I need to quantify betulinic acid from my sample.
HPLC condition: C18 reversed-phase column (250 mm × 4.6 mm i.d.) with 5 μm pore size column , the mobile phase was isocratic acetonitrile-water with pH3 (72:28, v/v), UV detector at λ = 230 nm. Flow rate and injection volume were 0.5.0 ml/min and the injection volume was 10 μl.
I spiked BA standard into my sample, the peak at 5.1 min was increased, but the peak at 4.9 and 5.1 were overlapping. Does anyone know how to adjust the HPLC condition to separate it?
The peak corresponding to your desired compound is very sharp and clear. You will just have to try different combinations of mobile phase and flow rates to separate the overlapping peaks.
Dear Oscar Chan ,
you have so many options available but I guess changing a isocratic flow to gradient should be your first option. You do not need to do much and as I know what does it mean being lazy just apply gradient.
Best,
Michal
Hi-
Thanks for listing your chromatography conditions
Please verify the flow rate for your column as it reads "0.5.0"- I will assume 0.5 mL/min, which seems low for such a column, I usually run them closer to 1 mL/min.
Given a flow rate of 0.5 mL/min, the compound isn't retaining much on the column, so there is little opportunity to resolve your compound from the impurities- neither the compound nor the impurities have much interaction with the column, so little chance for separation.
To cause more retention, reduce the amount of acetonitrile- this is the strong solvent which causes the compounds to elute from the column. By reducing the concentration, you are making the solvent system weaker, and causing the peaks to elute later. Try reducing the acetonitrile to 70%, or a bit lower since you have some resolution now.
I also suggest running blanks, and also running a positive control of betulinic. The positive control verifies everything is working, and verifies the retention time.
I also question whether 230 nm is the best wavelength, ans there aren't many chromophores in that molecule. You may find more sensitivity at 220 nm, or lower. I didn't see what compounds were being used to buffer at pH 3, but you may be able to use a lower wavelength, as you are running isocratic, so you won't see baseline drift from buffers that absorb UV light.
I do suggest that you look up the theory of chromatography.
Please be cautious. Their are countless invalid published HPLC methods on the web for betulinic acid and it appears that you may have copied one. Several researchers with no HPLC training have copied the same method over-and-over, citing each other's work, but the methods do not retain the sample on the column (elutes at void volume). Some very basic training in HPLC would allow you to spot these fake methods, avoiding the creation of more false methods and accumulation of invalid data. Training is needed.
Please take a look at your results. You have no retention of the sample on the HPLC column. *Look at your Tzero and K prime values. Training in the basic fundamentals of liquid chromatography are needed to use the HPLC. These basics will help you understand what is happening so you can think about how to revise the method to retain the sample(s).
- A few hints: If you wish to use UV detection, 210 nm (4nm) might be a better choice. Instead of a mobile phase with mostly ACN, perhaps a gradient of ACN/ acidified water (with phosphoric acid) or Methanol/ acidified water (freshly made with phosphoric acid) on your C18 column.
Here is free training article link to help you (follow the internal article links for more info): "Do your HPLC Methods Meet Basic Requirements? HPLC Training: RETAIN, SEPARATE and RESOLVE"; https://hplctips.blogspot.com/2019/12/do-your-hplc-methods-meet-basic.html
The two factors of column selection (polar and non-polar) and the size of the coated silica particles in the fixed phase are more influential in this case than others. Also, using the suitable solvents is not ineffective to type of the compounds in the analyte.
Hi Oscar Chan ,
Is the chromatogram in the picture the spiked with BA?
If not, you have also a big tailing problem and could be due to several reasons. You should consult an expert otherwise with an approach trial & error you lose time and money.
This peak is looking good. For a better separation, you can decrease the percentage of acetonitrile or change the acetonitrile with methanol . also try to increase temperature of a column hopefully it will give good separated peaks
This peak is looking good. For a better separation, you can decrease the percentage of acetonitrile or change the acetonitrile with methanol . also try to increase temperature of a column hopefully it will give good separated peaks Aftab Yaseen
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