Determine the Stern-Volmer quenching constant (K_SV = 105.0 M⁻¹) and bimolecular quenching rate constant (k_q = 5.68 × 10⁹ M⁻¹·s⁻¹) for the collisional deactivation of quinine sulfate fluorescence by chloride ions in 0.050 M H₂SO₄ at 25.0 °C using spectrofluorometry. Explore interactive analytical gravimetry, Class A volumetric glassware and density determination.
Ready to dispense 0.01 M of liquid.
Adjust [Cl⁻] V, then weigh the dispensed aliquot.
Mass observations are determined by the high-precision thermodynamic equation of state for pure liquid water. Readings reflect thermal equilibrium and analytical balance force restoration.
['The linear Stern-Volmer relationship applies strictly to purely dynamic collisional quenching at low quencher concentrations ([Q] < 0.05 M).', 'At high ionic strengths or elevated quencher concentrations ([Q] > 0.1 M), kinetic sphere-of-action and static quenching contributions cause upward curvature in F₀/F plots.', 'Bimolecular rate constants extracted from steady-state intensity require independent time-resolved lifetime determination (τ₀).'] The physical chemistry simulation model is parameterized for quinine sulfate dihydrate in 0.050 M H₂SO₄ at 25.00 °C (λ_ex = 350.0 nm, λ_em = 450.0 nm, fluorophore concentration c = 1.00 μM, unquenched lifetime τ₀ = 18.50 ns). The unquenched reference intensity is normalized to F₀ = 100.00 AU. Collisional quenching by chloride ions ([Cl⁻] ∈ [0.0020, 0.0050, 0.0100, 0.0150, 0.0200, 0.0250] M) follows F₀ / F = 1 + K_SV [Cl⁻] with literature benchmark K_SV = 105.0 M⁻¹. Measured fluorescence intensities are F = [82.64, 65.57, 48.78, 38.83, 32.26, 27.59] AU, corresponding to intensity ratios F₀ / F = [1.2101, 1.5251, 2.0500, 2.5753, 3.0998, 3.6245]. Ordinary least-squares regression yields an empirical slope m = 104.979 M⁻¹ (difference = 0.0199% from 105.0 M⁻¹) and Y-intercept b = 1.00024 with R² = 0.9999999. Combining K_SV with τ₀ = 18.50 ns extracts an empirical bimolecular quenching rate constant k_q = 5.675 × 10⁹ M⁻¹·s⁻¹, in exemplary concordance with the theoretical Smoluchowski diffusion-controlled rate limit in aqueous solution (k_diff ≈ 7.4 × 10⁹ M⁻¹·s⁻¹, quenching efficiency γ = 77%).
Provides monochromatic excitation at λ_ex = 350.0 nm (xenon arc lamp source) and collects orthogonal right-angle fluorescence emission at λ_em = 450.0 nm with high quantum efficiency photomultiplier detection.
10.00 mm internal optical path length cuvettes polished on all four optical faces to allow right-angle 90° excitation and emission light paths with negligible scattering and autofluorescence.
Maintains the sample solution at a constant temperature of 25.00 ± 0.02 °C to eliminate thermal quenching effects (temperature coefficient ≈ −1.5% per °C).
Allows precise preparation of primary standard quinine sulfate dihydrate solutions in 0.050 M H₂SO₄ and volumetric addition of standardized potassium chloride quencher stock solutions.
High-purity reference fluorophore (fluorescence quantum yield Φ_f = 0.546 in 0.050 M H₂SO₄, unquenched lifetime τ₀ = 18.50 ns) prepared at 5.0 μM to avoid inner filter effects.
Analytical reagent grade KCl dissolved in 0.050 M H₂SO₄ matrix at 0.500 M stock concentration, used to prepare calibration standards from 0.0020 M to 0.0250 M [Cl⁻].
Determine the Stern-Volmer quenching constant (K_SV = 105.0 M⁻¹) and bimolecular quenching rate constant (k_q = 5.68 × 10⁹ M⁻¹·s⁻¹) for the collisional deactivation of quinine sulfate fluorescence by chloride ions in 0.050 M H₂SO₄ at 25.0 °C using spectrofluorometry. Suggested independent volumetric settings: 0.002, 0.005, 0.01, 0.015, 0.02, 0.025 M.
Prepare a 5.0 μM working solution of quinine sulfate dihydrate in 0.050 M H₂SO₄. Verify that the peak absorbance at 350.0 nm is below 0.05 AU to eliminate inner filter effects (primary self-absorption of excitation light) and secondary emission reabsorption across the 10.00 mm optical cell.
Prepare six 25.00 mL volumetric flasks, each containing an identical 5.00 mL aliquot of 5.0 μM quinine sulfate stock (final fluorophore concentration 1.00 μM). Using a calibrated micro-burette, deliver calculated volumes of 0.500 M KCl stock into the flasks to obtain chloride quencher concentrations [Cl⁻] = [0.0020, 0.0050, 0.0100, 0.0150, 0.0200, 0.0250] M. Dilute to the mark with 0.050 M H₂SO₄ and mix thoroughly.
Power on the spectrofluorometer xenon arc lamp and allow 30 minutes of thermal and lamp arc stabilization. Zero the detector against a 0.050 M H₂SO₄ solvent blank. Insert the unquenched fluorophore standard ([Cl⁻] = 0.000 M) into the Peltier-controlled cell at 25.00 °C. Adjust PMT gain and excitation/emission slit widths (5.0 nm bandpass) so that the unquenched emission intensity F₀ reads exactly 100.00 AU at λ_em = 450.0 nm.
Rinse the four-sided quartz cuvette twice with the first quenched solution ([Cl⁻] = 0.0020 M), wipe all optical faces with lint-free optical lens tissue, and place it in the sample chamber. Allow 3 minutes of thermal equilibration at 25.00 °C. Record steady-state emission intensity F at 450.0 nm with integration time of 2.0 seconds. Repeat sequentially for all six quencher concentrations.
Compute the fluorescence intensity ratio F₀/F for each quencher concentration [Cl⁻]. Verify that F₀/F increases monotonically with quencher concentration, from F₀/F = 1.210 at 0.0020 M to F₀/F = 3.625 at 0.0250 M.
Plot the fluorescence intensity ratio F₀/F on the Y-axis against chloride concentration [Cl⁻] on the X-axis. Perform ordinary least-squares linear regression to extract the slope K_SV and Y-intercept. Combine K_SV with the unquenched singlet lifetime τ₀ = 18.50 ns to calculate the bimolecular quenching rate constant: k_q = K_SV / τ₀. Compare k_q with the theoretical Smoluchowski diffusion-controlled rate limit.
Points are your recorded gravimetric observations. The regression line validates the linear mass-volume proportionality and extracts density.
| # | Chloride Quencher Concentration [Cl⁻] (M) (M) | Relative Fluorescence Emission Intensity F (450 nm) (AU) | Quencher Concentration [Cl⁻] (M) | Fluorescence Ratio F₀/F (dimensionless) | Remove |
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