## Results and Discussion
Differences were also detected in the high-field regions 0.5-2.5 ppm and 3.0-4.0 ppm, which corresponded to the regions for amino acids and carbohydrates [25; 26]. Signals for amino acids and carbohydrates display characteristic amine protons, methyls, mething groups and anomeric protons (for carbohydrates) which were detected in most of the polar and medium-polar solvent extracts [28]. However, peak congestion in these regions makes the assignment of compounds very challenging. In the present study, 10 amino acids and two carbohydrate constituents were identified in these spectral regions. The identification was further supported by 2D experiments (\(J\)-resolved, heteronuclear multiple-bond correlation, (HMBC) spectroscopy). Acetone, chloroform and hexane extracts of _C. calcitrans_ were among the solvent extracts that showed signals for carotenoids, specifically the regions 6.0-7.0 ppm (olefinic protons) and 0.8-2.5 ppm (aliphatic protons) [24]. Generally, there are two types of carotenoids, which include one that forms from only carbon and hydrogen and are called carotenes, whereas another type, which contain oxygen, are called xanthophylls [29; 30]. The proton resonances assignable to olefinic protons were observed at 6.75, 6.66, 6.58, 6.41, 6.13, and 6.07 ppm for toxanthin, 6.20-6.70 ppm for canthaxanthin and astaxanthin, and 6.67-6.57 ppm for lutein. Multiplet signals were observed between 6.10-6.40 ppm for zeaxanthin. Fucoxanthin and astaxanthin identification have been confirmed by 2D NMR. The \(J\)-resolved spectrum observed signals at 2.07 and 2.01 ppm corresponding to methyls proton of fucoxanthin and astaxanthin, respectively (Figure S1). From the HMBC spectrum, a 2 \(J\) correlation between the proton signal at 2.07 ppm (H-19) to the carbonyl signal at 200 ppm (C-8) and 3 \(J\) correlation between the proton at 3.78 (H-3\({}^{\prime}\)) ppm to methine carbon signal at 65 ppm (C-5\({}^{\prime}\)) in cyclohexene ring could be seen which confirmed the assignment of fucoxanthin (Figure S2A). As for astaxanthin, correlations were observed between the proton signal at 2.01 ppm (H-17) with the methyl carbon at 17 ppm (C-2) in cyclohexene ring (Figure S2A). Unfortunately, none of the 2D experiments were able to confirm precisely the resonances of the zeaxanthin, canthaxanthin, lutein and violaxanthin due to their low concentrations in the extracts. The \({}^{1}\)H NMR spectra of the hexane extract showed signals characteristic of fatty acids. The signals were close to the reported signals for PUFAs, such as arachidonic acid [C20:0], \(\alpha\)-linolenic acid [C18:3], linoleic acid [C18:2], stearic acid [C18:0], oleic acid [C18:1] and palmitic acid [C16:0] [25; 26]. The signals corresponding to \(\alpha\)-linolenic acid [C18:3], linoleic acid [C18:2], and oleic acid [C18:1] were deduced based on the presence of the olefinic protons (5.30-5.40 ppm), protons attached to the bisallylic carbons (129-132 ppm). The \(J\)-resolved spectrum showed a multiplet signals of the olefinic protons at 5.30-5.40 ppm and the HMBC spectrum revealed that these signals were correlated to methylene carbon signals at 25-27 ppm which signified the characteristic of PUFAs (Figures S1 and S2). The difference in double-bond positions as well as double-bond configuration (i.e., either cis or trans) were also considered in order to determine the fatty acid profile. Moreover, to identify tentatively the presence of palmitic acid, stearic acid and also arachidonic acid, the signal of methylene group (CH\({}_{2}\)) protons, observed at 1.20-1.40 ppm, was used to discriminate the fatty acid components. The assignment of the fatty acid \({}^{1}\)H NMR resonances was further supported by 2D NMR data as shown in Figure S3 and S4. The spectral features and the chemical shift assignments were compared with the previous reported literature. Several resonances belonging to chlorophyll \(c_{1}\) (Chl \(c_{1}\)) and chlorophyll \(a\) (Chl \(a\)), which are accessory pigments, were seen in many diatoms.