Chromatography Forum

Hi everyone,

I need a procedure to measure the tocols activity after few days the tocols standard prepared. Its mentioned that then tocols conc in standard stock solution need to re-adjust the conc. of standard tocols. And I found one journal said like this...

Standards will be standardized using spectrophotometric measurements at the following wavelengths: 292 nm (α-tocopherol, α-tocotrienol), 296 nm (β-tocopherol, β-tocotrienol), 297 nm (γ-tocotrienol, δ-tocotrienol) and 298 nm (γ- tocopherol, δ-tocopherol). Absorbance difference (A-A0) of each working solution is measured with a spectrophotometer at defined above wavelength. A is the absorbance of a standard solution and A0 is absorbance of blank ethanol. The concentrations of standard solutions, made in ethanol matrix, will be corrected based on tabular data for extinction coefficient (E1cm 1%) of 75.8, 89.4, 91.4, 87.3, 86.0, 86.2, 91.0 and 85.8 for α-tocopherol β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol and δ-tocotrienol, respectively. Concentrations will be calculated from peak area determined by the spectrophotometer and linear regression.

How to calculate the conc. using spectrophotometer. IS there any source that give full explanation about this. Hope will get reply from you all.Thank You

Calculating a concentration from an extinction coefficient is straightforward. You measure the absorbance and divide by the extinction coefficient to get the concentration. The units used here are non-standard, but I would assume that "(E1cm 1%)" refers to a 1% solution in a 1 cm path length.

So, measure the absorbance difference, divide by the fudge factor given, and the result should be your concentration in %.

hi good morning.

So, measure the absorbance difference, divide by the fudge factor given, and the result should be your concentration in %.[/quote]


sorry, I didn't get the idea. Can you please explain further the steps. New to this . Thank You

First off, let me apologize for using local (American) idioms. It was late and I was trying for some humor*.

Try this:

So, measure the absorbance difference, divide by the extinction coefficient given for the compound of interest, and the result should be your concentration in %.

the "E1cm1%" means (as far as I can tell) the absorbance that a 1% solution would have in a 1-cm cell. As I said, this is non-standard terminology.

So, using alpha-tocopherol as an example: Your blank has an absorbance of 0.01AU. Your sample solution has an absorbance of 0.768AU (both measured at 292 nm). The difference is 0.758. Divide that by 75.8 and you get 0.01% as your concentration.


*"Skinner's Constant (the Fudge Factor): That quantity which, when multiplied by, divided by, added to, or subtracted from the answer you get, gives you the answer you should have gotten." http://www.freewebs.com/leiora/thelaws.htm

Now clear. another question

"" you get 0.01% as your concentration."" .so it means 0.01g of tocopherol in 100ml of ethanol.
therefore, the concentration is 0.1mg/ml. am I correct?

Thanks for your knowledge

That would be my guess.

However, as I said before, the terminology is non-standard. Did the original reference intend percent as w/v (weight/volume) or as w/w (weight/weight)? If it's the latter, then you would have to divide by the specific gravity of the solvent.

I am working in the same way....
And I have a related question: if I measure the absorbance with a 0.1cm path cell way.... Then, should I to divide the absorbance difference by 10 and then by the fudge factor???

Thank you!!!

Why don´t you guys take a good look at the Beer-Lambert law?

Thank you. I had done it.

Yup. I did it.

The formula is conc. in microgram per mililiter, C.

C= (A x 10^4)/(E1%, 1cm)

A=absorption value of each tocols in stock solution
E1%, 1cm, value according to the tocol

So all you need to do is find out what they mean by %, for instance via units given for C values or E values. By the way, a w/v can not be, legitimatly, expressed as %.

That formula took from BSEN12822:2000 Foodstuff- Determination of vitamin E by high performance liquid chromatography - measurment of alpha , beta, delta and gamma tocopherol.
The link below is the scanned formula. It measures the conc. in ug/ml

http://imageshack.us/photo/my-images/192/formulaif.png/

Thank You