The calibration curve in spectrofluorimetric analysis becomes non-linear when
(a)molecular weight of analyte is high
(b)intensity of light source is high
(c)concentration of analyte is high
(d)molar absorptivity of analyte is high
Answer
Answer: C ✓ checked by 4AB · confidence high
The source book printed no answer; this one was worked out and checked — see the explanation.
Printed A; the loss of linearity at high concentration is the inner-filter effect.
Explanation
The fluorescence intensity is $F = K\phi P_0\,(1 - 10^{-\varepsilon b c})$. Only when the absorbance $\varepsilon b c$ is small (below about 0.05) does this reduce to $F \approx 2.303\,K\phi P_0\,\varepsilon b c$, which is directly proportional to concentration. As the analyte concentration rises, the absorbance is no longer small. Most of the exciting light is absorbed near the front of the cell and part of the emitted light is re-absorbed (inner-filter effect, self-quenching), so the calibration curve bends towards the concentration axis and can even fall. Molecular weight has no bearing on linearity. A more intense source raises F in proportion to $P_0$ without destroying linearity. A high ε only lowers the concentration at which curvature sets in; along any one calibration curve ε is fixed, and it is the rising concentration that pushes εbc out of the linear range. Answer: (c).