i have tried different mobile phase systems but some of the alkaloids seems to co-elute causing the need for further separation. i need suggestions for mobile phase systems for this plant
Did you use a gradient elution? What kind of column did you try? Unfortunately, all chromatographic problems are different. Therefore, your question can not be answered in this general form.
Pennington, L. J. and Schmidt, W. F. Belladonna Alkaloids and Phenobarbital Combination Pharmaceuticals Analysis I: High Performance Liquid Chromatographic Determinations of Hyoscyamine-Atropine and Scopolamine. J. Pharm. Sci. 71: 951-953. 1982.
Did this research a long time ago, and the chemistry could probably be very similar for your alkaloids.
My experience is that since the C18 is bound to an HPLC column via Si-O bonds and because C18 groups are quite large and bulky, the un-reacted Si-OH sites are what makes HPLC of alkaloids more difficult than some other organic compounds. An alkaloid can adsorb and/or desorb from the hyrdrophilic SiOH phase instead of partitioning on and off the hydrophobic C18 phase.
We found adding e.g. 1% (CH3)4N+ ions (tertramethylammonium chloride) to the aqueous component of the mobile phase was sufficient to return alkaloids to the hydrophobic partitioning present in reverse phase HPLC. Do not know if people use "reverse phase" anymore in describing HPLC anymore. . .
Walter
You may also consider that in many cases preparative TLCs may give help in resolving mixtures of very similar compounds.
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