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Chapter 25: Q29P (page 710)
What does it mean for a separation procedure to be ”rugged” and why is it desirable?
A separation procedure to be rugged and why is it desirable.
It is desirable because that small changed conditions do not change the final outcome of separation.
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Retention factors for three solutes separated on anon-polar stationary phase are listed in the table. Eluent was a 70 : 30 (vol/vol) mixture of 50 mM citrate buffer (adjusted to pH with) plus methanol. Draw the dominant species of each compound at each pH in the table and explain the behavior of the retention factors.
25-3 What length of column packed withparticles is needed to yield the same plate number as in Fugure 25.12? How long woald the separation take?
Chromatography–mass spectrometry. Cocaine metabolism in rats can be studied by injecting the drug and periodically with drawing blood to measure levels of metabolites by HPLC–mass spectrometry. For quantitative analysis, isotopically labelled internal standards are mixed with the blood sample. Blood was analysed by reversed-phase chromatography with an acidic eluent and atmospheric pressure chemical ionization mass spectrometry for detection. The mass spectrum of the collisionally activated dissociation products from the m/z 304 positive ion is shown in the figure on the next page. Selected reaction monitoring (m/z 304 from mass filter Q1 and m/z 182 from Q3 in Figure 22-33) gave a single chromatographic peak at 9.22 min for cocaine. The internal standard -cocaine gave a single peak at 9.19 min for m/z 309 (Q1) 182(Q3).
(a) Draw the structure of the ion at m/z 304.
(b) Suggest a structure for the ion at m/z 182.
(c) The intense peaks at m/z 182 and 304 do not have isotopic partners at m/z 183 and 305. Explain why.
(d) Rat plasma is exceedingly complex. Why does the chromatogram show just one clean peak?
(e) Given that -cocaine has only two major mass spectral peaks at m/z 309 and 182, which atoms are labelled with deuterium?
(f) Explain how you would use -cocaine for measuring cocaine in blood.
Spectrum for Problem 25-25.
Left: Mass spectrum of collisionally activated dissociation products from m/z 304 positive ion from atmospheric pressure chemical ionization mass spectrum of cocaine.
Right: Chromatograms obtained by selected reaction monitoring. [Data from G. Singh, V. Arora, P. T. Fenn, B. Mets, and I. A. Blair, “Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry Assay for Trace Analysis of Cocaine and Its Metabolites in Plasma,” Anal. Chem. 1999, 71, 2021.]
To which kinds of analytes do these liquid chromatography detectors respond?
(a) ultraviolet
(b) refractive index
(c) evaporative light scattering
(d) charged aerosol
(e) electrochemical
(f) fluorescence
(g) nitrogen chemiluminescence
(h) conductivity
The chromatogram in Box 25-3 shows the supercritical fluid chromatography separation of seven steroids monitored by three detectors.
(a) In the middle chromatogram, ultraviolet detection provides near universal response for the steroids, whereas in the lower chromatogram the ultraviolet detector provides a selective response for a few of the steroids. How can ultraviolet detection act as either a selective or universal detector?
(b) Why is a sloping baseline observed at 210 nm, but the baseline is flat at 254 nm?
(c) Use the baseline disturbance early in the 254 nm chromatogram to measure tm. How does the measured value compare with that predicted using Equation 25-5 given that the column is 25 × 0.46 cm and the flow rate is 2.0 mL/min.
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