Measuring absorbance with coloured sample - (Jun/23/2009 )

Hi,

I am trying to measure absorbance (say at 540nm), but the sample I am using has an intense colour which interferes with the absorbance reading.
I have tried using a colour control, but I am not sure if this is giving me a true result.

How am I able to do this?

what is causing the color? what color?

is the solution translucent or opaque?

if the color is from a component in the medium that the sample is in then you can blank the spectrophotometer against the medium.

you can try diluting the sample (and medium as blank).

any attempt to read the sample hinges on your ability to prepare a proper blank.

also...

if you are comparing to a standard curve then you may want to prepare the standards in the same medium.

mdfenko on Jun 23 2009, 11:51 PM said:

i am growing some cells in medium and then adding an extract (sample). Then i am adding some reagent and trying to read the absorbance from that. The extract has the colour. I have tried a colour control (medium + extract only), but i don't think this is giving a true result based on other experiments.

i have blanks and negative controls already etc.

Could you resuspend the cells in a clear solution, like a buffered saline? Also, you can get most culture media without phenol red, which is the main source of the media's color.

Pipette Dude on Jun 24 2009, 03:32 AM said:

the color control should also contain whatever the reagent is made in to bring final volumes and dilutions to the same as the sample. the only difference should be the reagent itself.

even so, if the color is too intense and/or dense then you may have to find another modification or method to assay.