1. Robert E. Synovec CPAC Department of Chemistry University of Washington 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 (  -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 >  -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 Diameter4.6 mm10  m x 100  m Flow Rate1 mL/min10 nL/min Waste Generated 500 L/year5 mL/year Sample Used10  L1 nL or less Pump Pressure1000 psi5 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. Separation Channel Sample By-pass Sample Inlet Mobile Phase Inlet Outlet Detection Region 100  m 10  m An integrated LC device: sampling, separation and detection Typical channel dimensions 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.

9. 3 injections, 2 nL each 46810 0 40 80 Time, min Absorbance, mAU On-Chip Automation Provides Reproducible Injection 8 mM Bromocresol Green 5 mM phosphate mobile phase, pH 7, 40 nL / min PDMS channel 100  m x 10  m x 23 cm Valcor Solenoid Absorbance Detection Ocean Optics SD2000

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

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

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

14. 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 Upstream Downstream 04008001200 20 60 100 140 180 Signal (mrads) Time (Seconds) PEG 106 PEG 11840

15. Ratio of the Upstream and Downstream Position Signals for PEG 106 and PEG 11840 Independent of Concentration 0 0.2 0.4 0.6 0.8 1.0 02004000200400600 Time (Seconds) Ratio PEG 106PEG 11840 Ratio signal as a function of time is independent of concentration Diffusion and molar mass information

16. 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. Sugar Analysis using  -MWS 20  L off-chip sample injection at 1 ppth 3 injections for each sample 200400600 0.40 0.45 0.50 0.55 0.60 Ratio Molar Mass (g/mol) Glycerol Deoxyribose Glucose Sucrose Lactose Raffinose Various sugars are readily distinguished on-chip, suitable for real-time analysis of bio-related processes

18. Process Monitoring with the  -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 100200300400500 0.36 0.38 0.40 0.42 0.44 0.46 0.48 0.50 Ratio Molar Mass (g/mol) Gly Gly-Gln Gly-Gly-His Gly-Gly-Tyr-Arg

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 Sample From Fermentation Process Clean sample for Analysis by LC, etc… To Waste or Recycle Mobile Phase Quantitative (reproducible) Robust Simple Applies laminar flow fluidics with diffusion control.

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

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

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!