A couple of years ago, I performed intrinsic fluorescence spectroscopy on enzyme solutions (including Pectinex Ultra Clear and Pectinex Ultra SPL), using the following settings:

• Excitation: 280 nm
• Emission: 300–500 nm
• Data interval: 0.2 nm
• Scanning speed: medium
• Spectral bandwidth: 5 nm (excitation and emission)
• Buffer: Citrate buffer, pH 4

At that time, I obtained the expected fluorescence spectra, with a peak around 340 nm and intensity reaching up to ~1000 a.u.

Recently, I repeated the exact same measurements using the same instruments (recently changed lamp), same buffer, same enzymes (both from the original stock and freshly purchased one), and same settings. However, the fluorescence intensity is now ~20 times lower. The spectral shape is similar, but the signal is significantly reduced.

I verified the instrument’s performance (Raman peak check, wavelength accuracy, etc.), and everything appears to be functioning correctly.

Does anyone have experience with this and where the issue could be? Any ideas on further troubleshooting would be greatly appreciated.