[gingi85]

Hey all!

I am using a single quad MS system. Does it make sense that I would need to inject a full nmol of analyte to get a half decent looking mass spectrum? That seems like an aweful lot, but I'm not sure what I can do to decrease that amount?

Another question: While I am getting the expected target m/z for my molecule my isotopomer distribution is incorrect and varies from injection to injection. Any ideas as to what might be going on?

[MOTOBALL]

Sensitivity depends on,

1) method of ionization (ESI or APCI)

2) mode of ionization (+ve or -ve)

3) structure of analyte

4) composition of solvent

5) sometimes flow rate

6) MW of analyte

7) m/z scan range

I think that you are getting variable/incorrect isotopic distributions is because the signal (m/z value) is "dropping out", i.e. is intermittent during the data acquisition; this may go back to 4) above.

If you care to fill in some of these blanks, and especially post the spectrum, I'm sure that somebody will be able to advise.

[gingi85]

Thanks for your reply. Here is some more information:

1) ESI

2) Both. Although, slightly better sensitivity in negative

3) A series of modified nucleotide tirphosphates that have been labeled with a fluorescein-type dyes.

4) The analytes are dissolved in a triethylammonium bicarbonate buffer of an unknown concentration. This is essentially an LC/MS system for which I have removed the column. I am injecting about 10uL of the analyte solution. The mobile phase is 0.1% formic acid solution.

5) 0.5mL/min

6) 1300-1600g/mol.

7) 500-2000m/z

The analyte in the attachment has a molecular formula C48H49N8O26P3S3. The first attachment is in negative mode spectrum. The two large cluster of peaks correspond to the singly and doubly charged ions. The second attachment is in positive mode. I am assuming the peaks I am seeing are the result of triethylamine adducts from the buffer. 

Any advice would be greatly appreaciated!

[Babcock_Hall]

I am an occasional consumer of mass spectral information, not an expert by any means.  Are you doing selected ion monitoring?  It seems as if this might improve one or both of the problems you mentioned.

[MOTOBALL]

Your update of info. is good.  Initially, let's disregard considerations of HPLC separation, just focus on flow-injection analysis (FIA).

1) Since you get better sensitivity in -ve mode, you should go to using the same mobile phase as the solution in which the analyte is dissolved.  You want -ve ions, so no acid additives !!

2) Just for set-up purposes using flow-injection analysis as you are at the moment, can you acquire data in the continuum mode where each scan is loaded into the same box ??  In other words you get only a single scan from each sample injection.  This will let you see the isotopic ratios quite clearly.

3) ESI proceeds more readily with a solvent system of low surface tension (MeOH/H2O, 90:10 v/v) than one with a high surface tension (MeOH/H2O, 10:90 v/v).  Can you include some MeOH (as much as you can without causing precipitation) in your mobile phase for these scouting expts ??  The fact that you are running in a purely aq. mobile phase (no organic component) is consistent with my thoughts regarding the signal drop-out.  Water has a very high surface tension and does not want to fission down to a cascade of increasingly smaller droplets in  a smooth fashion.  Much more of a pop/splatter type of thing.

4)  Set up to do FIA, with modified conditions as above.  Drop the flow rate to 10-20 uL/min, and tune the ESI/source parameters whilst observing the signal intensity and peak shape.

5) when you are satisfied with (4), go to HPLC if you need separation of a mixture.

If you need identification, then full-scan is required; but, as BabcockHall points out, maximum sensitivity is achieved by selected ion monitoring.

Good Luck tomorrow; let us all know how it goes.

[gingi85]

Thanks, all for your replies.

MOTOBALL, I'm not sure what you mean by "loading each scan into the same box." I'm not sure if I can set it to only do a single scan per injections, since it is constantly cycling through the scans for the duration of the run. I'll take a look...

I will also make sure to add some sort of organic solvent to by mobile phase and see if that gives me better results.

[MOTOBALL]

Data acquisition in the continuum mode scans over the specified m/z range, but instead of generating say 50-100 scans using FIA, all spectra are essentially superimposed one on top of the other.  You end up with 50-100 scans acquired, but the output is a single spectrum with (usually)much improved S/N.

[MOTOBALL]

Any progress yet ??

I should have stated in a previous reply that one uses the MS instrument in the multi-channel analyzer (MCA) mode to acquire low-level signals superimposed on top of each other, i.e. one single spectrum generated from many scans into one "box".

The continuum mode, per se, does not improve S/N but does allow the isotope ratios be seen more accurately.