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Agilent Technologies — Technical Presentation (February 2019)

43 slides from Agilent Technologies's technical presentation, Feb 21, 2019

  1. Slide 1: Updating Old Methods: Is the Gain Worth the Pain?

    Updating Old Methods: Is the Gain Worth the Pain? Becoming a Better Chromatographer HPLC educational webinar Mark Powell Applications Engineer Columns and Supplies Technical Support

  2. Slide 2: Why Would You Consider Updating? What Is the Goal?

    Why Would You Consider Updating? What Is the Goal? • Do I need to update? • Reduce analysis time • Productivity (free up time for other tasks) • More analyses (free up instrument time) • More samples (higher throughput) • Improve resolution • Improve sensitivity • Increase column life • Save solvent

  3. Slide 3: What Can Be Changed? Summary of Allowable Adjustments: USP <621>

    What Can Be Changed? Summary of Allowable Adjustments: USP General Chapter <621> Parameters for System Suitability Isocratic Gradient Particle Size L/dp: -25% to +50% No Changes allowed or Column Length N: -25% to +50% Column Inner Diameter Flexible, w/ constant linear velocity No Changes allowed Fl

  4. Slide 4: What Are the Column Options?

    What Are the Column Options? • Smaller particle size • Higher efficiency -> shorter column -> faster method • Increase resolution • Better sensitivity • Consider pressure limit of instrument • Smaller diameter • Solvent savings • Depends on instrument configuration and plumbing • Bonded phase • Matc

  5. Slide 5: Agilent InfinityLab Poroshell Particle Sizes

    Agilent InfinityLab Poroshell 120 Particle Sizes 0.75 µm 0.5 µm 0.35 µm 1.7 µm 2.5 µm 1.2 µm 0.35 µm 0.5 µm 0.75 µm InfinityLab Poroshell 120 InfinityLab Poroshell 120 InfinityLab Poroshell 120 1.9 µm 2.7 µm 4 µm Highest UHPLC UHPLC performance at Improved HPLC performance lower pressure performance

  6. Slide 6: Particle Size: When to Use What Size

    Particle Size: When to Use What Size Technique / product Performance 4 µm Improved HPLC performance Pressure rating: 600 bar Typical pressure: Often < 200 bar Efficiency: ~200% of 5 μm totally porous HPLC 2.7 µm UHPLC performance at lower pressure Pressure rating: 600 bar Typical pressure: 50% of su

  7. Slide 7: Decreasing Particle Size Increases Efficiency

    Decreasing Particle Size Increases Efficiency DAD1 A, Sig=240,8 Ref=off (AEM_PLUTO_TRANSFER\CATECHINS_19UM-P120-ECC18_2016-05-24_10-52-29.D) mAU Columns: 50 1.9 µm Agilent InfinityLab Poroshell 120 EC-C18, 2.1 x 50 mm, 1.9 um 40 30 Agilent InfinityLab Poroshell 120 EC-C18, 2.1 x 50 mm, 2.7 um 20 Agi

  8. Slide 8: Agilent InfinityLab Poroshell Phases

    Agilent InfinityLab Poroshell 120 phases Best for high and Best for Best for low pH Best for more HILIC for polar Best all around mid pH mobile alternative Chiral phases mobile phases polar compounds compounds phases selectivity Poroshell 120 Poroshell 120 Poroshell 120 Poroshell 120 Poroshell 120 P

  9. Slide 9: Benefits of Transferring to Agilent InfinityLab Poroshell

    Benefits of Transferring to Agilent InfinityLab 120 Poroshell • Superficially porous particles • Can run at high speed without sacrificing resolution • Existing instruments can turn over more samples per hour • 30-50% increase in efficiency near the backpressure of a 3.5-5.0 µm particle • Boosts the

  10. Slide 10: Van Deemter Equation

    Van Deemter Equation ℎ = 𝐿 Τ𝑁 • A term: eddy diffusion and flow h = A + B / u + C u distribution B • Particle size & packing quality important • Narrow particle size distribution • B term: longitudinal diffusion • Diffusion in the mobile phase • C term: mass transfer h • shorter diffusion paths…

  11. Slide 11: Van Deemter Equation: Eddy Diffusion

    Van Deemter Equation Eddy diffusion weddy ~ λ dp λ: Quality of column packing Differences in diffusion paths due to: Different paths Poor column packing Broad particle size distribution

  12. Slide 12: Van Deemter Equation: Longitudinal Diffusion

    Van Deemter Equation Axial or longitudinal diffusion Increase in peak width due to self-diffusion of the analyte At low flow the analyte remains in the mobile phase for a long time • High increase in peak width • Increased height of a theoretical plate Flow

  13. Slide 13: Van Deemter Equation: Resistance to Mass Transfer

    Van Deemter Equation Resistance to mass transfer wC ~ dp2 Different diffusion paths Totally porous particle (TPP) Superficially porous particle (SPP)

