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Extinction coefficient


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#1 rajaram

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Posted 07 May 2009 - 07:27 PM

Hi friends... I have few queries to be solved...
1.How to calculate extinction coefficient for hemocyanin at 335nm?

For example:
hemocyanin concentration was calculated using an extinction coefficient (ΕmM1cm) of 17.26, calculated from (E1%1cm) = 2.83 (Nickerson & Van Holde 1971) on the basis of a functional subunit of 74 000 (Antonini & Brunori 1974).

2.If we have extinction coefficient, How to calculate molar concentration?
3.How consistant is this quantificaiton of biochemical compounds based on extinction coefficient?
I am unacquainted to this UV spectroscopy, please explain with funtamental concepts.
Thank you for reading my query

#2 Gerard

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Posted 10 May 2009 - 01:07 AM

1.How to calculate extinction coefficient for hemocyanin at 335nm?
The extinction coefficient is a measured value and can't be calculated.

2.If we have extinction coefficient, How to calculate molar concentration?
E=mM/cm
measured extinction value divided by extinction coefficient gives mmol compound

3.How consistant is this quantificaiton of biochemical compounds based on extinction coefficient?
The extinctioncoefficient is a constant, this means the consistancy of the quantification depends on the error in the measurement itself
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#3 rajaram

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Posted 10 May 2009 - 03:48 PM

Dear Mr, Gerard,

Thank you very much for your prompt reply.
The following lines i've just copied from a Scientific paper, in this i could not understand E17.26 from 2.83...

"hemocyanin concentration was calculated using an extinction coefficient (ΕmM1cm) of 17.26, calculated from (E1%1cm) = 2.83 (Nickerson & Van Holde 1971) on the basis of a functional subunit of 74 000 (Antonini & Brunori 1974)."

#4 Gerard

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Posted 11 May 2009 - 01:59 PM

I dont understand it either.

I would calculate is as follows

1%=10g/1000ml=10/74000*1000 mmol/l=0.135mmol/l
E(0.135mM)1cm = 2.83

E(1mmol)1cm = 2.83*(1/0.135)=21.0
Ockham's razor
Pluralitas non est ponenda sine necessitate
-- "You must assume no plural without necessity".

#5 rajaram

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Posted 12 May 2009 - 11:44 AM

May be the %extinction coefficient 2.83 is wrong. Instead it would have been 2.33, if so, the answer is 17.2. By formula 2.83 gives 21.0. you are right.




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