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Agilent Technologies — Technical Presentation (November 2018)

37 slides from Agilent Technologies's technical presentation, Nov 27, 2018

1. Slide 1: Why They Matter: An Introduction to Chromatography Equations

   Why They Matter An Introduction to Chromatography Equations Paul Altiero Applications Chemist, Columns & Supplies 27 November 2018 1 11/26/2018 An Introduction to Chromatography Equations Agilent Restricted

2. Slide 2: Objectives of Talk

   Objectives of Talk • Chromatography is a physical process • Much can be described with simple equations • Understanding the process simplifies Method development, Troubleshooting, Predicting behavior, etc. Page 11/26/2018 An Introduction to Chromatography Equations Agilent Restricted 2

3. Slide 3: Topics

   Topics • Chromatographic Process • Isocratic Resolution • Particles and Pressure • Van Deemter Equation • Gradient Resolution • Method Conversions Page Demystifying Chromatographic Process Agilent Restricted 3

4. Slide 4: Chromatographic Process

   Chromatographic Process • Partition between mobile phase and stationary phase (K = Cs/Cm) • Description of the separation: Rs – Resolution N – Column Efficiency, Plates k, k’– Retention Factor, Capacity Factor α – Selectivity tret – Retention time Page Demystifying Chromatographic Process Agilent Re

5. Slide 5: Definition of Resolution

   Definition of Resolution tR-2 - tR-1 ∆ tR Rs = = (w2 + w1)/2 w Resolution is a measure of the ability to separate two components Page Demystifying Chromatographic Process Agilent Restricted 5

6. Slide 6: Resolution

   Resolution … Determined by 3 Key Parameters – Efficiency, Selectivity and Retention The Fundamental Resolution Equation ∆tR = w N = Column Efficiency – Column length and particle size a = Selectivity – Mobile phase and stationary phase k = Retention Factor – Mobile phase strength Page Demystifying C

7. Slide 7: Parameters Affecting Resolution (Rs)

   Parameters Affecting Resolution (Rs) • Retention Factor (k): describes how well an analyte is retained by the stationary phase, expressed as a ratio of column volumes, can be adjusted by making changes to the organic strength of the mobile phase • Selectivity or Separation Factor (α) • Column Effici

8. Slide 8: Parameters Affecting Resolution (Rs)

   Parameters Affecting Resolution (Rs) • Retention Factor (k) •Selectivity or Separation Factor (α): This is the ratio of retention factors for two adjacent peaks. Larger α values indicate better separation. Selectivity can be adjusted by changes to either the mobile phase or the stationary phase. • C

9. Slide 9: Parameters Affecting Resolution (Rs)

   Parameters Affecting Resolution (Rs) • Retention Factor (k) •Selectivity or Separation Factor (α) • Column Efficiency as Theoretical Plates (N): As the number of plates increase, peaks become thinner and sharper, which improves resolution. Plates are often described by their height (H),or Height Equ

10. Slide 10: Chromatographic Profile

    Chromatographic Profile Equations Describing Factors Controlling RS Retention Factor (tR-t0) k= t0 Selectivity α = k2/k1 Theoretical Plates-Efficiency N = 16(tR / tW-base)2 N = 5.56(tR / tW-1/2)2 Page Demystifying Chromatographic Process Agilent Restricted 10

11. Slide 11: Resolution as a Function of Selectivity, Column Efficiency, or Retention

    Resolution as a Function of Selectivity, Column Efficiency, or Retention 7.00 Selectivity Affects Resolution Most 6.00 • Change bonded phase • Change mobile phase α 5.00 Increase N Resolution 4.00 Increase Alpha 3.00 Increase k' 2.00 N 1.00 k 0.00 1 2 3 Rs = N½/4 • (a-1)/a • k’/(k’+1) 4 5 6 7 8 9 10

12. Slide 12: Different Mobile Phases May Give Different Selectivity

    Different Mobile Phases May Give Different Selectivity mAU 700 O 600 50/50 MeOH/HOH Me 500 ... Anisole 400 Phenethanol ... 300 200 p-F-Phenethanol …..... 100 Toluene ... 0 0 2 4 6 8 10 12 14 16 18 min mAU 700 600 41/59 ACN/HOH 500 OH 400 300 200 100 0 0 2 4 6 8 10 12 14 16 18 min ZORBAX® SB-C18 4.6

13. Slide 13: Selectivity Differences Across InfinityLab Poroshell Bonded Phases

    Selectivity Differences Across InfinityLab Poroshell Bonded Phases mAU 3 4 7 150 InfinityLab Poroshell 120 EC-C18 8 100 1 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 50 2 5 0

