*3.3. MIR Spectroscopy*

The MIR region is the most relevant for the spectral analysis of milk, since strong characteristic absorption bands associated with fundamental vibrational and rotational stretching modes of the main milk constituents molecules, including proteins, saccharides, fats, etc., are manifested in this part of the optical spectrum. At the same time background scattering on the milk particles is not so prominent because the ratio of the fat globules and casein micelles sizes to the working wavelengths is much lower than in the NIR region. Analytical methods based on MIR spectroscopy have been used for the laboratory testing of milk since sixties [68] and are well established now [69]. Novel approaches are constantly developing [50,69,70] because this technique gives the most precise and detailed information about sample chemical composition; some examples of the most remarkable applications of MIR spectroscopy for the analysis of milk are summarized in the Table 5.

Because milk is an extremely complicated multicomponent scattering medium the straightforward determination of the constituents' concentrations from the intensity (or area) of the characteristic absorption peaks in spectra according to the classical Beer-Lambert law is impossible in most cases. Therefore, various multivariate chemometrics algorithms for spectral calibration and data analysis are applied: Partial Least Square Regression (PLS) and Discriminant Analysis (PLS-DA), Principal Component Regression (PCR), Principal Component Analysis (PCA), Artificial Neural Networks (ANN), Soft Independent Modeling of Class Analogy (SIMCA), Multivariate curve resolution alternating least square (MCR-ALS), etc. [71,72].

It is very important to mention that MIR spectroscopy is an immensely powerful tool not only for the quantitative determination of the basic milk constituents or residual amounts of antibiotics and disinfectants (see Table 5), but also widely used for detecting milk adulteration, when original proteins and fats are replaced with vegetables products, or foreign, often dangerous substances (sucrose, starch, urea, melamine, formalin, etc.) are deliberately and illegally added to milk and other dairy products by fraudsters to extend the shelf life or falsely increase the claimed protein and fat content.



 errors of determination for the concentrations of components (in %) are presented if not specified otherwise; detection limits are in

Fourier transform infrared (FTIR) spectrometers are usually used for the analysis of milk and dairy products in the MIR region; highly specialized platforms are available on the market, for example MilkoScan® series by FOSS (Hillerød, Denmark). The main obstacle for wider implementation of this technique is strong MIR water absorption [82]; so very thin optical cuvettes or, which is more preferable, attenuated total internal reflectance (ATR) accessories have to be used [62]. The main element of an ATR accessory is an optically dense IR crystal with a high refractive index which is simultaneously transparent in a working spectral range (Figure 4). An incident IR beam travels through the crystal and undergoes multiple total internal reflections. The surface of the crystal is in direct contact with a liquid medium where an evanescent electromagnetic wave propagates along the border. In regions of the spectrum where the liquid absorbs electromagnetic energy of the evanescent wave the IR beam in the crustal is attenuated.

**Figure 4.** Measuring MIR absorption spectra using ATR setup.

Because the penetration depth of the evanescent wave is limited to a few micrometers, IR radiation interacts with the liquid sample in a very thin near-surface layer. As a consequence, the spectra are very sensitive to the presence of fat globules or fat biofilms in the boundary layer that forms at the interface between the milk and the crystal which serves both as radiation waveguide and sensing element [83]. In combination with such factors as high-cost spectral equipment, complicated maintenance, cleaning, calibration, and sample preparation procedures, this fundamental limitation makes FTIR/ATR spectroscopy almost exclusively off-line, laboratory technique not suitable for real-time in-line monitoring in milking machines on farms or milk processing equipment on production facilities [65].
