Newbie question FAME detection GC/MSD

[redpanda]

I'm a new postdoc fresh out of grad school and joined a lab that has a "new" Agilent 5975c-MSD that has been a giant paper weight for the last two years (well maintained, just not used). I have limited experience with GC - mostly working with analysis of amino acids under the supervision of people with far more experience than I have. Most of my mass spec background is in LC - metabolomics.

We are a lipid lab and I wanted to utilize this instrument to analyze fatty acids. My first attempt yielded mixed results. I ran the Supelco FAME 37 ( in methylene chloride) which I diluted in hexane in the following concentrations 1mg/ml, 100ug/ml, 10 ug/ml, 1ug/ml and 100 ng/ml.

I ran the method that was already loaded for FAME's

Column: HP-5MS 30 m x 0.25 mm x 0.25 μm
Injection: 1 ul
Method: 50oC 1 min, 25oC to 175oC, 4oC/min 235oC
Split: 1/50
Carrier: He


Two problems:
1) My chromatogram only had 28 peaks corresponding to 24 FAMEs. Most of the longer chain FAMEs were missing >20:0 and the 18:0 was also missing.

2) I only had peaks from the sample at 1mg/ml. I ran the samples in order of lowest concentration to highest - so this would have been the last vial to run in the series. Total volume was 100 ul in a vial with 100 ul insert.

I am a little lost, I am used to LC where I can tweak the method a bit and get better results. I tried a few tweaks, but nothing seamed to work. Any suggestions?

Thanks

[Peter Apps]

With your 50:1 split, your 1 mg/ml mixture puts 20 ng on the column, which should (and does) give a detectable peak. At 100 ug/ml you have 2 ng on column, which should give a decent peak if the MS is tuned properly (and there is nothing else wrong), 0.2 ng will be pushing the limits for a FAME which fragments into several ions with no one large selective ion that you can monitor. So if you want to get peaks all the way down to your most dilute standard, use splitless injections. That you do not see a 2 ng peak suggests that they system could do with some maintenance.

I suspect that some of the peaks in the mixture overlap completely on the DB5 column.

Peter

[MSCHemist]

We had a thread a few pages down on FAME's on a db-5

viewtopic.php?f=2&t=23740

[James_Ball]

With your 50:1 split, your 1 mg/ml mixture puts 20 ng on the column, which should (and does) give a detectable peak. At 100 ug/ml you have 2 ng on column, which should give a decent peak if the MS is tuned properly (and there is nothing else wrong), 0.2 ng will be pushing the limits for a FAME which fragments into several ions with no one large selective ion that you can monitor. So if you want to get peaks all the way down to your most dilute standard, use splitless injections. That you do not see a 2 ng peak suggests that they system could do with some maintenance.

I suspect that some of the peaks in the mixture overlap completely on the DB5 column.

Peter

Splitless injection or splitless with a uniliner even for maximum sensitivity, or first try going to 5:1 split which should improve your sensitivity 10X and see how that goes.

If you really want to separate all the isomers you will need a specialty column, the db-5 phase just won't quite do it.

This is what we have used in the past to separate the cis-trans isomers. http://www.restek.com/catalog/view/1439

[Consumer Products Guy]

1. I'd extract ions of 74 and 87, should show in the saturated FAME, so one can readily identify those.

2. You didn't list an inlet temperature, was wondering if you need to increase that. And that can affect inlet discrimination for the bigger molecules.

I say this, because we have no trouble getting C18 and C20 (and larger) FAME through Agilent GC-MSD here.

I'd also check your ramp rate of 40°C/min at the end, as depending on Agilent GC model and temperature range, may not be able to keep up with this fast a ramp, even though you can enter it. Check your operating manual, lists which rates and temperature ranges are compatible with standard oven.

This sounds like much more of a "GC Issue" than a "GC-MSD Issue". Once you get the molecules through the column end: you're there.