Calculating Antibody concentration from HPLC chromatogram

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Chemgod1
Chemgod1's picture
Calculating Antibody concentration from HPLC chromatogram

Hi,
I have purified an IgG using AKTA/FPLC and ran the purified product on HPLC. I need to calculate how much antibody I have from the Peak Area (which is in mAu*min). I know the flow rate but I don't have the extinction coefficient for my IgG. I guess I can estimate the extinction coefficient for IgG. But can someone please tell me How I can calculate it using the arithmetic. Thanks a lot.

qiang wang
qiang wang's picture
Most mammalian IgG have

Most mammalian IgG have protein extinction coefficient in the range of 12-15.
to estamite, you may assume it as ~14

qiang wang
qiang wang's picture
I found this booklet may be
R Bishop
R Bishop's picture
Concentration (mg/ml) =

Concentration (mg/ml) = Absorbance at 280 nm divided by absorbance coefficient.
Note this assumes the path length is 1cm, which is true of most spectrometers
Rb
 

Chemgod1
Chemgod1's picture
Thank you all for your help

Thank you all for your help in advance.
So If my HPLC has a flow cell with 0.2cm instead of 1cm for the path length, how do I incorporate that in?? Do I multiply the final mg/ml with 0.2? That does not make sense, since my units comes out to be mgxcm/ml ?
What, I have is Peak area in (mAUxmin units) from my chromatogram. I know that 1AU=1000mAU=14mg/ml, but 14 is for ext. coeff. percent (e%) which is based on 1% (1g/100ml) solutions. So I've been told to use 1.4mg/ml instead.
Therefore, if I have an antibody peak area of 1000 mAUxmin, then that would mean I have 1.4mg/ml of antibody? and if my flow cell is not 1cm, but 0.2cm then what??
Here is what I have in terms of units only, please correct me:
(mAUxmin) X (flow rate in (ml/min)) = mAUxml
(mAUxml) X (1mg/1.4mAU) = mgxml ???
???
 

R Bishop
R Bishop's picture
Chemgod1,

Chemgod1,
You are close. You actually want to divide by 0.2 cm not multiply.
The absorbance (A) = extinction coefficient (E) * Concentration (C) * Light path length (L)
A = e C L
ε is the extinction coefficient has the units liter mole-1 cm-1
Now if you work through your formula cm cancels and leaves you with (mg/ml)
Its been a while for me, so please check my math!
Rus