515 hplc pump

Preparative HPLC was generally carried out using Quaternary Gradient Module 2545, Photodiode Array Detector 2998, Autosampler 2707, Waters Prep Degasser and Waters Fraction Collector III. Software: Waters ChromScope v1.40 Beta (Waters, Milford, MA, USA). Stationary phase: Nucleodur^® C18 HTec, 5 µm, 250 × 21 mm, mobile phase: binary gradient of water (A) and acetonitrile (B) at a flow rate of approx. 15.5 mL/min.
Separation of fraction SE6 yielded 1 (118 mg) and 2 (1.2 mg). 3 (11 mg) was obtained from SE7, 4 (5.3 mg), 5 (13 mg) and 6 (0.65 mg) from fraction SE8. Subfractionation of MPLC fraction M2 and M3 led to 15 (39 mg). 16 (0.5 mg) was obtained from M4, and M5 yielded 17 (0.6 mg), 18 (0.9 mg) and 19 (0.3 mg).
Fractions N3 to N5 obtained from FCPC and SE3 were further purified using the following system: Two Waters 515 HPLC Pumps, Waters Pump Control Module II, Degasys DG-2410 (Uniflows, Tokyo, Japan), Waters 996 PDA Detector, software: Waters ChromScope v1.40 Beta Software (Waters, Milford, MA, USA), stationary phase: Eurospher 100 C18, 250 × 21 mm; 7 µm (VDS Optilab, Germany), mobile phase gradient: water (A), acetonitrile (B), flow: 10 mL/min, injection volume: 1 mL. 7 (0.12 mg), 8 (0.9 mg) and 9 (1.7 mg) were isolated from N3, N5 and N4 resp. Compound 14 (11 mg) was isolated from SE3 along with 10 (3.9 mg), 11 and 12 as a mixture (4.3 mg) and 13 (2.7 mg).

Short chain organic acids (citric, tartaric, malic, succinic, lactic, formic and acetic acids) were quantified by an isocratic HPLC system with conductivity detection [31 (link)]. The system consisted of two Waters 515 HPLC pumps, a Waters 717 autosampler (Waters Corp., Milford, MA, USA), an LBK chromatography oven model 2155 (Pharmacia, Sweden), and a Milton Roy Conductomonitor III conductivity detector (LDC, FL, USA). The column was an exclusion column Phenomenex Rezex ROA-Organic AcidH + 8%, 300 (l) × 7.8 mm (i.d.) (Phenomenex, Torrance, CA, USA). The mobile phase consisted of 2.5 mM solution of tri-fluoroacetic acid (TFA; 0.4 mL min⁻¹). A solution consisted of 2.5 mM TFA, 20 mM bis-[2-hydroxyethyl]imino-tris-[hydroxy-methyl]methane (bis-tris buffer), and 100 mM EDTA was added at the column out-let to increase the detection sensitivity. A flow rate of 0.4 mL/min and 60 °C of column temperature was applied, the injection volume was 40 µL. Samples and standards were filtered through 0.45 µm nylon membranes, and analyses were carried out in duplicate. The identification of each compound was carried out by comparing retention times of the peaks in distillates with those previously obtained by the injection of standards. The results were expressed in mg of compound per liter of spirit.