Although dissolving sample compounds in the eluent is the basic procedure of HPLC, with some samples this is not possible due to their solubility or the sample preparation method required. When excessive differences in solvent strength or pH between the eluent and the sample solvent arise, this can cause problems such as split peaks and broad peaks. This is considered to be caused by dispersion phenomena or varying degrees of dissociation of the analyte, as a result of the sample solvent being temporarily replaced by the eluent in the column. Examples of samples which gave this type of problem are discussed below.

The chromatogram (A) represents 2µL of an aqueous solution of a sample injected, and a shoulder is observed.

As the pH was different between the sample solution and the eluent, 4µL of this sample was injected after diluting it by a factor of 2 with the eluent to reduce the pH difference. The peak shape was much improved as in chromatogram (B).

Other ways to overcome this type of problem include reduction of the sample volume and dilution of the concentrated solution with the low strength eluent. If peak deterioration is observed, you should always confirm the sample solution has the same characteristics as the eluent. If not, you should always consider the alternatives discussed above.