How Raman Scattering Works

When monochromatic light strikes a molecule, most photons scatter elastically at the same wavelength — this is Rayleigh scattering. A tiny fraction, roughly one in ten million, scatters inelastically at a shifted wavelength. This inelastic process is Raman scattering, and the shift encodes the vibrational frequencies of the molecule’s chemical bonds.

The energy-level picture

In quantum mechanical terms, an incident photon excites the molecule to a short-lived virtual energy state — not a real electronic level, but a transient distortion of the electron cloud. The molecule immediately relaxes by emitting a photon. If it returns to the same vibrational ground state, the emitted photon has the same energy as the incident photon (Rayleigh). If it returns to a higher vibrational level, the emitted photon has less energy and therefore a longer wavelength: this is Stokes Raman scattering, the dominant process at room temperature. If the molecule was already in an excited vibrational state before the collision, it can return to the ground state, emitting a higher-energy (shorter wavelength) photon: anti-Stokes Raman scattering.

The Raman shift

The Raman shift is expressed in wavenumbers (cm^-1) relative to the excitation wavelength, making spectra independent of the laser wavelength used. Each chemical bond — C-H, C=O, N-H, C-C, and so on — vibrates at a characteristic frequency, producing peaks at predictable Raman shifts. The complete pattern of peaks is a molecular fingerprint: two compounds with different structures will produce different Raman spectra, even if they look identical by color or physical appearance.

Why 785 nm excitation is common in process Raman

The choice of excitation laser wavelength involves trade-offs. Shorter wavelengths produce stronger Raman signal (intensity scales as 1/λ⁴) but also excite more fluorescence, which can overwhelm the weak Raman signal. Near-infrared excitation at 785 nm dramatically reduces fluorescence from biological matrices and colored process liquids while still providing good signal. The K LAB ExPro R series uses a 785 nm laser and covers 100-3600 cm^-1, capturing the full fingerprint and CH-stretch regions relevant to bioprocess monitoring of glucose, lactate, and viable cell concentration in bioreactors.

Advantages for in-process measurement

Raman spectroscopy requires no sample preparation, is non-destructive, and works through glass or sapphire windows — making it well suited to real-time monitoring of closed bioprocesses. Water, a major component of biological broths, is a very weak Raman scatterer, unlike in infrared spectroscopy where water absorption is overwhelming. Chemometric models trained on reference data can extract analyte concentrations directly from the Raman spectrum in seconds, enabling closed-loop feedback control during cell culture runs.