Chromatography Forum

Hello all,

I have recently setup a method for an acidic compound ICG, using a 2.1x100mm 3.0micron C18 column.
My mobile phases are A) 10mM Ammonium Acetate pH 3.0 B) ACN
My gradient is as follows.

T0 40%B
T1 40%B
T1.5 90%B
T2.5 90%B
T3 40%B
T6 40%B

Any ideas as to why this is coming off with separate retention times? I have played with the gradient as well as isocratic, but still observe this effect.
I did prepare this 10,000ng/mL Standard in mobile phase, with the exact composition as starting T0.

Thanks,

Does anybody have an idea what this could be or how to resolve?

Do you know the purity of the material that you used to prepare the standard ? and does it correspond to the peak areas by any chance ?

Peter

Blank run free of this small peak ?

Hi Pepter,

Yes the blank is indeed a blank, the purity is greater than 90%

What could be going on?

Hi Pepter,

Yes the blank is indeed a blank, the purity is greater than 90% The ratio of peak areas is about 95% in the larger peak, 5% in the small one

What could be going on? You have up to 10% of an impurity - and an unknown peak at about 5% of total area - maybe that is a clue

Peter

Yes but the impurity will not be having the same molecular weight and transition ions.

Also, this peak is not evident in the other published methods with samples as low as 1ng/mL.

Additionally, as I play with the gradient , as well as injection volumes and flow rates, fragmentor voltage, and collision energy

this ratio changes...

I had a tough time initially with optimizer because the software wants to optimize the first peak automatically, so I set abudance limits for

the precursor and product ions, then the optimizer optimized the fragmentor voltage to around 190 and CE at around 37...

Did you check info about degradation products of ICG ? (make a test vial that will be expose to intensive light than make a analyze of it)
No chance to add confirmatory ion for ICG, only one product - 330.2 ?
Does this impurity scale with change of ICG concentration ?
What is your qqq resolution unit/wide/widest ?


Optimizer:
Option where you give the retention time and window for your compound doesn't work ?

Iam not an expert but maybe some questions will help you to find answer.

Are you sure your impurity won't have the same mass and transition ions? If the impurity is a close relative of the target compound, for example with an extra fairly labile group, it may show up as a source fragment (i.e. it may form in source a fragment that is identical to the target compound). Similarly (but more rarely), if the change in the impurity is something that causes relatively little mass change (eg -COOH to -CONH2), it will still be close enough for an isotope peak of the impurity to fall in the window for the expected parent, and it may undergo the same fragmentation.

Remember, too that the relative peak areas of the impurity and the target compound are not necessarily a good measure of the relative abundance of the two in your sample; they will probably have different ionisation efficiencies.

This is really quite annoying, I am not able to setup a quant method because of this.

Interesting thing is that the MS2 Fullscan method comes up with only 1 peak.

However, when I run optimizer using the precursor ion, two peaks become evident.

Something seems to tell me that there is a problem with the Product Ion Scan, whereby the precursor is fragmented , and then the multiple peaks are evident as in the image above.

Does this extra peak gives a shoulder? or is it completly seperated?

Seems that it is an inevitable impurity. Should not affect my Quant.

Seems that it is an inevitable impurity. Should not affect my Quant.

But you still need to take the (im)purity of the standard into account when you run your calibration.

Peter

Have you tried modifying the pH of the mobile phase?

I did change the Ammonium Acetate Buffer to a pH to 5.0 using Formic Acid.