The Standardized Determination of Hemoglobin

The fundamental quantity to be used in any standardized spectro-photometric determination of hemoglobin should be the quarter millimolar extinction coefficient because of the four Fe atoms per hemoglobin molecule. This quantity is independent of the molecular weight of hemoglobin. Consequently, a redetermination of the molecular weight will not change the value of the extinction coefficient to be used and will not affect hemoglobin concentrations when these are expressed in quarter millimoles per liter. 

If we now introduce t, the true extinction coefficient of HiCN, keeping in mind that c milligram-atom Fe/liter corresponds to c quarter millimoles Hb/liter, we may change Eq. (5) to  

The true extinction coefficient thus equals the experimentally obtained value plus a correction for non-HiCN iron and minus a correction for non-HiCN extinction. 

Even if it were possible to evaluate the factor AD/c accurately, it would not be advantageous to apply this correction to the experimentally obtained extinction coefficient. In routine hemoglobin photometry some non-HiCN extinction will always be present. Thus, using an extinction coefficient influenced by a similar factor will at least partially eliminate the non-HiCN extinction error from the determination of hemoglobin concentrations. 

It is therefore obviously justified to adopt iron determination as the basic method for the evaluation of emcNi this method being the one with the most solid experimental foundation presently available. The methods 

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