Chromatography Forum

I'm dealing with HPLC to analyze gamma aminobutyric acid (GABA) with the following method:
-Sample pretreatment: 100ul GABA + 100ul 0.2 M carbonate buffer pH 9.8 + 200ul Dansyl chloride (8g/l) --> mix and heat block at 80 degree for 40 min---> add 8ul Acetic acid (10%).---> mix then centrifuge (12.000 rpm for 5 min)
-Mobile phase: Eluant A: THF/ Methanol/50mM sodium acetate pH 6.3 (5/75/420)
Eluant B: Methanol
-Column: OptimaPak C18 (4.6 X 150mm)
-Elution conditions: Equilibration 6 min (20%, B)
Gradient 20min (20-80%, B)
Cleaning 3 min (100%, B)
flow rate: 1ml/min
measurement : UV 286nm

But there is one thing happen.
With small concentration of GABA I got the higher peak than the higher concentration.
Have you ever face this problem before?
Can anyone explain for me about this. Why did it happen and how to solve the problem?
Sometime I obtain peak, but sometime doesn't, why?

Please help me (
Thank you so much.....

If this were my problem, I would begin with a single standard solution of GABA, somewhere in the middle of your concentration range and run the derivatization a number of times (6 x should suffice to check for consistency). If you see major variations, check for autosampler problems, then take close look for errors on the derivatization (mixing time, pipetting, etc.).

If you get consistent results, then repeat with standards at the top and bottom of your linear range.

what is your sample solvent?

maybe try also this:
mix the sample and the buffer well before adding the dansyl chloride

@Dr. Tom jupille
I did 1 single standard gaba concentration (10mM) for 3 times but I got the same results (same peak height and retention time) ---> it's ok
But I'll try again as you recommended me and check whether the auto sample is right or not

@Dr. Hollow
I dissolved solid gaba by HPLC water
I'll try again as you recommended

I wonder if the problem might relate to high analyzed concentration or not
Because I analyzed gaba at concentration of 5mM, 10mM, 25mM (final concentration in the mixture prepared sample)

Thank you so much
I will try again and
I would like to have your advice if it still have any problem