HPLC: Reversed Phase, Gradients and Method Development · seed 1 · A4, ink-friendly. The answer key prints on its own page for grown-ups.

Tuning a liquid separation

Science · Chemistry · ages 19-21
Name ______________________   Date ____________

  1. For comparable neutral compounds in the same conventional reversed-phase method, which group usually elutes earlier?

    • The less hydrophobic compounds
    • The more hydrophobic compounds
    • All compounds at the same time

  2. Why does a packed HPLC column need a substantial pressure difference?

    • To turn the liquid sample into vapour before separation
    • To drive liquid through packing that resists flow
    • To make retention times independent of solvent composition

  3. An HPLC peak must contain exactly one compound, and matching its retention time to a standard proves its identity.

    Circle one:   True   False

  4. A weak acid has pKa 4. Its neutral form is more retained than its anion. Both pH 2 and pH 6 are compatible with the method. Which trial favours greater retention?

    • Use pH 6 to favour the neutral form
    • Use pH 2 to favour the neutral form
    • Use either pH; both give the same acid ionisation

  5. In water/acetonitrile reversed-phase HPLC, early compounds need weak solvent but late compounds are excessively retained. Which programme is a reasonable trial?

    • Begin with high organic content, then decrease it
    • Keep the low organic content fixed for the whole run
    • Begin with low organic content, then increase it

  6. Increasing acetonitrile fraction shortens retention in a water/acetonitrile reversed-phase method. Column, temperature and flow stay fixed. Which explanation fits the taught solvent-strength relation?

    • More acetonitrile gives greater elution strength
    • More acetonitrile gives weaker elution strength
    • Composition changes only viscosity, not elution strength

  7. For a weak base, pH well below the pKa of its conjugate acid favours the neutral base.

    Circle one:   True   False

  8. An established gradient has acceptable early peaks but excessively retained late peaks. Which controlled trial tests a stronger finish?

    • Change initial composition, pH and flow together
    • Keep the finish fixed and increase sample mass
    • Raise final organic fraction; keep the start fixed
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Answer key

For grown-ups. Fold this page away before handing over the rest.

Tuning a liquid separation W1-mt_hlJl6nXQqT-s1

  1. The less hydrophobic compounds · Less hydrophobic neutral compounds usually have weaker retention under these conditions.
  2. To drive liquid through packing that resists flow · Fine packing creates flow resistance. The pressure difference drives the mobile phase through it.
  3. False · Compounds can co-elute. Retention-time agreement supports an assignment under matched conditions but does not prove it.
  4. Use pH 2 to favour the neutral form · pH 2 is below pKa 4, favouring the neutral acid. The question states that this form is more retained.
  5. Begin with low organic content, then increase it · A low-organic start can retain early compounds. More organic solvent later usually reduces late retention. Check the resulting resolution.
  6. More acetonitrile gives greater elution strength · More acetonitrile commonly strengthens elution in this reversed-phase system and reduces retention.
  7. False · Lower pH favours the protonated, charged form of the base. Higher pH favours its neutral form.
  8. Raise final organic fraction; keep the start fixed · Changing the final organic fraction tests the finish while limiting other changes. All required separations still need checking.
Worksheet · LightMySky