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A reliable method for the separation of fluorescein dyes from their impurities was developed using high performance liquid chromatography and involved a μBondapak C18 reverse phase column and mixtures of methanol and ammonium acetate buffer. This technique was used to verify the purity of commercial products as well as to aid in the development of an empirical theory related to retention of halogenated fluorescein dyes by reverse phase columns.
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