Unwanted peaks in bank

[rc_12321]

Hi, friends , I have been facing this problem for the last one month.We received this method from the supplier .The method details as follows,

Mobile Phase-A: 1.2 gm of Monobasic Potassium phosphate in 1000 mL of water adjusted pH to 2.7
Mobile Phase-B:Acetonitrile

Column:Zorbax-SB-C18 :Temperature-30 C;
Gradient:Time/% B-0/3, 3/3 , 20/50, 28/50, 32/70, 60/70, 65/3 70/3

Wave length -230 nm

Blank: Mobile Phase-A :B (90:10)

The probelm is that we are observing a lot of junk peaks in the blank injection.We tried all options including even passivating the HPLC system.After passivation junk has been reduced and every thing is good except that one blank still eluting at the RT of active .We communicated to the supplier but they told us they had never faced this kind of problem .

What could be the reason for this ? Please help us.

[Uwe Neue]

Most likely, the junk peaks come from the mobile phase. There are 50 million posts on the Chromforum that deal with the same situation. Check our related posts.

Contaminants are most likely in your A mobile phase. But before you investigate this, make a blank run - no injection. If your system does not let you do this, you can run a duplicate gradient with a single injection. Just copy the entire gradient table again to your original gradient table and run the thing again, including the reequilibration procedure between normally two runs. If you do not get any peaks in the fake second run, the junk if coming from the injector, and you need to see what you can do to clean it.

If the junk pattern is similar (maybe just a bit less) in the fake second run, then it is coming from the mobile phase. You can prove this further by varying the equilibration time with the initial mobile phase composition. If the patter increases with increasing equilibration time, it is coming from the initial solvent composition.

Now you need to figure out the cause of this. You need to examine EVERYTHING that your mobile phases come in contact with... Containers, degassers, stirrers, filters... whatever, plus change the source of the chemicals, such as the source of the water, the organic solvent, buffers (less likely in your case) etc.

Sometimes thinking about the problem up front can lead quickly to a possible source, such as cheap chemicals, unsuitable filters etc.

[tom jupille]

To follow up a bit on Uwe's (correct) answer, we've put together a "mini-tutorial" on this problem:
http://www.lcresources.com/more_resourc ... hp?f=3&t=5

You will have to register (free) to access the tutorial.