Slide 1: Getting Off to a Good Start
Getting Off to a Good Start Isocratic method development Paul Altiero Applications Chemist, CSD February 13, 2020 1 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in diagnostic procedures. DE322025463
Slide 2: Isocratic Method Design and Development
Isocratic Method Design and Development Column and Scouting Sample Introduction: Resolution: Gradients: Chemistries: What and Equations Mobile Choose Your Why and Impacts Phases and Bonded Phase Mechanics Wisely 2 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in di
Slide 3: Isocratic Method Design and Development
Isocratic Method Design and Development Column and Scouting Sample Introduction: Resolution: Gradients: Chemistries: What and Equations Mobile Choose Your Why and Impacts Phases and Bonded Phase Mechanics Wisely 3 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in di
Slide 4: What is an Isocratic HPLC Method and Why Do I Want to Use One (or not)?
What is an Isocratic HPLC Method and Why Do I Want to Use One (or not)? • An isocratic separation is: (one where) “the composition of the solvent remains constant throughout the separation”1. • Benefits of an Isocratic Method – Simple to adjust – No baseline drift – No Re-equilibration time – Not im
Slide 5: Getting Started
Getting Started • Define the objective – What are the goals of the separation? Resolving multiple, critical pairs or just one component from matrix? – Is speed/throughput important? – What are the requirements around LoQ, accuracy, and precision? – How do we assess system/method performance? • Gathe
Slide 6: Getting Started (Continued)
Getting Started (Continued) • List Known Challenges – examples include: – Analytes with very similar structures/properties – Compatibility of analytes and detector – lack of chromophores, poor ionization – Additives/buffers – solubility of buffers, interference from additives, for example, TFA • Pla
Slide 7: 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, – Ruggedness (RSD 0.1 - 0.25%) – Robustness • Absolute
Slide 8: Isocratic Method Design and Development
Isocratic Method Design and Development Column and Scouting Sample Introduction: Resolution: Gradients: Chemistries: What and Equations Mobile Choose Your Why and Impacts Phases and Bonded Phase Mechanics Wisely 8 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in di
Slide 9: Fundamental Resolution Equation – Isocratic Separations
Fundamental Resolution Equation – Isocratic Separations Rs ¼(N) = 1/2 (a - 1) k a 1+k a = Selectivity – increase by changing bonded phase and mobile phase N = Plates – increase by using longer column or reducing particle size k = Retention – increase by changing bonded phase and mobile phase Does no
Slide 10: Factors that Maximize Isocratic Resolution Between Peaks
Factors that Maximize Isocratic Resolution Between Peaks Increase Decrease %organic retention k • Change the chemistry of the Change relative mobile or stationary phase a peak position • Change %organic Increase • Change column N retention time dimensions or flowrate faster than • Decrease particle
Slide 11: Selectivity Impacts Resolution the Most
Selectivity Impacts Resolution the Most 7.00 Rs = N½/4 • (a-1)/a • k’/(k’+1) 6.00 Increasing N Increasing Alpha 5.00 Increasing k' Resolution 4.00 3.00 2.00 1.00 0.00 Alpha: 5000 10000 15000 20000 25000 Plates: 1.10 1.35 1.60 1.85 2.1 k 2.0 4.5 7.0 9.5 12.0 Selectivity impacts resolution most • Chan
Slide 12: The Van Deemter Equation for Band Broadening
The Van Deemter Equation for Band Broadening 𝐵 𝐻 = 𝐴 + + 𝐶𝑢 𝑢 Where A, B, and C represent a different set of constants for a particular solute, column, and set of experimental conditions1. The A term is eddy diffusion and is independent of linear velocity. The B term is the longitudinal…
Slide 13: Optimal Flow Rates for Agilent InfinityLab Poroshell Columns
