My samples are from hexane fractions but I already redissolved them in methanol. my compounds for sure are non polar, so what mobile phases and column
Dear Sir. Concerning your issue about the mobile phase and column used for Hexane fractions extracts. Phytochemical analysis of chloroform and methanol extracts of P. longum leaves was performed for the presence of alkaloids, phenolic compounds, tannins and saponins (Harborne, 1998; Falodun et al., 2008; Edeoga et al., 2005). Test solution of 50 mg/ml was prepared in acetone to perform the analysis. Presence of alkaloid in the chloroform and methanol extracts of P. longum was tested by following the method of Falodun et al. (2008). One ml of the test solution was treated with a few drops of Dragendorfs reagent. Presence of alkaloid was also tested by treating 1 ml of test solution with Mayer’s reagent. The presence or absence of alkaloids in the extracts was observed on the basis of change of colour of the test solution. Chloroform and methanol extracts were separated by column chromatography using glass columns. Silica gel (60-120 mesh / 230-400 mesh, Merck, Mumbai) slurry prepared in the respective solvent (hexane / petroleum ether) was loaded in the column. Plant sample was dissolved in minimum quantity of solvent (chloroform / ethyl acetate) and adsorbed on silica gel. Then solvent was air dried and introduced into the column. Chloroform extract was separated using petroleum ether-ethyl acetate-methanol as mobile phase while methanol extract was separated with two different mobile phases, a) hexane–chloroform and b) hexane-ethyl acetate-methanol. The fractions were collected under gravitational flow. The first few fractions were collected with non polar solvents (hexane / petroleum ether). The solvents were used as a gradient system with 10% increase in each step (Table 2 a, b, c). The fractions obtained from first step column chromatography from choloform extract were further fractionated in the same way using mobile phase petroleum etherdichloromethane-ethyl acetate-methanol as a gradient system with 10% increase in each stepI think the following below link may help you in your analysis:
http://shodhganga.inflibnet.ac.in/bitstream/10603/5449/9/09_chapter%203.pdf
Thanks
Normal phase for column.
Increase the polarity step-wise starting from hexane.
https://chem.libretexts.org/Demonstrations_and_Experiments/Basic_Lab_Techniques/Packing_Columns/Packing_Normal_Phase_Columns
I would suggest starting with something you are sure will not cause precipitation, avoid using too much water. You mention your sample is soluble in methanol, meaning that they exhibit some afffinity or polar solvents. In that case, I might start with a 65% aqueous mobile phase and run a standard gradient to 100% organic. I also run much stronger elutropic gradients for water-insoluble compounds. For the analysis of fatty acids, I usually start with 90/10 MeOH:IPA and go to 10:90 over several minutes. A standard C18 column will get the job done in most situations. If you're concerned about clogging a column, it is best to work with a stronger solvating method and go backwards (adding increasing amounts of weak solvent)until you achieve separation.
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