November 1, 2016 (Vol. 36, No. 19)

Implementing New Technology Enables Low-Cost, High-Throughput Protein Purification

The growing demand to rapidly purify vast numbers of proteins is driving development of high-throughput, automated protein purification methods. In these automated methods, multiple channels for parallel chromatography are deployed, with UV monitoring of individual channels to control the onset and end of purification.

Chromatography (HPLC) is used for protein purification, where the detection of a mixture of components is commonly done with spectroscopy (primarily absorption and, sometimes, fluorescence). Current HPLC detectors frequently use deuterium lamps as a light source because of the high light output in the UV wavelengths as well as excellent stability of light and relatively long life.

However, advancements in LED technology are providing for alternative solutions that match or exceed the performance of traditional UV lamps, while also reducing system costs for instrument manufacturers. Deep UV (UVC) LED solutions have enabled HPLC manufacturers to replace deuterium lamps and offer lower cost detectors for fixed wavelength applications like protein purification.

In chromatography applications that only require a few fixed wavelengths for detection, manufacturers can reduce system costs by as much as 75% by pursuing a UVC LED-based approach. This reduction in cost is due to the design simplicity of UVC LED-based detectors, which require simple electronics, power supplies, and photodiodes for signal detection. This is in contrast to the deuterium lamp-based instruments, which require expensive power supplies and monochromators for analysis.

In addition to the lower initial cost, UVC LEDs offer many other performance benefits over deuterium lamps. Because of the monochromaticity of LEDs, instrument designers can select the wavelength to match the absorption spectrum of the compound of interest. Compared to the broader spectrum of deuterium lamps, UVC LEDs also offer more irradiance at a specific wavelength in the deep UV range (Figure 1).

Another key benefit of LED-based detectors is stability of light. The stability of light output is defined as fluctuations in light output over short periods of time (i.e., the duration of a measurement). High light output stability of a UV light source in HPLC ensures detection of lower concentrations of compounds. Since minor fluctuations in light intensity can increase the noise, stability of the light intensity in tens of ppm (parts-per-million) is required for the HPLC light sources.

Fluctuation is evaluated by peak-to-peak intensity variations within a 30 second time window, and averaged over 15 minutes or longer. A comparison of UV lamps indicates that deuterium lamps have the best stability (i.e., lowest fluctuation) when compared with other conventional UV lamps. AlN-based UVC LEDs exceed the stability of high-end deuterium lamps with a peak-to-peak fluctuation lower than 50 ppm (0.005%). The superior stability (lower fluctuation) of UVC LEDs leads to lower detection limits in HPLC.

Deuterium lamps require a warm-up period of up to 30 minutes to allow the lamp to reach thermal equilibrium. For this reason, most lamps are left on while not in use so that the instrument is ready as needed—wasting much of the lamp’s useful life. LEDs offer longer life and instantly turn on, ensuring that LED lifetime is not wasted in warm-up.

Lifetime considerations are significant in HPLC systems, as frequent light source replacement could lead to laboratory bottlenecks and extra costs. A standard 30W deuterium lamp has an average lifetime of about 3,000 hours. With lifetimes ranging from 3,000 to 8,000 hours depending on operating conditions, UVC LED matches or exceeds the lifetime of a deuterium lamp. All of this extended life is useful for measurement, decreasing operation, and maintenance costs for the instrument.

In addition, their smaller footprint enables ease of scale-up as the number of channels is increased and the emission from UVC LEDs can be easily fiber coupled, which is an advantage in applications where the flow cell needs to be isolated to avoid solvent explosion, toxicity issues, or alternatively, when the flow cell is placed in an oven.

Granted, LEDs are anything but new—the technology has always offered a smaller footprint, ease of alignment, and enhanced end-user productivity. New high-performance UVC LEDs hold the key to higher performance with lower cost in these applications.

As the adoption of UVC LEDs increases in new chromatography systems, manufacturers are leveraging the benefits around cost, size, and performance to offer innovative new detectors that will revolutionize genomics research. 

Figure 1. Irradiance comparison of a UVC LED with a peak at 255 nm versus a typical deuterium lamp

Hari Venugopalan, Ph.D. ([email protected]), is director of global product management at Crystal IS.

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