1. Liquid Chromatography 2: New Technology Lecture Date: April 3 rd, 2007

2. Outline of Topics: New LC Technology  Waters UPLC – ultra-high pressure chromatography  Monolithic stationary phases: –Dionex ProSwift –Phenomenex  Eksigent Technologies 8-channel HPLC  NanoStream 24 column HPLC

3. 10 min 1980’s to present day 3.5 - 5µm spherical micro-porous 1500-4000 psi (106.4-283.7 bar) 50,000 - 80,000 plates/meter 3.9 x 300mm Early 1970’s 10µm Irregular micro-porous 1000-2500 psi (71-177 bar) 25,000 plates/meter 3.9 x 300mm 10 min Particle Size Evolution Late 1960’s 40µm pellicular non-porous coated 100-500 psi (7.1-35.5 bar) 1000 plates/meter 1m columns 10 min Waters

4. UPLC  UPLC TM : Ultra-performance LC  The Science of UPLC TM –Theory –Packing particle developments –Applications Waters

5. Smaller Particles  Smaller particles provide increased efficiency  With smaller particles this efficiency increase extends over a wider linear velocity  This provides the ability for both added resolution and increased speed of separation  Particles are central to the quality of the separation The evolution of the van Deemter plot Waters

6. Faster Chromatography Can Reduce Resolution * 50 mm column * Higher Flow Rates 2.0 mL/min 0.0 1 2 3.0 mL/min. 1 2 Time in Minutes 3.0 Fails Rs Goal of 3 Limitation 5um Reversed Phase Column “Compressed Chromatography” Run time is reduced, but resolution is lost!

7. UPLC Separations Waters

8. Achieving Speed without Compression Peak Capacity = 153 AU 0.00 0.05 0.10 Minutes 0.005.0010.0015.0020.0025.00 30.00 2.1 x 50 mm, 5 µm Peak Capacity = 123 AU 0.00 0.05 0.10 Minutes 0.005.0010.0015.0020.0025.0030.00 2.1 x 50 mm, 1.7 µm 6x Faster 3x Sensitivity AU 0.00 0.05 0.10 Minutes 0.000.501.001.502.002.503.003.504.004.50 5.00 Waters

9. UPLC and Chromatographic Speed Future: 1.7µm hybrid particle 2.1x100 up to 15,000 psi Typically 230,000 plates/meter Combining Speed, Sensitivity and Resolution Quality LC data, faster Minutes 1.00 0.000.200.400.600.80 1 minute Waters

10. Fundamental Resolution Equation In UPLC™ systems, N (efficiency) is the primary driver Selectivity and retentivity are the same as in HPLC Resolution, Rs, is proportional to the square root of N If N ↑ 3x, Rs ↑ 1.7x R s = N 4 (  -1  ) k k+1 ( ) System Selectivity Retentivity Efficiency Waters

11. Improving Resolution with Smaller Particles (Using constant column lengths) Efficiency (N), is inversely proportional to Particle Size (d p ): Rs ↑ 1.7X N ↑ 3X,dp ↓ 3X, Waters

12. Efficiency, N is inversely proportional to the square of Peak Width, W Peak height is inversely proportional to peak width Relationship between Peak Width and Efficiency for Constant Column Length Rs ↑ 1.7X,N ↑ 3X,dp ↓ 3X,T ↓ 3X, Sensitivity ↑ 1.7X Waters

13. Back Pressure at Constant Column Length Back Pressure is proportional to Flow Rate, F, and inversely proportional to the square of Particle Size: Optimal Flow Rate is inversely proportional to Particle Size P ↑ 27Xdp ↓ 3X, Waters

14. Summary of Effects at Constant Column Length Resolution Improvement Speed Improvement Sensitivity Improvement Back Pressure 1.7 vs. 5 µm particles 1.7x3x1.7x25x 1.7 vs. 3 µm particles 1.3x2x1.3x6x Waters

15. Fixed Column Length: Flow Rate Proportional to Particle Size AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.002.004.006.008.0010.0012.00 15.00 4.8 µm, 0.2 mL/min, 354 psi AU 0.000 0.010 0.020 0.030 0.040 0.050 Minutes 0.001.002.003.004.005.00 6.00 Theory: 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure 1.7 µm, 0.6 mL/min, 7656 psi 2.1 x 50 mm columnsWaters

16. HPLCUPLC™ Cycle time (min) 273 # of Samples Run per Year 10,00090,000 Productivity Improvements  UPLC™ gives 70% higher resolution in 1/3 the time  Target resolution is obtained 1.7x (+70%) faster  Method development up to 5x faster  Assume that an HPLC is running about 67% of the year, or 4,000 hr: Waters

17. Gradient Peak Capacity tgtg w w w w w Gradient Duration Peak Width w ↓, P ↑ Peak capacity is a measure of the separation power of a gradient on a particular column. Waters

