Agilent Technologies, Inc. · NYSE:A
Agilent Technologies — Technical Presentation (March 2020)
36 slides from Agilent Technologies's technical presentation, Mar 12, 2020
Slide 1: Gradient Design and Development
Gradient Design and Development Paul Altiero Applications Chemist, Agilent 12 March 2020 1 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 2: Gradient Design and Development
Gradient Design and Development Development: System Transferring Introduction: Scouting Configuration: Gradients to What and Gradient to Measure Different Why Analytical Delay Volume Columns Method 2 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 3: Gradient Design and Development
Gradient Design and Development Development: System Transferring Introduction: Scouting Configuration: Gradients to What and Gradient to Measure Different Why Analytical Delay Volume Columns Method 3 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 4: What is Gradient Elution?
What is Gradient Elution? A separation that occurs by continuously increasing the solvent strength of the mobile phase mAU 120 1. Hydroquinone 2. Resourcinol 100 3. Catechol 4. Phenol 80 5. 4-Nitrophenol 6. p-cresol 60 7. o-cresol 8. 2-Nitrophenol 40 9. 3,4 di methyl phenol 10. 2,3 di methyl phenol
Slide 5: Gradient Elution for Reversed-Phase HPLC
Gradient Elution for Reversed-Phase HPLC Increasing the solvent strength = Increasing the % organic in the mobile phase Linear solvent strength gradient = % per min is a constant 90% 90 70% = 80% %ACN 50% t G = 40 min. 30% 10 t G = 2%/min. 0 10 20 30 40 min. } } } } t 1 = t 2 = t 3 = t 4 F
Slide 6: Gradient Elution Analysis of a Complex Sample
Gradient Elution Analysis of a Complex Sample EU banned Azo colorants in textiles Column: Poroshell 120 EC-C18, Sample: 1. 1,4-phenylendiamine 14. 2,4-dimethylaniline 3.0 x 150 mm, 2.7 μm (p/n: 693975-302) 2. 4-methoxy-m-phenylenediamine 15. o-dianisidine Column temperature: 40 ℃ 3. 4-methyl-m-pheny
Slide 7: Three Major Reasons to Choose Gradient Elution
Three Major Reasons to Choose Gradient Elution 1. Faster separation of samples having components that vary in polarity. 2. To separate mixtures having a large number of components 3. Consistent, better peak shape throughout the run Page 7 For Research Use Only. Not for use in diagnostic procedures.
Slide 8: Gradient Separation Reduces Analysis Time and Improves Resolution
Gradient Separation Reduces Analysis Time and Improves Resolution – Even with the Shortest Columns Columns: Poroshell 120 EC-C18, 3.0 x 30 mm, 2.7 µm Mobile Phase: A: 10 mM ammonium acetate, pH= 6.8, B: Acetonitrile. Temperature: 30 °C Mobile Phase: A: 20 mM Phosphate buffer, B: Acetonitrile. Temper
Slide 9: Gradient Separation is Faster than Isocratic
Gradient Separation is Faster than Isocratic Separation of acetaminophen impurities on Poroshell 120 EC-C18 Isocratic elution: Gradient elution: 85% aqueous/15% ACN 5–50% ACN in 5 min Column: Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm Mobile Phase: A: 10 mM ammonium acetate, pH 6.8 B: Acetonitrile 2
Slide 10: Gradient Design and Development
Gradient Design and Development Development: System Transferring Introduction: Scouting Configuration: Gradients to What and Gradient to Measure Different Why Analytical Delay Volume Columns Method 10 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 11: Gradient Separations: Instrument Impact on Column Performance
Gradient Separations Instrument Impact on Column Performance . Dwell volume Extracolumn volume . Number of scans or points Data sampling (or acquisition) rate 11 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 12: Instrument Impact on Column Performance
Instrument Impact on Column Performance Dwell volume . • Dwell volume = volume from Dwell formation of gradient to the column volume . Number of scans or points 12 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 13: Determining the Dwell Volume of Your System
