of downstream to upstream signals • Ratio is independent of concentration • Predict molar mass: calibration required for each class of compounds • Tune range by flow rate and detection positions

17. Raffinose 0.60 Lactose 0.55 Sucrose Glucose Ratio 0.50 Deoxyribose 0.45 Glycerol 0.40 200 400 600 Molar Mass (g/mol) Sugar Analysis using m-MWS Various sugars are readily distinguished on-chip, suitable for real-time analysis of bio-related processes • 20L off-chip sample injection at 1 ppth • 3 injections for each sample

18. 0.50 0.48 0.46 Ratio 0.44 0.42 0.40 0.38 0.36 100 200 300 400 500 Molar Mass (g/mol) Process Monitoring withthe -MWS“Can readily monitor peptide or protein synthesis” • Amino Acid and Peptide solutions for monitoring protein synthesis or digestion • Quickly able to detect addition or subtraction of amino acids Gly-Gly-Tyr-Arg Gly-Gly-His Gly-Gln Gly

19. Bio-Fermentation Processes There is a need for reliable sampling and on-line LC

20. On-Line Monitoring of Bio-Fermentation, etc. Applying Micronics H – Filter for Sampling Clean sample for Analysis by LC, etc… Mobile Phase • Quantitative • (reproducible) • Robust • Simple Applies laminar flow fluidics with diffusion control. Sample From Fermentation Process To Waste or Recycle

21. Process Monitoring: Coupling LC on-a-chip and the -MWS to MicroreactorsAqueous and Non-aqueous Polymerizations, Bio-reactions, etc.

22. Glass Chip Applications Glass Chip Prototype • Integrated chip-based LC and CE with -MWS detection • Process monitoring of organic reactions • HPLC detector for aqueous and non-aqueous separations

23. Conclusions LC analyzers and NeSSI • Miniaturized analytics • On-line monitoring • Relatively fast cycle time • Demonstrated modularity • More informative process control • Decrease in maintenance costs More work needs to be done!