Spectrophotometry Fundamentals Questions and Answers

Answer: C

Both dilution and a shorter path reduce the absorbance in proportion, bringing the reading into the range where photometric error is least. Slit width and lamp current affect the light available rather than the absorbance, and moving off the peak works but sacrifices the flat region that makes the reading insensitive to wavelength error. Two of the Beer-Lambert law's three terms are under the operator's control, and that is where the remedy lies. A limit that survives making every external component ideal is an intrinsic one, and worth knowing for that reason.

Answer: D

Concentration falls fourfold and absorbance follows in proportion:

\[ A = \frac{1.8}{4} = 0.45 \]

giving about 0.45, squarely in the band where photometric error is least. A twofold dilution would have left it at 0.9, still usable but further up the curve. Choosing a dilution factor that lands the reading in the low-error band, rather than merely reducing it, is what makes the second measurement worth making.

Answer: A

Curvature towards the concentration axis at high absorbance means the reading is lower than proportionality predicts, which stray light and molecular interaction both produce. A wrong blank shifts the whole line, reversed cells add a constant offset, and a lamp change would produce a step rather than a curve. The direction in which a calibration departs from a straight line identifies the mechanism, and bending downward at the top is the classic signature of these two.

Answer: B

An intercept means something absorbs even at zero analyte, so the blank is not accounting for everything the standards contain. Varying absorptivity and stray light bend the line rather than shifting it, and a wrong path length changes the slope. Whether a defect changes a line's slope, its intercept or its straightness distinguishes three quite different causes, and that is the first question to ask of a calibration that looks wrong. Framing the question as which quantity the change acts upon makes the answer follow without calculation.

Answer: C

With one beam any change in source output or detector response appears directly in the reading, and a lamp's output drifts as it warms and over its life. A reacting sample is possible but would affect only that sample, saturation does not drift, and a wavelength shift would be abrupt. The remedy is to re-zero on the blank frequently, or to use a double-beam instrument where the drift cancels in the ratio.

Answer: D

A difference between cells on one solution can only come from the cells themselves - unmatched wall thickness, fingerprints, scratches or residue. Lamp warm-up, slit width and wavelength would affect both cells equally. Isolating a variable by keeping everything else the same is the whole of this diagnosis, and the fact that only the cell changed between the two readings names the cause without further work. A single measurement chosen to respond to the one asymmetry beats several that respond to shared behaviour.

Answer: A

Two faces of a cell are polished and in the beam, and the other two are frosted for handling - grease on an optical face both absorbs and scatters, producing an error that no blank corrects. Strength, heat and markings are not the reason. A design that makes the correct handling obvious by feel is worth respecting, and it is a good example of an instrument's ergonomics carrying part of its accuracy.

Answer: B

Light scattered out of the beam is missing from the detector just as absorbed light would be, and the instrument cannot tell the difference. The particles may absorb a little but scattering dominates, and refractive index and detector response are secondary. The remedy is filtration or centrifugation before measurement, and the fact that the instrument measures attenuation rather than absorption specifically is worth remembering whenever a sample is not perfectly clear.

Answer: C

When a substance exists in two interconverting forms, there is generally a wavelength at which both have the same absorptivity, so the total absorbance there is independent of how the two are proportioned. It is neither a maximum, an equivalence point nor an error minimum. Measuring at such a point gives the total concentration regardless of the equilibrium, which is a useful trick when the ratio of forms cannot be controlled. A limit that survives making every external component ideal is an intrinsic one, and worth knowing for that reason.

Answer: D

Differentiating the spectrum suppresses slowly varying backgrounds and enhances sharp changes, so a narrow band riding on a broad scattering slope becomes measurable. Total absorbance is what the undifferentiated spectrum gives, and neither abundance nor stray light is emphasised. The price is a worse signal-to-noise ratio, since differentiation amplifies noise as readily as it amplifies structure, which is why derivative methods need a clean spectrum to begin with.