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Agilent Technologies — Technical Presentation (December 2018)
40 slides from Agilent Technologies's technical presentation, Dec 11, 2018
Slide 1: Conquer Method Variability
Conquer Method Variability Evaluate Variables During Method Development Rita Steed LC Columns Application Engineer December 11, 2018 1 December 11, 2018 Conquer Method Variability Agilent Restricted
Slide 2: Common Separation Goals and Method Performance Criteria
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 • Ruggedness • Inj. Repeatability: areas, Tf, (RSD 0.1 - 0.25%) • Robustness • Absolute retention factor
Slide 3: Terms and Definitions
Terms and Definitions Robustness Ruggedness “is a measure of [an analytical procedure] to “reproducibility of results when a method is remain unaffected by small, but deliberate performed as written under actual use variations in the method parameters”2 conditions”1 • Prerequisite for a rugged metho
Slide 4: Why Incorporate Ruggedness and Robustness?
Why Incorporate Ruggedness and Robustness? Scenario: You’re trying to reproduce an experiment in a journal article but have been unsuccessful. You contact one of the authors, Marco the Magnificent. You explain your dilemma and ask him to help you understand why it’s not working. His response: “OF CO
Slide 5: It's Science Not Magic: How to Test Ruggedness
It’s Science Not Magic How to Test Ruggedness Ruggedness Assess method performance in two or more different labs—ideally over time Lack of ruggedness is attributable to insufficient documentation, differing practices, reagents, apparatus, and/or instrumentation Ruggedness Example: Column Lot 1. Test
Slide 6: Lot-to-Lot Reproducibility Improves Method Ruggedness
Lot-to-Lot Reproducibility Improves Method Ruggedness 1 pH 3 Retention, k´ Selectivity, a RSD Lot 1 Lot 2 Lot 3 % RSD Lot 1 Lot 2 Lot 3 % 2 3 1. Cefotaxime 1.51 1.48 1.42 3.1 - - - - 4 2. Cefoxitin 4.08 4.02 3.88 2.6 2.70 2.72 2.73 0.56 3. Cefamandole 8.17 8.04 7.74 2.8 2.00 2.00 1.99 0.29 0 5 10 15
Slide 7: Help Ensure Method Reproducibility: Test at Least 3 Lots
Help Ensure Method Reproducibility Test at Least 3 Lots Poroshell 120 EC-C18 4.6 x 50mm 2.7um, PN 699975-902* Sample: Alkylphenones Mix, PN 5188-6529, 2ul injection Rs=1.99 Agilent 1290, 254,4 80Hz, 25C mAU 65% MeCN:35% H20 w/0.1% Formic Acid 500 B14272 400 300 200 100 0 0 0.5 1 1.5 2 2.5 min mAU Rs
Slide 8: More Reliable Results
Slight Change to Method More Reliable Results Poroshell 120 EC-C18 4.6 x 50mm 2.7um, PN 699975-902* mAU Rs=2.56 Sample: Alkylphenones Mix, PN 5188-6529, 2ul injection 400 Agilent 1290, 254,4 80Hz, 25C 300 B14272 60% MeCN:40% H20 w/0.1% Formic Acid 200 100 0 0.5 1 1.5 2 2.5 3 3.5 min mAU Rs=2.54 400
Slide 9: Determining Robustness
Determining Robustness Systematically vary separation parameters and measure effects on Rs • Incorporate parameter ranges into written method to allow flexibility – Include precautionary statement if needed • Helps minimize or avoid many ruggedness problems - but not all Robustness Example: % Organi
Slide 10: How Much Resolution is Necessary?
