nanoLC-MS Peak Broadens at Low Concentrations

Discussions about GC-MS, LC-MS, LC-FTIR, and other "coupled" analytical techniques.

4 posts Page 1 of 1
I am developing an LC-MS method for a dibenzazepine derivative and am looking for some assistance creating an acceptable peak. I am using a Thermo Ultimate 3000 UHPLC system, a Thermo EasySpray C18 column/ionization source, and a Q Exactive HF-X MS. Normally this system is for proteomics, but I have used it for small molecule work in the past with good results. Additionally, I am working with a purified solution of my compound.

Below are two EICs of my compound. The top figure is from a 20 ul injection of 100 nM solution. This looks like an okay but overloaded peak to me. The bottom figure is either a 2 ul injection of 100 nM or a 20 ul injection of 10 nM, both generate the same result. As you can see, the peak has broadened into obscurity. What could be causing this an how could it be corrected?


Compound Information:
I cannot give the exact compound, but I can say that it is dibenzazepine with an additional modification. The modification contains an aromatic region. The entire compound contains no acidic or basic residues. It is obviously very nonpolar.

Other Relevant Info:
-Buffer A is 0.1% FA in H20, buffer B is 0.1% FA in ACN. The above figures used a 5%-95% B gradient over 1 hour at 0.35 nL/min
-The UHPLC is plumbed with a C18 trap column for desalting such that sample is first loaded on the trap column, then the flow is reversed and sample flows off the trap towards the resolving column and MS.
-The loading buffer is 2% ACN 0.1% FA

What I've tried (none of it made any difference):
-Removed and retightened all lines in the fluid path
-Replaced all solvents and glassware
-Replaced column/ion source
-Replaced trap column
-Spiked in other small molecules to look at peak shape. They are fine so I don't think it's a dead volume.
-Tried using 10 mM Ammonium Formate with 0.1% FA as buffer A
-Tried spiking peptide digest into the sample
-Tried using MeOH instead of ACN in buffer B

Any thoughts or help would be much appreciated. I can provide some additional info if it will help.
I would like to see the respective LC-UV traces to determine where this peak broadening occurs.
Suspect that it is post - LC system.
JMB wrote:
I would like to see the respective LC-UV traces to determine where this peak broadening occurs.
Suspect that it is post - LC system.

Unfortunately, there is no UV module in this system.
I’m wondering if, at the lower loading, there is not enough of the analyte to coat all active metal sites.
So the drug sticks initially, and is slowly bled off by subsequent LC flow.
At the higher loading, the metal sites are coated instantaneously and the vast majority of the analyte does not see any uncharted active sites.
Can you change source parameters (temp; voltages etc) to to improve the profile?

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