HPLC方法开发中如何有效选择色谱柱.ppt

An Efficient Approach to Column Selection in HPLC Method Development Craig S. Young and Raymond J. Weigand Alltech Associates, Inc. 2051 Waukegan Road ? Deerfield, IL 60015 Phone: 1-800-ALLTECH ? Web Site: roduction 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 ponents? ? Number pounds present? ? Sample matrix? ? pK a values of ponents? ? 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 ponents? ? Partial resolution? ? Fast analysis? ? Economy (low solvent usage