Optimal Flow Rates for Agilent InfinityLab Poroshell 120 Columns Smaller particles have higher optimal flow rates • Operating below the column’s 105.0 Maximum efficiency for optimal flow rate negatively each column impacts efficiency (and 95.0 resolution) 85.0 • Operating above the optimal flow rate
Slide 14: Improve Performance by Reducing LC System Volume
Improve Performance by Reducing LC System Volume Agilent 1290 Infinity LC System: Agilent 1290 Infinity LC System: Default stacking and capillary tubing with LC System rack and ultra-low configurations Solvent tray dispersion optimizations Solvent tray Needle seat capillary: 0.12 x Needle seat capil
Slide 15: Improve Performance by Reducing LC System Volume
Improve Performance by Reducing LC System Volume Use smaller internal diameter capillaries and a smaller volume detector flow cell Effect of Capillary id + Flow Cell Volume on Efficiency 16000 5% ↑ pressure 34% ↑ N 16% ↑ N 14000 k’=6 k’=3 12000 10000 83% ↑ N k’=1 8000 6000 4000 2000 0 butylparaben n
Slide 16: Sample Considerations – Mobile Phase Diluents and Solubility
Sample Considerations – Mobile Phase Diluents and Solubility Analytes not very soluble in water Increasing sample solvent strength Nnap = 13387 1.5 μL injection of sample diluted 1:10 in water Nnap = 11906 1.5 μL injection of sample diluted 1:10 in mobile phase Nnap = 7857 1.5 μL injection of sample
Slide 17: Sample Injection Volumes Can Affect Peak Shape and Resolution
Sample Injection Volumes Can Affect Peak Shape and Resolution 16 µL injection • Injection volumes contribute to overall system volume 8 µL injection • Keep injection volumes to a minimum, while retaining solubility 0.5 µL injection mAU 250 N=8394 amitriptyline butylparaben N=13641 naphthalene 200 15
Slide 18: Isocratic Method Design and Development
Isocratic Method Design and Development Column and Scouting Sample Introduction: Resolution: Gradients: Chemistries: What and Equations Mobile Choose Your Why and Impacts Phases and Bonded Phase Mechanics Wisely 18 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in d
Slide 19: Column Choices – Which Particle Type to Choose?
Column Choices – Which Particle Type to Choose? 11.59 mAU 100 Totally porous particle 12.67 16.43 16.15 80 29.29 ZORBAX Eclipse Plus C18 15.24 11.11 4.6 x 250 mm, 5 µm 9.71 20.68 60 23.07 40 Run time: 35 min 20 0 5 10 15 20 25 30 min 7.03 mAU Superficially porous particle 2.31 250 200 • Pressure: 32
Slide 20: Poroshell Technology – What Makes it Better?
Poroshell Technology – What Makes it Better? Totally porous particle (TPP) Superficially porous particle (SPP) • Analytes travel though the particle more efficiently, improving peak shape and resulting in faster run times. Poroshell is made of a solid core • High efficiency allows you to use a large
Slide 21: Column Choice: Evaluate Different Bonded Phases
Column Choice: Evaluate Different Bonded Phases • Bonded phase affects selectivity (alpha) • Different interactions for polar and nonpolar compounds • Exploit other interactions with bonded phase • Changing the bonded phase can improve selectivity/resolution, and reduce analysis time. • Having diffe
Slide 22: 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 InfinityLab Poroshell 120 Phenyl Hexyl 3 4 mAU 7 150 8 100 1 2 56 50
Slide 23: Importance of Alternate Selectivity Chemistries
Importance of Alternate Selectivity Chemistries • Three 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 C, 254 nm 23 February 13. 2020 Getting Off to a Good Start For R
Slide 24: Polar Embedded Phase for Alternate Selectivity
Polar Embedded Phase for Alternate Selectivity Beta blockers mAU InfinityLab Poroshell 120 EC-C18 1. Atenolol 2 2. Pindolol 300 6 3. Naldolol 200 5 4. Metoprolol 1 5. Acebutolol 100 3 4 7 6. Propranolol 7. Alprenolol 0 -100 0 2 4 6 8 10 12 min mAU 2 InfinityLab Poroshell 120 Bonus RP 300 6 5 200 1 P
Slide 25: Agilent InfinityLab Poroshell 120 Portfolio
