Peak due to gradient

[stelef]

Dear All,
I am using a gradient program (Solution A: 2.5 mL Triethylamine in 1L of water, pH 3 adjust with trifluoroacetic acid and Solution B: MeOH). In diluent blank and instrument blank appears a peak which has the same RT with one of my impurities. I tried to change the gradient and the flow rate, but the peak and the peak of the impurity are affected the same way. Have you any idea how can I solve this problem?

[Gerhard Kratz]

First of all increase reequilibration time, than try to replace Triethylamine by another ion pair reagent and than replace MeOH by Acetonitrile.

[stelef]

Thanks a lot! I will try it and in case of failure I will ask again for help

[Alera]

Can you give more details, i.e. gradient conditions, detection wavelength, RT of the peak (chromatogram is better). Why are you using this mobile phase system (wouldn't be my first choice)? Do you have flexibility to use a different column?

[stelef]

My gradient is: Time (min): 0-5-10-20-35-40-50-60-65-80 Sovent B concentration (%): 7-7-20-30-50-50-70-70-7-7, the peak comes at ~38 min, wavelenght: 254nm. I have read about this mobile phase in a paper, I tried it, and except this problem I have good peak separation.

[Alera]

Obviously, you are dealing with a "system" peak, most likely it comes from one of the components of the mobile phase (triethylamine, TFA, MeOH or even water), especially if you see it in a blank (0uL) injection.

Two ways to solve the problem - figure out which reagent produces the peak and try to replace it with different lot/better grade. Second choice -separate it chromatographically and live with it.

Couple of notes - your gradient looks a little bit complicated to me (too many steps). You should be able to replace it with a simpler one, like 7-70%B in 5-50 (or 60) minutes. It would be more reproducible in general and between different systems.

The simplest thing you can try to separate the peaks is to change selectivity by adding some ACN to your MPB (say try 25 and 50% ACN in MeOH).

Let us know how it goes.

[stelef]

Thanks a lot!

[DR]

Could easily be a water contaminant. Search this board for "Empore extraction disk" for posts on how to clean up the water before making the A-phase, or for a slightly less effective solution - works only if you have a high pressure mixing system - put a guard column in the flow path between the A pump and mixing chamber.

[carls]

Stelef,

If the intensity of the interfering peak increases with increasing equilibration time (try different equilibration times, e.g. 10, 15, 20, 25min) then the contaminant is in your mobile phase. If it doesn't increase with increasing equilibration time then the problem is elsewhere.

I have most commonly found the problem to be in the aqueous component. You can determine which is contaminated by switching the organic to ACN. Its very unlikely both ACN and MeOH will have the same impurity.