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After poisonous melamine and cyanuric acid appeared in milk in China (Box$\mathbf{11}\mathbf{-}\mathbf{3}$) in $\mathbf{2008}$, there was a flurry of activity to develop methods to measure these substances. An analytical method for milk is to treat 1volume of milk with 9 volumes of ${\mathbf{H}}_{\mathbf{2}}{\mathbf{O}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{3}}\mathbf{CN}\mathbf{(}\mathbf{20}\mathbf{:}\mathbf{80}\mathbf{vol}\mathbf{/}\mathbf{vol}\mathbf{)}$to precipitate proteins. The mixture is centrifuged for $\mathbf{5}\mathbf{min}$to remove precipitate. The supernatant liquid is filtered through a $\mathbf{0}\mathbf{.}\mathbf{5}\mathbf{-}\mathbf{μ³¾}$filter and injected into a HILIC liquid chromatography column (TSK gel Amide-stationary phase) and products are measured by mass spectrometry with selected reaction monitoring (Section $\mathbf{22}\mathbf{-}\mathbf{5}$). Melamine is measured in positive ion mode with the transition$\mathbf{\text{³¾/³ú127→85}}$. Cyanuric acid is measured in negative ion mode with the transition $\mathbf{m}\mathbf{}\mathbf{/}\mathbf{2128}\mathbf{→}\mathbf{42}$.

(a) Write the formulas for the four ions and propose structures for all four ions.

(b) Even though milk is a complex substance, only one clean peak is observed for melamine and one for cyanuric acid spiked into milk. Explain why

Question:The graph shows retention data from a${\mathbf{C}}_{\mathbf{8}}$silica column with an acetonitrile/water mobile phase.

(a) What mobile phase composition provides greatest retention$\mathbf{\left(}\mathit{k}\mathit{\right)}$for the components? Least retention? Coelution $\mathbf{(}\mathbf{equal}\mathbf{}\mathit{k}\mathit{}\mathit{)}$of two components?

(b) Predict the retention time of each peak at $\mathbf{40}\mathbf{\%}\mathbf{}\mathbf{and}\mathbf{}\mathbf{60}\mathbf{\%}\mathbf{}\mathbf{acetonitrite}$. Draw a chromatogram (a "stick diagram" representing each peak as a vertical line) of the separation at each mobile phase composition.

(c) Would$\mathbf{60}\mathbf{\%}$acetonitrile yield adequate resolution?

(d) Assuming Gaussian peaks, does the separation at$\mathbf{60}\mathbf{\%}$acetonitrile have the attributes of a good separation?