Simple determination of carboxyhemoglobin by double wavelength spectrophotometry of absorbance difference and the comparison with gas chromatographic method

This paper describes the spectrophotometric determination of carboxyhemoglobin (CO-Hb) in blood on the basis of double wavelength spectrophotometry of absorbance difference. Absorbance measurements are made in the 500–600 nm region at a blood dilution of 100–200-fold. Blood is diluted with a solution containing Na₂S₂O₄ to provide two components of CO-Hb and deoxyhemoglobin (deoxy-Hb). Absorbance difference at the two wavelengths at which deoxy-Hb has the same absorbance reflects only the CO-Hb component because the opposite component is nulled out of the mixture. After measurement of the absorbance difference, the measuring solution is saturated with CO gas to make all Hb derivative CO-Hb and remeasured at the same wavelengths. The percent of CO-Hb is considered the absorbance difference ratio. Results obtained by the present method was in satisfactory agreement with gas chromatographic data in blood not containing methemoglobin (Met-Hb). Comparative experiments using the gas chromatographic method and the present method were performed with samples containing Met-Hb. However, while there is a deficiency in the gas chromatographic method when the samples contain Met-Hb, the results of the present method were in close agreement with theoretical values when samples are mixed with CO-Hb, O₂-Hb and Met-Hb. Advantages of this method are that it is simple and accurate, standard curve or equation for calculation and accurate dilution are not necessary.