Chromatography Forum

How to firgure out the content of a substance from its peak?

I have know the peak of lutein in the graph and know the Ret time, area, height, area %, height % of that peak.

How to calculate the content of lutein in the sample from the area % and height % ?

I have to hand in this result tommorow. Help me as quick as you can.

Thanks

Please note: I'm a GC guy and I have no experience with LC but as your request seems to be very urgent I think it's okay to just share my thoughts.

Whenever I have to do quantification I take a calibration curve of my analyte to determine the linearity the detector has for my compound. That way I found e.g. that my TCD gives a too high response for very small amounts of water in solvents.

Generally speaking, quantification is a difficult task:

If your detector gives you an (about) equal response for every compound of your sample (including the solvent) and the detector was not saturated (i.e. the main peak(s) are cut off) then just go for the 100 percent method:

The percentage your peak of interest has from the total area of your chromatogram (i.e. the added area of all of your peaks, baseline-corrected) is the percentage of your analyte in the sample.

If your detector was saturated for the other components, height is the way to go assuming your peak of interest is not cut off. That is an older technique coming from the days when integrators were rare (and unreliable) devices so the determination of the area was difficult (cutting out the 'peaks' from the paper trail and weighting them on the analytical balance was one of the more arcane area determination techniques) while measuring the height of a peak just takes a ruler.

The most simple approach (which I found to be quite robust) is the addition method: After you've analyzed your sample you spike it with an additional amount of the compound of interest. Assuming your detector response is linear, your detector doesn't get to full scale etc. you can (somewhat) safely determine the content of your component.

No matter which way you prefer a calibration curve is the way to go for a sensible quantification IMHO.

If you don't have time (or access to the instrument) check your previous runs if you've chromatograms of samples which contained a known amount of lutein.

You will need something to compare your values against. Unless you have determined the response of a standard of known concentration (I guess that's what a standard is), you have no way of calculating the concentration of your sample.

As HPLC is a relativ method you have to use a standard of known content.

make an injection of a lutein solution of known concentration and measure it just the same way as you did with your sample.
Adjust the concentration of the standard solution so you will somewhat similar
peaks heights

Calculate the concentration of your sample according to the equation:

Area/Concentration=constant (if the injection volumes of smpl and std are identical)

Thank for your help..
Please give me more detail help.
I already have had the pure lutein, pure beta-caroten and pure lycopen that I dillute and run HPLC. (so I have had a chromograph of a known concentration sample)
So please give me some formula, so I can calculate the concentration of lutein in the sample through the area% and height% of the peak of lutein in the chromograph of the sample and the chromograph of the pure dilluted lutein.
Thanks

although this is basic knowledge I will help you.

Valid only if injection volumes are identical!

1) Conc(lut_smpl) = (Area(smpl) * Conc(std) / Area(std)

2) %Lut = Conc(lut_smpl) * 100% * V_smpl * dilution_fact / wheight_smpl

Conc(lut_smpl) = lutein conc in injected sample (mg/ml)
V_smpl = Volume in which your sample was initialy disolved (ml)
wheight_smpl = inital wheight of your sample (mg)
dilution_fact= V2/V1... e.g. if you took 5 ml (=V1) and diluted it to 100 ml (=V2) -> 100/5=20

Thank Hollow very much
Your latter post only give details for your former post and that is what I need.
Thank you very much.