Traceability in fluorometry: Part II. Spectral fluorescence standards.

The need for the traceable characterization of fluorescence instruments is emphasized from a chemist's point of view, focusing on spectral fluorescence standards for the determination of the wavelength- and polarization-dependent relative spectral responsivity and relative spectral irradiance of fluorescence measuring systems, respectively. In a first step, major sources of error of fluorescence measurements and instrument calibration are revealed to underline the importance of this issue and to illustrate advantages and disadvantages of physical and chemical transfer standards for generation of spectral correction curves. Secondly, examples for sets of traceable chemical emission and excitation standards are shown that cover a broad spectral region and simple procedures for the determination of corrected emission spectra with acceptable uncertainties are presented. With proper consideration of the respective measurement principle and geometry, these dye-based characterization procedures can be not only applied to spectrofluorometers but also to other types of fluorescence measuring systems and even to Raman spectrometers.

Capillary isoelectric focusing with UV-induced fluorescence detection.

Low-molecular-mass fluorescent compounds excitable in the near UV region with suitable acidobasic and electrophoretic properties are suggested as isoelectric point (pI) markers for isoelectric focusing (IEF) with UV photometric and UV excited fluorometric detection. The experimental set-up of capillary IEF with UV excited fluorometric detection and properties of new UV-induced fluorescent pI markers are given. The pI values of 18 new pI markers determined independently of IEF methods range from 2.1 to 10.3. The examples of separation of new pI markers together with derivatized proteins by capillary IEF with photometric or fluorometric detection are presented.