Fluorescein 5-isothiocyanate → 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)benzoic acid

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Gentamicin (1mM) and fluorescein isothiocyanate (FITC) (1.25mM) were allowed to react in 0.05M sodium carbonate/bicarbonate buffer, pH 9.0, for 2 hours at room temperature. 2ml of the reaction mixture was applied to a column (1 × 97cm) of G-15 grade Sephadex and eluted with carbonate/bicarbonate buffer as above, with a flow rate of 1.8ml/h. Column fractions (1.8ml) were assayed for gentamicin content by radio-immunoassay and for fluorescein content by fluorimetry. FIG. 1 of the accompanying drawings shows the result. Unreacted gentamicin left the column first. The FTC-G product exited next, well separated from the gentamicin peak. Unreacted FITC was very strongly held on the G-15 column and was not eluted.

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