title: Relative and absolute determination of fluorescence quantum yields of transparent samples.
abstract: Luminescence techniques are among the most widely used detection methods in the life and material sciences. At the core of these methods is an ever-increasing variety of fluorescent reporters (i.e., simple dyes, fluorescent labels, probes, sensors and switches) from different fluorophore classes ranging from small organic dyes and metal ion complexes, quantum dots and upconversion nanocrystals to differently sized fluorophore-doped or fluorophore-labeled polymeric particles. A key parameter for fluorophore comparison is the fluorescence quantum yield (Φf), which is the direct measure for the efficiency of the conversion of absorbed light into emitted light. In this protocol, we describe procedures for relative and absolute determinations of Φf values of fluorophores in transparent solution using optical methods, and we address typical sources of uncertainty and fluorophore class-specific challenges. For relative determinations of Φf, the sample is analyzed using a conventional fluorescence spectrometer. For absolute determinations of Φf, a calibrated stand-alone integrating sphere setup is used. To reduce standard-related uncertainties for relative measurements, we introduce a series of eight candidate quantum yield standards for the wavelength region of ∼350-950 nm, which we have assessed with commercial and custom-designed instrumentation. With these protocols and standards, uncertainties of 5-10% can be achieved within 2 h.
authors.affiliation: Bundesanstalt für Materialforschung und-prüfung BAM, Berlin, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Würth
authors.forename: Markus
authors.initials: M
authors.lastname: Grabolle
authors.forename: Jutta
authors.initials: J
authors.lastname: Pauli
authors.forename: Monika
authors.initials: M
authors.lastname: Spieles
authors.forename: Ute
authors.initials: U
authors.lastname: Resch-Genger
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D002855:Chromatography, Thin Layer; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: D005456:Fluorescent Dyes