DETERMINATION OF MULTIPLES QUALITY PARAMETERS OF COCOA PRODUCTS USING QUANTITATIVE 13C SOLID STATE NMR

Quantitative Nuclear Magnetic Resonance (qNMR) spectroscopy in solution has been widely used in multiple chemical analysis. On the other hand, applications of quantitative solid-state (SS-qNMR) is much more limited. The ¹H qNMR, the major technique in solution, is rarely used in SS-qNMR because of the strong dipolar interaction, which limits the resolution even when high speed magic angle probes and sophisticated pulse sequences are used. Therefore, most SS-qNMR applications are based on ¹³C nuclei. When comparing to ¹H SS-NMR, ¹³C SS- qNMR have the advantage of higher resolution and the chemical shift anisotropy and heteronuclear dipolar interactions can be suppressed by the use of decoupler and magic-angle spinning (MAS) at moderate spinning speed. However, contrary to the acquisition of ¹H signal, the measurement of ¹³C signals is a longer experiment, due to its longer longitudinal relaxation time (T1), low magnetogyric ratio and low natural abundance. To get around these limitations, the majority of ¹³C acquisitions are done via cross polarization (CP) sequence, which transfer magnetization from the abundant ¹H to the rare ¹³C nuclei, via dipolar interaction. Nonetheless, the CP sequence is not interesting for the quantitative study of heterogeneous products, containing solid and liquid phases, because it enhances the solid-state signals, but not the liquid phase signal, because of its lack of dipolar interaction. For quantitative measurements of heterogenous products like chocolates, cocoa butter etc, that contain liquid and solid phases, the alternative presented here is the use of a direct excitation of ¹³C transition pulse sequence, with high power decoupling and MAS, called HPDEC sequence. The acquisition of such quantitative ¹³C HPDEC spectrum enables the determination of several physical and chemical properties of the cocoa-based products through only one analysis. The spectra can offer qualitative information regarding the crystalline form and the isothermal crystallization of the triacylglcerides (TAG) molecules, as well as quantitative information such as the presence of sucrose and the solid fat content (SFC) of the sample. These are important quality control parameters for the chocolate industry, that is demanding fast and multiple analysis to develop new high quality and cheaper products to deal with the increase in cocoa beans prices, associated with a higher demand and a lower supply.

For quantitative analysis, the HPDEC sequence uses a 90° pulse, acquisition time of 50 ms, decoupler power of 70 W, and recycle delay D1 equal to 850 s that is equal to 5T1 and 3 kHz spinning frequency. The T₁ was determined with Inversion-Recovery (IR) pulse sequence. The 3 kHz spinning frequency, decoupling power and recycle delay were used to avoid sample heating, which melts the fat content of cocoa products.

The possibility of assessing multiple physical and chemical properties through the ¹³C spectrum is associated with the assignments of the different chemical groups present in the sample. For example, the study of the solid fat content (SFC) is done via the ratio of the areas of the signals at 30 and 34 ppm, which are assigned to the methylene groups present in the liquid and solid portion of the TAG molecules respectively. The difference in chemical shift observed for the same groups, in liquid and in solid phases, is the result of the trans configuration observed for the rigid portion of the TAG molecules, which results in the signal at 34 ppm. Other non-fat component signals like sucrose, present in chocolates samples, are associated with other peaks in the spectrum. Based on the signal areas it is possible to determine the various physical and chemical properties mentioned above. The use of a SS-qNMR ¹³C spectrum in the determination of, for example, the SFC is, therefore, based on physical and chemical properties of the sample, contrary to the stablished methods, which are based only on physical properties, and result in SFC values that are similar to the ones obtained via the stablished protocols. However, the proposed method has the advantage of determining other samples properties all in the same analysis, such as the crystalline polymorph present in the sample as well as the sweetness level, according to the quantity of sucrose present regarding the quantity of fat present in the sample. Therefore, the proposed ¹³C SS-qNMR method can be a useful method for the rapid analysis of several quality parameter of chocolates and others cocoa-based products as well for other foods products that are rich in fats like butter and margarines.