How Much Resolution is Necessary? Aim for Rs 2.0 between all analytes Baseline Resolution Rs = 1.5 Insufficient Rs, compromises accuracy, precision, robustness, and ruggedness Initial resolution can decrease due to changes in separation variables Build in robustness so Rs is small when sepa
Slide 11: Experimental Variables That Impact Resolution
Experimental Variables That Impact Resolution Column Sample • Column lot* • Injection volume • Solvent strength Mobile Phase • Buffer pH Instrument • Buffer concentration • Column temperature • Ionic strength • Detector flow cell volume* • % organic modifier Gradient • Dwell volume* • Gradient steep
Slide 12: Experimental Variables That Impact Resolution
Experimental Variables That Impact Resolution Column • Select high-quality column manufacturer • Select column with long lifetime at desired pH • Assess lot-to-lot reproducibility • Compare retention, selectivity, resolution, peak width and symmetry. • Method Validation Kit Mobile Phase Lifetime of
Slide 13: Mobile Phase: Aqueous Component
Mobile Phase: Aqueous Component Experimental Variables That Impact Resolution Column Your opportunity to improve Mobile Phase robustness and ruggedness • Aqueous component – Importance of buffers – Selection considerations – pH – Concentration • Organic component Sample Instrument Gradient separatio
Slide 14: Buffered Mobile Phase - Importance
Buffered Mobile Phase - Importance Control Retention of Ionizable Analytes BUFFERS: Provide effective means for varying and controlling pH Improve retention, peak width, and symmetry (especially for pH 3) Minimize or eliminate column-to-column differences Eliminate differences in water pH Allow ef
Slide 15: Buffer Comparison
Buffer Comparison 0.1 % formic acid pH 2.7 1. SMR 2. TPC Buffer Type 10 mM NH4HCO2 pH 3.0 3. SDD 4. FZD 5. SMMX • Can affect Rs and 6. PYM 10 mM NH4HCO2 pH 3.4 column lifetime 7. OXA 8. SDMX • MS or other detector 9. SQZ 10. DFZ 10 mM NH4HCO2 pH 3.8 10 mM CH3COONH4 pH 4.8 min 10-40 %B/12 min @ 2 mL/
Slide 16: pH and Resolution
pH and Resolution Test for pH Robustness Buffer pH • Select based on desired pH and optimum buffer pH range • Measure pH of buffer solution before mixing with organic modifier • Compare resolution at desired pH ± 0.1–0.2 pH units Remember – Even small variations can have an affect. *DryLab simulatio
Slide 17: Changes in Buffer Concentration
Changes in Buffer Concentration Retention, Peak Width and Peak Shape 10 mM Phosphate 25 mM Phosphate 8637I 8637I Buffer Concentration & Ionic Strength • Start at 20 – 25mM • Avoid overshooting/ readjustment when pHing • Compare Rs at desired concentration ± 5–10mM Column: ZORBAX Eclipse XDB-C8, 4.6
Slide 18: % Organic Modifier
% Organic Modifier Baseline Resolution Rs ≥ 1.5 2(RTB – RTA) Rs = Target ≥ 2 WA + WB ∆ RT WA WB 1. W. Long, Best LC Practices for Efficient LC Operations; Par 3: Making LC method better (Webinar Series), Agilent Technologies, September 19, 2017. 18
Slide 19: Small Changes Can Affect Resolution
Small Changes Can Affect Resolution ZORBAX Rapid Resolution Eclipse XDB-C8, 4.6 x 75 mm, 3.5 µm Mobile Phases: 59% MeOH Rs (5,6) = 2.5 A: 25 mM phosphate, pH 7.00 (10 mM TEA) B: methanol (10 mM TEA) Flow Rate: 1.0 mL/min Temperature: 25°C controlled Injection: 5 L Detection: 275 nm Rs (5,6) = 1.7 S
Slide 20: The Sample
The Sample Experimental Variables That Impact Resolution • Column • Mobile phase • Sample • Injection volume ruggedness – Issue – Vinj is increased to improve (S/N) ratio – Issue – Decrease column size – Solution – Compare resolution, peak shape and repeatability at 20% and 200-500% Vinj • Use minim
Slide 21: 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 22: Test For Injection Volume Robustness
Test For Injection Volume Robustness Column: ZORBAX Rapid Resolution Eclipse XDB-C8 4.6 x 75 mm, 3.5 µm Rs(6,7) = 1.6 10 µL Mobile Phase: 44% 25 mM phosphate, pH 7, 56% methanol Flow Rate: 1.0 mL/min Temperature: 25°C Detection: UV 250 nm Sample: 1. ketoprofen 2. ethyl paraben Rs(6,7) = 2.1 3. hydro
Slide 23: Increasing Sample Solvent Strength
Sample Considerations - Diluents and Solubility Analytes not very soluble in water Increasing Sample Solvent Nnap = 13387 1.5 μL injection of sample diluted 1:10 in water Nnap = 11906 1.5 μL injection of sample Strength diluted 1:10 in mobile phase Nnap = 7857 1.5 μL injection of sample diluted 1:10
Slide 24: Instrument
Instrument Experimental Variables That Impact Resolution • Column • Mobile Phase • Sample • Instrument • Column temperature • Detector flow cell volume • Gradient Separations 24 December 11, 2018 Conquer Method Variability Agilent Restricted
Slide 25: Adequate Temperature Control
