[ninona]

Hi guys,
I'm monitoring the formation of nitrophenol with the spectrophotometer.
In literature is recommended to use 348 nm but I have only the 405 nm filter.
Do you think can be ok as well.?
Can be the sensitivity decreased?
danke

[russellm72]

Ich habe keine ahnung aber....

I wouldn't of thought it had that much of a absorption at that wavelength?

What are you making nitrophenol from?

R.

[ninona]

Thanks,
I'm measuring the esterase activity on p-nitrophenylacetate----->nitrophenol
I checked the spectrum of nitrophenol on line  at 348 nm gives a logarithm epsilon = 3.627. At 405 nm is 2.252.
What do you think?
danke
tchuss

[russellm72]

Ok so if I am right your checking an enzyme that cleaves the acetate group affording the phenol.

Ok I'm a chemist so this is how I'd do it.

I'd make up equimolar solutions of p-NO2PhAc and p-NO2PhOH and inject an equal ammount down an HPLC column say monitoring at 254nm. This will show you the difference in UV absorbtions. Then you can do your reaction and monitor by HPLC. You'll easily separate these two components on a ODS (reverse phase) analytical column.

Of course, if your on a tiny scale your method may be better but I haven't much experience with UV spectroscopy apart from a fleating visit when I was making CyDyes.

R.

[ninona]

ja, i'm in a biology lab.
We don't have HPLC right here. The method i'm doing is easy and fast, but the only problem is this about the right wavelenght.
I will try to use some positive controls, we will see
danke
ciao
N.

[ARGOS++]

Dear Ninona,

As you may see on the Slide number 14 of this reference, that you lose quite some sensitivity, but you can real use your Filter of 405 nm.

The Reference:

http://personal.ecu.edu/gemperlinep/Research/PDF_slides/Batch.pdf

I hope this may be of any help to you.

Good Luck!
                    ARGOS⁺⁺