The True Power of Fluorescence A-TEEM Spectroscopy

The application of the simultaneous absorbance-transmission and fluorescence excitation-emission matrix method (A-TEEM) can be used for absorbance measurements or fluorescence EEM for multicomponent analysis, but its real power is derived from the fact that the collected EEM are corrected for the inner filter effects.

To access the true power of fluorescence A-TEEM spectroscopy, multivariate software methods need to be employed to monitor component concentration and reveal unexpected compounds in a mixture. This novel method is applied in different application domains that span from chemistry to water analysis to the pharmaceutical domain.

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Introducing A-TEEM

The A-TEEM patented method can rapidly adapt to wide concentration ranges and complex mixtures. It deals with a five-dimensional data set, three for the fluorescence EM and two for the absorbance. Furthermore, the CCD base of detection is particularly treatable for real-time analysis and, thanks to the low free light optics, can reach a high chemical sensitivity.

Absorbance provides information on the components that absorb light but do not fluoresce and information needed to correct the fluorescence spectra for the sample concentration-dependent inner filter effect. These measurements and corrections are performed in a matter of seconds.

Fluorescence is a highly sensitive technique. The inner filter effect is a well-known physical phenomenon where the detected fluorescence signal loses linearity at increasing concentrations on the sample due to the absorption and reabsorption of photons in the sample observation area. Consequently, samples should always be diluted to ensure proper fluorescence signals.

Quantum Dots

Quantum loads that are absorbed over a broad range and photoluminescence emissions over a narrow range can be tuned depending on the material from which the quantum dot is made and the size of the quantum dot. The applications of quantum dots include, for example, in vivo imaging light emitting devices, photodetection, or solar energy conversion.

By avoiding the use of the toxic heavy metal lead and cadmium, indium phosphide-based quantum dots are a promising alternative that retains the spectral range of size tenable emission while expanding the scope of applicability of soluble semiconductor emitters.

Fluorescence EEM was first collected on undiluted samples of quantum dots to find the optimal excitation and emission spectral ranges.

The inner-filter effect can significantly affect fluorescence emission spectral profiles, distorting the general shape, shifting the spectral position of the peak maximum and decreasing the emission intensities. Thus, when recorded with a standard spectral flow meter, the detected fluorescence does not coincide with the true fluorescence emission of the sample, even for small fluoro concentrations.

These fluorescence spectra distortions can be critical in display lighting and bioimaging application where precise knowledge of the quantum dots emission properties are required.

Water Quality Control

Drinking water treatment plants that primarily use surface water sources are regulated because certain water components are a precursor to toxic disinfection by-products that may react over time in the distribution system with disinfectants.

These components, namely dissolved organic carbon, are commonly subject to significant variation in an often unpredictable pattern associated with rainfall, snow melt, and other events. A-TEEM meters can be used to monitor the concentration and composition of dissolved organic matter in a typical surface water source.

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EEM provides a wealth of information about dissolved organic matter, which can be very difficult to interpret. To evaluate the quantitative changes in the EEM associated with the treatment, Perfect Analysis is applied to all samples to compose the excitation spectra, emission spectra, and concentration loading for the main fluorescent components.

The data stream platform performs all the necessary spectral correction, quickly assembles EEM into convenient data set objects to easily manage labels, access cases and classes, and include or exclude data from the analysis with a simple click.

Solo from Angen Vector provides the graphical interface to quickly manage and analyze EEM data, create and apply models, and interpret results. The dashboard provides the latest readings, time series, tables, or trend analysis removals percentages.

All these parameters and reports on fit statistics and residual evaluation are useful for system performance monitoring, contamination detection, and early warning alerts.

Most surface water treatment plants remain prone to contamination by petroleum-derived carcinogens as they lack a detector upstream at the intakes.

Early spill warnings require that water-soluble fluorescence or chromophoric components - including benzene, toluene, ethylbenzene and xylene, the so-called BTEX - be discriminated from the fluorescent backgrounds of the natural dissolved organic matter components.

Benzene is believed to be the most toxic BTEX component and has a low fluorescent quantum yield in water. In contrast, typical surface water dissolved organic matter components has higher fluorescence quantum yield than BTEX compounds.

