UV-Vis Binding Fit Calculator

Signal Selection and Baseline Control

Choose a wavelength where the complex shows a clear, monotonic absorbance change. Record A0 from the zero-ligand sample, then keep slit width, integration time, and temperature constant during the titration. Baseline drift can bias 1/(A-A0), so blank-correct solvent and cuvette contributions before fitting. Average replicate scans to reduce noise, and exclude points near detector limits or where A is not greater than A0. Use consistent cuvette orientation.

Linearization and Parameter Meaning

For a 1:1 host-guest equilibrium, the Benesi-Hildebrand plot linearizes the titration by regressing y = 1/(A-A0) against x = 1/[L]. The slope and intercept describe how quickly the signal approaches saturation, and their ratio gives Kb. Because x depends on molar concentration, confirm the selected unit and ensure the free ligand is close to the added ligand under your conditions. Avoid precipitation and inner-filter effects.

Assessing Fit Quality and Leverage

Fit diagnostics indicate whether the linear model is reasonable. R2 near 1 suggests the transformed points follow a straight line, but also check leverage at low concentrations where 1/[L] becomes large. RMSE reports scatter in y-units, so rising RMSE often signals noisy ΔA values. If one point shifts the slope materially, repeat it again, or deselect it with the Use checkbox and record the justification.

Thermodynamics and Practical Interpretation

Amax is estimated from the intercept, representing the limiting absorbance at high ligand for a saturated 1:1 complex. Amax should align with the plateau region; if it is extreme, your data may not reach saturation. The calculator reports ΔG as -RT ln(Kb), so enter temperature instead of a default. Binding constants from UV-Vis are conditional and vary with pH, ionic strength, and solvent composition.

Reporting, Archiving, and Comparability

For reporting, include the raw titration table, the selected points, and the fitted line parameters. Exporting CSV helps you archive datasets beside instrument files and re-fit later using alternative approaches. The PDF snapshot supports lab notebooks, peer review, and fast sharing with collaborators. When comparing ligands, keep wavelength, concentration range, and path length consistent so Kb values remain comparable. For screening, emphasize repeatable workflows, clean blanks, and clear documentation across many samples.

FAQs

1) What data format works best for this fit?

Use paired columns of ligand concentration and absorbance at one wavelength. Include A0 from the zero‑ligand sample, and select only rows where A is greater than A0 for stable inversion.

2) Why does the tool skip points with A ≤ A0?

The model uses 1/(A−A0). If A is equal to or below A0, the denominator is zero or negative, producing undefined or misleading transformed values.

3) How many points should I include?

Use at least three points, but eight to twelve evenly spaced concentrations usually improves stability. Include low, mid, and near‑plateau regions, and avoid extreme outliers.

4) What does R² tell me here?

R² reflects linearity of the transformed Benesi‑Hildebrand plot, not the raw curve. A high value suggests consistency with a 1:1 model, but always inspect for leverage at low concentrations.

5) Can I compare Kb between experiments?

Yes, if conditions match: wavelength, temperature, buffer, ionic strength, and solvent. Report those settings with Kb because optical binding constants are conditional and method dependent.

6) What if my system is not 1:1?

If stoichiometry differs, the linear plot may curve or give unstable Amax. Consider alternative models or nonlinear global fitting, and treat the reported Kb as an approximate screening metric.