Luminescence wavelength standards

Table II. Luminescence Wavelength Standards--Organics In Solution and Inorganic Ions In Glass Matrices...

Requirements for standards used In macro- and microspectrofluorometry differ, depending on whether they are used for Instrument calibration, standardization, or assessment of method accuracy. Specific examples are given of standards for quantum yield, number of quanta, and decay time, and for calibration of Instrument parameters. Including wavelength, spectral responslvlty (determining correction factors for luminescence spectra), stability, and linearity. Differences In requirements for macro- and micro-standards are considered, and specific materials used for each are compared. Pure compounds and matrix-matched standards are listed for standardization and assessment of method accuracy, and existing Standard Reference Materials are discussed. 

Definition and Uses of Standards. In the context of this paper, the term "standard" denotes a well-characterized material for which a physical parameter or concentration of chemical constituent has been determined with a known precision and accuracy. These standards can be used to check or determine (a) instrumental parameters such as wavelength accuracy, detection-system spectral responsivity, and stability (b) the instrument response to specific fluorescent species and (c) the accuracy of measurements made by specific Instruments or measurement procedures (assess whether the analytical measurement process is in statistical control and whether it exhibits bias). Once the luminescence instrumentation has been calibrated, it can be used to measure the luminescence characteristics of chemical systems, including corrected excitation and emission spectra, quantum yields, decay times, emission anisotropies, energy transfer, and, with appropriate standards, the concentrations of chemical constituents in complex S2unples. 

Requirements of Standards. The general requirements for luminescence standards have been discussed extensively (3,7-9) and include stability, purity, no overlap between excitation and emission spectra, no oxygen quenching, and a high, constant qtiantum yield independent of excitation wavelength. Specific system parameters--such as the broad or narrow excitation and emission spectra, isotropic or anisotropic emission, solubility in a specific solvent, stability (standard relative to sample), and concentration--almost require the standard to be in the same chemical and physical environment as the sample. 

In order to obtain true emission and excitation spectra it is uaially necessary to apply conections for variations in excitation intentity and the wavelength sensitivity of the detection system. The correction needed may be calculated by comparing the instmment response for a standard compound of known corrected ectral characteristics with that of the sample under study, although q)ectrofluorimeters have been described which fully electronically compensate for intensity and wavelength response of the system Comparison of the area under the corrected emission spectrum with that of various standard fluorescence compounds allows the quantum yield of the luminescence process to be calculated ... 

Luminescent standards have been established for use in calibrating fluorescence spectrometers and have been suggested for Raman spectroscopy in the past (18). The standard is a luminescent material, usually a solid or liquid, that emits a broad reproducible luminescence spectrum when excited by a laser. Once the standard is calibrated for a particular laser wavelength, its emission spectrum is known, and it can provide the real standard output , d)i(AF) depicted in Figure 10.8. In practice, a spectrum of the standard is acquired with the same conditions as an unknown then the unknown spectrum is corrected for instrument response function using the known standard... 

Figure 10.11. Observed and corrected emission curves for two luminescent standards. Raw curves were recorded for coumarin 540a soiution excited by 514.5 nm light, and for Kopp 2412 glass excited by 785 nm light. Raman shift is stated relative to the appropriate laser wavelength. Corrected output was calculated by comparison to a standard tungsten source. All curves are normalized to their maximum output. See Reference 20 for details. Spectrum A was determined on a Dilor X-Y spectrometer, B was acquired with a Chromex 2000.

The application of NIR luminescent materials depends on the availabiUty of robust, cost-effective excitation sources and light detectors for such materials. For NIR luminescent lanthanide complexes, it is especially the relatively limited choice of accessible detectors that has slowed their investigation and further development. Most standard spectrofluorimetric and microscopic equipment are equipped with detectors that are mainly sensible in the visible. An extension of the spectroscopic sensitivity of this equipment farther into the red usually does not go to wavelengths longer than 850 nm (the limit of typical multi-alkali photocathodes). We will briefly discuss some recent developments in excitation sources and detectors that are applicable in particular to NIR luminescent lanthanide complexes. 

Broadband ultraviolet excitation light source causes easily difficulties with the choice of materials. UV is heavily absorbed by most of the optical materials. This naturally lowers transmission of the optical components, and also very easily causes unwanted luminescence emission. Suitable materials are often more expensive and harder to machine than the standard optical glasses. Mostly, the other demands for the optics are not very strict, and a single lens can replace the objective lens. This can be manufactured out of fused silica at relatively low cost. Fused silica is highly transparent at UV wavelengths above 300 nm and is usually quite pure with only minute traces of background creating luminescent materials. 

One of the unique uses of an array-detector spec-Irofluorometer is the production of total luminescence spectra. Such a spectrum is a plot of the emission spectrum at every excitation wavelength usually presented as a three-dimensional plot. Total luminescence spectra can be obtained in a conventional manner with a standard photomultiplier transducer, although collection of the spectra involved are quite time-consuming. Here, the emission spectrum is obtained at one excitation wavelength. Then, the exeitation monochromator is moved to another wavelength and the emission spectrum scanned again. A. computer stores the various spectra and presents the total three-dimensional luminescence display. 

Quantum yields of luminescence (Reisfeld, 1972) were determined in standard references, varying the excitation wavelength for emission of samarium(III) and emission of europium(UI) in phosphate glasses, and for 7/2 emission of gadolinium(III) and emission of terbium(ni) in borate glasses. 

A = absorbance c = concentration d = path length / = frequency 1,5 = absorption dissymmetry ratio glujjj = luminescence dissymmetry ratio I = light intensity J = total angular quantum number t = time = extinction coefficient A = wavelength a = standard deviation. 

The simplest method for the measurement of the luminescence quantum yield of a luminophore is based on the comparison with a standard species with known quantum yield. The luminescence quantum yield of such reference compounds are practically independent on the excitation wavelength, hence they can be utilized for the whole spectral range of their absorption. 

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