I am having difficulty to separate very close peaks which are isomers. Currently I am using Xbridge Waters C18 column (4.5 mm, 150 mm and 5 micrometer). I used solvent A as water (0.1% acetic acid) and solvent B as Acetonitrile (0.125% acetic acid). My solvent system now 0-5 min (10-12% B), 5-40 min (12-15% B). The flow rate 1.0 mL/min. Here I attach my chromatogram. The peaks are flavonoids-glycosides. Please help me. Thank you.
Double the length of the column; decrease the size of the packing phase...
Thank you for the suggestion Dr. Alan. The longest column that I have is 25cm. I will try to reduce the size of packing phase. Are there any other ways for me to improve the separation?
@ Shazeli There is usually much less you can do with playing with the conditions such as temperature (ramp) or carrier gas type or flow rate. I think you need a radical change here not a minor one. You can change the liquid phase on the column also. Many years ago I separated easily all the C4 and C5 hydrocarbon isomers by playing around with the solid-liquid phases and conditions. I'll look up what I did there (in my Ph.D thesis) in terms of the column. I know it's a different challenge to yours but the principles are roughly the same.
OK - I used 20% benzyl cellosolve on Chromosorb P (brickdust) in 20 foot long columns. Probably not applicable to your situation. I changed the carrier gas to hydrogen (instead of nitrogen) for increased sensitivity and ran at room temperature. But this gives you an idea of what I tweaked from the original and ridiculous AgNO3 on I think the same packing material (Chromosorb P).
Dr Alan, I tried 25cm column. The result was bad and 15cm column apparently shows better peaks. So I decided to stick to 15cm column. I tried to prepare mobile phase water in acidic condition with pH 3. The peaks improved but not fully separated. The HPLC that I use now dont have oven temperature so I cant adjust the temperature. I'm thinking to change mobile phase ACN to mixture of ACN and Methanol but not sure the ratios to start with. Here the current chromatogram with for solven B (ACN) 0-3 min (5%), 3-5 (5-11%), 5-10 (11-11.05%), 10-19 (11.5-12%), 19-32 (12-12.1%)
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