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?

 

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)

 

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

 

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.

 

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.