Silica gel 60 f254 hptlc glass plates

HPTLC separations were run on 20 cm × 10 cm normal phase Silica gel 60 F254 HPTLC glass plates (Merck, Darmstadt, Germany). Extracts (20 μL/band) were sprayed with compressed air as 8 mm bands onto the HPTLC plate by a Linomat 5 TLC sampler (CAMAG, Muttenz, Switzerland), with 14 mm distance from each side, and a minimum distance of 3 mm between tracks at 8 mm distance from the bottom. HPTLC separation was carried out with n-hexane-ethyl acetate-acetic acid (60:36:4, v/v/v) in an Automated Multiple Development chamber (AMD2, CAMAG, Muttenz, Switzerland) up to a migration distance of 80 mm, which took 20 min. Plates images were captured with a TLC Visualizer Documentation System (CAMAG, Muttenz, Switzerland) operated with winCATS software (CAMAG, Muttenz, Switzerland). Images were processed and evaluated using VideoScan Digital Image Evaluation software (CAMAG, Muttenz, Switzerland).

TLC analysis was performed on a CAMAG TLC system (Muttenz, Switzerland) and visionCATS software. HPTLC Silica gel 60 F254 glass plates 20 cm × 10 cm from Merck (Darmstadt, Germany) were used as a stationary phase. The plates were activated at 60 °C for one hour before use. Before TLC analysis, extracts were put at room temperature for 2 h. Using the CAMAG Linomat 5 semi-automatic sampler, 7 μL samples were applied using a 100 μL syringe. Samples were applied with 8 mm bandwidth and 8 mm from the bottom of the plate. The number of samples on each plate differed between medicinal plants. The syringe was washed with purified water between each sample. The CAMAG ADC2 automatic developing chamber was saturated with the mobile phase ethyl acetate:water:formic acid 80:10:10 (v/v/v) for 30 min and then developed until 80 mm height. The same mobile phase has been used in two previous studies analyzing Salvia officinalis L. extracts and Artemisia species, and it is also stated as the TLC method for analyzing Calendula flowers by the European Pharmacopoeia [39 ,66 ,67 ]. After development, the plates were visualized in the TLC Visualizer 2 under UV wavelengths of 366 nm, 254 nm, and white light. TLC was performed only on simple extracts made from one medicinal plant.

All organic solvents were of LC-MS² grade. DEAE sephadex A-25, was obtained from Pharmacia Biotech AB, Uppsala, Sweden. Preparative C18 125°A 55–105 µm, was obtained from Waters GmbH, Eschborn, Germany. Gangliosides GM3, GM2, GD2, GD3, and GD1a were obtained from Matreya, USA. Isotopically labelled D5-GM3 and D5-GM1 were purchased from Avanti Polar Lipids, and isotopically labelled D3-GM2 and D3-GD3 from Cayman Chemicals. GM1a was derived from FIDIA, Italy and Cronassial (bovine brain gangliosides containing mainly GM1a, GD1a, GD1b, and GT1b) from the company Dr. Madaus & Co, Cologne, Germany. HPTLC silica gel 60 F254 glass plates, Merck KGaA (Darmstadt, Germany). Plexigum (Poly(Isobutyl methacrylate)), SigmaAldrich (St. Louis, Missouri, USA). Mouse anti-human GD2 IgG2a antibody, clone 14. G2a, cat. #554272, BD Pharmingen (Franklin Lakes, New Jersey, USA). Alkaline phosphatase-conjugated goat anti-mouse IgG+IgM (H+L), cat. #115-055-068, Jackson ImmunoResearch Laboratories Inc. ((Farmington, CA, USA). SIGMAFASTTM BCIP^®/NBT (5-bromo-4-chloro-3-indolyl phosphate/Nitroblue tetrazolium) tablets, Sigma Aldrich (St. Louis, Missouri, USA).