Nated sample (6 g) was subjected to 1D separation employing two 40 g silica gel flash columns (particle size 35?0 ). Both the silica columns had been equilibrated with 4 column volumes of chloroform prior to loading the samples. The curcuminoids had been detected at 244 and 360 nm using a peak width of 30 seconds and threshold of 0.05 AU. The separation of curcuminoids was completed in 125 min using a gradient plan of solvent A (chloroform) and solvent B (methanol) having a flow rate of 40 mL/min. Four important peaks were separated along with minor peaks (Fig. 1a). The gradient elution of columns was performed in accordance with Table 1. A total of 69 fractions of 42 mL every single have been collected throughout the elution method. All fractions have been analyzed by HPLC and fractions containing peaks 16?9, 20?two, 25?8 and 33?6 were pooled, concentrated below vacuum to acquire compounds (1?). two.4. Separation of curcuminoids by pseudo 2D hyphenated chromatographic approach Pseudo two dimensional separation of curcuminoids was carried out making use of silica (40 g) and diol (30 g) columns in series. Impregnated sample (six g) was loaded; and column was eluted with chloroform for the separation of curcuminoids as pointed out above. The solvent gradient made use of for pseudo 2D separation is shown in Table 1.1349151-98-9 custom synthesis In this experiment, 71 fractions of 42 mL every single have been collected and analyzed by HPLC; fractions 8?4, 15?three, 24?6 and 28?32 yielded 4 compounds (Fig.tert-Butyl pent-4-ynoate Chemscene 1b).PMID:23812309 These grouped fractions were pooled and concentrated below vacuum to get crystalized compounds (1?). Employing UV spectral information for compounds (1?) were tentatively identified as curcumin, demethoxy curcumin (DMC), bisdemethoxy curcumin (BDMC) and dihydrobisdemethoxy curcumin (DHBDMC) (Fig. 1b).J Chromatogr B Analyt Technol Biomed Life Sci. Author manuscript; readily available in PMC 2014 October 15.Jayaprakasha et al.Page2.five HPLC evaluation All fractions obtained from 1D and pseudo 2D experiments were dissolved in acetone and subjected to HPLC analysis. The HPLC method consisted of Agilent HPLC 1200 series (Foster City, CA) system consisting of a degasser, quaternary pump, auto-sampler, column oven, and photodiode array detector. Elution of curcuminoids was carried out with gradient mobile phases of (A) 3 mM phosphoric acid in water and (B) acetonitrile using a flow price of 0.7 ml/min at 30 using Gemini C18 (Phenomenex, Torrance, CA) column. Curcuminoids have been determined working with the gradient: 75 to 45 A from 0 to five min, 45 to 20 in 5?2 min, 20?0 in 12?0 min and 10?five in 20?five min and maintained for isocratic run for 5 min to equilibrate the HPLC column. Data was processed employing the CHEMSTATION computer software (Agilent, Foster City, CA) (Fig. 2). 2.six. 1H NMR calibration and Quantization NMR spectrometer (JEOL USA, Inc., Peabody, MA, USA) operating at a frequency of 400 MHz for protons equipped using a five mm multinuclear inverse probehead and NM-ASC24 sample changer was employed for acquisition of NMR spectra. 1H NMR spectra obtained in the single pulse sequence have been utilized to ascertain the purity of the isolated compounds. Recognized quantities (three? mg) of isolated compounds (1?) have been dissolved in 525 of DMSO-d6 separately and compound (four) in 525 of acetone-d6 and transferred to five mm NMR tubes. An external coaxial glass tube (OD two mm) containing 60 0.012 3(trimethylsilyl) propionic-(2,two,3,3-d4) acid sodium salt (TSP-d4) remedy in D2O was inserted into NMR sample tube for quantitative reference. The TSP-d4 concentration inside the tube was pre-calibrated utilizing a.