Chromatography Forum

Hi,
At first, I determined a sample by Shimadzu 10A(made in Japan), I found an odd peak just elute after the main peak, which is about 10%（account by area normalization method）
With same column (C18), mobile phase, temperature and same sample but different Liquid Chromatograph—Dionex U3000, I try it again, the peak just disappear.
I am not sure about the result. So, today I use another chromatograph
(Agilent 1100), and the peak appear again(about 10%)
So I want to know, Does different chromatograph causes different results(I mean the peak numbers)?
Anyone help me?

Gradient
Time ACN(0.1%TFA) Water(0.1%TFA)
0 35% 65%
10 47% 53%
10.1 35% 65%
15 35% 65%
shimadzu chromatogram

Dionex chromagogram

I was wondering if you are using a gradient method. Chromatographs you have tried have different dwell volumes which will result in different "real" gradients.

It would be more helpful if you let us know more about your method. An embedded chromatogram would be fantanstic.

I was wondering if you are using a gradient method. Chromatographs you have tried have different dwell volumes which will result in different "real" gradients.

Can't that be done isocratic? Doesn't look too challenging...

I was wondering if you are using a gradient method. Chromatographs you have tried have different dwell volumes which will result in different "real" gradients.

It would be more helpful if you let us know more about your method. An embedded chromatogram would be fantanstic.

I've pasted the chromatogram and the graident.The peak 2 in Dionex chromatograph is 0.04%(realated area), well, it's up to about 10% by Algient 1100 and Shimadzu. I can't uederstand......

The first place I would look is at the wavelength and bandpass settings on the three systems, particularly if you are on a sloping region of the absorbance spectrum for that impurity peak.

Hi, Tom
Thank you for your advice.
I've checked the wavelength and bandpass settings, there is nothing wrong. I also checked the DAD chromatograms again, the wavelength I chose is right. I mean it's not on the sloping region.

Ok, next question: what size column and what flow rate?

If you are using a 150 x 4.6 mm column at 1 mL/min flow rate, for example, then your column dead time will be around 1.5 minutes, which means that the neither peak is well retained, in which case a dwell volume difference (as suggested by rwang) could result in "funny" peak shapes and responses.

A good rule of thumb in gradient LC is that the first peak of interest should elute no earlier than 3x the dead time in order to get away from t0 anomalies.

In addition to the comments/suggestions above, I’d suggest you to lengthen the equilibration time, because I don’t think your column is equilibrated in just 5 minutes.
Finally, you’ll need to start with a lower ACN concentration and make the gradient even shallower in order to retain the compounds adequately and thereby achieve a reasonable separation.

Best Regards

yeah, I think the stationary phase can't retain the compounds.But when I reviewed literatures I got this method, I still can't figure out why they chose this methdo. And do you really think that dead volume can cause a peak so "big"(about 10%) ? I'm not sure about that.
Latter I will change a better method.
Thank you !

yeah, I think the stationary phase can't retain the compounds

I’m sure it can, but as mentioned earlier you need to start with a lower ACN concentration.

Best Regards

As was stated - you should start with lower AcN concentration something like below. Also increase your equilibration time.


Gradient
Time ACN(0.1%TFA) Water(0.1%TFA)
0 50% 50%
15 35% 65%

Thank you all, I'll try it latter.