What is A-TEEM spectroscopy?

A-TEEM spectroscopy refers to the ability to simultaneously acquire Absorbance, Transmittance and a fluorescence Excitation Emission Matrix (A-TEEM) of a particular sample. HORIBA pioneered this technique with the patented Aqualog and Duetta system, which combines A-TEEM spectroscopy with simultaneous multichannel CCD detection to provide extremely fast results.

A-TEEM spectrometers can be used for fluorescence EEMs or for absorbance measurements for multi-component analysis, but its real power is derived from the fact that the EEMs collected by the instrument are corrected for inner filter effect. This means they are true and accurate representations of the molecules of interest over a much broader and more useable concentration range (typically up to ~2 absorbance units). Therefore, these EEMs allow for much more precise fingerprinting than is possible with an EEM collected from a traditional scanning fluorometer. A-TEEM spectroscopy is now bringing the fluorescence technique into the true analytical market and it has been demonstrated that it can, in some cases, replace traditional instruments like an HPLC or mass spectrometer as a simpler, faster and less expensive analytical tool.

To access the true power of fluorescence A-TEEM spectroscopy, one needs to employ multivariate software methods such as Principal Components Analysis (PCA), Classical Least Squared (CLS) method and Parallel Factor Analysis (PARAFAC).

Most components of CDOM have broad overlapping fluorescence excitation and emission spectra in the UV and visible range. Many sample measurements are used to create a model and then use chemometrics to get scores of each component in an individual sample. The very unique thing about a fluorescence EEM is that it can be used as a molecular fingerprint. Changes in the emission spectrum, the excitation spectrum, or both can be tracked very easily using this 3D fluorescence method for water analysis as well as many other applications.

One common application for EEMs, and especially for A-TEEM spectroscopy is 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. A-TEEMs are used to identify the presence of each at very low concentrations, typically in the ppb range.

Most components of CDOM have broad overlapping fluorescence excitation and emission spectra in the UV and visible range. Many sample measurements are used to create a model and then use chemometrics to get scores of each component in an individual sample. The very unique thing about a fluorescence EEM, corrected for IFE, is that it can be used as a precise molecular fingerprint. Changes in the emission spectrum, the excitation spectrum, or both can be tracked very easily using this 3D fluorescence method for water analysis.

EEMs are also used to study petrochemicals, drugs, proteins and food science, including wine, beer, and many other beverages. Below is an example of an Italian wine sample measured before and after a week-long oxidation treatment (exposure to air). The EEM and corresponding absorbance spectra give a fingerprint of the wine that show changes to the fluorescence spectral shape and intensity for components such as caffeic acid, flavanols, epicatechin, gentisic acid, and anthocyaninie.

By using chemometric analysis, the individual EEMs are also used to characterize single walled carbon nanotubes. (HORIBA App Note: Fluorescence Spectra from Carbon Nanotubes with the Nanolog, n.d.) The carbon nanotube, which is a rolled graphene sheet, can be single walled or multiwalled (multiple sheets rolled). (Dresselhaus, 2000) (O'Connel, 2002) Only single walled carbon nanotubes (SWCNTs) emit photons due to their semiconductor properties.

The helical folding angle also effects the absorption and emission wavelengths of the SWCNT. (Bachilo, 2002) Depending on if the graphene sheet is rolled horizontally or at an angle, the carbon structure differs. The specific geometry can be described in terms of the wrapping vector containing the length (the tube’s circumference) and a helix angle a (ranging from 0 to 30°) and so the two numbers (n,m) are used for SWCNT definition. The helix angle in terms of (n,m) is:

In the case of SWCNTs, the semiconductor absorbs between the c2 and v2 energy levels, where the electron hole is passed down as shown in Fig. 34. A photon is emitted at the band gap, or c1-v1 energy level. These conduction bands and emission bands depend on the diameter of a carbon nanotube, which can also be described as an exciton Bohr radius. The smaller the radius, the higher the energy (or shorter the wavelengths) of light absorbed and emitted. This is due to the quantum confinement effect, which dictates that the wavelength of light emitted is restricted by the size of the particle, or in this case, the diameter of the tube. (O'Connel, 2002) (Dresselhaus, 2000)

With the rise of protein production using mammalian cell culture, it has become increasingly important to control the quality of the cell culture media for use in production processes.

Cell culture media are usually prepared as aqueous solutions, and should provide everything a cell line needs for optimal cell growth as well as product yield and quality.

In any given bioreactor process it is important to identify the proper type of cell culture medium and its quality, because even subtle variations in composition could have a noticeable impact on the growth rate of the cell culture and its yield.  Therefore, the composition and quality of cell culture media in bioreactors must be tightly controlled in order to maintain an optimal bioreactor process. As a result, methods of identifying and analyzing the quality of cell culture media have become an important focus in this field.

A synchronous scan is when the excitation monochromator scans at the same time as the emission monochromator and the fluorescence emission is read out. Typically, one can set an offset between the excitation and emission monochromators that matches the Stokes Shift (difference between excitation and emission peaks). These types of synchronous scans have been historically used for component analysis, but due to the more modern instruments for measuring EEMs with CCD detectors, the EEM gives more information and takes the same amount of time.

An offset of 0 nm can be set so that the excitation and emission are scanning together at the same wavelengths. This is what is called right angle light scattering, or RALS, and results in what is really a right-angle reflectance spectrum. This type of synchronous scan measures the reflected or scattered light from the excitation.