UV-Vis Spectrophotometer Resources

In the most basic terms, spectrophotometers enable photometric comparisons of relative light intensities across the ultraviolet and visible spectrums. Directing a controlled, constant intensity light source (halogen, deuterium, xenon) across the spectrum or at a specific wavelength through a sample easily can confirm known or calculate unknown characteristics of the sample. The incident light (I₀) can be redirected backward as reflection, suffer an energy loss as absorption, and pass through transparent or translucent samples as transmission. Samples that are in liquids need to be placed into a test tube or cuvette composed of plastic, glass, or quartz that is compatible with the sample and the measurement wavelength, and placed into a holder in the sample compartment before a measurement can be made. 

Spectrometers can have different optical setups with a single, double (or split), or dual beam setup. The simplest and most economical units rely on a single beam of light that travels from the wavelength selector (monochromator or filter system) to the detector, but requires separate measurements of the baseline solvent blank to compare with the measurement of the sample. A double beam unit splits the light beam into a reference beam and a sample beam, enabling measurement of a blank and a sample in the sample compartment under the same instrument conditions with a single detector that alternates data collection between the reference and the sample. A dual beam setup uses two beams and two detectors in parallel to measure the sample and reference simultaneously.  

Samples in solid or solution form can reflect the incident light used for spectrophotometric analysis. When a surface is very smooth, it can produce a mirror-like, specular reflection, where the incident ray of light reflects as a single ray of light at a reflection angle that equals the incident angle. Rough surfaces and those that scatter light inside the sample instead produce a diffuse reflection where even a single angle of incidence can result in a reflection that dissipates through a broad distribution of angles. Measuring the transmission of light of these materials using a traditional sample holder will result in only a fraction of light reaching the detector, leading to reduced spectral quality and information loss. However, using an integrating sphere accessory enables all light passing through the sample to be collected for total transmission measurements by positioning the sample at the entrance point or for total or diffuse reflectance measurements by positioning the sample at the exit port.