Glossary of UV Vis Spectrophotometry
This glossary helps to explain some of the technical terms we use when describing our spectrophotometers and colorimeters. This may be useful information to help determine which instrument is most suitable for your needs.
Often abbreviated to Vis. A visible spectrophotometer or colorimeter typically measures in the visible region of the electromagnetic spectrum (340 - 750 nm), although Biochrom spectrophotometers can measure up to 1100 nm (the near infrared region). If your samples are coloured this is an indication that they will absorb visible light. If you do not need to measure in the UV region (<340 nm) a visible spectrophotometer or colorimeter may be a more economical alternative.
Often abbreviated to UV/Vis or UV-Vis. UV-Visible spectroscopy offers the maximum flexibility and is suitable for applications in the wavelength range 190 to 1100 nm. In UV/Visible spectroscopy the UV region is considered to be any wavelength less than 340 nm. Nucleic acid, purified proteins and other organic molecules are often measured in the UV region. Another name for a UV/Vis spectrophotometer is UV/Vis spectrometer. Special cells and cuvettes have to be used in UV/Vis spectrophotometers.
Deuterium arc lamps measure in the UV region 190 - 370 nm. As Deuterium lamps operate at high temperatures, normal glass housings cannot be used for the casing. Instead, a fused quartz, UV glass, or magnesium fluoride envelope is used. When run continuously typical lamp life for a Deuterium lamp is approximately 1000 hours, however this can be extended by up to a factor of three using PTR technology. Deuterium lamps are always used with a Tungsten halogen lamp to allow measurements to be performed in both the UV and visible regions.
Also known as quartz Iodine lamps, these measure most effectively in the visible region from 320 - 1100 nm. Instruments that only use Tungsten halogen lamps as the light source will only measure in the visible region. Using Press to Read technology the lamp life of Tungsten halogen lamps can be extended to approximately 3000 hours.
Xenon flash lamps provide a high energy light source with a short warm up time and long lamp life. They measure in both the UV and Visible regions of the electromagnetic spectrum from 190 - 1100 nm. Xenon lamps typically provide 80 flashes per second giving them their distinctive bird-like noise.
Produces light at a single wavelength, without the need for a monochromator. Lamp life is almost infinite and LED sources are stable with little variation in bandwidth making them an attractive, low cost solution for simple applications.
Press to Read (PTR)
Biochrom press to read (PTR) technology helps to prolong lamp life and reduce the cost of ownership by ensuring that the lamp is only on when a measurement is being taken.
Optical Beam Configuration
These are the simplest and most economical instruments. In a single beam spectrophotometer all of the light passes through the sample holder. Measurements are made by placing a reference in the sample holder, which is measured to standardize the instrument. This reference value is subtracted from subsequent sample measurements to remove effects from the solvent and the cell.
Also known as dual beam spectrophotometers or split beam spectrophotometers with Reference Beam Compensation (RBC). The light from the source is split into two paths with approximately 70% of the energy from the monochromator passing through the sample and the rest going to a separate feedback detector. This corrects for variations in energy and improves measurement stability and reproducibility. As with single beam instruments, all of the light passes through the sample holder and measurements must be made first by placing a reference in the sample holder to correct for solvent and cell effects.
Spectrophotometers using a split beam configuration and diode array detection do not need a lid to protect the ingress of light into the sample chamber. See FAQ. This is particularly useful feature when measuring samples in test tubes.
In a double beam spectrophotometer the beam from the light source is split in two. One beam illuminates the reference cell holder and the other illuminates the sample. This configuration enables reference correction to be applied continually throughout a measurement and is commonly used when the sample and reference change over time e.g. kinetics measurements. Although advances in electronics have greatly improved the reliability of single and split beam instruments, double beam instruments still provide the greatest accuracy.
Also known as spectral bandwidth or bandpass, this relates to the physical size of the slit from which the light passes out from the monochromator. Bandwidth is classically measured using the full width half height method but for instruments with bandwidths <4 nm this can also be done using the peak and valley ratio of a toluene in hexane scan.
The narrower the bandwidth, the greater resolution will be achieved in measurements and scans improving peak separation, identification and quantification. Some pharmaceutical and chemical applications specify that a bandwidth <2 nm should be used. To achieve full European Pharmacopoeia compliance a bandwidth ≤1nm is required with a toluene in heaxane ratio of >2.0.
