Calculating concentration from peak area

[BathASU]

Please could someone explain where im going wrong with calculating the concentration of my antibody using the peak area and the beer lambert law.

Do I use the peak area as my absorbance? the units are in mAu/s ?

So my values are

Absorbance 0.882 (taken from area of peak in mAU/s)
Extinction coefficient 215130
Path length 1 (10mm)
Mr of antibody 145531.5

Absorbance (Au) = extinction coefficient x path length (cm) x concentration (mol/L)

so (0.882/215130x1) = 0.00000409

0.00000409 x 145531.5 = 0.59mg/ml which is wrong as i know my antibody should be around 21mg/ml.

Please could someone work throught the equation step by step

Thankyou

[rwjohnson]

Hi. I would be interested in learning more about your analysis; it sounds interesting. I have never calculated Beers law directly when quantifying via chromatography. Actually, I never calculated Beers law using spectroscopic techniques either. I usually prepare a calibration curve to bracket the expected concentrations.

I do notice that you don't have an injection volume in the calculation.

Ron J

[BathASU]

I think I may have solved the problem now.

The software im using was reporting the response in mV and not mAU. I think I have worked out the conversion factor and the results are in the right ball park now. Will prepare some samples tomorrow and run them on a UV spectrometer and the HPLC system and hopefully they should match up perfect, can only hope...

[BathASU]

Hi Ron, the antibody samples im analysing come in glass vials and are reconstituted with a fixed volume. The manufacturers however, tend to overfill the vials so the correct doseage can be withdrawn.

Hence I cannot just use a calibration curve as I cannot assume the stated concentration is corrrect.

As for the equation not taking into account the injection volume, indirectly it does, the more thats injected the greater the peak area will be.

Thanks for showing an interest

[rwjohnson]

This sounds interesting. Do you calculate your extinction coefficient yourself or is available from the antibody supplier?

Ron J.

[BathASU]

No I've been unable to find it anywhere in the literature, just used the Expasy protparam online tool to calculate it based on the amino acid sequence

[rwjohnson]

That is a pretty cool website. I have never had an opportunity to work on proteins.

I'm still not understanding how you can get a concentration of your sample without taking inj volume into the equation. The concentration that the detector sees will not be the same as the solution concentration. I would be interested in knowing how the spectrometer and the HPLC detector compare.

Ron J

[HW Mueller]

Also, I didn´t see the flow factor anywhere. the correct calculation was discussed some time ago, here.

[rwjohnson]

Mr. Mueller,

I would like to review that discussion if you suggest how to find it. I'm not familiar with quantifying without calibration standards. Is this technique common? I mostly deal with manufactured things but I can see how it would be tedious to require standards for biological research.

Ron J

[ScottHorn]

Is the antibody you're working with polyclonal or monoclonal? If monoclonal, what isotype? In my experience there is very little variation in the extinction coefficients of antibodies within the same isotype, so if you know the isotype you can just use a published extinction coefficient for that isotype.

[HW Mueller]

HW Mueller [ Jump to post ] Posted: Fri Nov 28, 2008 8:29 am


Replies: 10
Views: 1352

... is to use a known absorption (extinction) coefficient to estimate the peak area in order to have an idea about possible material ...

A search of the archives yielded the following, among others:

Forum: Liquid Chromatography Topic: Using peak area to estimate concentration

Uwe Neue [ Jump to post ] Posted: Tue Nov 25, 2008 12:54 pm


Replies: 10
Views: 1352

... concentration in the sample. The peak area is proportional to the mass injected, and by knowing the extinction coefficient, you can calculate this mass as follows: ...


If you don´t find enough info, let us know, so that I can look up my material, the application can be a bit tricky.
I used it only to help guess whether I am loosing material somewhere. HPLC should be calibrated with a standard.