Chromatography Forum

Hello every one,

i am trying to develope HPLC method for B carotene. there is a good peak on chromatogram but peak has a shoulder and i am strugling to get rid of it. In following link If you see the peak shoulder for peak MDA 3.650, my problem is pretty much same as shown in figure though in my case i dont have 2 peaks.



Chromatographic conditions i use are;

Mobile phase: Methanol: dichloromethane (75:25)
Flow rate: 1.8 ml/minute
Detection wavelength: UV-absorption 450 nm
Temperature: 40C°C
Injection volume: 20µl
Run time: 10 minutes
Retention time: 5 minutes
Column: Agilent eclipse XDB-C18, 5μ Particle size, 4.6 x 150 (L x ID mm)

It will be very helpful to know how can get rid of shoulder peak or if that much of peak shoulder is acceptable

Thanks in advance

So you are looking for more resolution? I would suggest first try dropping your flow rate to 1 mL/min and see if it gets better. If you need more try 80:20 methanol/DCM.

I don't see a "shoulder" on the MDA peak. If you are referring to the small peak at 4 minutes, that's not a shoulder, it's another peak. Both of the big peaks *do* tail quite a bit, however.

Slowing the flow rate down as mardexis suggested may help a bit, but I wouldn't expect it to make a huge improvement. Going to a weaker mobile phase (cut back a bit on the dichloromethane) may also help. You might also want to look at some different columns to see if you can get more favorable selectivity.

Is your baseline this "dirty" also? In this example it just looks like the Analyte is sitting on a series of "dirt" peaks.
If your chromatogram is cleaner and the small peak has the same spectrum as the MDA peak it might be caused by a mobile phase sample solvent mismatch.

Lowering the temperature may also improve your resolution. I think 40°C is quite high.

I think most people misunderstood the question - you just gave an "example" chromatogram (but not the one you are actually getting), showing SIMILAR degree of separation, and you were asking whether it was sufficient or not. It looks like separation is sufficient for most practical purposes (quantitation of the main peak), but keep in mind, if you are developing a method and you are getting this separation on a brand new column, the method is not going to be very rugged.

I would try the advices that you got in order to increase retention on the column. It WILL make your peak wider using isocratic conditions, so think about using a gradient method.