Explore our resources below to support your UV-Vis spectroscopy needs. Learn more about our instruments and their features, specifications, and applications in our broad range of product literature and webinars. Understand how and when to use absorbance, spectral bandwidth, transmittance, and reflectance characteristics to maximize the capabilities of your UV-Vis instrument.
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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.
When samples are irradiated with light, they selectively absorb incident light at specific wavelengths. The wavelength with the highest absorbance (λmax) is typically used as the analytical wavelength and expressed in nanometers (nm). Absorbance measurements are simple to take and are used to generate spectrum curves.
Absorption can provide direct and indirect options for calculating concentration. An example of direct measurement for a protein is at A280, which then allows the calculation of the protein concentration (c) based directly on the sample absorbance (A) at 280 nm and the protein-specific extinction coefficient (ε) using the Beer-Lambert equation c = A / (ε × L), where L is the pathlength. An indirect measurement, such as a colorimetric assay, relies on the generation of a standard curve and a reaction that produces a color change proportional to amount of protein in the sample.
The slit size coming out of the monochromator governs the spectral bandwidth (or bandpass) of the instrument and affects the ability to discriminate between two neighboring peaks (spectral resolution), the intensity of light that reaches the detector, the measurement time, and the signal-to-noise ratio. A narrower bandwidth spectrophotometer can increase resolution and the signal-to-noise ratio at the expense of measurement duration; a more economical instrument with a wider bandwidth can decrease time-to-results at the expense of decreased resolution and increased background noise. To meet the performance and bandwidth requirements for all global pharmacopoeias including United States Pharmacopoeia (USP), European Pharmacopoeia (EP) and Japanese Pharmacopoeia (JP), you can choose the Thermo Scientific Evolution Pro instrument, or choose the Evolution One series models to meet all requirements for USP and EP.
Transmittance measures the amount of light that passes through a sample and can provide a quantitative information based on known reflective and transmissive properties of a material. Transmittance values are typically reported as a percentage, comparing the ratio of the light reaching the detector to the incident light entering the sample. A sample that is perfectly transparent transmits all the light (100% T); a completely opaque sample transmits none of the light (0% T). This measurement can be used to calculate the concentration of chemicals in solution, test the clarity of water and thin films, assess the grade of products like maple syrup, and more.
To calculate transmittance (T), the amount of light exiting the sample (I) is divided by the amount of incident light entering the sample (I0): T= I/I0. Typically, these measurements are reported as percentage transmittance by multiplying by 100: %T= 100(I/I0). There is a logarithmic relationship between absorbance and transmittance, where A = - log10T: when absorbance equals 0, transmittance is 100% T and when absorbance equals 1 transmittance is 10%.
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.