# Haemoglobin literature spectra does not match

I am studying haemoglobin absorption spectra in its oxygenated and deoxygenated state and I compiled values from different sources including:

• A PhD thesis by van Assendelft [1], using extinction coefficients in (mmol/L)-1/cm

• A book compiling almost one century of research on the topic by the same laboratory that helped standardizing the haemoglobin cyanide standard [2]. Extinction coefficients in the same unit as [1], numerical values are very close.

• Coefficients compiled by Scott Prahl[3]. The unit is the same but there is a factor 3.7 up to 4.0 between his data and that of Assendelft and Zijlstra.

• Coefficients measured by Kolyva[4].

All the published coefficients are defined as base 10 extinction coefficient. That is if I is the light intensity, they are expressed in Beer-Lambert law as:

$$I = I_0 \cdot 10^{-c\cdot \varepsilon \cdot l}$$

Where $$l$$ is the light path length (cm), $$c$$ the concentration in absorbing compound (mol/L) and $$\varepsilon$$ the molar extinction coefficient (L/mol/cm).

How comes there is a 3.7-4.0 factor between Prahl and Kolyva data on the one hand and Assendelft and Zijlstra on the other hand?

At first I thought of some $$log(10)\approx2.3$$ difference, as if some of the author forgot to precise that they used base $$e$$ extinction coefficient at some point but it does not fit. If anyone has an idea on the topic, the help would be most appreciated.

Further information

The cyanmethaemoglobin method is often used for the determination of the haemoglobin concentration of a solution. In particular, its absorbance at 540nm is known to be 11.00L/mmol/cm[5] which is in agreement with Assendelft and Zijlstra published data.

### References

1. O.W. Assendelft, Spectrophotometry of Haemoglobin Derivatives, 1970. ISBN: 902320560X.
2. W.G. Zijlstra, A. Buursma and O.W. van Assendelft, Visible and Near Infrared Absorption Spectra of Human and Animal Haemoglobin, 2000. ISBN: 9067643173.
3. Scott Prahl, Optical Absorption of Hemoglobin, 1999, https://omlc.org/spectra/hemoglobin/
4. C. Kolyva, Systematic investigation of changes in oxidized cerebral cytochrome c oxidase concentration during frontal lobe activation in healthy adults, 2012. DOI: 10.1364/BOE.3.002550. Coefficients are in MEDIA 1.
5. F. Meng, Determination of extinction coefficients of human hemoglobin in various redox states, 2017. DOI: 10.1016/j.ab.2017.01.002. Table 1.
• In Scott Prahl's article, he discusses that some authors consider equivalents and some use molar values and that this would create a 4x difference in the values. Have you factored this in? Mar 9 '20 at 16:43
• @Tyberius Thanks a lot, I had read it long ago and forgotten it by the time I came to look at those spectra once again. Thank you for the fresh look you had on the issue! I answered the question with your remark :) (Kind of weirdly, I had the reflex to open the different books and articles I had found here and there because they were "publications" but had not that of rereading Prahl's article which I classified unconsciously as a "blog post", thus less likely to contain valuable information. My bad.) Mar 10 '20 at 7:06

As stated by Scott Prahl :

One of the most confusing things about looking at hemoglobin (Hb) spectra is that the values are typically tabulated in equivalents. The term equivalent is used to indicate the amount of hemoglobin which contains 1 gm atom of Fe and combines with 1 gm molecule of O2 or CO. One equivalent of hemoglobin is assumed to be 64,500/4 or 16,125 gm. A concentration of 10$$^{-6}$$ equivalent is 16.125mg of hemoglobin per cc.

It seems that Assendelft and Zijlstra used the equivalent quantification of haemoglobin, meaning that all their extinction coefficients should be multiplied by 4. This leads to the following figure:

Sorry for not having read again Scott Prahl's description before posting. I read it several months ago, as I was gathering data from different source and forgotten it since...