A typical Excitation Emission Matrix (EEM)
A measurement becoming more widely used in the field of fluorescence spectroscopy is the excitation emission matrix, or EEM. An EEM is a 3D scan, resulting in a contour plot of excitation wavelength vs. emission wavelength vs. fluorescence intensity. EEMs are used for a variety of applications where multi-component analysis is required and are often referred to as providing a molecular fingerprint for many different types of samples.
Some of the first published uses of EEM spectroscopy were in the 1980’s where the technique was used to study tryptophan fluorescence in low density lipoproteins in human blood serum (Koller, 1986) and to investigate fluorescent components in human plasma from tumor patients (Leiner, 1986)
Traditional scanning fluorometers do not allow for the full power of EEM molecular identification due to three fundamental limitations. Firstly, traditional scanning spectrofluorometers cannot inherently compensate for varying sample concentrations of fluorescing molecules. The inner filter effect (IFE) is a well-known phenomenon that distorts the measured fluorescence spectrum of a molecule due to absorption that occurs at higher concentrations of sample (typically above 0.1 to 0.2 absorbance units). The only way to correct for IFE with a traditional fluorometer is to acquire a secondary measurement on a different absorbance spectrometer, and adjust the measured fluorescence signal accordingly. But this too is very tricky as the measurement does not occur simultaneously and with the same exact volumes. Therefor traditional scanning fluorometers lack true concentration independence, severely limiting the ability of a traditional EEM to accurately identify many samples.
Secondly, since fluorometers by definition measure fluorescence, they are missing important absorbance and color information for all non-fluorescent molecules which can be a critical part of a comprehensive multi-component molecular identification.
A recent development, simultaneous absorbance, transmission, and fluorescence EEMs acquisition (A-TEEM), is available on two HORIBA spectrofluorometers and solves many of the problems outlined above while correcting for the inner filter effect on the fly. Since the measurements are taken simultaneously, concentration variations do not exist.
Lastly, single channel scanning instruments are very slow, requiring many minutes to an hour to collect an entire data set. Therefore scanning fluorometers are limited to how much EEM data they can collect in a day and also to only working with samples that do not change during the EEM acquisition time.
The accuracy of EEM fluorescence, without IFE correction, is limited to samples with concentrations only at absorbance values less than about 0.1-0.2. To access the true power of fluorescence EEMs one needs to employ multivariate software methods such as Principal Components Analysis (PCA), Classical Least Squared (CLS) method and Parallel Factor Analysis (PARAFAC).
The inner filter effect
The inner filter effect is comprised of two processes the Primary Filter Effect (PIF) where the excitation light intensity is gradually diminished due to absorption as a function of the optical path length of the liquid sample before reaching the fluorescent volume and the Secondary Filter Effect (SIF), where the emitted fluorescence intensity is diminished due to reabsorption even by the portion of the sample that is not excited directly by the excitation beam.
Spectral consequence of the inner filter effect
EEMs are more and more used for quality analysis of water, specifically for the study of chromophoric dissolved organic matter, also called CDOM. Dissolved organic matter includes amino acids, humic acids, fulvic acids, and other examples of decayed matter in natural water sources, or disinfection byproducts of water treatment processes.
EEMs are used to identify the presence of each at very low concentrations, typically in the ppb range. Instrumentation for this application ideally measures both fluorescence spectra and absorbance simultaneously.
Because fluorescence is linear with concentration only at absorbance values less than about 0.1-0.2, higher absorbance samples must use corrections to the fluorescence intensity for inner-filter effects by measurement and application of the UV-visible absorbance spectrum.
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