Spectral analysis

The hemoglobin molecule exists in various forms: oxyhemoglobin (HbO2), reduced hemoglobin (HbR), carboxyhemoglobin (HbCO), sulfhemoglobin (HbS), methemoglobin (HbMet), and fetal hemoglobin (HbF). Each of these forms has its own light spectrum (R.g ,2.). The specific wavelengths at which measurements are made are generally chosen so that one wavelength is the maximum difference between the extinction coefficients of the moieties of interest and the other is an isosbestic point, i.e. the extinction coefficients are the same.

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Fig. 2 Spectral analysis of hemoglobin. Points A, B, and C identify three wavelengths where different Hb moieties have the same extinction coefficients (isosbestic points): A, triple isosbestic point at 548 nm (HbO2, HbCO, and HbR); B, double isosbestic point at 568 nm (HbO2, HbR); C, double isosbestic point at 578 nm (HBCO, HbR). (Reproduced with permission from Shapiro..., eta.L (19.94.,)..)

Modern co-oximeters are spectrophotometers that simultaneously analyze four hemoglobin moieties: reduced hemoglobin, oxyhemoglobin, carboxyhemoglobin, and methemoglobin. As illustrated in Fig 3, the basic components of a co-oximeter are a light source, a series of lenses, filters, mirrors that focus the light beam, a cuvette or sample chamber, a monochromator that isolates the wavelengths of interest using filters or gratings, and photodiode detectors that emit electrons in proportion to the amount of light striking their surfaces. The emitted electrons (current) are fed to a circuit whose output is proportional to the relative absorbance of the sample.

Fig. 3 Basic components of the co-oximeter. A, light source; B, lens and mirror; C, monochromator; D, beam splitter; E, reference detector; F, cuvette; G, sample detector; H, temperature-regulated block. (Reproduced with permission from SMplr£,,§Lai (1994))

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