Many laboratories in the life sciences require fast, accurate, and reliable measurements. This is especially true in high performance liquid chromatography (HPLC) and DNA concentration and purity measurements, all of which rely on quantitative analysis by absorption spectroscopy. Leading instrument manufacturers for these applications are looking for alternative sources such as UV-C (C-band UV LEDs in the 100-280 nm range) as a means of meeting the new needs of their end users. UV-C LEDs offer these manufacturers the opportunity to provide miniaturization and low-cost solutions to differentiate their products and increase market penetration.
UV LEDs in other frequency bands have been widely used in some applications. The use of UV-C LEDs is limited by LED efficiency, especially at the upper limit of the spectral band.
The UV-C LEDs at the upper limit are very useful in many test and measurement-centric applications in the life sciences. Laboratories are looking for small instruments that cost less than comparable large instruments to increase productivity and laboratory performance. Emerging technologies are miniaturizing instruments, which not only reduces costs, but also reduces laboratory use. Low-cost, small-sized LED-based instruments allow researchers to perform routine measurements on their benches. At the same time, when full-spectrum measurements are required, researchers can use more expensive full-spectrum UV lamps at the center of the lab, which reduces lab bottlenecks and increases productivity.
In the past, instrument manufacturers were hindered by the lower performance of UV-C LEDs on the market during development. However, with the advent of higher performance components, manufacturers can use LED capabilities to address these trends with new instrument models. In this article, some applications of UV-C LEDs will be discussed.
UV-C LED facilitates fixed wavelength detection in HPLC
High performance liquid chromatography (HPLC) is a separation technique that introduces a sample mixture into a column. Since the components in the sample solution have different partition coefficients between the mobile phase and the stationary phase, when the relative motion is performed in the two phases, the components are moved at a moving speed after repeated adsorption-desorption processes. A large difference is produced, which is separated into individual components and sequentially flows out of the column. The detection of these components was mainly carried out by using an absorption spectrum of an ultraviolet spectrophotometer. HPLC is commonly used for routine process monitoring, quality control, and biotechnology research in protein purification, pharmaceutical and beverage manufacturing.
Current HPLC detectors typically use xenon lamps as their primary source. The HPLC manufacturer chose a xenon lamp because of the high stability of the light output during the measurement period. In HPLC, the high light output stability of the UV source ensures that lower concentrations of compounds are detected. Compared to other UV lamps such as Xenon flash lamps or mercury lamps, the stability of xenon lamps is increased by two orders of magnitude.
LED achieves stability requirements
The new high-performance UV LEDs are equivalent to high-end xenon lamps with peak fluctuations below 0.005%. UV-C LEDs provide similar sensitivities while reducing the cost and size of the overall instrument for fixed wavelength detection. In this way, when the manufacturer needs a single or a few fixed wavelengths, it can help the end user to use the laboratory area reasonably. In addition, LEDs offer a longer life and turn them on immediately, ensuring that LED life is not wasted in preheating, unlike xenon lamps. In addition, light from the LEDs can be easily fiber coupled, which is advantageous in applications where isolation of the flow cell is required. Manufacturers can choose UV-C LEDs as an alternative source for fixed wavelength detectors and build more cost effective systems.
For fixed wavelength HPLC systems, the biggest difference in cost is usually due to the cost of the initial configuration as it includes the light source and other ancillary equipment. An HPLC system using an LED detector requires a power supply, a photodiode, and a beam splitter. The total cost of an HPLC detector system with LEDs is approximately $750. In contrast, HPLC systems using xenon light sources require more expensive equipment to build. The necessary power supplies are much more expensive and require space to store the lights. In addition, xenon lamps are broad-spectrum light sources that emit many wavelengths of light in the ultraviolet range. This requires an expensive filter and monochromator for fixed wavelength HPLC detection. Therefore, typical system costs are expected to be close to $4,000. Figure 1 shows a typical instrument design using a xenon lamp (a) and a UV-C LED (b).
Figure 1 shows that the optical path of a fixed wavelength HPLC detector using a xenon lamp source (a) is a bit more complicated than that of a fixed wavelength HPLC detector using UV-C LED (b).
Reduce the cost of DNA purity measurements
Another example of the use of UV-C LEDs to be discussed next is DNA concentration and purity measurements. DNA extraction ensures the integrity of biological research and can affect many areas such as biotechnology, forensics, genomics research and drugs. This includes the detection of genetic disorders, the production of DNA fingerprints, and the production of genetically engineered organisms.
In these applications, the key to increasing productivity and reducing costs is the speed and accuracy of the measurements. DNA and protein have absorption peaks at 260 nm and 280 nm, and the absorbance at these wavelengths determines the concentration of DNA and protein, respectively, and the ratio of absorbance determines the purity of the DNA sample. Spectrometers for DNA concentration and purity measurements rely on xenon flash lamps, which provide instant on/off, allowing rapid evaluation of high linearity measurements over a wide concentration range.
While broad-spectrum UV lamps (such as Xenon flash lamps) can produce sufficient light at multiple wavelengths (most of which can be seen in the visible spectrum), only specific wavelengths of light can be used for single parameter measurements. Since DNA purity is determined by absorption measurements made at 260 nm and 280 nm, other components, such as filters and mirrors, must be used to filter out unwanted wavelengths before the broad spectrum light is illuminated into the sample. Xenon flash lamps also require high voltages and increase the protection of electronic equipment during lighting. These expensive electronic devices, along with other optical components, quickly increase the overall cost of the instrument.
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