Determining the Dwell Volume of Your System • Look it up in the LC manual or follow the procedure below • Replace column with short piece of HPLC stainless steel tubing • Prepare mobile phase components A. Water - UV-transparent B. Water with 0.2% acetone - UV-absorbing • Monitor at 265 nm • Run gra
Slide 14: Measuring Dwell Volume (VD)
Measuring Dwell Volume (VD) Best straight line fit through linear trace Extension of original baseline 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. 14 For Research Use Only. Not fo
Slide 15: Efficiency Yield vs. Dispersion in LC Systems
Efficiency Yield vs. Dispersion in LC Systems 100 mm, 1.8 µm columns, k*=4.8 30000 N estimated, 5 sigma 25000 20000 15000 4.6 mm 10000 3.0 mm 5000 2.1 mm 0 1.0 mm 0 10 20 30 40 50 60 Dispersion, 5 sigma Use the largest columns suitable for the application requirements -- they are less affected by ex
Slide 16: Gradient Design and Development
Gradient Design and Development Development: System Transferring Introduction: Scouting Configuration: Gradients to What and Gradient to Measure Different Why Analytical Delay Volume Columns Method 16 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 17: 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 column. Colum
Slide 18: Gradients are Critical Tools for Faster Methods
Gradients are Critical Tools for Faster Methods • Run a scouting method 5% to 95% organic (reversed phase) • Quick evaluation: how much of the gradient is occupied 𝛥𝑡𝐺 – ≤ 25% isocratic is recommended 𝑡𝐺 𝛥𝑡𝐺 – ≥ 40% gradient is recommended 𝑡𝐺 mAU 150 100 Ideal for gradient method 50 0 min…
Slide 19: Step 1: Choose Shorter Efficient Column
Step 1: Choose, Shorter Efficient Column Perform Gradient Scouting from 5%B-100% in 15 min Sample: 1. Aldicarb sulfoxide, Column: InfinityLab Poroshell 120 EC-C18, 2. Oxamyl, 3. Methomyl, 4. Aldicarb 4.6 x 100 mm, 2.7 m sulfone, 5. Carbofuran-3-hydroxy, 6. Aldicarb, 7. Propoxur, Gradient time: 15 m
Slide 20: Step 2: Reduce Gradient Range to Minimize Time
Step 2: Reduce Gradient Range to Minimize Time Adjust gradient from 5%B-80% in 10 min Sample: 1. Aldicarb sulfoxide, 2. Oxamyl, Column: InfinityLab Poroshell 120 EC-C18, 3. Methomyl, 4. Aldicarb sulfone, 4.6 x 100 mm, 2.7 µm 5. Carbofuran-3-hydroxy, 6. Aldicarb, 7. Propoxur, 8. Carbofuran, 9. Carbar
Slide 21: Step 3: Finalize Your Results
Step 3: Finalize Your Results Increase starting % organic and reduce time Column: InfinityLab Poroshell 120 EC-C18, Sample: 1. Aldicarb sulfoxide, 2. Oxamyl, 4.6 x 100 mm, 2.7 µm 3. Methomyl, 4. Aldicarb sulfone, Gradient: 15 – 80% 5. Carbofuran-3-hydroxy, 6. Aldicarb, 7. Propoxur, 8. Carbofuran, 9.
Slide 22: Gradient Scouting Works for Any Sample
Gradient Scouting Works for Any Sample Gradient from 5%–100% in 10 min for acetaminophen Column: Poroshell 120 EC-C18, 4.6 x 50 mm 2.7 µm Mobile phase: A: 10 mM ammonium acetate, pH 6.8 B: acetonitrile Flow rate: 1.5 mL/min Temperature: 30°C Sample: 1.4-aminophenol 2.Acetaminophen 3.4-propionamidoph
Slide 23: Optimizing Gradient from 5%–50% to Reduce Wasted Time
Optimizing Gradient from 5%–50% in 5 min to Reduce Wasted Time Column: Poroshell 120 EC-C18, 4.6 x 50 mm, 2.7 µm Mobile phase: A: 10 mM ammonium acetate, pH 6.8 B: Acetonitrile Flow rate: 1.5 mL/min Temperature: 30°C Sample: 1.4-aminophenol 2.Acetaminophen 3.4-propionamidophenol 4.2-acetamidophenol
Slide 24: Final Optimization to Reduce Time
Final Optimization to Reduce Time Gradient from 5%–50% in 3 min Column: Poroshell 120 EC-C18, 4.6x50mm, 2.7µm Mobile phase: A: 10 mM ammonium acetate, pH 6.8 B: Acetonitrile Flow rate: 2.0 mL/min Temperature: 30 °C Sample: 1.4-aminophenol 2.Acetaminophen 3.4-propionamidophenol 4.2-acetamidophenol 5.
