Chromatography Forum

A girl helped me run HPLC of three samples of know concentration of lutein, lycopen, and beta-caroten and six other samples that I have to find their concentration of carotenoids.
When she dilluted lutein, lycopen and beta-caroten, she forget to write down how she had dilluted lycopen and beta-caroten. So now I know only one sample of known concentration. That is lutein.
Is there anyway to find out the concentration of lycopen and beta-caroten from the chromograph of three samples: lutein, lycopen and beta-caroten.

What happened to me?

Do you have any stock solutions of known concentration?
Otherwise, if you have an RI detector, the response of these things should be close enough to estimate the concentration of one peak with another.

Depending on what your UV/VIS detector was set to, you can use the published spectra of the compounds to get the relative absorbtivities. For instance at 474 nm lutein in heptane has A=0.236 for a 1 cm path, and at 472 nm lycopene has A=0.345, and at 436 nm beta-carotene has A=0.196. I got this data from a couple sources; see my article in American Laboratory News Nov. 2005. You can search americanlaboratory.com.

Do you have any stock solutions of known concentration?
Otherwise, if you have an RI detector, the response of these things should be close enough to estimate the concentration of one peak with another.

If I have an RI detector, then how to estimate the concentration of one peak with another?

Depending on what your UV/VIS detector was set to, you can use the published spectra of the compounds to get the relative absorbtivities. For instance at 474 nm lutein in heptane has A=0.236 for a 1 cm path, and at 472 nm lycopene has A=0.345, and at 436 nm beta-carotene has A=0.196. I got this data from a couple sources; see my article in American Laboratory News Nov. 2005. You can search americanlaboratory.com.

Thank you for your help, I will search for your article.

If I have an RI detector, then how to estimate the concentration of one peak with another?

You know the concentration of lutein, and you have a peak area. Use that peak area/concentration to determine the concentration of your other peaks based on the area.
Is your method isocratic?

Thank noser very much
My method is isocratic.
I have ever thought of that but I feel unsure about it.
Now, thank again.

Depending on what your UV/VIS detector was set to, you can use the published spectra of the compounds to get the relative absorbtivities. For instance at 474 nm lutein in heptane has A=0.236 for a 1 cm path, and at 472 nm lycopene has A=0.345, and at 436 nm beta-carotene has A=0.196. I got this data from a couple sources; see my article in American Laboratory News Nov. 2005. You can search americanlaboratory.com.

I have found your article. Is the title "A method for the determination of carotenoids and fat-soluble vitamins".
I want to know more about your method. Please can you explain more detailed and easier? Then how can I apply your method in my situation?
Thank you very much.

If you still have your stocks somewere, just put an aliquot in spectrophotometer and read absorbance at characteristic wavelength (or better take a spectra). Then using Lambert-Beer law and known molar extinction coefficient for b-carotene and lycopene calculate concentration.

Also: make fresh standards with known concentrations and run them again on HPLC.

Regards.

area = (extinction coefficient) * (concentration in sample) * (method-related constant). The method-related constant has all the stuff like flow rate, injection volume, cell path length, detector units, etc. but it is the same for every peak in the chromatogram. Since you know area, extinction coefficient and concentration for lutein, you can solve for the constant. You have the area, extinction coefficient, and constant for carotene, so solve for concentration. The only problem is that the extinction coefficient is dependent on the wavelength and solvent, and if the reference data is at a different wavelength than your experiment, your calculation will have a significant error.

In my previous post, I forgot to mention that the absorbances were for a concentration of 1.0 µg/mL.

If you have specific questions about the article, I would be happy to answer them. You may email me at mark.tracy@dionex.com