2.2.1. 2D 1H,1H-COSY
COSY is one of the simplest and most frequently used 2D NMR experiment [123]. It shows the homonuclear coupling of nuclei (i.e., 1H-1H) separated by up to several covalent bonds. The pulse sequence consists of a 90° excitation pulse followed by a specific evolution time (t1), a second pulse, and finally a measurement period (t2, not to be confused with relaxation rates or times). The second pulse can be 90° or 45° or 135°, depending upon the specific requirements, and respectively yield COSY [124], COSY-45 or COSY-135 functionality (see [125,126,127]). A two-dimensional Fourier Transform (FT) yields the final spectra and shows the frequencies for proton (1H) or carbon (in the case of carbon detection) along both axes. There are two types of peaks; (I) Diagonal peaks, which represent the peaks of the conventional 1D spectra, and (II) cross-peaks, which have different values in the two frequency axes and are therefore off the diagonal. These off diagonal cross-peaks are the most important pieces of information as they mark correlations between pairs of nuclei due to through bond magnetization transfer. This helps in identifying which atoms are connected [128], critical for structural elucidation of both known molecules and unknown molecules in solution [129]. By implementing phase-cycling [130,131], it is also possible to distinguish different types of coupling and yields further helpful information about the chemical structure of a molecule [132]. As an example, the use of the COSY experiment was presented in the work of Zheng et al. [88]. The main goal of their work was to investigate potential biological differences and compare the pharmacological effects between Danggui (an herbal drug used in traditional Chinese medicine) and European Danggui. For that, Zheng et al. treated blood deficiency rats with Danggui and European Danggui and collected samples of their serum and urine. The samples were later measured using 1H-CPMG-NMR, 1H-NOESYPRESAT-1D, 1H,1H-COSY, and 1H,13C-HSQC, and then compared to equivalent spectra from untreated rats. The results showed that exposure to Danggui and European Danggui altered the levels of 18 different metabolites, such as lactate, nicotinamide, glycerol and formate, which were involved in a total of seven different metabolism pathways. Additionally, it was proven that Danggui and European Danggui have different chemical compositions, with Danggui having better blood-enriching effects than European Danggui.