Spectrophotometry Fundamentals Questions and Answers

Answer: A

The interferogram must be sampled at exactly known path differences, and a helium-neon laser's own interference fringes provide a ruler far finer than any mechanical encoder. It does not measure absorbance, align the beam or illuminate the sample. Using a known wavelength as a length standard is what gives these instruments their excellent wavelength accuracy, and it is why they need no external wavelength calibration at all.

Answer: B

The Jacquinot advantage refers to the large circular aperture such an instrument can use in place of a narrow slit, admitting far more light. The Fellgett or multiplex advantage is the separate benefit of measuring all wavelengths at once. Nyquist concerns sampling and Doppler concerns motion. Two distinct advantages with two distinct names, and confusing them muddles an explanation of why the technique won.

Answer: C

Also called the Fellgett advantage, it is that every wavelength contributes to every point of the interferogram, so each is measured for the whole duration rather than for a brief moment as a scanning instrument passes it. Multiple samples, detectors and scan averaging are separate ideas, though averaging is a real and additional benefit. Where a detector's noise rather than the source's brightness dominates, measuring everything all the time is worth a great deal, which is exactly the infrared case.

Answer: D

Secondary conformation is best determined by Circular dichroism.

Circular dichroism measures the different absorption of left- and right-circularly polarised light. In the far ultraviolet (about 190 to 250 nm) the peptide backbone gives characteristic spectra for alpha-helix, beta-sheet and random coil, so CD estimates the secondary structure of a protein in solution quickly and with little material.

Fluorescence spectroscopy reports on the surroundings of tryptophan and tyrosine side chains, mostly tertiary structure. Electron microscopy images particles but does not resolve secondary structure in solution, and analytical ultracentrifugation gives mass, shape and association state.