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- Posts: 1
- Joined: Sat Jan 26, 2019 3:46 pm
I am a undergraduate student working on the bioactivity of certain herbs and plants. I have been trying to further separate my extract using HPLC to test separate compounds for activity, but I have encountered some problems adjusting my gradient.
The chromatogram is shown here: . I am using a C18 sunniest column with10.0 mm x 150 mmParticle size 5 µm
The gradient is as such with A: 0.1% TFA Milli Q and B 0.1% TFA Acetonitrile.
Time A (%) B (%)
0 55 45
25 10 90
25.1 0 100
30.0 0 100
30.1 55 45
(Recalibrate for 10 minutes at 45% B)
I'm mainly interested in the 6 peaks labeled in the picture, and I have collected all of them together.
however, I am not sure how to cleanly separate them after. I have so far tried:
(a) A gentler gradient (55%B to 90%B over 25 minutes) but the peaks are slightly broader and still kind of overlapping
=> I am not sure if I am understanding the concept of gradient elution properly, because I presumed that a gentler gradient would have given me better resolution (by increasing k) . Perhaps changing the starting %B is a problem?
(b) isocratic gradient at 30% -> The main peaks (Peak 2 and Peak 5, Peak 6) are nicely separated but analysis time is long and peaks are broad to the point that small peaks (Peak 1,3, 4) cant be detected properly.
Any advice or even a general direction would be greatly truly appreciated.