However there is a trade off, as reducing the bandwidth reduces the energy passed to the detector therefore instruments require a longer measurement time to achieve an acceptable signal to noise ratio. Depending on the application, an instrument with a larger bandwidth may be a more economical alternative.
Methods of Spectrophotometric Measurement
Single Wavelength Measurement
Also known as Fixed Wavelength. This is the simplest application of a spectrophotometer and allows for measurement of the Absorbance or Transmittance (percentage of Transmission %T) at a specified wavelength. All Biochrom spectrophotometers and colorimeters can perform single wavelength measurements.
Multi Wavelength Measurement
This is an extension of single wavelength measurements where sample absorbance is recorded at multiple wavelengths. Mathematical manipulations of this data can often reveal details about the sample's composition or purity. Nucleic acid measurement at 260nm and 280 nm is an example of a multi wavelength application to check sample purity. Absorbance at multiple wavelength used in combination with a mathematical formula is often used to define color intensity in e.g. wine.
Concentration using a Factor
The concentration of a sample is determined by multiplying the Absorbance value by a specific factor. Concentration using a factor utilizes Beer Lambert Law where concentration is proportional to absorbance.
Concentration using a Standard Curve
Where a specific factor is not known, the concentration of a sample can be determined through constructing a standard curve by measuring the Absorbance of standards that contain known concentrations of the analyte. Biochrom spectrophotometers offer a variety of curve fitting algorithms.
Kinetic measurements are often used for enzyme kinetics measurements where the Absorbance of a sample at a given wavelength is measured over a specified time. The change in Absorbance and slope of this plot provides details about the rate of reaction. Using Biochrom instruments, kinetic measurements can be performed from seconds through to hours. Kinetic measurement often requires a temperature control accessory to hold the sample at constant temperature.
Also known as spectral scanning or wavescan measurements. The Absorbance or % Transmittance of a sample is measured over a specified wavelength range. The position and magnitude of these peaks and valleys will give an indication of sample composition and purity.
Life Science Methods
A range of Biochrom spectrophotometers contain dedicated built-in Life Science methods. These include methods for the calculation of nucleic acid (DNA, RNA and Oligonucleotide) purity and concentration, plus determination of protein concentration using direct UV methods or using BCA, Biuret, Bradford and Lowry colorimetric protein assays.
Measurement of the Absorbance (also known as the optical density or OD) of a suspension of cells at 600 nm (OD 600nm) can give a researcher an indication of when cells are ready for harvesting e.g. E coli. It is important to note that for turbid samples such as cell cultures, the Absorbance (OD) measured is due to light scattering, and not due to molecular absorption. All Biochrom spectrophotometers can measure OD at 600nm.
Spectrophotometer Accessories and Connectivity
Biochrom cell changers hold up to 8 samples and can increase productivity by allowing for the automatic measurements of samples. Cell changers are required for parallel kinetics measurements.
Used with a flow cell this enables measurement of controlled sample volumes. Sippers are ideal for speeding up sample analysis e.g.for monitoring continuous production processes but they also for reduce sample handling when measuring toxic materials.
Applications that require sample measurements at non-ambient temperatures need some form of temperature control e.g. life science methods at 37 °C. Biochrom offers two methods for temperature control; electrical control (also known as Peltier control), which gives precise temperature control over a wide range of temperatures and water circulated temperature control (water jacket) an economical alternative for precise temperature control.
Print directly from the instrument. Options offered by Biochrom include built-in thermal printers, and connection to external devices such as chart recorders, or serial printers.
Stores data on the instrument's internal memory, allowing download or export to removable media e.g. SD card, Bluetooth® or USB mass storage devices for storage or data manipulation on a PC.
Full control of a spectrophotometer via a PC, including data export and analysis.
All Biochrom instruments are CE marked and are manufactured under a rigorous quality system.
The European Pharmacopoeia (EP) gives guidelines for product quality and measurement consistency. Biochrom spectrophotometers achieving EP compliance satisfy all EP requirements for UV/Visible spectrophotometers, including photometric and wavelength accuracy, spectral bandwidth etc. Users of these instruments can be confident that their results will be consistently of the highest quality. For full EP compliance a 1nm bandwidth instrument is required and a toluene in hexane ratio of greater than 2.0.
Instrument Qualification / Operational Qualification (IQ/OQ). This is a series of specialist test protocols to inspect, verify and document instrument performance and installation.
21 CFR Part 11 Compliance
For industries and users that require their samples to satisfy the FDA requirements for electronic records and electronic signatures, Biochrom offers 21 CFR Part 11 compliant software.
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