(a) Nonpolar aromatic compounds were separated by HPLC on an octadecyl$\left(C_{18}\right)$bonded phase. The eluent was 65 vol% methanol in water. How would the retention times be affected if 90% methanol were used instead?

(b) Octanoic acid and 1-aminooctane were passed through the same column described in (a), using an eluent of 20% methanol/80% buffer (pH 3.0). State which compound is expected to be eluted first and why.

role="math" localid="1656416023291" $$\begin{gathered} {\mathbf{CH}}_{\mathbf{3}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CO}}_{\mathbf{2}}\mathbf{H} \\ \mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{Octanoic}\mathbf{}\mathbf{acid} \\ {\mathbf{CH}}_{\mathbf{3}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{CH}}_{\mathbf{2}}{\mathbf{NH}}_{\mathbf{2}} \\ \mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{}\mathbf{1}\mathbf{‐}\mathbf{Aminooctane} \end{gathered}$$

(c) Polar solutes were separated by hydrophilic interaction chromatography (HILIC) with a strongly polar bonded phase. How would retention times be affected if eluent were changed from 80 vol% to 90 vol% acetonitrile in water?

(d) Polar solutes were separated by normal-phase chromatographyon bare silica using methyl t-butyl ether and 2-propanol solvent. How would retention times be affected if eluent were changed from 40 vol% to 60 vol% 2-propanol? (Hint: See Table 25-4.)

Part (a)

The retention time will be shorter if 90% methanol in water were used instead of 65%methanol in water because methanol is less polar than water.

So, the non-polar compounds would be more soluble in the mobile phase.

Part (b)

At pH=3, the octanoic is in neutral form, while the 1-aminooctane is in the form of cation. Cation form is insoluble in the non-polar phase. So, if octanoic acid and 1- aminooctane passed through the same column using an eluent of 20% methanol/80% buffer (at pH 3.0), the 1-aminooctane would eluated first.

Part (c)

If polar solutes were separated by hydrophilic interaction chromatography (HILIC) with a strongly polar bonded phase, the retention time would be longer if eluent were changed from 80 % to 90 % acetonitrile in water because the polarity of mobile phase decreases and the polar analytes are more strongly retained than non-polars.

Part (d)

If polar solutes were separated by normal-phase chromatography on bare silica (polar stationary phase) using methyl t-butyl ether and 2-propanol solvent (less polar mobile phase), the retention time would be shorter if eluent were changed from 40% to 60 % 2-propanol because 2-propanol has higher eluent strength than methyl t-butyl