What is a UV-Vis Spectrophotometer?
Spectrophotometry is a cross-disciplinary analytical method that allows the quantitative measurement of atoms or molecules in a test sample to be made based on the amount of light it absorbs at a specific wavelength. Scanning a range of wavelengths can reveal characteristic spectra for the identification of the components of the sample. There is a wide range of spectrophotometers available that can handle different types of measurements, different sample types and make use of different regions of the electromagnetic spectrum.
UV-Vis spectrophotometry allows measurements from a sample based on its absorption of ultraviolet and visible light.
Basic Operating Principles of UV-Vis Spectrophotometry
The electrons present in all atoms and molecules are located in discrete energy levels. These electrons can transition to higher levels by absorbing light of the required wavelength in the UV or visible range of the spectrum. Materials can be identified and quantitatively analyzed based on this specificity.
The spectrophotometer measures a beam of light from a light source that is either transmitted through liquid samples or reflected from solid surfaces. It calculates how much has been absorbed by comparing it to the intensity of a reference beam without the sample in place. Liquid samples are held in a transparent container or cuvette. According to the Beer-Lambert Law, absorption is directly proportional to the concentration of the sample, if the distance travelled by the light through the sample and the sample???s absorption characteristic is known. Thus, accurate absorption measurements are essential for quantitative measurements. Several factors, including the optical geometry and components used, can influence these measurements.
For example, since the intensity of the light sources can vary with time, the way in which the reference and sample beams are compared is important. The basic configurations are single beam and double beam. In single beam instruments, there is a single position for both the sample and reference. The reference is measured first and then replaced with the sample for the sample measurement. This cannot account for any variations in light intensity between the two measurements. With double-beam systems both the reference and the sample are in the sample compartment at the same time and the incident beam is split so that part passes through the reference and part passes through the sample, essentially giving simultaneous measurements.
Components in a UV-Vis Spectrophotometer
- Light source: Most UV-Vis spectrophotometers use a combination of Deuterium and Tungsten lamps for good intensity across the wavelength range. More recently Xenon lamps have been introduced, mainly in lower specification systems.
- Monochromator: This is used to select the wavelength of interest. It can range from a simple filter in a single wavelength system to a diffraction grating of various degrees of sophistication in scanning systems.
- Mirrors, slits, and lenses. These are used to direct the light beams inside the spectrophotometer.
- Sample compartment: Most samples in UV-Vis spectrophotometry are contained in a cuvette, a sipper, or on a microplate. Autosamplers may be available for automated analysis.
- Detector: The light is detected by a diode sensor, a Photo-multiplier tube (PMT) or photodiode array, each providing rapid acquisition with high sensitivity across the entire spectrum of interest.
UV-Vis Spectrophotometers from Lambda
Lambda is one of the industry-leading suppliers of UV-Vis spectrophotometers for traditional chemistry, biochemistry, life sciences, microbiology, pharmaceutical research, food & agriculture, cosmetics, materials science, petrochemistry, and quality control. We supply a world-class range of Hitachi instruments, from the entry-level U-5100 to the research-grade U4150. If you would like to learn more about the spectrophotometers that we have available at Lambda, simply contact a member of the team today.