VisualChem The interactive chemistry textbook
中文

Chloride quenching of quinine fluorescence

Excite acidic quinine with a pulsed laser, record the fluorescence decay and fit its lifetime; repeat at several chloride concentrations, obtain the quenching rate constant from a Stern–Volmer plot, and compare it with the diffusion limit.

Difficulty ●●● ≈ 40 min

Aim Measure the fluorescence lifetime of quinine in 0.5 M sulfuric acid and the bimolecular rate constant for quenching by chloride, and decide whether the quenching is dynamic or static.
337 nm pulsed excitation · detection at 480 nm · computed with the parameters of Gutow (2005)

Time-resolved fluorometer: quinine and chloride

Fluorescence decay
020406080100120140time / ns123lg counts
  • signal
  • exponential fit to the tail
  • instrument response

This shot: τ = 20.6 ns (fitted over 420 points)

steady-state fluorescence F/F₀ = 1.000

Stern–Volmer plot
00.020.040.06[Cl⁻] / (mol L⁻¹)01002003004001/τ / (10⁶ s⁻¹)
  • 1/τ
  • diffusion limit 8RT/3η

Measure at least two concentrations to draw a line.

Parameters from Gutow (J. Chem. Educ. 2005): quinine’s lifetime in 0.5 M H₂SO₄ is 20.4 ns, chloride quenches with 5.2 × 10⁹ M⁻¹ s⁻¹, and the instrument response is about 2.5 ns; the diffusion limit of 5.8–6.5 × 10⁹ M⁻¹ s⁻¹ is the range he computed from the solution viscosity. Each decay is a single exponential convolved with a Gaussian instrument response, with square-root-of-counts noise from a deterministic hash; the fit uses only the tail from 5 ns after the peak, as a student would by hand. The “dark complex” option exists only for comparison — chloride does not act that way.

Procedure

  1. Choose the 0 mM sample and press “Fire the laser and fit”. On the log scale the tail of the decay is a straight line whose slope gives the lifetime τ₀.
  2. Measure the 5, 10, 20, 30, 40, 50 and 60 mM chloride samples in turn, recording the fitted lifetime each time.
  3. Read the Stern–Volmer plot: the slope of 1/τ against [Cl⁻] is k_q and the intercept is 1/τ₀. Calculate K_SV = k_q τ₀.
  4. Compare k_q with the violet band (the diffusion limit 8RT/3η): does nearly every collision quench?
  5. Switch to the “dark complex” and measure again; compare how the lifetime and the steady-state fluorescence change, and decide which kind of quencher chloride is.

What to notice

  • Without chloride the lifetime is about 20 ns; 60 mM chloride shortens it to about 3 ns.
  • 1/τ against [Cl⁻] is a straight line with slope about 5 × 10⁹ M⁻¹ s⁻¹, close to but a little below the diffusion limit.
  • With the hypothetical complex the fluorescence dims while the lifetime stays put — the opposite of what chloride does.

In a real lab

  • The nitrogen laser and the photomultiplier both run on kilovolt supplies, and the laser beam is invisible ultraviolet: wear UV-blocking goggles throughout and keep the beam enclosed behind non-reflective walls.
  • The instrument response of about 2.5 ns broadens fast decays: when the lifetime is only a few nanoseconds, fit an exponential convolved with the instrument response.
  • Quinine keeps for more than a year in acid and its fluorescence is not quenched by dissolved oxygen, so the samples need no degassing; the sulfuric acid stock is concentrated enough to burn if not washed off promptly.

Related lessons

All experiments