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From outer space to inner space, new manufacturing methods and advanced components change what we can see
Maybe more than anything else, scientists explore nature with light. Light-scattering detectors—also known simply as light detectors or photon detectors—provide one useful type of instrument. As Alan Rawle, applications manager at Malvern Instruments (Westborough, Mass.), explains, “They are actually detecting light rather than scattered light per se.” The light that these devices can detect and how they do it help scientists see and quantify things more completely than ever in some applications.
In comparison to a charge-coupled device (CCD) detector in a camera, these light detectors “are extremely diverse and relate to the detection wavelength or wavelength range,” Rawle says. “One could look at anything from infrared (IR) detectors used in smoke cameras to devices used to examine the scattered light from the dust in Saturn’s rings, allowing composition and size data to be simultaneously extracted.”
Malvern’s Viscotek GPC/SEC systems and detectors can also be used in “many applications in molecular mass measurement of polymers,” Rawle says.
Evolving equipment
When asked about the biggest changes in light-detector technology, Rawle answers, “Nothing radical—evolutionary rather than revolutionary.” He adds, “For example, now we have blue LEDs, and lower wavelengths give increased scattering.” The detectors also improve with advances in semiconductor technology. “Detector areas have become larger and more sensitive, allowing very low light levels to be examined,” Rawle explains.
The detector itself is also evolving. “The biggest move forward in the past 20 years was from conventional photon multiplier tubes—PMTs the size of a baby’s arm with poor quantum efficiency—to avalanche photodiode detectors—APDs, which are smaller and have much higher quantum efficiencies— and photodiode arrays,” Rawle explains.
Moving to APDs allows these detectors to collect adequate signals without increasing the laser power. “More powerful lasers or laser diodes were used in the past to compensate for low signal, such as situations with small particles, low concentrations or poor optical contrast,” Rawle points out. “Indeed, this is the defined space for many biological and protein applications, but more powerful lasers risked heating the sample or damaging the sample.”
The improvement in quantum efficiency with APDs, says Rawle, “has meant smaller systems—less bench space—with just as good or better performance than the antiquated PMT-based equipment.” He adds, “As an example one can measure the size of a small surfactant molecule—say, Triton X-100, which is about 8 nanometers across—in 30 seconds or so.”
Analyzing adulterants in foods
Light-scattering detectors image a wide range of wavelengths and particles, including the dust in Saturn’s rings. (Image courtesy of NASA/JPL-Caltech/Space Science Institute.)Keeping foods and medications safe is a key objective, and one that is an ongoing challenge. It can be addressed with a combination of evaporative light-scattering detection (ELSD) and other technologies, such as ultraviolet (UV) detection and mass spectrometry (MS). “The combination of ELSD with UV detection and MS detection allows for a more complete profile of samples to understand products and any impurities that are found in the samples,” says Viet Pham, senior product marketing manager at Waters (Milford, Mass.).
One nutraceutical designed to improve the health of joints often contains Vitamin D3 and glucosamine. “The properties of these compounds make simultaneous analysis difficult,” Pham says. “Vitamin D3 is highly nonpolar and UV-absorbing, and glucosamine is highly polar with no chromaphore, and each component is typically present in very different concentrations.” ELSD plus MS analysis can measure Vitamin D3, glucosamine and other components.
In analyzing oils, especially olive oil, scientists measure triacylglycerol (TAG). “Traditional HPLC methods for the analysis of TAGs are very long—up to two to three hours—and require a complicated extraction technique before samples can be analyzed,” Pham says. “By using Waters ACQUITY UPC2 with multidetection—ELSD, photodiode array, MS—analysts are able to perform identification and quantitation of their samples in a single run.”
To get the most from the light detection, users need specific controls. “The Waters ACQUITY UPLC ELSD provides flexibility and fine control of temperatures for the nebulizer and drift tube to enable analysts to optimize conditions to maximize sensitivity and selectivity,” Pham explains. “The ELSD flow path has been optimized for the lowest dispersion to allow scientists to take advantage of the peak widths found in UPLC methods.”
Protein power and more
The Waters ELSD Model 2424 uses evaporative light-scattering detection to analyze many samples, including foods and medicines. (Image courtesy of Waters.)Biological research benefits from today’s light detectors in many ways. “Interesting new uses of light-scattering detectors include size and charge analysis of proteins, exosomes and lipid particles,” says Richard Jones, product manager at Beckman Coulter Life Sciences (Indianapolis, Ind.).
Light detectors turn out to be especially useful in developing new therapeutics. “As a protein therapeutic is being handed off from R&D to the development team, protein multimerization and aggregation state are important parameters to be examined,” Jones explains. “Light scattering provides a very rapid method to examine protein size and molecular weight.”
Light detectors can also measure the zeta potential—the charge difference between the surface of a particle and the liquid around it. “The rapid examination of zeta potential is important to gain an understanding of the impact of drug formulation on the propensity of a protein to stay in solution,” Jones explains.
Exosomes are vesicles—usually less than 100 nanometers in diameter— that cells create and secrete. “Many recent companies have been founded with the aim of designing and manufacturing therapeutic exosomes for delivering therapeutics to specific cell types,” Jones says. Beckman’s DelsaMax PRO Zeta Potential Dynamic Light Scattering Analyzer has been used to characterize the zeta potential of some therapeutic exosome preparations.
The DelsaMax PRO has 32 detectors. According to Jones, “Two major recent advances in light-scattering technology are detector number and pressurization of samples.” For the DelsaMax PRO, says Jones, having so many detectors provides two key benefits: “The first is that if any particular detector generates spurious data, it can be quickly noted as an outlier from the other detectors; the second is that the measurement times can be dramatically reduced, thus preserving the integrity of proteins during the application of electric potential.” With previous technology, measuring the zeta potential would probably significantly damage a cell.
More medicines
Light detectors can be used to study medicines in more ways than ever, and researchers keep finding new ones. Scientists from Switzerland and the United States used light-scattering technology to measure the excipients— inert substances—in drug formualtions.1 In particular, they looked at polysorbates (PS), and tested them with three technologies, including mixed-mode (MM)-ELSD and the fluorescence micelle assay (FMA); the results showed that some technologies work better for some uses than others. As the scientists wrote: “For PS20 degraded by chemical oxidation, quantitation results were lower for FMA than MM-ELSD, while the opposite trend was observed with base hydrolysis.”
In developing vaccines, additives can improve efficacy. These adjuvants can be liposomes, which are spherical sacs made of lipids. The amount of lipid in the liposomes can affect a vaccine’s efficacy and stability. U.K. scientists created an HPLC-ELSD “method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics).”2 They used the system to quantify four lipids, and concluded: “HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.”
The ongoing advances in light detectors, especially ones that improve the ability to work with biological molecules, promise to push this technology into even more medical applications. Some of the same advances will also make this technology more useful in basic research. The use of light as a fundamental method of detection in science will not be going away anytime soon, and advances like the ones described here show us even more of what technology can light up around us—whether the light is used to make better medicines or explore nature.
References
- Lippold, S.; Koshari; S.H. et al. Impact of mono- and poly-ester fractions on polysorbate quantitation using mixed-mode HPLC-CAD/ELSD and the fluorescence micelle assay. J. Pharm. Biomed. Anal. 2016; doi: 10.1016/j.jpba.2016.09.033.
- Roces, C.B.; Kastner, E. et al. Rapid quantification and validation of lipid concentrations within liposomes. Pharmaceutics 2016; doi: 10.3390/ pharmaceutics8030029.
Mike May is a freelance writer and editor living in Florida. He can be reached at [email protected].