For the most accurate quantitative determination of an analyte by UV-Vis, at which wavelength should you measure absorbance?
AAny wavelength where the analyte shows some absorption
BThe wavelength of minimum absorbance, to stay within the linear Beer's law range
CThe wavelength of maximum absorbance (λmax)
D254 nm, the standard UV analytical wavelength
Measuring at λmax maximizes sensitivity (highest absorbance per unit concentration, improving signal-to-noise) and minimizes errors from small wavelength calibration errors — at the absorption peak, the curve is flat, so tiny wavelength deviations produce negligible absorbance changes. At non-peak wavelengths, the absorbance curve is steep and small wavelength errors cause larger errors in the measured absorbance.
Question 2 True / False
A double-beam UV-Vis spectrophotometer is more accurate than a single-beam instrument primarily because it can measure two analytes simultaneously.
TTrue
FFalse
Answer: False
The key advantage of a double-beam instrument is that it measures the reference (blank) and sample simultaneously by splitting the beam, so fluctuations in lamp intensity cancel out in the absorbance ratio. The ability to measure two analytes simultaneously (diode-array instruments) is a separate feature unrelated to beam splitting. Single-beam instruments must measure blank and sample sequentially, making them vulnerable to drift between the two measurements.
Question 3 Short Answer
A mixture contains two analytes, A and B, both of which absorb in the visible region but with different spectra. You measure the total absorbance at two wavelengths: 450 nm and 600 nm. How do you determine the concentration of each analyte?
Think about your answer, then reveal below.
Model answer: At each wavelength, the total absorbance equals the sum of contributions from A and B (Beer's law is additive). Using the known molar absorptivities of pure A and B at both wavelengths, set up two equations in two unknowns (concentrations of A and B) and solve the system.
This is multicomponent analysis. If εA1 and εB1 are molar absorptivities at λ1, and εA2 and εB2 at λ2, then: A_total(λ1) = εA1·[A]·b + εB1·[B]·b and A_total(λ2) = εA2·[A]·b + εB2·[B]·b. With two equations and two unknowns, the system is solvable as long as the two analytes have sufficiently different spectral profiles (if εA1/εB1 = εA2/εB2, the equations are linearly dependent and the system cannot be solved).