I am confused with literature data about H2O2 extinction coefficient. I found that the values of H2O2 extinction coefficient depends on wavelength, but in literature, it could be found different values for the same wavelength. For instance, at 240 nm 43.6 M-1cm-1, 39.4M-1cm-1, 36M-1cm-1 than at 230 nm 81M-1cm-1, 71M-1cm-1 etc.
I recommend to determine the extinction coefficient of hydrogen peroxide for your own set-up with synthetic standards, because different factors (T, pH, wavelength, etc.) can have an influence. Prepare a known stock using molecular weight for H2O2
of 34.02 g/mol and a density of 1.463 g/ml (Merck Index). Absorbance
measurements can then be made from diluted standard solutions of H2O2 diluted from a fresh 30% stock (Fisher) - be careful to use fresh solutions, as H2O2 degrades over time. Then you can determine the extinction coefficient according to Beer's Law.
It can also be pH-dependent and 'stabilizer' addition dependent - so you will want to check those parameters as well. I too have found this frustrating with other molecules.
I recommend to determine the extinction coefficient of hydrogen peroxide for your own set-up with synthetic standards, because different factors (T, pH, wavelength, etc.) can have an influence. Prepare a known stock using molecular weight for H2O2
of 34.02 g/mol and a density of 1.463 g/ml (Merck Index). Absorbance
measurements can then be made from diluted standard solutions of H2O2 diluted from a fresh 30% stock (Fisher) - be careful to use fresh solutions, as H2O2 degrades over time. Then you can determine the extinction coefficient according to Beer's Law.
I completely agree with both Jack as well as Christoph. There is nothing better than preparing your own standard curve and determine extinction coefficient based on all the conditions you are following for the assay. We do often observe slight variation in wavelength at which molecule or the complex absorb maximally (based on the conditions one uses and sometimes even the make of Spectrophotometer). Accordingly, you can determine the actual wavelength at which you can record the measurements as per the protocol and conditions you are using.
I use 43.6 M-1cm-1 (at 240 nm) , I checked this value many times in thiol peroxidase reaction, in which 2 moles of thiol correspond to 1 mole of H2O2. I registered thiol decrease in presence of enzyme and H2O2, and 43.6 M-1cm-1 worked exactly.
Comment update, February 7, 2020: Using 43.6 M-1cm-1 for 240 nm, we calculated molar extinction coefficients of H2O2 at other wavelengths, it can also be useful (the table of results is in the file attached). For 250 nm, the results are also presented in other article in English - https://rdcu.be/b4LjP ("A comparative study of catalase activity in Culiseta annulata (Schrank) and Culex pipiens L. (Diptera, Culicidae)")
Sorry, but also I have a problem. Also I found and used 43.6have M-1 cm-1 because I checked this value searching on the browser, but I didn't understand why if I perform only one measure at 240nm I obtain (for my solution) A= 0.745, instead if I try to record the spectra with spectrophotometer I did not have the same result and I did not observ a clear peak. Maybe I make a mistake somewhere: for the measurement I diliute 1ul of H2O2 in 500uL of water.L Can someone help me?
Dear Annalisa,
maybe you should first try to make a 1 mol/dm-3 H2O2 solution. After that you should follow the Lambert-Beer equation in order to calculate the molar extinction coefficient for H2O2 at 240 nm (A= ε * c * l).
The recipe for preparation of hydrogen peroxide solution is attached in this message.
I hope that this will help you. Good luck!
Milan
43.6 M-1cm-1 means that 1 mM solution of H2O2 has A=0.0436 (if l=1 cm). Therefore, If the value for an aqueous solution of H2O2 is of 0.745, you have 0.745/0.0436 = 17.087 mM H2O2. If you previously diluted H2O2 500-fold, your initial solution is 17.087*500 = 8543.5 mM H2O2 that corresponds to highly concentrated hydrogen peroxide. Hydrogen peroxide solution spectrum is without a clear peak, extinction is simply higher at a shorter wavelength, see an attached file.
There are no clear peaks in hydrogen peroxide spectrum, all the epsilon and wavelength to quantify it are just considered as operational values to quantify. They are effective but you are not measuring at a specific wavelength. It is like when you measure turbidity for bacteria, the values that you obtain are just a good esteem.
H2O2's eps changes with the wavelength as shown in the attached figure from (R.F. Beers, Jr., I.W. Sizer, A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase, J Biol Chem 195 (1) (1952) 133-40.). According to this reference, H2O2 eps at 240 nm is 38 M-1.cm-1. The value of eps will be useful to check the concentration of a given H2O2 solution (e.g. commercial 30% solutions after dilution). You should note that H2O2 concentration reduces over time as the solution gets older, hence you should check the concentration of the stock (i.e. 30% H2O2) regularly and perhaps a best method is to dilute the solution and measure its absorbance (e.g. at 240 nm) but you should have confidence on the value of eps to use. I present here example of calculation of absorbance at 240 nm after diluting a 30% H2O2 stock by adding 0.5mL from the stock solution to 100 mL of DI water. If eps@240nm = 38 M-1.cm-1 (above reference) the absorbance of the diluted solution should be 1.851 and if eps@240nm=43.6 M-1.cm-1 (given by A. V. Razygraev), the absorbance of the diluted solution should be 2.123. Which value of eps to use is your call but the main message is make sure you have confidence on the value you use taking into account the conditions under which you are doing the measurement (pH, T, quality of diluting water, age of the H2O2 stock, conditions of storage in a fridge or otherwise, was the stock bottle left open for extended time, were there impurities mistakenly introduced to the solution etc....).
Yes, in different conditions you may have different molar extinction coefficients for the same diluted compound (at the same wavelength). Probably, this is true for hydrogen peroxide. When I checked 43.6 M-1cm-1 for 240 nm in glutathione peroxidase reaction, I obtained an excellent concordance of GSH decrease and H2O2 concentration. However, you may found other extinction coefficients, for example 39.4 M-1cm-1 (in paper Nelson, D. P., & Kiesow, L. A. (1972). Enthalpy of decomposition of hydrogen peroxide by catalase at 25 C (with molar extinction coefficients of H2O2 solutions in the UV). Analytical biochemistry,49(2), 474-478). Interestingly, Nelson, D. P., & Kiesow, L. A. obtained roughly the mean of two values they found earlier in literature (36 M-1cm-1 and 43.6 M-1cm-1)
Hello! I've been facing the same issue. I have an old H2O2 solution and I need to find out its concentration, but I'm not sure about which extinction factor to use...
All the answers here are helpful.
Chedly Tizaoui you said that I can choose which value of eps to use as long as I make sure you I have confidence on the value, taking into account conditions such as pH, T, quality of diluting water, age of the H2O2 stock, conditions of storage, etc. But how do I know how to choose an eps value based on these factors? The solution that I have is 35% (1L total), it has been stored protected from light, but not in a fridge (ambient temperature, which vary), it is a few years old and I don't know the pH (but I can measure it).
It solely depends on the protocol you are following [provided you have used all the conditions specified in the paper from where you picked up the protocol].
I am sure Dr. Tizaoui, will give a better answer.
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