18. UPLC Applications: High Resolution Peptide Mapping AU 0.00 0.02 0.04 0.06 0.08 AU 0.00 0.02 0.04 0.06 0.08 Minutes 0.005.0010.0015.0020.0025.0030.0035.0040.0045.0050.0055.0060.00 UPLC™ 1.7 µm Peaks = 168 P c = 360 2.5X increase HPLC 4.8 µm Peaks = 70 P c = 143 Waters

19. Particle Size Evolution Waters

20. Technology Requirements  Small particle, rugged chemistries  High pressure fluidic modules (up to15,000 psi)  Reduced system volumes and optimized flow paths  Reduced cycle time, minimum carryover autosampler  High speed detectors, optical and mass Waters

21.  Requires innovation in every aspect of column: –Sub 2µm particles  Porous for optimum mass transfer  New patented bridged hybrid particle required for pressure tolerance and outstanding chromatographic performance  Innovative sizing technology for narrow particle size distribution –Column hardware  New patented frit technology to retain particles  New end fittings for high pressure/low dispersion operation –Packing technology  New column packing processes to optimize stability Waters Technology Requirements

22. Creating a New Particle Technology AdvantagesDisadvantages Inorganic (Silicon) Mechanically strong High efficiency Predictable retention Limited pH range Tailing peaks for bases Chemically unstable Polymer (Carbon) Wide pH range No ionic interactions Chemically stable Mechanically ‘soft’ Low efficiency Unpredictable retention Hybrid (Silicon-Carbon) Particle Technology Waters

23. Bridged Ethane-Silicon Hybrid Particles Anal. Chem. 2003, 75, 6781-6788 Si SiSi O O O O O C CC C C C Bridged Ethanes in Hybrid Matrix - Strength, - Great Peak Shape - Wider pH Range Waters

24. HPLC and UPLC TM 2.1x100mm 4.8µm HPLC 0.30 AUFS Time in Minutes 0.010.0 Rs = 4.71 Rs = 9.15 2.1x100mm 1.7µm ACQUITY UPLC More Resolution ACQUITY UPLC TM 0.30 AUFS 10.0 Rs = 1.86 Rs = 2.30 8 Diuretics + impurity Waters

25. 10.0 0.30 AUFS Rs = 4.71 Rs = 9.15 2.1x100mm 1.7µm ACQUITY UPLC 0.33 AUFS Time in Minutes 0.03.5 Rs = 3.52 Rs = 1.82 2.1x30mm 1.7µm ACQUITY UPLC Scaled Gradient Same Resolution as HPLC, Less Time ACQUITY UPLC TM Waters HPLC and UPLC TM

26. Drawbacks to UPLC Cost Solvent mixing problems Lack of variety in commercial columns at 1.7 um Baseline ripple – real data from GSK: HPLC UPLC

27. Monolithic Stationary Phases Basic Idea:

28. Monolithic Stationary Phases Basic Idea: Organic vs. inorganic

29. Comprehensive 2D LC ● 2D LC: two LC experiments run back-to-back, with the effluent from the first LC column broken into pieces and injected on a second LC column

30. Micro-HPLC Development of precision microfluidic systems for drug discovery and miniaturized medical devices microfluidic flow control microscale pumping Microfabrication In other words, miniaturize the entire LC system

31. Advantages of Miniaturization: Increase in the number of parallel analyses Decrease in analysis time Decrease in sample/reagent consumption Increase in integrated system functionality Barriers to Microscale HPLC Poor control of low flow rates Loss of separation efficiency from instrumental components Low sensitivity for absorbance detection Eksigent Technologies: “Express”

32. Microfluidic Flow Control Precise control of flow rate (1 nl/min to 100 µl/min) Ability to pump against substantial back pressures (to 10,000 psi or more) Active feedback for identification -and prediction- of leaks or blockages Virtually instantaneous response to step changes in flow rate setpoint

33. Microfabrication Detectors and Column

34. microscale flow control increases in separation speed, system component optimized to minimize extra column variance. Advances allow typical gradient methods to be run at injection-to-injection cycles 4-6 times faster than conventional analytical HPLC without a loss in resolution. This speed is a result of higher resolution in microscale formats, coupled with extremely rapid gradient mixing and column re- equilibration times. column flow rates from 200 nl/min up to 20 ul/min. Eskigent Express

35. High Throughput HPLC: Eksigent Express 800 56 Chromatograms 10 Minutes 50 x.300 mm; 5  m Luna C18(2) Gradient: 65  95 % ACN in 25 s Hold for 20 s; Equilibrate: 20 s 12  L/min

36. Another Example: Nanostream  PLC

37. 24 UV absorbance detectors 8-head Autosampler Stationary phase – 10  m (Van deemter plot!) Column Length – 80 mm Equivalent i.d. – 0.5 mm Injection volume 0.4-1.0  L Nanostream  PLC

38. Further Reading  Many other new LC technologies are being developed  For more recent developments, see: –A. Chem. Annual Reviews