  14. Slide 14: Van Deemter Curves

    Van Deemter Curves Optimize your flow rate for Agilent InfinityLab Poroshell 120: • For 2.1 mm id, we suggest 0.42 mL/min • For 3.0 mm id, we suggest 0.85 mL/min • For 4.6 mm id, we suggest 2 mL/min Overlay of van Deemter plots: the optimal flow rate for Agilent InfinityLab Poroshell 120 is faster t

  15. Slide 15: Faster Ibuprofen Analysis on Poroshell 120 EC-C8

    Faster Ibuprofen Analysis on Agilent InfinityLab Poroshell 120 EC-C8 4.6 x 50 mm, 2.7 µm Poroshell 120 EC-C8 USP Ibuprofen Oral Suspension (L7) 2.4 minutes 630 mL (0.7 mL/L phosphoric acid in water 4 mL/min 4.6 x 50 mm, 2.7 µm 370 mL acetonitrile 419 bar Benzophenone / ibuprofen (0.48 mg/mL) Rs = 12

  16. Slide 16: Transfer of Isocratic USP Methods (Naproxen)

    Transfer of Isocratic USP Methods (Naproxen) Parameters for System Suitability Isocratic Gradient Particle Size L/dp: -25% to +50% No Changes allowed or Column Length N: -25% to +50% L (mm) dp (μm) L/dp % N % <621> compliant 150 5 30,000 100% 10,639 100% Yes 150 4 37,500 125% 19,054 179% Yes 100 4 2

  17. Slide 17: Scaling USP Naproxen Method from 5 µm TPP to 4 µm SPP

    Scaling USP Naproxen Method from a 5 µm TPP to 4 µm SPP System Suitability Method Requirement: N > 4000, Rs > 11.5 mAU Agilent ZORBAX Eclipse Plus C18 5µm 4.6 x 150 mm 60 N = 10639 USP Prescribed Column 50 40 Rs = 13.7 L/dp = 30,000 30 20 10 0 0 1 2 3 4 5 6 7 8 9 min mAU InfinityLab Poroshell 120 4µ

  18. Slide 18: Scaling USP Naproxen Method from 5 µm TPP to 2.7 µm SPP

    Scaling USP Naproxen Method from a 5 µm TPP to 2.7 µm SPP System Suitability Method Requirement: N > 4000, Rs > 11.5 mAU N = 10639 Agilent ZORBAX Eclipse Plus C18 5µm 4.6 x 150 mm 60 Rs = 13.7 USP Prescribed Column 50 40 L/dp = 30,000 30 20 10 0 0 1 2 3 4 5 6 7 8 9 min mAU 30 N = 21046 98%↑N Infinit

  19. Slide 19: Retention Factor - Gradients

    Retention Factor - Gradients tG  F k = * S    Vm With gradient separations, the retention factor is influenced by • F = flow rate • tG = gradient time (minutes) • ΔΦ = change in volume fraction of B mobile phase • Vm = column volume • S = constant (4 - 6 for small molecules, 10 - 1000 for pepti

  20. Slide 20: Retention Factor - Gradients

    Retention Factor - Gradients tG  F k = * S    Vm

  21. Slide 21: Maintaining k*

    Maintaining k* Keep relative peak position and shorten analysis Any decrease in Can be offset by a proportional • Column length • Decrease in tG or F • Increase in %B • Column volume (i.d.) • Decrease in tG or F • Increase in %B • %B (same column) • Decrease in tG or F tG • F k*  S •  • Vm 21

  22. Slide 22: Reduce Analysis Time, Keep Gradient Steepness the Same

    Reduce Analysis Time, Keep Gradient Steepness the Same Sample: 1. Aldicarb sulfoxide, 2. Oxamyl, 3. Methomyl, 4. Aldicarb sulfone, 5. Carbofuran-3-hydroxy, 6. Aldicarb, 7. Propoxur, 8. Carbofuran, 9. Carbaryl, 10. Methiocarb, 11. 11. ISTD (BDMC) Column: Agilent ZORBAX Eclipse Plus-C18 Column: Agilen

  23. Slide 23: Gradient Method Transfer– 4.6 x 150mm, 5 µm Agilent ZORBAX SB-C18

    Gradient Method Transfer– 4.6 x 150mm, 5 µm Agilent ZORBAX SB-C18 23 February 21, 2019 For Research Use Only. Not for use in diagnostic procedures.

  24. Slide 24: Maintaining Peak Position & Resolution

    Maintaining Peak Position & Resolution Have shortened column & gradient time – need to do so by the SAME factor 1/3 column length – 1/3 gradient time ex: RRHT column – 4.6 x 50 mm, 1.8µm, SB-C18 24 February 21, 2019 For Research Use Only. Not for use in diagnostic procedures.

