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Virtual On-Site: Advanced LC Method Development

Who should take this course?

If you've ever wished that HPLC method development were more logical and methodical, this is the course for you. Attendees should have at least one year of HPLC experience in the laboratory and at least some involvement in developing new methods or troubleshooting older ones. This course is for chromatographers who want to develop better methods faster and less expensively.

What does it cover?

The course begins with a thorough review of basic chromatography parameters and their relationship as the basis for a rational approach to extract as much useful information as possible from a minimum investment in experimental time and effort. It answers questions such as:

  • what is the best packing particle size and column geometry based on user needs?

  • how do I transfer legacy HPLC methods to UHPLC?

  • how do I make UHPLC methods backward compatible with older HPLC systems?

  • can I do the separation isocratically or will I need a gradient?

  • how do I decide what type of column to use?

  • what's the best solvent?

  • how important is pH?

  • how do I optimize a gradient profile?

  • when to I need to use ion-pair chromatography?

  • should I use HILIC or normal-phase instead of reversed-phase?

  • how do I ensure my method is robust?

  • how much can I allow my users to adjust method parameters without having to revalidate

  • and much, much more!

When is the course available?

 "The course is presented for groups of 5 - 30 people using Citrix GoToMeeting service. Virtual On-Site is six 2.5-hour sessions (typically 2 or 3 sessions per week). The usual lead time to schedule a course is approximately 4-6 weeks. 

Call us at (925) 297-5374 or email to to arrange timing, pricing, and content.

What topics are covered?

The course content can be tailored to your specific needs. A typical schedule looks like this:

Section 1. Getting Started
  • Overview: UHPLC vs. HPLC
  • Setting analytical goals
  • Method Development strategies: OFAT vs. QbD
  • Selecting a detector
  • Estimating LOD/LOQ
Section 2. Review of HPLC & UHPLC Basics
  • Basic measurements: k', alpha, N, Rs, TF
  • Overall method development strategy: k', alpha, N (in that order!)
Section 3. Columns
  • Evolution of HPLC/UHPLC
  • Parameters affecting N
  • Particle size effects
  • Impact of particle size on hardware
  • Totally porous vs. solid-core particles
  • Impact of silica purity
  • Bonded-phase column chemistry
Section 4. Strategy: reversed-phase of neutral molecules
  • Mechanism of reversed phase and its impact on selectivity
  • Initial column and mobile phase selection
  • Controlling k'
  • Controlling alpha: mobile phase strength
  • Controlling alpha: temperature
  • Controlling alpha: mobile phase type
  • Controlling alpha: column type
  • Developing orthogonal methods
Section 5. Strategy: reversed-phase of ionizable molecules (acids or bases)
  • Effect of pH on retention & selectivity
  • Choice of buffer: identity & concentration
  • Dealing with tailing problems
Section 6. Strategy: Ion-pair & mixed-mode chromatography
  • Mechanism of ion pair
  • Choice of ion-pair reagent type and concentration
  • Problems with ion-pair
  • Alternatives to ion-pair: mixed mode columns
Section 7. Gradient separations
  • Why/when to use gradients
  • Similarities between gradient and isocratic
  • Controlling gradients: steepness
  • Controlling gradients: range
  • Controlling gradients: shape
  • Differences between gradient and isocratic: the linear solvent strength model
  • Selecting initial gradient conditions
  • Dwell volume issues
  • Baseline drift & noise issues
Section 8. Quality by Design
  • What is QbD
  • Combining selectivity parameters
  • Semi-automated method development
Section 9: Strategy: Normal-phase & HILIC
  • Normal-phase mechanism
  • Normal-phase vs. reversed-phase selectivity
  • Polar bonded-phase columns
  • Normal phase solvent selection
  • Alternatives to normal phase
  • What is HILIC and what is it good for
  • Initial column and mobile phase selection
  • Controlling k' (mobile phase water content)
  • Controlling alpha: mobile phase strength
  • Controlling alpha: mobile phase type
  • Controlling alpha: pH
  • Controlling alpha: column type
  • "Gotchas" (differences) between HILIC and reversed-phase
Section 10: Chiral / Ion-Exchange / Prep Scale-up
  • Overview of chiral separations
    • Protein columns
    • Cavity-type columns
    • Pirkle-type columns
    • Macrocyclic antibiotic columns
  • Overview of ion-exchange chromatography
    • Ion exchange capacity
    • Ionic strength
    • pH
    • Buffer type
    • Column type
    • Organic modifiers
    • Overall strategy
  • Scale-up
    • The touching-bands model

Section 11: UHPLC-HPLC method transfer

  • Equipment considerations: pressure, extra-column volume, detector sensitivity
  • Gradient equipment issues: dwell volume, mixing volume
  • Scaling parameters
  • Method adjustment vs. modification
  • "Gotchas": stationary phase chemistry, temperature control, selectivity/pressure effects, "good column hygiene"
Section 12: Quality Issues
  • Validation overview
  • Method limits
  • System suitability
  • Precision / imprecision
  • Method adjustment vs. modification
  • Documentation
Section 13. LC-MS
  • Overview of quadrupole MS operation
  • LC-MS compatibility issues
  • Strategy for adapting LC-UV methods to LC-MS
  • Using internal standards
Section 14. Sample Preparation
  • Overview of sample prep techniques
  • "Dilute 'n shoot"
  • "Crash 'n shoot"
  • Liquid-liquid extraction
  • Solid-phase extraction
  • Selecting an SPE cartridge
  • Controlling the extraction process
  • Direct-injection / column switching
Section 15. Biopolymer Separations
  • Denaturing vs. non-denaturing methods
  • Reversed-phase strategies
  • Similarities and differences with small-molecule strategy
  • Ion exchange strategies
  • Hydrophobic interaction strategies
  • Gel filtration strategies

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