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![[Figure 5]](vi0157fig5mag.jpg)
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Figure 5
(a) Fluorescein emission spectra recorded in various conditions (×10 means scaled up by a factor of 10). Several observations can be made: (i) the fluorescence intensity decreases when the pH is lowered; (ii) the peak wavelength is dependent on pH, but also on temperature and crystalline environment; (iii) the shoulder corresponding to the contribution of the mono-anionic form of fluorescein can be clearly identified at cryo-temperatures, but not at room temperature. (b) Relative contribution of the shoulder peak measured as a function of pH at 100 K. pH values are reported as measured at room temperature. Values correspond to the mean of four (respectively three) independent measurements for films of Tmpk mother liquor (respectively for Tmpk crystals). The length of error bars corresponds to twice the r.m.s. deviation from these measurements. Buffers were sodium citrate (pH < 6.0), MES (pH 6.0) and HEPES (pH 7.5). The contribution from the shoulder peak was calculated by subtracting the experimental spectra (with background subtracted and peak surface normalized to 1) to a Gaussian curve initially obtained by fitting the part of a pH 7.5 emission spectrum corresponding to the di-anionic contribution. At each pH, the Gaussian curve was shifted to match the experimental spectrum at the peak wavelength. The obtained difference spectrum was then summed in a fixed wavelength range covering the shoulder peak. At the used concentration of fluorescein, residual inner filtering effects were small. However, these effects, if present, would vary with pH and would imply a slight degradation in the `contrast' of the titration curve. |
![[Figure 5]](vi0157fig5.jpg)
 | JOURNAL OF APPLIED CRYSTALLOGRAPHY |
ISSN: 1600-5767
