Help me to improve my signals in LC MS :)

[KevinBe]

Hi All,

So here is another question from the neophyte that I am.

We got a new LC MS installed two days ago (soo it has all the right specs at the miment) and I've been playing with it and I find the signals a bit too low to my liking.

Column: 150x4.6mm 5µm C18 - 40°C
Flow : 0.8ml/min

MS: DUIS
Scanning: 100 to 450 m/z in positive mode

Solvents: A H2O with 0.1% HCOOH, B: MeCN with 0.1% HCOOH (I tried with MeOH + 0.1% HCOOH with similar results).

Run: 5% B at 0 min - 95%B at 9.5 min. Pressure of about 75 bars

I'm trying to observe fluorinated esters and they are basically merely sticking out of the baseline...

Should I:

Get another column? (100x2.1mm 3um?)
Slow down the flow rate? To what? I guess I'll have to extend the run too...
Scan from 120 m/z? Is this a limitation that you can't see "small" molecules?

I could also start playing with the horizontal position of the spray nozzle, but I'm not sure ...

Thank you so much for any help you could offer to a newbie!

[James_Ball]

Is it a single quad?

Not sure about the low mass analytes on those, but with MRM I run Picrate with good sensitivity. Full scan LCMS normally has more background to deal with, especially at low masses.

Definitely go with lower flows as the ESI is sensitive to the concentration of analyte in the effluent flow so same ng of analyte with lower flow will be more sensitive. Also with less flow you bring in less of any contaminates in the mobile phase so less background too. For MS to work you have to evaporate all of the mobile phase, so lower flows helps make that more efficient also.

Another thing that helps is the smaller diameter and smaller particle size column should give a sharper/taller peak for the same mass of analyte which will make it more visible above the background.

[H.Thomas]

Dear Kevin,

a few answers to your questions (just my personal opinion...):

> Should I:
>
> Get another column? (100x2.1mm 3um?)
> Slow down the flow rate? To what? I guess I'll have to extend the run too...

Definitely you should get a 2 mm column. Though the MS will work with 0.8 ml/min, sensitivity will improve with smaller flowrate. And you save money on solvents

Run time does not necessarily increase if you use the smaller diameter column. You should get similar results with a 4.6 mm column at 0.8 ml/min and a 2.1 mm column at ~ 0.2 ml/min (assuming same phase and particle size). With the 4.6 mm column reducing the flow will degrade your separation.

> Scan from 120 m/z? Is this a limitation that you can't see "small" molecules?

You can see small molecules. But your solvents and additives also give signals in this low mas range. You should use the scan mode only for method development. Look in the postrun at the selected ion traces of your fluorinated esters. Now you will see much less noise and more signal. If you acquire your chromatograms in SIM mode, you will have even better S/N.

[KevinBe]

Hi,

Thank you so much for answering the question of the neophyte that I am!

Yes it’s a single quad.

1. I thought that the full ion scan was the way to go, a bit like what I do in GC-Ms when I try to identify unknown compounds in a reaction mixture. I guess it’s harder to do that by HPLC?

What’s your standard cutoff when you scan? 150?

I will definitely order a 5cm x 2.1mm with 3uM particules and see if it helps. Thought the column guard already adds 2 extra cm

I still have a few questions:

2. I have a dual ionisation source. The Corona needle is at 4.5kV and I noticed that when my flow was at 0.5mL/min I have basically 0 corona current! It comes back to about 5uA when I flow at 0.2mL… I guess that basically tell me that I can’t go much higher than 0.2mL without flooding the needle. My drying gas is at 15L/min.

3. Very often my most intense ion for some compound is (M+CH3CN)+ - is there a way to avoid this adduct? Imagine if I didn’t see a nice peak in the scan mode… how could I guess that this is the ion I should put in my SIM?

Thanks a lot!

Kevin

[H.Thomas]

Hi Kevin,

I'm not familiar with the DUIS-Interface, I just use ESI. But it would surprise me, if you can't go higher than 0.2 ml/min. You may ask a Shimadzu Application Scientist about that - they're usually very helpful.

You don't want to perform quantitative work in scan mode. SIM will improve sensitivity by orders of magnitude.

To get rid of CH3CN-adducts is simple - use methanol

To find the ions you are looking for: set a small scan range around the molar mass of your analyte, eg. MW±40 or 50.

[KevinBe]

Thanks a lot! I really appreciate you help!