Chromatography Forum

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.

Did you set up the method with standard?

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.

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?

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.

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

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?