Agilent InfinityLab Poroshell 120 Portfolio Best for low pH Best for High pH Best for Best for Polar Best All Round Mobile Mobile Alternative Best for Chiral Analytes Phases Phases Selectivity InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshell InfinityLab Poroshe
Slide 26: Isocratic Method Design and Development
Isocratic Method Design and Development Column and Scouting Sample Introduction: Resolution: Gradients: Chemistries: What and Equations Mobile Choose Your Why and Impacts Phases and Bonded Phase Mechanics Wisely 26 February 13. 2020 Getting Off to a Good Start For Research Use Only. Not for use in d
Slide 27: Starting Point Scouting Gradient
Starting Point Scouting Gradient • A good starting point for work is a scouting gradient. • The conditions recommended by John Dolan are 5–95% acetonitrile, low pH, and are dependent on the column length. • Where 10 cm columns are chosen, use a 10 minute gradient. • This example shows a 150 mm colum
Slide 28: Estimating Isocratic Conditions from Our Scouting Gradient Results
Estimating Isocratic Conditions from Our Scouting Gradient Results For a scouting gradient on a 4.6 x 100 mm column, from the previous table. Where the run time is 10 minutes and the gradient goes from 5% to 95%. The starting percentage of organic can be estimated as: %𝐵 = 9.5 𝑡𝑎𝑣𝑔 −…
Slide 29: Exploring Organic Modifiers
Exploring Organic Modifiers Why? • It’s easy – ACN and MeOH are readily available • Works on any bonded phase – optimize separation no matter the column choice MeOH – Higher pressure, generally better peak shape with bases, protic solvent Acetonitrile – Aprotic, wider UV window, stronger than MeOH “
Slide 30: 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 a mobile phase pH that will help optimize retention and selectivity. • Noncharged analytes have better retention – For example, acids at low pH and bases at mid
Slide 31: 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 3 250 200 1 5 6 10 mM HCO2NH4 8 150 1. Procainamide 100 3 4 2. Caffeine 50 0 3. Acetyl Salicylic Acid 0 0.5 1 1.5 2 2.5 3 3.5 min 4. Hexanophenone Deg. mAU 2 7 pH 4.8 5. Dipyrimadole
Slide 32: Simplify Solvent Handling
Simplify Solvent Handling • Keep solvent concentrations constant and solvent vapors out of the lab. • Venting valve for mobile phase, time strip • Safer handling, easier to grab InfinityLab • Prevents tubing from twisting solvent bottle and Stay Safe • Can be used on any system cap 32 February 13. 2
Slide 33: Tips for Robust Methods
Tips for Robust Methods • Always start method development with a new column • Select columns with robust properties at the pH of the method • Choose a high-quality column with a long lifetime • Consider batch-to-batch reproducibility • Consider scalability of particle sizes and chemistries for downs
Slide 34: Ensure Proper Column Connections
Ensure Proper Column Connections Bad Ferrule cannot seat properly Poor fitting connections • Will broaden or split peaks, or cause tailing • Will typically affect all peaks, but especially early eluting peaks Too long • Can cause of carryover Bad Mixing Chamber mAU 140 120 100 One bad connection 80
Slide 35: InfinityLab Quick Connect and Quick Turn Fittings
InfinityLab Quick Connect and Quick Turn Fittings • Spring loaded design • Easy, no tools needed • Works for all column types • Reusable • Consistent ZDV connection Quick Connect fitting • Finger tight up to 1300 bar • Hand tighten the nut, then depress the lever Quick Turn fitting • Finger tight up
Slide 36: 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 37: Conclusions
Conclusions • Resolution is a common method development goal ‒ Selectivity is a main driver of resolution • Column choices – Superficially porous particles speed up analysis – Explore alternate selectivity to increase resolution • Method conditions – Consider the selectivity effects of mobile phases
Slide 38: 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