  25. Slide 25: Gradient Transfer: 4.6 x 250 mm, 5 µm to 4.6 x 100 mm, 2.7 µm

    Gradient Transfer: 4.6 x 250 mm, 5 µm to 4.6 x 100 mm, 2.7um Mobile phase: Sulfadiazine, A: 0.1% formic acid in water Sulfathiazole B: 0.1% formic acid in ACN Sulfapyridine Sulfamerazine, mAU Sulfamethazine, 11.596 40 mL Sulfamethazole, Time %B 100 Sulfamethoxypyridazine, 12.674 0 8 Sulfachloropyrid

  26. Slide 26: Resolution

    Resolution Baseline separations for rugged methods h Rs = 1.5 t If we consider peaks of equal height: • 1 - minimum for a measureable separation • 0.6 - required to discern a valley between two equal-height peaks • 1.5 - considered to be a baseline separation • 1.7 or greater - desirable for rugged

  27. Slide 27: Notoginseng Analysis: 4 µm Poroshell and 5 µm Totally Porous

    Notoginseng Analysis: 4 µm Agilent Poroshell and 5 µm Totally Porous Same dimensions, Same conditions, higher resolution mAU Agilent InfinityLab 2 1 Notoginsenoside R1 80 Mobile phase: Poroshell 120 EC-C18, 2 Ginsenoside Rg1 4 4.6 x 150 mm, 4 µm 3 Ginsenoside Re A) water 60 4 Ginsenoside Rb1 W1/2= 0

  28. Slide 28: Notoginseng Analysis: Speed Optimization

    Notoginseng Analysis: Speed Optimization Gradient for 4.6 × 150 mm: mAU Agilent InfinityLab Time (min) %A 60 Poroshell 120 EC-C18, Rs= 2.31 1.0 mL/min 0 81 4.6 x 150 mm, 4 µm Initial Pressure: 70 min 40 135 bar 12 81 20 60 64 0 61 10 10 20 30 40 50 60 min 65 10 mAU Agilent InfinityLab 66 81 Poroshel

  29. Slide 29: Notoginseng Analysis: Speed Optimization

    Notoginseng Analysis: Speed Optimization • Flow rate increased from 1 mL/min to 1.5 mL/min (1.5 / 1.0 = 0.67) mAU •60 Gradient Poroshell 120 EC-C18, 4.6 x 150 mm, 4time um pointsRs= decreased 2.31 proportionally 1.0 mL/min •40 45 * 0.67 = 30 minutes Initial Pressure: 70 min 135 bar 20 0 10 20 30 40

  30. Slide 30: Achieving Better Sensitivity

    Achieving Better Sensitivity Totally porous 3.5 µm to 2.7 µm superficially porous ZORBAX Eclipse Plus C18, 3 x 100 mm, 3.5 µm Same method can be used Ibuprofen: with either column due PW1/2=0.014 to similar selectivity S/N=182 nc=43 2.7 µm columns: • Taller, narrower peaks • >40% more sensitivity, a

  31. Slide 31: Amino Acid Analysis on Poroshell 120 HPH-C18

    Amino Acid Analysis on Agilent InfinityLab Poroshell 120 HPH-C18 Column: Agilent InfinityLab Poroshell Agilent InfinityLab HPH-C18 or Agilent ZORBAX Eclipse Poroshell HPH-C18 Plus C18 4.6 x 100 mm, 2.7 µm Column Temperature: 40 °C Mobile Phase A: 10 mM Na2HPO4: 10 mM Na2B4O7, pH 8.2: 5 mM NaN3 Mobil

  32. Slide 32: Amino Acid Analysis on HPH-C18 in pH 8.3 Phosphate Buffer

    0 0 0 0 20 40 80 40 60 80 20 40 60 80 20 40 80 60 20 60 mAU mAU mAU mAU 0 0 0 0 1.070 1.061 1.081 1.076 1.546 1.589 1.656 1.637 2 2 2 2 3.381 3.403 3.421 3.442 4 4 4 4 4.049 4.073 4.091 4.107 4.290 4.319 4.342 4.366 4.451 4.481 4.503 4.532 4.715 4.742 4.763 4.793 5.060 5.079 5.105 5.123 5.385 5.415

  33. Slide 33: Competitor High pH Column vs Poroshell 120 HPH-C18: Stress Test

    Competitor High pH column vs Agilent InfinityLab Poroshell 120 HPH-C18: Stress Test A: 10 mM ammonium bicarbonate, pH 10 B: acetonitrile Injection 1 5-95%B over 5 minutes 2 3 7 1 5 6 4 1.5 2 2.5 3 3.5 4 4.5 min Other column Injection 500 Loss of resolution after Only 500 injections! 1.5 2 2.5 3 3.5

  34. Slide 34: Solvent Use

    0 0 0 20 40 60 80 20 60 80 20 40 60 80 40 100 120 100 120 mAU mAU mAU Solvent Use 0.459 0.440 0.599 0.590 1 0.979 1.029 1.092 2 1.503 1.602 1.701 1.666 1.755 2 2 2 2.751 3 2.926 2.963 4 3.139 3.286 3.389 3.508 5 6 3.578 3.551 3.750 3.801 7 8 3.958 4 4 4 3.999 3.995 4.184 4.153 4.379 4.434 9 4.500 4.

