For H202 oxidatative stress assay on endothelial cell. How to prepare 250 µM hydrogen peroxide from 30% H2O2 solution, using sigma's hydrogen peroxide ? Thanks in advance.
It depends on the type of "percentage" your stock solution has.
In the Sigma datasheet it says "% (w/w)" = weight per weight, which means that the solution contains 30 g H2O2 per 100 g of solution. The density of this solution is 1.11 g/ml, so 100 g have a volume of 90.09 ml. So 1 L contains 333 g of H2O2 (30 g / 90.09 ml x 1000 ml) which is a molar concentration of 9.79 mol/L (333 g/L / 34.01 g/mol).
To prepare a 250 uM solution take 0.0255 mL (25.5 uL) of your 30% solution and fill to 1 L with water (250 uM x 1000 ml / 9.79 M).
If it is w/v = weight per volume, then the calculated concentration is 8.82 M (= 300 g / L / 34.01 g/mol). In that case you would need 0.0283 mL (28.3 uL) for 1 L.
30% is an approximate value. You can quantify [H2O2] in water by Uv-vis, extinction coefficient is 19.6+-0.3 at 254 nm
http://www.h2o2.com/technical-library/physical-chemical-properties/radiation-properties/default.aspx?pid=65&name=Ultraviolet-Absorption-Spectrum
Regardless of the method used to prepare the solution, it is advisable determine its concentration experimentally. The reason is the hydrogen peroxide is relatively labile.
To do so, read a diluted solution in any spectrophotometer at 240 nm (use a quartz cuvette or any UV-transparent material), divide the absorbance by its molar extinction coefficient (Ɛ240nm = 0.0394 mM-1 cm-1), and multiply by the dilution.
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