This is my HPLC chromatogram of standard Silymarin solution. In your opinion what is the problem?
mobile phase was methanol-water (50:50).
Are you using a column similar to the one mentioned in the reference below, see link? If so then you need to include a buffer in the mobile phase like 0.05 M KH2PO4 adjusted at pH 2.3. Make up the buffer in the aqueous portion of the solvent (then filter if not using HPLC grade buffers) and then combine with the methanol. If this mixture turns cloudy this means the buffer precipitated out and you can't use this mixture (try again with less methanol).
It also appears that your column may be overloaded try using a smaller amount of Silymarin and/or smaller injection volume (10uL). Keep in mind Silymarin is a mixture and you should see more than one peak. This could be the reason the larger peak is tailing. Also check to make sure all the HPLC fittings are snug and made up made properly. Be sure to keep the amount of tubing between the column and detector at a minimum.
The more closely you follow the details in the link the better your chances of success. I also suggest increasing the length of the run, at least doubling it based on the article in the link.
Article A validated stability-indicating HPLC method for simultaneou...
I think the Silymarin standard powder is OK; as per the link I gave, there are at least 7 peaks from Silymarin. You need to improve your chromatographic conditions to see all of them.
Dr.Levison
I will do again tomorrow and I'll tell you the result.
thank you
Dr.Bruce Levison
please guide me. you see 3 chromatogram showing standard Silymarin solution (50 ppm), 100 ppm and my silymarin sample, respectively. As you noted above, more than one peak can be seen in these chromatographs. We used 150*4.6 mm, 5 micrometer column.
It appears that you are running a gradient and not letting your column equilibrate long enough between runs to get consistent or reproducible results. Try letting the column equilibrate in the initial conditions for 15 minutes (10 column volumes) before you make the next injection.
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