  35. Slide 35: System Considerations

    System Considerations Dwell Volume: from formation of gradient to top of column -minimize for faster equilibration and more efficient gradient formation Dwell Extra Column Volume from injection Volume to detector (flow cell) outside of the Extra Column column Volume -minimize to reduce band broadeni

  36. Slide 36: Paraquat and Diquat on ZORBAX Eclipse XDB-C8

    Paraquat and Diquat on ZORBAX Eclipse XDB-C8 Ion Pairing Conditions 1 Conditions: Column: ZORBAX Eclipse XDB-C18 4.6 x 150 mm, 5 mm Mobile Phase: 70% water + heptanesulfonic acid phosphoric acid to pH = 3.0 :30% methanol 2 Temperature: 30ºC Flow Rate: 1 mL/min Detection: UV 257 nm (Paraquat) UV 308

  37. Slide 37: Agilent InfinityLab Poroshell 120 HILIC-Z Approach

    Agilent InfinityLab Poroshell 120 HILIC-Z Approach • No loss in throughput: InfinityLab x10 2 +ESI MRM Frag=100.0V CF=0.000 DF=0.000 CID@30.0 (183.1 -> 157.1) Pesticide_2.1x100 (P120-ZIC HILIC_TCW290B_#21)_#41.d 1.1 Poroshell HILIC columns operates as 1.05 1 2.246 1 1 quickly and reliably as reverse

  38. Slide 38: Updating Chiral Separations

    Updating Chiral Separations Best for Chiral InfinityLab Poroshell • 4 chiral stationary phases on 2.7µm Agilent Poroshell particles Chiral-V 2.7 µm • Fast, efficient analysis using superficially porous particles (<5 min!) InfinityLab Poroshell • High efficiency provides superior peak shape and resol

  39. Slide 39: Fast, High Efficiency Chiral Separations

    Fast, High Efficiency Chiral Separations 39 February 21, 2019 Title Confidentiality label Regulatory statement (if applicable)

  40. Slide 40: Tips for Method Transfer: Adjusting Flow

    Tips for Method Transfer Adjusting flow for different column diameters 2 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟𝑐𝑜𝑙𝑢𝑚𝑛 2 𝐹𝑙𝑜𝑤𝑐𝑜𝑙𝑢𝑚𝑛 1 × = 𝐹𝑙𝑜𝑤𝑐𝑜𝑙𝑢𝑚𝑛 2 𝑑𝑖𝑎𝑚𝑒𝑡𝑒𝑟𝑐𝑜𝑙𝑢𝑚𝑛 1 2 2.1 𝑚𝑚 1 𝑚𝐿/𝑚𝑖𝑛 × = 0.21 𝑚𝐿/𝑚𝑖𝑛 4.6 𝑚𝑚

  41. Slide 41: Tips for Method Transfer: Adjusting Injection Volume

    Tips for Method Transfer Adjusting injection volume 𝑉𝑚 = 𝜋 × 𝑟 2 × 𝐿 × ~0.6 𝑉𝑜𝑙𝑢𝑚𝑒𝑐𝑜𝑙𝑢𝑚𝑛 2 𝐼𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛𝑐𝑜𝑙𝑢𝑚𝑛 1 × 𝑉𝑜𝑙𝑢𝑚𝑒𝑐𝑜𝑙𝑢𝑚𝑛 1 = 𝐼𝑛𝑗𝑒𝑐𝑡𝑖𝑜𝑛𝑐𝑜𝑙𝑢𝑚𝑛 2 Original 4.6 x 250 mm: ~2.5 mL Transferred to 2.1 x 100 mm: ~0.21 mL 0.21 𝑚𝐿 30 µ𝐿 × = 2.5…

  42. Slide 42: Agilent LC Column Navigator

    Agilent LC Column Navigator http://navigator.chem.agilent.com/

  43. Slide 43: Contact Agilent Chemistries and Supplies Technical Support

    Contact Agilent Chemistries and Supplies Technical Support 1-800-227-9770 Option 3, Option 3: Option 1 for GC and GC/MS columns and supplies Option 2 for LC and LC/MS columns and supplies Option 3 for Sample preparation, filtration and QuEChERS Option 4 for Spectroscopy supplies Available in the USA

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