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what is OD in terms of HPLC?

calculation

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4 replies to this topic

#1 neuron

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Posted 14 March 2013 - 11:56 PM

what does this mean-

"To the solution X was added a solution of 5 eq. of Y in 10 μl THF. The reaction was kept at room temperature for 6 hours. Next, the product was isolated using preparative HPLC to give 15 O.D.A260 of compound".

what is 15 O.D.A260? As far as I know about HPLC, we collect fractions based on the retention time, how do collect fractions based on OD?

#2 bob1

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Posted 15 March 2013 - 12:16 AM

Exactly what you said - it is the fraction, they have collected it to give an OD of 15 -so probably an awful lot of protein going through.

#3 mdfenko

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Posted 15 March 2013 - 05:16 AM

(i'm really picking nits here) od at 260 would be for nucleic acids

Edited by mdfenko, 15 March 2013 - 07:15 AM.

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#4 neuron

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Posted 17 March 2013 - 08:05 PM

Yes mdfenko, you are right , it is about nucleic acids. @bob, I checked about yields of nucleic acids, they calculate in terms of OD, if you just check synthesis of nucleotides, you will get to know. I am still struggling to calculate concentration, when od is given.

#5 bob1

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Posted 18 March 2013 - 12:16 AM

For dsDNA the calculation is quite simple e.g. (copied from cold spring harbour protocols)

Example of Calculation

A sample of dsDNA was diluted 50X. The diluted sample gave a reading of 0.65 on a spectrophotometer at OD260. To determine the concentration of DNA in the original sample, perform the following calculation:

  • dsDNA concentration = 50 μg/mL × OD260 × dilution factor

  • dsDNA concentration = 50 μg/mL × 0.65 × 50

  • dsDNA concentration = 1.63 mg/mL






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