Solvent, analyte same Rt?( HPLC)

[LG]

Hi All,

It's my first topic to be posted I'm trying to resolve a problem connected to solvent interfering with analyte. It seems to have the same eluting characteristics with one of the component of the mobile phase. I can't tell which one, because I don't have the sources to resolve this matter. The peak area of the solvent is about 1500 and the standard is just 2000. Their RT's are exactly the same. Unfortunately the concentration of this Impurity is quite low, 100ppm. I checked whether it's a carry over but it's not. The needle was washed several times before trying a new injection of standards. The mobile phase consist of THF, ACN and ammonium buffer, adjusting the pH with ammonia. I might try to check with different manufacturers to replace the buffer components one by one..or try a different column?
Any ideas?
Thanks in advance,
L.

[danko]

What happens if you only inject pure water?
Is it a gradient elution or an isocratic one? If the former, would you mind describing the gradient scheme? Finally, please state the column dimensions, flow rate and last but not least; the retention time of the peak/s in question.

Best Regards

[LG]

Firstly, thank you Dancho for the quick reply.
There are the answers:
Isocratic elution, C18 250*4,6mm, 5um, 100A
Rt around 4min, 1ml/min
I haven't tried to inject pure water, but I will.

Thanks,
L.

[Alera]

Mobile phases and detection wavelength would be helpful as well. How do you know it is something in the mobile phase, have you done a blank (0uL) injection? Can you provide a chromatogram?

[danko]

LG,
The fact that the elution mode is isocratic indicates that the problem originates from the sample solvent, rather than the mobile phase.
The other conclusion - that can be drawn from the supplied information regarding column dimensions and flow rate - is that the interfering peak is somewhat retained i.e. has hydrophobic properties.
So, if you inject both pure water (you’ll probably won’t see any peak) mobile phase (I wouldn’t expect any peak here either) and finally the sample solvent (blank) you’ll probably see the peak in the latter injection. Depending on what you see, the solution will be some kind of revising the solvent ingredients.

Best Regards

[annaand]

Why not to prepare samples by dissolving them directly in mobile phase. This way you shouldn't have any solvent peaks.
Anna

[LG]

Thanks to All,

I suspect it's coming from the mobile phase because it first appeared at the first blank injection and kept appearing since than. It's also just a bit smaller than my impurity in the standard. I dissolve the sample and standards in mobile phase which is my blank solution as well. UV cutoff at 254nm.
All the components are HPLC grade except the ammonia solution I used for adjusting the pH, but we normally use this particular solution and never had any problem with it, moreover of course I filtered the mobile phase.
Ordering ammonia solution from different manufacturer?

[LG]

..but Danko, maybe you are right and it's too hydrophobic and retains on the column. It needs to be flushed with a strong solvent I presume. I might tell you more tomorrow after I will have tried you have suggested. I'm quite new in this area, so thanks again for Everybody's help!

[Alera]

Hey LG,

I bet it comes from your injector, since you still see it even when you inject mobile phase. Have you tried another system? Do you have any flexibility to change the method? If you provide mobile phase composition and a chromatogram, I can make some suggestions how to tweak the method to separate the impurity from this system peak.
Rebuiliding the injector might be an easier way to go.

[LG]

Thanks Alera,
I couldn't make any progress today, unfortunately I had to deal with different difficulties. I'll post the chromatogram tomorrow. I haven't tried a different system so far...Flexibility? Within a certain limits I can change in order to be successful i have to I suppose...