solvent peaks elute rather than analyte

[jamie_sfs]

Hi everybody,

I'm a newbies of HPLC,hope expert here can give me some guidance.

I face a problem with solvent peaks elute rather than my samples.

Here is my sample

I couldn't identify which peak is my sample peak, all the major peaks were also found in my calibration and also methanol blank injection.

Here is chromatographic condition:

mobile phase: A - 0.1% Trifluoroacetic acid in water
B - Acetonitrile
Gradient : 5% B for 5 mins,then increasing to 95% B over 20 mins, hold at 95% B for 5 mins.
Temperature : 60 degree
flowrate: 0.4ml/min
injection volume : 2 μL
Detection : 250 nm (VWD)

I use methanol for sample extraction. All gradient used were HPLC grade.

[HW Mueller]

Did you set up the method with standard?

[jamie_sfs]

Yes.

My standard calibration range from 6~200 ppm.

I tried another gradient, and i got my sample peak,however result was much more lower than theoretical one.

[HW Mueller]

I don´t follow what you did. Standard was ok at different concentrations, after injecting sample you get massive carryover?
How do you know what the theoretical peak size should be?

[jamie_sfs]

Is actually the theoretical content of target component, usually referring from literature/journal.
Eg. Caffeine of soluble coffee : 3~5%

And my result only show 1% or lower.

[HW Mueller]

You didn´t answer a question.
You are basing your results on a sample whose content you don´t know?? With this "dirty" chromatogram?

[tom jupille]

jamie_sfs, you are not providing enough information for us to understand what you are doing.

What did your calibration runs look like? That means obtaining a standard of pure caffeine, preparing six standard solutions at different concentrations (covering the range of concentrations you expect to see), running each standard solution, and plotting peak area as a function of concentration. Can you link to the chromatogram of your highest-level standard so we can see what's going on?