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Liquid Chromatography 2: New Technology

Liquid Chromatography 2: New Technology. Lecture Date: April 3 rd , 2007. 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. 10 min.

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Liquid Chromatography 2: New Technology

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  1. Liquid Chromatography 2: New Technology Lecture Date: April 3rd, 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 10 min 10 min Waters Particle Size Evolution Late 1960’s 40µm pellicular non-porous coated 100-500 psi (7.1-35.5 bar) 1000 plates/meter 1m columns Early 1970’s 10µm Irregular micro-porous 1000-2500 psi (71-177 bar) 25,000 plates/meter 3.9 x 300mm 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

  4. Waters UPLC • UPLCTM: Ultra-performance LC • The Science of UPLCTM • Theory • Packing particle developments • Applications

  5. 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 Waters Smaller Particles The evolution of the van Deemter plot

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

  7. Waters UPLCSeparations

  8. 2.1 x 50 mm, 5 µm 0.10 Peak Capacity = 153 AU 0.05 0.00 0.10 2.1 x 50 mm, 1.7 µm Peak Capacity = 123 AU 0.05 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 Minutes 0.10 AU 0.05 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 Minutes Minutes Waters Achieving Speed without Compression 6x Faster 3x Sensitivity

  9. 1.00 0.00 0.20 0.40 0.60 0.80 Minutes Waters UPLC and Chromatographic Speed Quality LC data, faster Future: 1.7µm hybrid particle 2.1x100 up to 15,000 psi Typically 230,000 plates/meter Combining Speed, Sensitivity and Resolution 1 minute

  10. N ( ) ( ) k k+1  -1  Rs = 4 System Selectivity Retentivity Efficiency Waters 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

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

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

  13. Waters 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 dp ↓ 3X, P ↑ 27X

  14. Waters Summary of Effects at Constant Column Length

  15. 0.050 0.040 0.030 AU 0.020 0.050 0.010 0.000 0.040 0.00 1.00 2.00 3.00 4.00 5.00 6.00 Minutes 0.030 AU 0.020 0.010 0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 15.00 Minutes Waters Fixed Column Length: Flow Rate Proportional to Particle Size 1.5X Resolution 2.6X Faster 1.4X Sensitivity 22X Pressure 1.7 µm, 0.6 mL/min, 7656 psi Theory: 1.7X Resolution 3X Faster 1.7X Sensitivity 25X Pressure 4.8 µm, 0.2 mL/min, 354 psi 2.1 x 50 mm columns

  16. Waters 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:

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

  18. Waters UPLC Applications: High Resolution Peptide Mapping 0.08 HPLC 4.8 µm Peaks = 70 Pc = 143 0.06 0.04 AU 0.02 0.00 0.08 UPLC™ 1.7 µm Peaks = 168 Pc = 360 2.5X increase 0.06 0.04 AU 0.02 0.00 0.00 5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00 Minutes

  19. Waters Particle Size Evolution

  20. Waters 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

  21. Waters Technology Requirements • 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

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

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

  24. Rs = 2.30 0.30 Rs = 1.86 AUFS 0.30 Rs = 9.15 10.0 Rs = 4.71 AUFS 0.0 10.0 Time in Minutes Waters HPLC and UPLCTM 8 Diuretics + impurity 2.1x100mm 4.8µm HPLC 2.1x100mm 1.7µm ACQUITY UPLC More Resolution ACQUITY UPLCTM

  25. 0.30 AUFS 0.33 10.0 Rs = 9.15 Rs = 3.52 AUFS Rs = 4.71 Rs = 1.82 Waters 0.0 3.5 Time in Minutes HPLC and UPLCTM 2.1x100mm 1.7µm ACQUITY UPLC ACQUITY UPLCTM 2.1x30mm 1.7µm ACQUITY UPLC Scaled Gradient Same Resolution as HPLC, Less Time ACQUITY UPLCTM

  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. Eksigent Technologies: “Express” • 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

  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. Eskigent Express • 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.

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

  36. Another Example: Nanostream PLC

  37. Nanostream PLC • 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

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

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