Molecule higher-order overtones

The low correlation coefficient of 0.747 for the pH is caused by the small pH range of about 0.6 pH unit. On the other hand, determination of pH in cheese by NIRS is the result of many overlapping overtones that are also very weak. Consequently, it seems impossible to make a high precision calibration for the measurement of pH by NIRS. Mathematical manipulation of the raw NIR data of the cheese samples shows a high correlation or intercorrelation between the parameters — water sol. N/tot. N, TCA sol. N/tot. N, and water-soluble primary amines — and the total protein content. This is to be expected because during cheese ripening proteins are broken down to peptides and amino acids, mainly by enzymatic processes. Because the measurement of protein by NIRS is based on the absorption of casein molecules as well as a variety of peptides and amino acids, it is very difficult to resolve the protein absorption bands in a lot of smaller bands and to correlate these bands to the constituents from which they arise. The only possibility probably is to use higher order mathematical data transformations. 

Near-infrared absorption is therefore essentially due to combination and overtone modes of higher energy fundamentals, such as C-H, N-H, and O-H stretches, which appear as lower overtones and lower order combination modes. Since the NIR absorption of polyatomic molecules thus mainly reflects vibrational contributions from very few functional groups, NIR spectroscopy is less suitable for detailed qualitative analysis than IR, which shows all (active) fundamentals and the overtones and combination modes of low-energy vibrations. On the other hand, since the vibrational intensities of near-infrared bands are considerably lower than those of corresponding infrared bands, optical layers of reasonable size (millimeters, centimeters) may be transmitted in the NIR, even in the case of liquid samples, compared to the layers of pm size which are detected in the infrared. This has important consequences for the direct quantitative study of chemical reactions, chemical equilibria, and phase equilibria via NIR spectroscopy. 

The sensitivity of this technique is demonstrated by Fig. 1.9, which shows an overtone absorption line of the water molecule H2O, recorded with an unmodulated laser and with this modulation technique. The signal-to-noise ratio of the absorption measured with phase modulation is about 2 orders of magnitude higher than without modulation. The sensitivity reaches a maximum if the modulation frequency is chosen to be equal to the width of the absorption line. 

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