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An Efficient Approach to Column Selection in HPLC Method Development
Introduction
Common Mistakes in Method Development:
- Inadequate Formulation of Method Goals
- Little Knowledge of Chemistry of Analyte Mixture
- Use of the First Reversed Phase C18 Column Available
- Trial and Error with Different Columns and Mobile Phases
These Mistakes Result In:
- Laborious, Time-consuming Development Projects
- Methods that Fail to Meet the Needs of the Analyst
HPLC Method Development – A Proposed Procedure
At Your Desk
- Define your knowledge of the sample
- Define your goals for the separation method
- Choose the columns to be considered
In the Laboratory
- Choose the initial mobile phase chemistry
- Choose the detector type and starting parameters
- Evaluate the potential columns for the sample
- Optimize the separation conditions (isocratic or gradient) for the chosen column
- Validate the method for release to routine laboratories
Choosing the Appropriate HPLC Column Should Be Based Both Upon Knowledge of the Sample and Goals for the Separation
Benefits of this Approach Include:
- Small initial time investment
- Big time savings in the HPLC laboratory
- More “informed” approach to column selection
- More efficient than “trial and error” approach
Knowledge of the Sample Influences the Choice of Column Bonded Phase Characteristics
Knowledge of the Sample
- Structure of sample components?
- Number of compounds present?
- Sample matrix?
- pKa values of sample components?
- Concentration range?
- Molecular weight range?
- Solubility?
- Other pertinent data?
Column Chemistry :(bonded phase, bonding type, endcapping, carbon load)
Goals for the Separation Influence the Choice of Column Particle Physical Characteristics
Goals for the Separation
- Max. resolution of all components?
- Partial resolution?
- Fast analysis?
- Economy (low solvent usage)?
- Column stability and lifetime?
- Preparative method?
- High sensitivity?
- Other goals?
Column Physics :(particle bed dimensions, particle shape, particle size, surface area, pore size)
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