About 0-01 ml. of blood taken from a finger prick is dissolved in 10 ml. of 0-04 % ammonia solution. The solution is divided into two halves, and oxygen is bubbled through one half to convert any carboxyhaemoglobin into oxyhaemoglobin. The spectra of the two halves are then compared in a spectrophotometer, and the difference between them is used to estimate the carboxyhaemoglobin content of the blood either graphically or by calculation from a simple formula. Calibration is simple and need only be done once. A sample of blood can be analysed in about 20 minutes, which includes the time to collect the sample. The method is sensitive enough to be used for the analysis of solutions of blood containing less than 1 % carboxyhaemoglobin.Modem methods for the determination of carbon monoxide in blood have been critically reviewed by Douglas (1962) and by Cobum, Danielson, Blakemore, and Forster (1964), and may be classified as follows: manometric, volumetric, colorimetric, spectroscopic, and those based on the determination of the concentration of carbon monoxide in alveolar air. Only a few of these are sensitive enough to measure low concentrations of carboxyhaemoglobin and most either are time-consunming or require large samples of blood. The methods involving analysis of alveolar air are sensitive but cumbersome.The method described here has been developed to overcome these disadvantages; it is quick, accurate even at low concentrations of carboxyhaemoglobin, and requires only a minute sample of blood.
Principle of MethodThe basis of the method is that the sample, diluted in 0-04 % ammonia solution, is divided into two parts, from one of which the carbon monoxide is displaced by oxygen by bubbling oxygen through it. The solution containing carboxyhaemoglobin is placed in the sample beam of a spectrophotometer and the oxygenated sample in the reference beam, so that the quantity which the instrument records is the difference between the absorbance of the carboxyhaemoglobin and that of the equivalent amount of oxyhaemoglobin; this is used to determine the concentration of carboxyhaemoglobin in the untreated sample. This difference is measured near its maximum at 420-2 m,t and also, in order to define a baseline from which the height of the peak may be measured, at 414 m,u and 426 my. The total haemoglobin present is estimated from the difference between the absorbances of an oxygenated sample at 575 mix and 559 m,u, these being respectively the wavelengths of maximum and minimum absorbance of oxyhaemoglobin.The absorption spectra of oxyhaemoglobin and carboxyhaemoglobin, and the difference between the corresponding values of the absorbance of the two forms of haemoglobin, are shown in Figure 1. It should be noted that the peak of absorbance of carboxyhaemoglobin occurs at 419 3 m,u, i.e., at a slightly lower wavelength than the peak of the difference between the absorbances of the two forms. The peaks of the two quantities are, however, sufficiently close together for it to be possible to use measurements at 420 mu in...