Over recent years, UV/Visible spectrophotometry has also become even more popular in application areas such as molecular biology. This popularity is partly due to the simplicity of analysis, the non-destructive nature of the sampling, and the cost-effective nature of the technique. M Lee reports.
UV/Visible spectrophotometry has long been used as a universal laboratory tool covering a diverse range of applications from clinical studies to water quality monitoring to protein analysis.
The market expectations for a modern spectrophotometer are that it should be compact, easy to use, highly reliable, and have minimal running costs. Instrument performance, except for the most demanding applications, is now taken for granted.
Early spectrophotometers tended to be approximately one metre wide and half a metre deep, incorporated complex double-beam optics for stability, and a simple chart recorder with limited output options. Modern instruments are now far more compact and sophisticated. For example, the Ultrospec 100 pro Visible Spectrophotometer from Amersham Biosciences offers flash scanning due to a novel diode array design, incorporates a large graphical display, and stores up to 50 methods.
The instrument also outputs to a wide range of printers and can interface with a PC for advanced data manipulation and storage. However, itmeasures only 310 x 130 x 230 mm and weighs less than 2.0 kg.
Diode array technology enables all of the light to pass through the cuvette. This light is then focused onto a curved diffraction grating and split into its constituent components. Each wavelength then falls onto a separate pixel on the diode array detector. There are no moving parts in a diode array and a scan takes only a few seconds. Furthermore, the position of the cuvette relative to the lamp allows the use of an open sample compartment, which is very convenient if additional reagents are to be added to the sample while in situ.
Diode array technology was previously only available in spectrophotometers that are more expensive. However, it is now available in lower cost instruments and replaces traditional optical designs that require a physical movement in either the diffraction grating or a mirror to measure at a different wavelength. The movement associated with traditional optics makes scanning across wavelengths slower than is possible with a diode array design and there is also a risk of mechanical failure.
A previously common problem with UV spectrophotometers was the short lamp lifetime for the deuterium sources used to provide the energy at wavelengths below 340 nm. Xenon sources offer a cheaper alternative, have at least a 10-fold longer lifetime, and cover UV wavelengths above 230 nm and the visible spectrum up to 900 nm without compromising linearity. However, optimal performance is still provided by the deuterium/tungsten configuration and now the lifetime issue has been addressed by using apress to read' technology. Press to read technology ensures that the lamp is only used when taking a measurement and does not affect signal stability.
A further problem for the analyst can be the availability of sample. The extraction and purification of nucleic acid from cells is a standard procedure usually followed by checks on the purity and the yield. These purity and yield analyses are performed using a spectrophotometer.
A DNA mini-preparation may yield enough sample for several types of analysis; however, for total RNA or mRNA the usual yields are much less. To avoid dilution of the sample, quartzcapillaries and ultra-microvolume cells are now available to handle volumes less than 12 µl. To handle such low volumes the optical system must be optimised and the cells produced to very high standards.
More advanced applications also require complex calculations, a good example of this being the quantification of the fluorescent dyes incorporated into microarray probes. UV/Visible spectroscopy is used to calculate both the quantity of probe produced and the quantity of dye incorporated in to the probe. This ensures that the correct quantity of each probe is added into the hybridisation and provides the opportunity to balance the signal of each dye, thus making the process more reliable and robust. These calculations are now part of standard routines stored within some spectrophotometers including the Ultrospec 3100 pro UV/Visible Spectrophotometer.
Spectrophotometers have progressed in terms of sophistication, sample handling, and convenience. Modern instruments provide solutions to the questions raised by new application areas while their ease of use and range of specialist features has enabled once complex analyses to move into the field of routine spectroscopy. Technology advances mean spectrophotometers are becoming a sophisticated and reliable tool to meet the demands of the laboratory.
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M Lee is with Amersham Biosciences UK Limited,Little Chalfont, Buckinghamshire, UK. www4.amershambiosciences.com
