Although fluorescence spectroscopy is a highly sensitive method, it is still rather difficult to identify a minor fluorescent component whose fluorescent peak is overlapped and masked by a dominant fluorescent component in a sample solution. Herein, we describe a two-dimensional correlation spectroscopy (2D-COS) approach based on the Kasha's rule to solve the above common problem. We initially suppose that a sample solution contains the major component only, and the spectral behavior of the major component obeys the Kasha's rule. Then, the shapes of emission spectra obtained under excitation lights of different wavelengths remain invariant. Under this condition, the introduction of a minor fluorescent component can be reflected by the changes on the shapes of emission peaks in the series of emission spectra. Moreover, subtle changes, which are difficult to be found in the original spectra, can be clearly visualized as cross peaks in 2D asynchronous spectrum constructed using a series of emission spectra. In addition, we demonstrate that the intensities of cross peaks can be enhanced by changing the sequence of the series of emission spectra. We utilize the approach on an aqueous solution containing eosin Y and a trace amount of bromocresol green. The presence of bromocresol green with the concentration as low as 400 nM can be revealed via the cross peaks in the resultant 2D asynchronous spectra. In a preliminary study, we suggest that 2D disrelation spectrum might provide an alternative chance to reveal the presence of small amount bromocresol green.
Keywords: 2D-COS; Eosin Y; Fluorescence; Kasha's rule.
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