correcting for dilutions with spectrophotometer

[northernlad2690]

i wonder if anyone could offer some subtle insight,

 

I have a set of standards of substance X with an appropriate standard curve constructed from this info. The sample of unknown concentration is diluted 10X and thus has produced a much smaller absorbance reading as expected, but I'm stuck on how to correct for this dilution as i'm not sure whether to;

 

A) multiply the absorbance reading by 10 or

B) multiply by 10 the figure from the point on the graph where the diluted absorbance reading is.

[enzymepurifier]

First, does your diluted measurement fall within the high and the low points of the standard curve that you generated?

 

If so, you most certainly have to take into account the dilution that you made initially. Option "B" is correct in that you will have to multiply the output value by your dilution factor of 10.

 

e.g. I have a standard curve that measures 0.000 OD to 1.000 OD that generated a working concentration range of 0 mg/ml to 10 mg/ml. If I made a 10x dilution of my sample and it read 0.500 OD, the resulting concentration would be 50 mg/ml.

[BabcockHall]

If the reading produced a very low value of absorbance, it might not be trustworthy, and the measurement should be repeated if possible. I would reach the same conclusion if its value is lower than the lowest value of the standard curve, because interpolations are generally better than extrapolations. Setting aside issues of accuracy, I have found that being consistent in the way that I handle dilutions is helpful to avoid errors. I would calculate a value of the concentration from the standard curve (let's call this C1). Then I would calculate the concentration of the starting solution (C2) using C1(V1) = C2(V2). That is where the tenfold comes in. A common error is to use incorrect values of V1 and V2.