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[HPLC] How to Develop HPLC Methods ( 5 step )

 
Step 1
What are your goals? Define your requirements:
• What is the purpose of the method?
• What is the analyte and are there more than one?
• What is sample matrix for analysis?
• Is quantification required?
• What level of effort is appropriate and what resources are available?
• What are the desired method criteria, e.g. minimum resolution, maximum runtime, accuracy & precision?
 
Step 2
What do you already know? Assess the structure of the analyte(s):
• Molecular weight & polarity - Suitability for RP-HPLC conditions. 
• pKa Ka  - Is a buffer required  and if so at what pH?
• Solubility - Solvents for  sample preparation?
• Chromophores - UV detection possible? Assess interferences in the sample matrix. Assess previous work performed on the analyte(s)
 
Step 3
What sample(s) will you use to develop the method? Identify the required materials for developing the method. Source the required materials:
• Can they be purchased?
• Can analyte free sample matrix be obtained?
• Are stress studies required to generate degradation products? Prepare test sample(s) to be used for method development
 
Step 4
What conditions will you use for the method? From a standard set of initial conditions modify as necessary based on previous steps, e.g. the pH of the buffer. Run a full range gradient and from this determine whether isocratic or gradient analysis is preferable. Calculate: Isocratic - %B. Gradient -starting %B. For complex separations - screen different conditions and choose the most promising.
 
Step 5
What method parameters will you use? Optimise the conditions from step 4 to achieve the desired separation. Adjust conditions to achieve acceptable resolution for the critical pair:
• %B or gradient range & steepness.
• Temperature.
• Change organic solvent.
• Mix organic solvents.
• Adjust buffer pH. Define all method parameters.
 
 
 
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