Hello every one
I need to quantify EtBr in aqueous solution with expected concentration lesser than 0.5 microgram/ml. I have a UV/HPLC in my lab but I can not find a trusted protocol to perform the EtBr quantification .
I will be grateful for any help.
Use Beer-Lambert Law and extinction coefficient (ex.cof.) of EtBr in water ( E480 nm = 5450 1/M*cm (a))
Prepare a solution of EtOH. Use UV to measure its ABS at 480 nm. Do not forget to use distilled water or buffer as blank. if the ABS is higher than 3 dilute it 10 times. If it is still above 3 dilute again until you get a ABS below 3 and above 0.1. Than use Beer-lambert Law: C=Abs/(ex.cof.*width of vessel)*1/(dilution).
For instance, if you get an absorbance of 1.5 after diluting 10 times using 1cm wide vessel your concentration will be: c=1.5/(5450*1)*1/10= 2.75 * 10^-5 M = 27.5 uM
(a) Saucier, J. M., Festy, B. & LePecq, J.-B. (1971) Biochimie 53, 973-980.
thank you Osman Doluca for your kind reply , but im looking for HPLC method for Ethidium Bromide quantification .
Mehmet Gokhan Caglayan, I cant find this method in European Pharmacopoeia
thank you mr Mehmet gokhan . it is a clear protocol but until now i do not find the Eu.ph. 6th ed. i have to check it in the original reference .
i find a good protocol , and i think that i will try it , ( http://www.ncbi.nlm.nih.gov/pubmed/2865876 )
thank you again for your kind and useful help
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