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Reverse Phase HPLC. By Julian Chesterman Natalie McKenzie Harry Zhou. Where Is It?. Why Is It There?. Final Purification Intended to Reduce the Level of Insulin-like Components These are Otherwise Difficult to Remove by Conventional Chromatographic Procedures . Why Does It Work?.

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reverse phase hplc

Reverse Phase HPLC

By

Julian Chesterman

Natalie McKenzie

Harry Zhou

why is it there
Why Is It There?
  • Final Purification
  • Intended to Reduce the Level of Insulin-like Components
  • These are Otherwise Difficult to Remove by Conventional Chromatographic Procedures.
why does it work
Why Does It Work?
  • Non-Polar Stationary Phase, More Polar Mobile Phase
  • Highly Polar Particles Elute Rapidly
  • Polarity of Mobile Phase Reduced Gradually Through Addition of Acetonitrile
  • Insulin Elutes Before Insulin Derivatives at pH of 3-4
how does it work
How Does It Work?
  • 4 Steps in Operation
    • Loading
      • Insulin in Water Solution
    • Elution
      • Multiple Column Volumes of Solvent for Separation
      • Gradient vs. Step elution
    • Washing
      • One Column Volume of Low Polarity Solvent
    • Equilibration (aka Reset)
what are the design requirements
What Are the Design Requirements?
  • Superpro Specifies 4 Cycles Per Batch
  • Budgeted to Take 11.1 Hours for Entire Batch
  • Need to Meet Input Requirements of the Next Unit
  • 16 kg/batch of Insulin
how do you determine a size
How Do You Determine a Size?
  • Used a Paper by Eli Lilly Published in Journal of Chromatography (1989) on Scaling Up RP-HPLC for Insulin Production
  • Used Scale Up Data in Paper and Linear Regression to Extrapolate to Size Needed
  • 15 g insulin/L of CV
  • Mass of packing scaled from required CV
  • Elution flow rate changed in proportion to CV
  • Bed height determined from required CV and diameter of available columns
what is the final design
What Is the Final Design?
  • Recommend a Prochrom LC 1600 from Novasep with a 27 cm Bed Height
  • Using Agilent Zorbax C8 Stationary Phase (330 kg)
what are the operating conditions
What are the Operating Conditions?
  • Flow Rate: 720 L/Hr Solvent
  • Gradient Elution: 18 to 28 weight% Acetonitrile in Water over 6 Column Volumes (4.5 Hrs)
  • 0.25 Molar Acetic Acid Buffer
  • Pressure: 50 Bar
  • Total Solvent Required for One Cycle
    • 1450 L Acetonitrile
    • 62 L Acetic Acid
    • 2850 L
does it do anything else
Does It Do Anything Else?
  • Yes!
  • Dynamic Axial Compression
alternatives
Alternatives
  • None, It’s Integral
  • Gel Filtration and Ion Exchange Are Complementary, But Can’t Replicate It’s Effectiveness at Separating Highly Similar Proteins