Differences in absorbancy of compound in different media - (Dec/29/2008 )
I have some questions regarding the absorbance of some compounds in a spectrophotometer.
Guess I'll give an example.
Say Compound X absorbs at wavelength 300nm and I plot a standard curve (which is a straight line) of OD vs concentration using distilled H20 was the blank.
Next, in an experiment involving bacteria cultures in a yellow medium. I'd spin the cells down and measure the supernatant. This time, I use the yellow medium as my blank.
Will the absorbancy be the same? Say if I get a concentration of 25 arbitrary units at OD300nm of 1.2, will an OD of 1.2 in this yellow medium give a 25 too? Since I blanked it with a similiar medium.
Anyone with experience preparing a standard curve?
I would think that's okay if the medium and the water have no effect on the absobtion of Compound X.
I advice to prepair the standardcurve with medium to prevent the possibility of a different moluculair absorbance coefficient of the compound in water or medium
Whilst I don't think they'd be any difference since it's blanked and the machine is actually measuring the absorbance as if the Compound X is there ALONE.
Gerard, the standard if prepared using a medium, wouldn't it be of no use if in the real world we use different medium, colored or otherwise? It'll be a hassle to prepare each curve for different medium.
By how much will the absorbance coefficient affect the readings?
Most of the times there will almost no difference but you dont know.
A example that will go very wrong
Imaging you want to measure phenolthaleine a (acid/base indicator), the sample is in a medium at pH 8 and you prepare your standard in water pH6 what do you think will happen?
Expanding Gerard's comment, if compound X reacts with any component of the media, or with anything secreted by or attached to the organisms in the culture, its optical properties could change...
Thank you guys.
So from what I understand, IF the compound to be tested (and to be used for standard curve generation) does NOT react with the medium AND that the conditions of the standard curve generation are the same as in real life, then the absorbency wouldn't change.
Thanks, I'm on my way to get as many points as possible to get good R^2 value