14. Slide 14: Column Efficiency (N)

    Column Efficiency (N) N - Number of theoretical plates. We can increase N by increasing the length of the column or decreasing the size of the stationary phase particles. (1.8 µm > 2.7 µm > 3.5 µm > 5 µm > 10 µm) tR 2 N = 16 w = f(L, 1/dp) L = column length dp = particle size Page Demystifying Chrom

15. Slide 15: Column Efficiency (N)

    Column Efficiency (N) N - Number of theoretical plates. We can increase N by increasing the length of the column or decreasing the size of the stationary phase particles. (1.8 µm > 2.7 µm > 3.5 µm > 5 µm > 10 µm) p L = column length dp = particle size Page Demystifying Chromatographic Process Agilen

16. Slide 16: What About Pressure?

    What About Pressure? Pressure Increases with Decreasing Particle Size Equation For Pressure Drop Across an HPLC Column h L v DP = ✓ Many parameters influence q dp2 column pressure ✓Particle size and column DP = Pressure Drop length are most critical h ✓Long length and smaller = Fluid Viscosity parti

17. Slide 17: Columns Packed with Smaller Particles Provide Higher Efficiency

    Columns Packed with Smaller Particles Provide Higher Efficiency sub 2 micron 3 micron Large Small Particle Particle N N α 1/(dp) 5 micron P α 1/(dp)2 10 micron Velocity Page Demystifying Chromatographic Process Agilent Restricted 17

18. Slide 18: Van Deemter Curve

    Van Deemter Curve Factors Affecting N Plate Height H (L/N) H = L/N Large Small Particle Particle H = A + B/u + C u Resistance to Mass Transfer Linear Velocity u The smaller the plate height, the higher the plate number and the greater the chromatographic resolution Page Demystifying Chromatographic

19. Slide 19: Van Deemter Equation, Expanded

    Van Deemter Equation, Expanded Plate Height H (L/N) Where: 2𝑢 2 • H is plate height 2𝛾𝐷𝑚 𝜔𝑑𝑝 𝑅𝑑𝑓 𝑢 H = 2λ𝑑𝑝 + 𝑢 + ( 𝐷 + 𝐷 ) • λ is particle shape with 𝑚 𝑠 regard to the packing • 𝑑𝑝 is particle diameter • 𝛾, ω, and R are constants • 𝐷𝑚 is the diffusion coefficient of the…

20. Slide 20: Putting it Together: The van Deemter Equation

    Putting it Together The van Deemter Equation H = L/N Large Small Plate Height H Particle Particle H = A + B/u + C u Sum Curve: van-Deemter Resistance to Mass Transfer H min Longitudinal diffusion Eddy Diffusion u opt Linear Velocity u The smaller the plate height, the higher the plate number and the

21. Slide 21: Van Deemter Curve: Effect of Particle Size

    Van Deemter Curve Effect of Particle Size H = A + B/u + Cu Column: ZORBAX Eclipse XDB-C18 0.0030 Dimensions: 4.6 x 50 mm Eluent: 85:15 ACN:Water 0.0025 Flow Rates: 0.05 – 5.0 mL/min Temp: 20°C Sample: 1.0L Octanophenone in Eluent 5.0 m HETP (cm) 0.0020 5.0m 0.0015 3.5m 1.8m 3.5 m 0.0010 0.0005

22. Slide 22: 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% D = 80% %ACN 50% t G = 40 min. 30% D 10 t G = 2%/min. 0 10 20 30 40 min. } } } } Dt 1 = Dt 2 = Dt 3 = Dt 4 F

23. Slide 23: Resolution Relationship for Gradient Elution

    Resolution Relationship for Gradient Elution 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) Page

24. Slide 24: 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 Page 24 Demystifying Chromatographic Process Agilent Restricted

25. Slide 25: Changing Gradient Time to Affect Retention (k*) and Resolution

    100% B Changing Gradient Time to Affect tg= 5 Retention (k*) and Resolution 0% B tg F 100% B k* = S D%B Vm tg= 10 1/k* = gradient steepness = b 0% B 100% B DF = 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

26. Slide 26: Maintaining k*

    Maintaining k* - To Keep Relative Peak Position in a Chromatogram Unchanged and Shorten Analysis Any Decrease in Can be Offset by a Proportional • Gradient time • Decrease in D%B or Vm • Increase in F • Decrease in tG or F • Column volume (i.d.) • Increase in D%B • Decrease in tG or F • D%B (same co

