If it didn't happen before, I'd be less inclined to blame manual injection technque, and I'd look closly at the syringe first, espeially for a partially blocked needle ( liquid doesn't squirt straight out ), or a leak back up the plunger or at the needle seal ( if removable needle type ) .
Then I'd do an experiment, I'd inject replicates of 0.2, 0.5. 1.0, 1.5, 2.0 ul. I would be looking for trends. Your data is confusing, there is no trend, which suggests you may have a spurious factor, like leaking septum, or injector flows or temperature not consistent. How does the total area compare with previous times when it was working OK?. If time is important, select C12:0, C18:0 and increase oven temperature for a short 5 - 10 min isothermal analysis. Plot total area against injected amount.
Watch the total area ( excluding solvent ), that tells you how much has been injected, and make sure your instrument has not changed eg the split mode is on. I assume retention times are very constant, if not investigate - leak most likely cause. The fact that there is no trend suggests that a leak ( internal or external - septum, O-ring, bottom seal, syringe, column joint ) may be an issue. Check integration, make sure peak heights and peak areas correlate with earlier data.
I'd also consider using a less volatile solvent for FAMEs, iso-octane or n-Heptane worked OK for me, but if it worked OK in the past with hexane,
I wouldn't change solvent now, except perhaps to flush the syringe with if you still have problems. Make sure you syringe cleaning is OK by injecting solvent and flat-lining.
Please keep having fun,
Bruce Hamilton