Adequate Temperature Control Small Temperature Changes Can Cause Significant Changes in Rs 20°C Temperature • Lab temps can vary >±5°C or more • Column temp changes affect Rs and repeatability 25°C • Useful changing selectivity, retention & efficiency • Important parameter to control during MD & val
Slide 26: Temperature Can Optimize Resolution and Selectivity
Temperature Can Optimize Resolution and Selectivity Gradient of Ten Cardiac Drugs on SB-C18 RRHT 50°C Rs=1.29 0.5 1 1.5 2 2.5 3 3.5 4 min 4.5 60°C Rs=2.37 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min 70°C Rs=3.27 0.5 1 1.5 2 2.5 3 3.5 4.5 4 min CI0126C December 11, 2018 Conquer Method Variability Agilent Restric
Slide 27: Gradient Separations
Gradient Separations Experimental Variables That Impact Resolution • Column • Mobile Phase • Sample • Instrument • Gradient Separations • Dwell volume • Gradient steepness 27 December 11, 2018 Conquer Method Variability Agilent Restricted
Slide 28: Effect of Dwell Volume on Ruggedness
Gradient Separations Effect of Dwell Volume on Ruggedness Dwell volume = Volume from formation of gradient to column; Behaves as isocratic hold at gradient beginning • Measure instrument dwell volume • Assess effect of dwell volume on Rs during MD – To simulate larger VD, use initial isocratic hold
Slide 29: Instrument Dwell Volume Differences Can Cause Changes in Retention and Resolution
Instrument Dwell Volume Differences Can Cause Changes in Retention and Resolution 1100 Quaternary 400 bar 1290 Infinity II 1300 bar 1290 Infinity II 1300 bar with ISET 2.1x100 mm Zorbax Eclipse Plus, 1.8 µm column; Flow = 0.8 mL/min CI0126C December 11, 2018 Conquer Method Variability Agilent Restri
Slide 30: Gradient Steepness and Gradient Shape
Gradient Steepness and Gradient Shape • Gradient steepness – Change can affect resolution – Small changes likely due to instrument performance differences – Compensate for any dwell volume differences first – Compare resolution at desired gradient time and at tg ±10–20% • Gradient shape – Linear gra
Slide 31: Gradient Steepness Affects Retention (k*) and Resolution
% Gradient Steepness Affects Retention (k*) WJ and Resolution % tg F % k* = S Vm WJ 1/k* = gradient steepness = b % ΔΦ = change in volume fraction of B solvent % S = constant F = flow rate (mL/min.) WJ tg = gradient time (min.) Vm = column void volume (mL) % % • S 4–5 for small molecules WJ • 10 <
Slide 32: Summary
Summary When a method is not rugged and/or robust • Method can fail unexpectedly, halting production • “Method creep” • Risk of redeveloping method after validation • Compromise quality HPLC separation robustness & ruggedness review • Many variables to consider; some more apparent than others • Care
Slide 33: 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/GCMS Columns and Supplies Option 2 for LC/LCMS Columns and Supplies Option 3 for Sample Preparation, Filtration and QuEChERS Option 4 for Spectroscopy Supplies gc-column-support@Agilent.com
Slide 34: 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 35: Appendix
Appendix CI0126C December 11, 2018 Conquer Method Variability Agilent Restricted 35
Slide 36: Determining the Dwell Volume of Your System
Determining the Dwell Volume of Your System Replace column with short piece of HPLC stainless steel tubing Prepare mobile phase components A. Water -V-transparent B. Water with 0.2% acetone - UV-absorbing Monitor at 265 nm Adjust attenuation so that both 100% A and 100% B are on scale Run gradient p
Slide 37: Measuring Dwell Volume (VD)
Measuring Dwell Volume (VD) VD = tD x F 0 10 20 Time (min) tD • Intersection of the two lines identifies dwell time (tD) • Dwell volume is equal to product of the flow rate and the dwell time. CI0126C Conquer Method Variability Agilent Restricted 37
Slide 38: Buffer Preparation
Separation Ruggedness Buffer Preparation 1. Dissolve salt in organic-free water in 1- or 2-L beaker. Use appropriate volume to leave room for pH adjustment solution. Equilibrate solution to room temperature for maximum accuracy. 2. Calibrate pH meter. Use 2-level calibration and bracket desired pH.
Slide 39: Initial Column and System Equilibration
Using Buffers Successfully Initial Column and System Equilibration In an appropriate vessel, test highest % organic/buffer ratio to verify that buffer will not precipitate. With stirring, add organic to buffer first, not vice versa. Equilibrate column with, in order: • 100% organic modifier (if bran
Slide 40: Shutdown State and Instrument Flushing
Using Buffers Successfully Shutdown State and Instrument Flushing Shutdown State Next day use—using same buffers • Pump mobile phase very slowly (for example, 0.01 – 0.1mL/min). When flushing column or for longer term column storage • Flush with 20/80 organic/water, then 80/20 organic/water or 100%
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