Some Common-Sense Corollaries

The goal of most HPLC analysis is to separate analyte(s) from other components in the sample for accurate quantitation. Several corollaries are often overlooked by practitioners  

Sample must be soluble If it s not in solution, it cannot be analyzed by HPLC. Solubility issues often complicate assays of low-solubility analytes or component difficult to extract from sample matrices. Low recoveries often stem from poor sample preparation steps rather than the HPLC analysis itself. 

For separation to occur, analytes must be retained and have differential migration in the column Separation cannot occur without retention and sufficient interaction with the stationary phase. For quantitative analy- 

The mobile phase controls the separation Whereas the stationary phase provides a media for analyte interaction, the mobile phase controls the overall separation. In HPLC method development, efforts focus on finding a set of mobile phase conditions for separating the analyte (s) from other components. Exceptions to this rule are size exclusion, chiral, and affinity chromatography. 

All C18-bonded phase columns are not created equal and are not interchangeable There are hundreds of Cl 8 columns on the market. They vary tremendously in their retention and silanol characteristics.9 

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