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Agilent Technologies — Technical Presentation (March 2019)
23 slides from Agilent Technologies's technical presentation, March 2019
Slide 1: Finding the Perfect Match: Practical Advice on Column and Mobile Phase Selection
Finding the Perfect Match: Practical Advice on Column and Mobile Phase Selection Paul Altiero Applications Chemist, Agilent 1 For Research Use Only. Not for use in diagnostic procedures.
Slide 2: When a method is not rugged and robust
When a method is not rugged and robust • Method fails unexpectedly, halting production • “Method creep” • Risk of redeveloping method after validation • Compromise quality Studies estimate that only around 40% of published findings can be replicated reliably.1 For Research Use Only. Not for use in d
Slide 3: Examples of Common Separation Goals and Method Performance Criteria
Examples of Common Separation Goals and Method Performance Criteria Good System Suitability Parameters Method Performance Criteria • Resolution: 2 • Accuracy • Peak shape: USP Tf close to 1 (<2) • Precision • Injection Repeatability: areas, Tf, etc. (RSD • Ruggedness 0.1 - 0.25%) • Robustness • Abs
Slide 4: What Makes a Good Starting Point for RP Method Development?
What Makes a Good Starting Point for RP Method Development? 1. Smaller particles and superficially porous particles offer fast, efficient analysis 2. C18 column – most general purpose column choice 3. Simple mobile phase a) Formic acid or other additive in aqueous portion (buffer salts only if neces
Slide 5: The Future − Higher Efficiencies using SPP
The Future − Higher Efficiencies using SPP InfinityLab Poroshell 120 Additional efficiency can be generated through the use of superficially porous particles (SPP) rather than a totally porous particle (TPP) SPP particle For Maximum pressure Typical pressure Efficiency Similar to sub-2 µm totally ~1
Slide 6: Poroshell Technology – What makes it better?
Poroshell Technology – What makes it better? Totally porous particle (TPP) Superficially porous • Analytes travel though the particle more efficiently: improving peak shape and resulting in faster run-times Poroshell is made of a solid • High efficiency allows you to use a larger SPP (ie. 2.7um) for
Slide 7: Agilent InfinityLab Poroshell 120 Portfolio
Agilent InfinityLab Poroshell 120 Portfolio Best for low pH Best for high Best for Best for polar Best all around mobile pH mobile alternative Best for Chiral Analytes phases phases selectivity InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Porosh
Slide 8: Factors that Affect Resolution
Factors that Affect Resolution Resolution Efficiency Selectivity Retention 7.00 6.00 Increasing N Increasing Alpha 5.00 Resolution Increasing k' 4.00 3.00 2.00 1.00 0.00 Plates: 5000 10000 15000 20000 25000 Alpha: 1.10 1.35 1.60 1.85 2.1 k 2.0 4.5 7.0 9.5 12.0 Selectivity impacts resolution the most
Slide 9: Selectivity Differences Across InfinityLab Poroshell Bonded Phases
Selectivity Differences Across InfinityLab Poroshell Bonded Phases mAU 3 4 7 150 InfinityLab Poroshell 120 EC-C18 8 1 100 2,5 6 50 0 0 2 4 6 8 10 12 mAU 3 4 7 150 InfinityLab Poroshell 120 SB-C18 8 100 1 2,5,6 50 0 0 2 4 6 8 10 12 mAU InfinityLab Poroshell 120 Phenyl Hexyl 3 4 7 150 8 100 1 2 5 50 0
Slide 10: Importance of Alternate Selectivity Chemistries
Importance of Alternate Selectivity Chemistries • 3 compounds ˗ Same molecular weight ˗ Only differ by positional location of the functionality InfinityLab Poroshell 120 columns 4.6 x 50 mm, 2.7 µm 70:30 – MeOH/H2O, 1.5 mL/min, 40o, 254 nm 10 For Research Use Only. Not for use in diagnostic procedur
Slide 11: Selectivity Can be Controlled by Changing pH
Selectivity Can be Controlled by Changing pH Agilent InfinityLab Poroshell HPH-C18 4.6 x 50 mm, 2.7 µm mAU 2 300 7 pH 3 1. Procainamide 250 2. Caffeine 200 1 5 6 10 mM HCO2NH4 8 3. Acetyl Salicylic Acid 150 100 3 4 4. Hexanophenone Deg. 50 0 5. Dipyrimadole 0 0.5 1 1.5 2 2.5 3 3.5 min 6. Diltiazem 7
Slide 12: pH – A Method Development Tool for Ionizable Compounds
pH – A Method Development Tool for Ionizable Compounds • Ionizable compounds will be in a charged or uncharged state based on pH • Choose mobile phase pH to optimize retention and selectivity • Non-charged analytes have better retention – i.e. acids at low pH and bases at mid or high pH • Silanols o
Slide 13: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion • Dipole-Dipole • Hydrogen Bonding • Ionic (Coulombic) Interactions • Charge Transfer (π – π interactions) 13 For Research Use Only. Not for use in diagnostic procedures.
Slide 14: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion – due to the instantaneous positions of electrons around the solute and solvent. The strength of dispersion increases with polarizability. But, the critical point is that dispersive effects are non-specific, significa
Slide 15: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion • Dipole-Dipole – Large, permanent dipole moments will drive molecules to align for maximum electrostatic interaction. As an example, acetonitrile with the functional group -C≡N would be expected to have a dipole-dipo
Slide 16: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion • Dipole-Dipole • Hydrogen Bonding – these interactions occur either when a proton donor (acidic) solvent interacts with a proton acceptor (basic) solute or when a basic solvent interacts with an acidic solute. Exampl
Slide 17: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion • Dipole-Dipole • Hydrogen Bonding • Ionic (Coulombic) Interactions – occurs with charged (ionizable) compounds and solvents with large dielectric constants. Can also occur between charged analytes and charged bonded
Slide 18: Solvent Selection
Solvent Selection Hydrophobicity and more Solvent/Solute Interactions • Dispersion • Dipole-Dipole • Hydrogen Bonding • Ionic (Coulombic) Interactions • Charge Transfer (π – π interactions) – aromatic or saturated compounds may be either π-electron rich (a π-base) or π-electron poor (a π-acid). Phen
Slide 19: Gradients
Gradients Solvent Selection and Rate of Change R VN a* k* 4 k* - represents the fact that k changes constantly during a gradient D%B = difference between initial and final % B values tg F S = constant k* = F = flow rate (mL/min.) tg = gradient time (min.) S (D%B) Vm Vm = column void volume (mL) 19
Slide 20: To Increase Gradient Resolution by Changing Retention (k*) Use
To Increase Gradient Resolution by Changing Retention (k*) Use: tG • A longer gradient time F • A higher flow rate Vm • A shorter column %B • A shorter organic range tg F k* = S (D%B) Vm 20 For Research Use Only. Not for use in diagnostic procedures.
Slide 21: Solvent Selection: HPLC Nomograph
Solvent Selection HPLC Nomograph 21 For Research Use Only. Not for use in diagnostic procedures.
Slide 22: Resources for Support
Resources for Support • Agilent University http://www.agilent.com/crosslab/university • Tech support http://www.agilent.com/chem/techsupport • Resource page http://www.agilent.com/chem/agilentresources – Quick Reference Guides – Catalogs, Column User guides – Online Selection Tools, How-to Videos •
Slide 23: 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/GC/MS columns and supplies Option 2 for LC/LC/MS columns and supplies Option 3 for sample preparation, filtration and QuEChERS Option 4 for spectroscopy supplies Available in the USA & Canad
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