Beer Lambert Law
March 5, 2009 at 10:36 am #11007biomed7Participant
hi thjere i was wondering if someone could help as i am stuck. i need to convert my readings into micromol/min and i have been given that the extinction coefficient for NADH is 6.22*10^3l.mol^-1cm^-1
now I have started this by rearranging the beer lambert equation so
c = A / E *l
so for example one of my readings was for serum = 0.854 so i did
c = 0.854 / (6.22*1) = 0.137 and I am stuck there. I am not sure what units is the results and what i
do to convert it to micromols?? thanks in advance….
March 5, 2009 at 2:42 pm #89487blcr11Participant
So what happened to your exponent on the power of ten? I didn’t check the extinction coefficient (now preferably called the molar absorptivity) of NADH. I’ll take your word for it that it is 6.22E3 A/M cm. What you haven’t told anyone is what the volume of your sample of serum was and and if it was a dilution that was measured. What you’ve calculated (almost, anyway) is the concentration (in moles per L or molarity) of NADH in the cuvette.
If you really mean to calculate "micromoles per minute," like you might if you were calculating a clearance rate of a drug, for instance, then you need to say more about the volume of sample, whether it was diluted or not prior to being put in the cuvette (if it was diluted then you need to know the dilution factor so you can adjust the amount in the cuvette for having been diluted prior to measuring); and you need to be more enlightening about the time intervals involved in collecting the specimen(s). If this is taking blood samples at timed intervals to see how quickly something is cleared from the bloodstream, then you will need more than one point to get something to come out in units of micromol/min.
Assuming your cuvetted is a standard cuvette with a 1 cm path length (and almost all cuvettes are; some exotic ones have special lengths, but you almost never see them in general use) then the molar concentration of NADH in your cuvette is:
0.854 A/ 6220 A/M NADH = 1.372E-4 mole/L NADH or 0.1372 micromol/ mL
I’m assuming there is a one-to-one correspondence between moles of analyte and moles of NADH. If that’s not the case, then you need to give more details on what you are actually trying to measure. If your specimen was 1.0 mL of undiluted serum, then there is 0.1372 micromol in your specimen. If it was diluted by a factor of 100 (10 microliter of serum + 990 microliter of buffer, say) prior to measurement, then there is 13.72 micrmole of substance in the original specimen. More than that, no one can say without more information about the specifics of the sample.
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