I would like to know the different solvents and their concentration used in HPLC.
Use 70% Methanol for extraction of phenolics from plant materials.
For HPLC use A=Acetonitrile
B=0.1%H3PO4
Flow= 1ml/min and 280nm
use gradient elution with little optimisation
Gudluck
:)
Antioxidant activity, bioactive polyphenols in Mexican goats’
milk cheeses on summer grazing
Mario Cuchillo Hilario1, Claudia Delgadillo Puga1*, Arturo Navarro Ocan˜a2 and Fernando Pe´rez-Gil Romo1
Journal of Dairy Research (2010) 77 20–26.
Extraction
Three subsamples (20 g each) weighed in Erlenmeyer flasks with 150 ml of methanol, methanol:water (80:20) or acetone, were set for shaking for 24 hours at room temperature. All extracts were then filtered and washed (50 ml of respective solvent), then the filtrates were concentrated with a vacuum rotary evaporator (Büchi R-205, Labortechnik AG, Switzerland) at 30°C and 150 rpm. Further, extracts were frozen at -80°C, lyophilized (Labconco Freezone 6, Labconco Corp., Kansas City, MO, USA) and stored at 4°C for later analysis
Total polyphenol content and plant bioactive compounds (PBC)
Total polyphenol content (TPC) in the plant extract (methanol:water (80:20 v/v)) was determined by the Folin-Ciocalteu colorimetric method described by Taga et al. (1984). The concentration was calculated using gallic acid as standard, and the results were expressed as mg of gallic acid equivalents (GAE)/kg dry matter of plant extract. To determine flavonoid and hydroxycinnamic acid, 20 mg of each dry extract was assessed by HPLC according to Ubando-Rivera et al. (2005). An HPLC 1525 high-pressure binary pump (Waters Milford, USA) and Symmetry C18 column (5 µm steel 3.9 mm × 150 mm; Waters Milford, USA) were employed. Methanol:water (at a ratio of 70:30 v/v) and 0.16 M acetic acid (pH 2.4) were used as carriers at a flow rate of 1 ml/min. The oven temperature was held at 45°C whereas the detection was performed at 280 nm (486 Waters Milford, USA). The following substances dissolved in methanol were used as standards: catechin (2.21 mg/ml), epicatechin (1.25 mg/ml), gallocatechin (0.032 mg/ml), gallic acid (0.030 mg/ml), caffeic acid (0.032 mg/ml), cinnamic acid (0.038 mg/ml) and ferulic acid (0.031 mg/ml). Calibration curves were made for each standard using three dilutions (1:1; 1:3 and 1:5). Peaks were identified by the retention times of individual standard flavonoids and hydroxycinnamic acids, using a Brezze version 6.30 Waters Software. The concentrations of hydroxycinnamic acids were expressed as g/100 g while flavonoid concentrations were expressed as mg/100 g dry matter of extract. All analytical reagents and standards were from Sigma-Aldrich, Steinheim, Germany.
70:30 acetone and wate. 4 extractions for sample yields best outcome.
a crude extraction with acidified methanol may work to extract many phenols, but if you want to clean the extracts further to focus on specific compounds there you will need to have additional steps. it also depends on what plants and tissure you want to extract from. your question is very general. do you mind providing specifics? do you have standards? are you interested in specific compounds or are you interested in estimating total phenols? a UV-VIS method would work better for total phenols
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