Bias in the absorption coefficient determination of a fluorescent dye, standard reference material 1932 fluorescein solution

The absorption coefficient of standard reference material® (SRM®) 1932, fluorescein in a borate buffer solution (pH=9.5) has been determined at λ=488.0, 490.0, 490.5 and 491.0 nm using the US national reference UV/visible spectrophotometer. The purity of the fluorescein was determined to be 97.6% as part of the certification of SRM 1932. The solution measured was prepared gravimetrically by diluting SRM 1932 with additional borate buffer. The value of the absorption coefficient was corrected for bias due to fluorescence that reaches the detector and for dye purity. Bias due to fluorescence was found to be on the order of −1% for both monochromatic and polychromatic (e.g., diode-array based) spectrophotometers.