Absorption spectroscopy of haemoglobin species

This chapter is very, very loosely related to Section F8(ii) from the 2023 CICM Primary Syllabus, which expects the exam candidates to be able to "explain the oxyhaemoglobin dissociation curve and factors that may alter it". Instead of addressing these examinable syllabus items directly, the focus here is on the detection of normal and abnormal haemoglobin species.  In brief, haemoglobin concentration (normal and abnormal) is measured directly, using visible absorption spectroscopy. The local unit features a 128-wavelength spectrophotometer with a measuring range of 478-672 nm. Indeed, it is pleasing to point out that the reference manual for this machine indulges in a digression on the theoretical foundations of absorption spectroscopy.

Principles of absorption spectroscopy

Absorption spectroscopy is based on Lambert-Beer's law, which relates the properties of transmitted light to the properties of the substance through which it is transmitted. Specifically, the law can be defined as a statement...

The measured absorbance for a single compound is directly proportional to the concentration of the compound and the length of the light path through the sample

...or as an equation...

Lambert-Beer Law

The absorbance, in this scenario, is defined as the logarithm of the ratio of the light intensity before and after transmission through the compound. Each haemoglobin species has a different extinction coefficient (ε) and these are known from empiric measurements.

Obviously if you have a whole host of weird compounds all absorbing light, the total absorbance is the sum of the absorbances of each compound.

Spectrophotometry of haemoglobin in the ABG machine

The blood gas analyser liberates haemoglobin from red cells by shredding them with a 30Hz ultrasound beam, and then passes the resulting puree through a beam of near-infrared light.

spectrophotometric analysis  of haemoglobin species in the ABG analyser

The light is then separated into 128 beams using a concave grate/mirror, and sent into 128 individual light-sensitive diodes. Each diode is responsible for measuring a specific wavelength, within a range of 478-672 nm. Each diode subsequently reports on the light intensity at that wavelength. The higher the light intensity, the lower the absorbance (i.e. less of that wavelength was absorbed by the sample).

Thus, for each blood gas sample, 128 separate wavelength-absorbance measurements are taken, and a graph can be generated, where absorbance can be plotted as a function of wavelength:

absorption spectra or total haemoglobin concentration

The total absorbance is the sum of absorbance from all the haemoglobin species, all together.

Thus, total absorbance correlates with the total haemoglobin concentration.

The ABG machine reports this variable as ctHb, the concentration of total haemoglobin.

However, when the extinction coefficient (ε) of a haemoglobin species is known at every given wavelength, one can separate the species.

Spectrophotometry of different species of haemoglobin

The chapter by van Kampen and Zijlstra ("Determination of haemoglobin and its derivatives") from the 8th volume of "Advances in Clinical Chemistry" presents several graphs of various haemoglobin species, of which I have selected a particularly effective representative:

original graph of the absortpion spectra of haemoglobin species

Hb in the legend above is obviously haemoglobin, HbO2 is obviously oxyhaemoglobin and HbCO is carboxyhaemoglobin. Hi is "hemiglobin", which is what the men of learning used to call methaemoglobin. HiCN is hemiglobin cyanide, and coproporphyrin III is a breakdown product of bilirubin which is excreted in urine and faeces.

With some modifications, a messy graph of ε/λ relationships can be created:

messy diagram of absorption spectra for haemoglobin species

In a slightly neater interpretation, the graphs still don't gain any additional educational value. See?

tidy diagram of absorption spectra for haemoglobin species

That's right, useless. But the message (if there was one) is that the different haemoglobin species will each have some sort of influence on the total absorbance. According to Lambert-Beer's law, the contribution of each species at any given wavelength will be proportional to that species' concentration in the sample.

Absorption spectra of the total haemoblbin concentration

If one knows the expected contribution from known empirical measurements, one can determine the contribution from each haemoglobin species individually:

equation describing the analysis of haemoglobin species spectra

Rather than discussing maths which I don't fully understand, I will instead quote the ABL800 FLEX reference manual which says that the specific constants are "determined using Multivariate Data Analysis where the spectra of the calibration compounds were considered together with the reference values of the calibration compounds."


Device-specific information in all these ABG pages refers to the ABG machine used in my home unit.

Other machines may have different reference ranges and different symbols.

For my ABG analyser, one can examine this handy operations manual.

There is also an even more handy reference manual, but one needs to be an owner of this equipment before one can get hold of it. Its called the "989-963I ABL800 Reference Manual"

Nastuk, William L., ed. Special Methods: Physical Techniques in Biological Research. Elsevier, 1962.

von Kompen, E. J. "Spectrophotometry of hemoglobin and hemoglobin derivatives." Advances in clinical chemistry 23 (1983): 199.

Zwart, A. "Spectrophotometry of hemoglobin: various perspectives." Clinical chemistry 39.8 (1993): 1570-1572.

H Sobotka et al, "Advances in Clinical Chemistry"; Volume 8- specifically, the chapter by E.J. van Kampen and W.G. Zijlstra, "Determination of haemoglobin and its derivatives"

- The graphs from this chapter were reproduced above without any permission from the authors, but in the spirit of respect and admiration for their work.