Slide 25: Gradient Design and Development
Gradient Design and Development Development: System Transferring Introduction: Scouting Configuration: Gradients to What and Gradient to Measure Different Why Analytical Delay Volume Columns Method 25 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
Slide 26: Resolution
Resolution Efficiency Selectivity Retention Factors that Affect Isocratic Resolution 7.00 Increasing N 6.00 Increasing Alpha 5.00 Increasing k' Resolution 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 resolutio
Slide 27: Resolution Relationship for Gradient Elution
Resolution Relationship for Gradient Elution R VN α∗ k* 4 k* - represents the fact that k changes constantly during a gradient %B = difference between initial and final % B values 87 tg F S = constant (≈ 4 for 100 - 500 Da) k* = F = flow rate (mL/min.) tg = gradient time (min.) S (%B) Vm Vm = col
Slide 28: Maintaining k*
Maintaining k* To keep relative peak position unchanged while changing analysis parameters Any decrease in: Can be offset by a proportional: • Decrease in tG or F • Column length • Increase in %B • Decrease in tG or F • Column volume (i.d.) • Increase in %B • %B (same column) • Decrease in tG or
Slide 29: A Shorter Column (smaller Vm)
A Shorter Column (smaller Vm) Reduce run-time and improve resolution while maintaining constant N 4.6 x 150 mm, 5 m 4.6 x 75 mm, 2.7 m N = 12,000 10 N = 16,000 10 9 9 Gradient: 15 – 80%B in 5 min Mobile Phase: A:H2O ZORBAX Eclipse Plus-C18 Poroshell 120 EC-C18 B: Acetonitrile 4.6 x 150 mm, 5 µm 4.
Slide 30: Changing Gradient Time to Affect Retention (k*) and Resolution
100% B Changing Gradient Time to Affect Retention (k*) and Resolution tg= 5 0% B tg F 100% B k* = S %B Vm tg= 10 1/k* = gradient steepness = b 0% B 100% B = change in volume fraction of B solvent S = constant F = flow rate (mL/min.) tg= 20 tg = gradient time (min.) Vm = column void volume (mL) 0
Slide 31: Adapting Gradient Methods to Different Column Dimensions
Adapting Gradient Methods to Different Column Dimensions To adjust gradient methods to different column dimensions keep gradient steepness (b) the same. gradient S • • Vm 1/k* Steepness =b= tG • F S = constant F = flow rate = change in % organic tG = gradient time during the gradient run k*
Slide 32: Adjusting a Gradient from a 4.6 x 150 mm Column to a 2.1 x 100 mm Column
Adjusting a Gradient from a 4.6 x 150 mm Column to a 2.1 x 100 mm Column 4.6 x 150 mm 2.1 x 100 mm ΔФ = 40 (20% - 60%) ΔФ = 40 (20% - 60%) Vm = 1.5 mL Vm = 0.2 mL F = 1.0 mL/min F = 0.2 mL/min tG = 15 min tG = ? (10 min) 𝛥𝛷1 ·𝑉𝑚1 𝛥𝛷2 ·𝑉𝑚2 𝛥𝛷2 𝑉𝑚2 𝐹1 𝑏= = 𝑡𝐺2 = 𝑡𝐺1 · · · 𝐹1 ·𝑡𝐺1…
Slide 33: Adjusting a Gradient for Constant %B
Adjusting a Gradient from a 4.6 x 150 mm Column to a 2.1 x 100 mm Column for constant %B 2 %A %B 𝑡𝑜𝑙𝑑 𝑡𝑛𝑒𝑤 𝐹1 𝑑2 𝐿2 𝛥𝛷2 𝑡𝐺2 = 𝑡𝐺1 · · · 95 5 0 0 𝐹2 𝑑1 𝐿1 𝛥𝛷1 95 5 1 0.7 for constant %B 35 65 13 9.0 2 𝐹1 𝑑2 𝐿2 35 65 14 9.7 𝑡𝐺2 = 𝑡𝐺1 · · 𝐹2 𝑑1 𝐿1 0 100 14.5 10.0 4.6 x…
Slide 34: Adjusting a Gradient for Constant %B
Adjusting a Gradient from a 4.6 x 150 mm Column to a 2.1 x 100 mm Column for Constant %B 2 %A %B 𝑡𝑜𝑙𝑑 𝑡𝑛𝑒𝑤 𝐹1 𝑑2 𝐿2 𝛥𝛷2 𝑡𝐺2 = 𝑡𝐺1 · · · 95 5 0 0 𝐹2 𝑑1 𝐿1 𝛥𝛷1 95 5 1 0.7 for constant %B 35 65 13 9.0 2 𝐹1 𝑑2 𝐿2 35 65 14 9.7 𝑡𝐺2 = 𝑡𝐺1 · · 𝐹2 𝑑1 𝐿1 0 100 14.5 10.0 4.6 x…
Slide 35: 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 Available in the USA & Canada
Slide 36: For Research Use Only. Not for use in diagnostic procedures. DE5919560185
36 For Research Use Only. Not for use in diagnostic procedures. DE5919560185
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