Fluorescence Quantum Yield Calculator

Calculate fluorescence quantum yield, radiative rate, and quenching constants.

Fluorescence intensity F_sample (counts)

Fluorescence intensity F_reference (counts)

Absorbance of sample A_sample

Absorbance of reference A_ref

Quantum yield of reference Φ_ref

Quinine sulfate=0.54, Fluorescein=0.95, Rhodamine 6G=0.95

Fluorescence lifetime τ (ns, optional) (ns)

Please check your inputs and try again.

What Quantum Yield Measures

Quantum yield is the fraction of absorbed photons that are re-emitted as fluorescence. It is bounded between 0 and 1, and the shortfall goes into competing non-radiative pathways.

Φ = photons emitted / photons absorbed = k_r/(k_r + k_nr)

Pathway	Fate of the excited state	Effect on Φ
Fluorescence (k_r)	Photon emitted	The numerator
Internal conversion	Energy lost as heat	Reduces Φ
Intersystem crossing	To triplet state	Reduces Φ, may give phosphorescence
Quenching	Energy transferred to another molecule	Reduces Φ

Because the relative method compares against a reference of known quantum yield, both must be measured under matched conditions — same excitation wavelength, similar absorbance, and correction for refractive index if the solvents differ.

Why Absorbance Must Stay Low

Both sample and reference should have absorbance below about 0.1 at the excitation wavelength. Above that, the inner filter effect distorts results: the front of the cuvette absorbs most of the light, so emission comes from a different region than the detector geometry assumes, and re-absorption of emitted photons compounds the error.

Fluorescence lifetime provides an independent check. Since Φ = k_rτ, a drop in quantum yield with unchanged lifetime indicates static quenching — a non-fluorescent complex forming — while a drop in both indicates dynamic collisional quenching. That distinction is the basis of Stern–Volmer analysis.

Worked Examples

Example 1: FITC dye: Fs=450, Fr=600, As=Ar=0.05, Φref=0.95

Φ=0.95×(450/600)×1

Result: Φ=0.7125 — good fluorescent dye

FITC actual Φ≈0.71 ✓ in pH 9 buffer

Example 2: Quenched probe: same setup but Fs=150

Φ=0.95×(150/600)×1

Result: Φ=0.2375 — 67% quenching

Stern-Volmer analysis would give quenching constant

Example 3: Distinguishing quenching mechanisms

Φ halves, τ unchanged

Result: Static quenching

A non-fluorescent complex removes molecules from the emitting population. Those still fluorescing behave normally, so lifetime is unaffected.

Example 4: Dynamic quenching

Φ and τ both halve

Result: Collisional quenching

The quencher deactivates excited molecules during their lifetime, shortening it. Both quantities fall together, which is the Stern–Volmer signature.

Common Mistakes

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Using absorbance above 0.1

The inner filter effect distorts both intensity and apparent yield. Dilute until absorbance at the excitation wavelength is below roughly 0.1.

⚠️

Not matching absorbance between sample and reference

The comparison assumes equal photon absorption. Mismatched absorbance must be corrected for explicitly in the ratio.

⚠️

Ignoring refractive index differences

When sample and reference are in different solvents, the ratio requires a refractive index correction term. Omitting it introduces systematic error.

⚠️

Assuming quenching always shortens lifetime

Static quenching forms a non-fluorescent ground-state complex, reducing intensity while leaving the lifetime of remaining molecules unchanged.

Frequently Asked Questions

Stern-Volmer quenching?

F₀/F = 1 + K_SV[Q] where K_SV = k_q×τ₀. Plot F₀/F vs [Q]: linear = dynamic quenching (collisional). Upward curve = mixed static/dynamic. K_SV gives bimolecular quenching rate k_q (~10¹⁰ L/mol/s for diffusion-limited). Used to study protein binding, oxygen sensing, FRET.

FRET efficiency from lifetime?

FRET efficiency E = 1 - τ_DA/τ_D. Donor lifetime decreases when acceptor is close. R = R₀(1/E - 1)^(1/6) gives distance. Förster radius R₀: 1-10 nm range. FRET = 'molecular ruler' for nanometer-scale distances in biology.

What is fluorescence quantum yield?

The fraction of absorbed photons re-emitted as fluorescence. It ranges from 0 to 1, with the remainder lost to heat, intersystem crossing or quenching.

Why must absorbance be kept below 0.1?

To avoid the inner filter effect, where the front of the cuvette absorbs most of the excitation light and emitted photons are re-absorbed, distorting the measurement.

How do I tell static from dynamic quenching?

Measure lifetime as well as intensity. Dynamic quenching shortens lifetime; static quenching leaves it unchanged because it removes molecules entirely from the emitting pool.

Why use a reference standard?

Absolute quantum yield measurement requires an integrating sphere. The relative method compares against a well-characterised standard under matched conditions, which is far more accessible.

Formula Explorer connections

Interpretation: This relationship converts chemical amount, mass, composition or balanced-equation ratios into a reaction quantity. Assumption: Use a balanced reaction, consistent units and the correct molar mass. Purity, side reactions and limiting reagents can change experimental results.

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