RAPID DETERMINATION OF CARBOXYHEMOGLOBIN BY ABSORBANCE DIFFERENCE BETWEEN DOUBLE WAVE LENGTH

Abstract

A spectrophotometric determination of CO-Hb content was investigated on the basis of double wavelength spectrophotometry. Absorbance differences at the two wavelengths at which O₂-Hb has the same absorbance reflect only the CO-Hb component because the O₂-Hb component is nulled out from the mixture of O₂-Hb and CO-Hb. After measurement of absorbance difference, the measuring solution was saturated with pure CO and remeasured. CO-Hb % was calculated from the ratio of the absorbance differences. Since temperature significantly influences the spectrum of hemoglobin pigment, the wavelength pair changed with temperature. For accurate measurement, it was necessary to control temperature of diluting solution before dilution. Liberation of CO from CO-Hb was observed at more than 80% CO-Hb, and 5% and 2% liberation from 100% CO-Hb occured at 100-fold dilution with 10mm cuvette and 20-fold dilution with 2 mm cuvette respectively. Advantages of this method are that the standard curve or equation for calculation and accurate dilution are not necessary because the interfering component is nulled out and 100% CO-Hb of the standard sample is made with the same sample ; and measurement is very rapid, taking less than 2 min.