I have crude plant extracts of which were separated 5 polyphenols I have to separate and purify these 5 fractions (cynarin,chlorogenic acid,1,3-Dicaffeoylquinic acid,and cynaroside).
I don't see why a humble C18 should not do the trick...
Depending on how complex the fractions are and your instrument setup, try a 4.6 x 100 mm, 2.1mu or even 4.6 x 250mm column
Indded a C18 should do, with acidified water (acetic acid or formic acid at low conc).to acetonitrile or methanol as the gradient; which dicaffeoylquininc, there are many of them, therefore this may require more than UV-Vis.
Hi Abdulelah,
Let's first check your listed target compounds:
Cynarin
Chlorogenic Acid
1,3-Dicaffeoylquinic Acid (CAS: 19870-46-3, the synonym name is Cynarin that actually is the first compound, the API of nature medicine Echinacea from the extraction of herb Echinacea roots).
Cymaroside
So, actually you have three target compounds. To separate them, Phenomenex Gemini C6-Phenyl LC column is one of best choices. And this column is very stable in pH 1.5 to 11.0 and under 100% aqueous conditions; the moderate hydrophobic retention, high H-bond accepting capacity and high steric selection for structure isomer selection can help you to separate your target compounds. Run LC gradient, 0% B to 95% B.
A: H2O-MeOH + HAc (to make pH around 5)
B: MeOH + TEA (elution as IPC mechanism)
The mobile phase is good for both HPLC and LC-MS/MS, and good for both MS APCI and ESI.
Hope it is helpful to you.
RP-C18 Columns with a precolumn with the same phase should work. 4.6 and a longer lenght (250 mm) should work better.
If you have a UPLC you could improve your separation and time of chromatogram using a narrow column, i.e. 2.1 diameter (and less legth, i.e. 100 mm.).
Other options could be a biphenyl.
Moreover, a good option for Catechins is a diol phase column with fluorescence detection.
You can use RP-C 18 column (125 × 4.6 mm length, 5 µm particle size) dear with solvent system solvent A - acetic acid:water (2:98, v/v), whereas solvent B - 1% acetic acid:acetonitrile (1:1, v/v). You can also try formic acid instead of acetic acid and you can also vary the concentration of acetic acid and formic acid.
C18 with acidified H2O to 100% acetonitrile in a gradient system, 4.6 x 250mm or 4.6 x 150mm columns.
I separated polyphenols on RP C18 analytical column 250 mm x 4.6 mm, 5 µm particle size. The mobile phase consisted of solvent A (1 %, v/v solution of orthophosphoric acid in water) and solvent B (acetonitrile). Gradient elution start with 98% A to 0%A
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