Great question — you’ve noticed a real difference between how spectrophotometric assays are done in cuvette-based UV–Vis vs. plate-reader formats. Here’s the reasoning behind why FRAP assays with microplate readers almost always use a fixed wavelength (593 nm or sometimes 615 nm) instead of scanning:

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1. Chemistry of the FRAP assay is well established

The FRAP assay depends on the reduction of the Fe³⁺–TPTZ complex to Fe²⁺–TPTZ.

The Fe²⁺–TPTZ chromophore has a very strong and sharp absorbance maximum at 593 nm (sometimes reported slightly shifted depending on buffer/pH/instrument).

Because this λmax is already known and consistent across conditions, researchers don’t need to re-scan every time — they can just measure at 593 nm where the signal is strongest and most specific.

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2. Plate readers are designed for throughput, not scanning

UV–Vis spectrophotometers (with cuvettes) can easily perform full spectral scans with high resolution. This is common during method development or if the chromophore’s absorbance spectrum is unknown.

Microplate readers, however, are optimized for high-throughput measurements at defined wavelengths. While some advanced readers can do spectral scanning, it is slower and less sensitive because of optics and signal averaging across wells.

For routine assays like FRAP, using a fixed wavelength makes plate-based assays much faster and more reproducible.

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3. Standardization and comparability

Because FRAP is widely used, the λmax has been standardized at 593 nm in the original Benzie & Strain (1996) method. Many later adaptations sometimes use 595 nm or 615 nm, but the principle is the same: stick to a known absorption maximum.

By always using the same wavelength, results across different labs, instruments, and studies remain comparable.

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4. Signal-to-noise considerations

Measuring at λmax maximizes absorbance changes while minimizing background interference.

Scanning across multiple wavelengths in a microplate format could introduce more variability (due to edge effects, pathlength differences, lamp fluctuations).

A fixed-wavelength measurement gives higher precision for quantitative assays.

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✅ In short:

In FRAP assays, the absorbance maximum of Fe²⁺–TPTZ is already well characterized (around 593 nm). Microplate readers prioritize speed, reproducibility, and throughput, so they usually measure directly at this known λmax rather than scanning. Scanning is mostly needed during method development, not for routine applications of an established assay.