Q9) Different concentrations of fluorescein isothiocyanate (FITC) were prepared
ID: 1046683 • Letter: Q
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Q9) Different concentrations of fluorescein isothiocyanate (FITC) were prepared and analyzed with fluorescence and the following calibration curve was made based on the data. The curve begins to decrease with higher concentration due to the phenomenon of, "self absorption." ITC Standard Curve E00 400 200 100 O.D0 50,00 100,00 152.00 31D2 FITC Concentratian luM (a) Why does the signal decreasing present a problem to confidently determine the concentration of an unknown quantity of FITC? Hint: think about what the concentration would be if an unknown gave an emission signal of 400 (b) How could you ensure that you had determined the correct concentration from this curve? Hint: it will involve making another solution)Explanation / Answer
(a) The emission signal at 400 corresponds to about 55 uM, if you see the graph carefully same signal strength is see also for a solution at 250 uM. Means, even if the concentration increases five time, the signal strength remain constant. If the unknown that you made had very high concentration the florescence reading will be much lesser than actual standard reading, gives you wrong results. So one should always measure multiple reading at different dilutions to calculate the unknown concentration, preferably at very low concentration where the plot is nearly linear.
(b) The correct concentration can be determined by measuring the fluorescence after diluting the sample, so that the fluorescence reading are located at near linear region of the standard curve. Again the error can be further reduced by measuring the reading at multiple dilutions.
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