A-TEEM enables a sensitive method for rapid reagent- and extraction-free detection of all BTEX compounds in a typical row of phase water with respective limits of detection and quantification of one and three milligrams per liter.

This A-TEEM and partial square analysis approach serve as an effective screening tool for all contamination in water sources. This method has a rapid data collection time, high sensitivity in parts per billion for oil and PAH compounds, and minimal sample preparation compared to conventional solid phase extraction and liquid or gas chromatographic analytical meters.

Wine Quality

Take a typical Chardonnay – rich, full-bodied, and buttery. Those qualities are not just a happy accident. The combination of various ingredients and control of those ingredients could be the difference between a celebratory toast and a glass of vinegar. The color and final composition of grape juice and wine are key quality characteristics associated with visual perception, taste, and mouth feel.

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Chromatographic analyses are costly, slow, and labor intensive, and reagent basic colorimetric assays are also slow and do not provide compound discrimination. The unique capacity of the multidimensional A-TEEM provides data set results using unsupervised, multivariate component analysis and calibrated list squares and regression meters for precise sample classification and final quantification.

In terms of application, this method can be used to characterize and classify juice and wine samples as a function of ripening and process-related parameters, including adulteration and storage issues such as oxidation and microbial spoilage, especially considering that grapes absorb many chemical compounds in smoke through their skin.

The level of contamination depends on the venue's proximity to the actual fire, the duration of the smoke in the venue, and the ripening stage of the actual grapes and grapevine variety. Moreover, fermentation storage can also have an impact.

Consumption of smoke-tainted wine typically involves quantifying key marker compounds like phenol, OM and P Cresol. Many smoke tank compounds exhibit high fluorescence quantum mills and distinct spectral features.

The Life Science Domain

Monitoring culture media conditions is essential for industries to improve cell proliferation for applications such as regenerative medicine and protein synthesis. Media stimulates growth and prolongs the viability of the cells. Carbohydrates in the form of sugars are the major source of energy.

Most media contain glucose and galactose. However, some contain maltose and fructose. Even subtle variations in composition could have a noticeable impact on the growth rate of the cell culture and its yield. Thus, identifying and analyzing cell cultural media is important.

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Analysis of this complex media is challenging because comprehensive analysis generally requires a combination of chromatographic separations and mass spectrometry. This can make protein analysis expensive and time-consuming.

Fluorescence can also help in biotechnological processes, as fluorescence measurements are based on fluorescent proteins and biological molecules which show endogenous fluorescence.

A-TEEM can monitor several important components to provide meaningful insight into the bioreactor condition. The A-TEEM meter allows users to profile free amino acids in cell culture media and reduces time and cost compared to conventional meters.

Also, PCA chemometric analysis provides easy classification. Researchers can successfully classify different media types using the combination of A-TEEM spectroscopy and chemometrics.

Insulin is a hormone made by the pancreas that allows the body to use sugar in food for energy or to store glucose for future use. Insulin helps prevent blood sugar levels from getting too high or too low, and many forms of insulin are used to treat diabetics.

As commercial insulin formulation is high in concentration, 4 milligrams per milliliter, the inner effect correction is very important for measuring the EM fingerprint.

The difference between short-acting and long-acting insulin is, in some cases, only one to three residues in the protein sequence. This difference, along with the controlled pH of storage and delivery, is used to either trigger or prevent the formation of insulin dimmers and examiners in the bloodstream.

The formation of these aggregates allows the body to absorb insulin more slowly, and the absence of aggregates makes it absorbs it more quickly. Insulin sequences that vary by only one or three amino acids can be differentiated by intrinsic fluorescence A-TEEM.

In conclusion, fluorescence is recognized as a highly sensitive tool for categorizing samples but combined with other measurement techniques, and it becomes even more powerful.

HORIBA has developed a multi-modal methodology based on fluorescence and absorption measurements to allow sample information to be obtained with more accuracy than a standalone fluorometer. Fast, sensitive and accurate, the A-TEEM meters means more possibilities for the scientific community and analytical world.

This information has been sourced, reviewed and adapted from materials provided by HORIBA.

For more information on this source, please visit HORIBA.

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