I would like to isolate more polar compound from the extract. In this regard, I am supposed to use HPLC for successful isolation of the compound (s). Can you please suggest me the procedure for preparative HPLC or suggest me good references for this protocol?
Your cooperation would highly be acknowledged.
I'm sorry, but this question can be answered in many ways-
Are you asking:
If number 2, which column are you using? One can run normal phase or reverse phase HPLC. I will assume C18, since this is by far the most common column, and it is run as reverse phase. It is also helpful to indicate how you have purified your mixture to this point, since the earlier steps give clues to the HPLC purification. For example, if the compound mixture was purified on a SCX (strong cation exchange column), I would know it is basic. Also, it is helpful to know if you think this is a known compound, or if you don't know what it is yet.
- You helped by indicating your compound is polar, suggesting an acidic, basic, or has several polar groups (such as hydroxyl).
- On C18, water is the "weak" solvent, and the organic solvent (usually methanol or acetonitrile) is the "strong" solvent. The strong solvent causes the compound to elute faster; increasing the concentration of the strong solvent makes the compound elute faster.
- Polar compounds interact less with C18 than non-polar compounds, and elute earlier. They elute with wealer solvents.
- Since you have a polar compound, I suggest adding a modifier such as formic acid, acetic acid, or TFA (trifluoroacetic acid). This helps to keep your compound as a sharp peak. A basic compound will elute as a salt, and elute faster since salts are more polar. An acidic compound will tend to remain as the "free acid" and elute a little later.
- Run an analytical HPLC with a scouting gradient and see where your compound elutes. Use the same sort of analytical column as your prep column, so the compounds elute the same way.
- You can this follow the procedure in the following link to work out a gradient for your prep column (the procedure should work even if not using an agilent system): https://www.agilent.com/cs/library/technicaloverviews/public/5991-2013EN.pdf
Thank you Jack Silver. I already run the analytical HPLC with Water and Methanol as solvent using C18 column. I have found the peak of the unknown compound. Now I want to purify the peak to isolate this unknown compound. Please tell me precisely as per the mentioned target.
That procedure is a bit long for a reply here, but please look at the link in the last bullet point in my reply above. That will guide you to a method for a prep column.
Dear Shahinozzaman first thing to do is to know which kind of compounds u can found in this spice and this by using the bibliography of the genus then you can look for the suitable conditions to use it with the HPLC experiments, u can change it (conditions or the column to obtain more clear profile)
when you get the best separation in HPLC profile u can start with the HPLC separation (preparative column)
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