27. Slide 27: Gradient Transfer Considerations

    Gradient Transfer Considerations •Keeping 𝑘 ∗ constant, substituting for the volume of the column and cancelling out constants we can establish the equality: 𝑭𝒏𝒆𝒘 𝑭𝒐𝒍𝒅 • 𝒕𝒏𝒆𝒘 ( 𝟐 )= 𝒕𝒐𝒍𝒅 ( 𝟐 ) 𝑳𝒏𝒆𝒘 𝒅𝒏𝒆𝒘 𝑳𝒐𝒍𝒅 𝒅𝒐𝒍𝒅 Page Demystifying Chromatographic Process Agilent…

28. Slide 28: Gradient Transfer Considerations

    Gradient Transfer Considerations •Rearranging to solve for the new time we get 𝑭𝒐𝒍𝒅 𝑳𝒏𝒆𝒘 𝒅𝒏𝒆𝒘 𝟐 •𝒕𝒏𝒆𝒘 = 𝒕𝒐𝒍𝒅 ∙ ∙ ∙ 𝑭𝒏𝒆𝒘 𝑳𝒐𝒍𝒅 𝒅𝒐𝒍𝒅 Page Demystifying Chromatographic Process Agilent Restricted 28

29. Slide 29: Maintaining k*

    Maintaining k* To Keep Relative Peak Position in a Chromatogram Unchanged and Shorten Analysis Any Decrease in Can be Offset by a Proportional • Gradient time • Decrease in D%B or Vm • Increase in F • Decrease in tG or F • Column volume (i.d.) • Increase in D%B • Decrease in tG or F • D%B (same colu

30. Slide 30: Gradient Transfer Considerations

    Gradient Transfer Considerations •Keeping 𝑘 ∗ constant, substituting for the volume of the column and cancelling out constants we can establish the equality: 𝑭𝒏𝒆𝒘 𝑭𝒐𝒍𝒅 • 𝒕𝒏𝒆𝒘 ( 𝟐 )= 𝒕𝒐𝒍𝒅 ( 𝟐 ) 𝑳𝒏𝒆𝒘 𝒅𝒏𝒆𝒘 𝑳𝒐𝒍𝒅 𝒅𝒐𝒍𝒅 Page Demystifying Chromatographic Process Agilent…

31. Slide 31: Gradient Transfer Considerations

    Gradient Transfer Considerations •Rearranging to solve for the new flowrate we get 𝒕𝒐𝒍𝒅 𝑳𝒏𝒆𝒘 𝒅𝒏𝒆𝒘 𝟐 •𝑭𝒏𝒆𝒘 = 𝑭𝒐𝒍𝒅 ∙ ∙ ∙ 𝒕𝒏𝒆𝒘 𝑳𝒐𝒍𝒅 𝒅𝒐𝒍𝒅 Page Demystifying Chromatographic Process Agilent Restricted 31

32. Slide 32: Gradient Transfer Considerations

    Gradient Transfer Considerations •Rearranging to solve for the new time we get 𝒅𝒏𝒆𝒘 𝟐 •𝑭𝒏𝒆𝒘 = 𝑭𝒐𝒍𝒅 ∙ 𝒅𝒐𝒍𝒅 Page Demystifying Chromatographic Process Agilent Restricted 32

33. Slide 33: Column Volume

    Column Volume •When developing methods, we regularly recommend flushing the column at a high %B for at least two column volumes. We also talk about re- equilibrating our columns for 3-6 column volumes at the end of our gradients. •But how do you calculate column volume? •Column volume can be calcula

34. Slide 34: Column Volume from Test Chromatogram

    Column Volume from Test Chromatogram Demystifying Chromatographic Process Agilent Restricted Slide 34

35. Slide 35: Column Volume by Geometry

    Column Volume by Geometry •Starting with the equation for the volume of a cylinder •𝑉𝑐𝑦𝑙 = 𝜋𝑟 2 d •Then adjust to allow for the space taken up by the particles and call it column volume •𝑉𝑐𝑜𝑙 = 𝜋𝑟 2 d ∙ 0.6 35 November 26, 2018 Demystifying Chromatographic Process Agilent Restricted

36. Slide 36: Summary

    Summary • Chromatographic Process • Isocratic Resolution • Particles and Pressure • Van Deemter Equation • Gradient Resolution • Method Conversions Page Demystifying Chromatographic Process Agilent Restricted 36

37. Slide 37: 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

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