Spectrophotometers: Finding Your Home in the Range (of Wavelengths)

Spectrophotometers: Finding Your Home in the Range (of Wavelengths)

by Caitlin Smith

There’s a good chance you don’t know how much of your daily life has been affected by the humble spectrophotometer. An instrument quantifies the absorbance or transmittance of light through a sample placed within the light path inside the spectrophotometer. The light-source wavelengths can be in the ultraviolet (UV), visible (VIS), near infrared (NIR) or infrared (IR) ranges. Bioapplications for spectrophotometry are diverse and include the quantification of nucleic acids and proteins, cell proliferation and cytotoxicity assays, ELISAs and enzyme kinetics. “From simple UV/VIS systems that measure DNA purity and concentration, to measurements on the newest cutting edge materials, these technologies are critical to the advancement of many important areas of our lives,” says Christopher Lynch, market development manager for food and safety at PerkinElmer. “From the time we wake up in the morning until we go to bed, there are few things we touch that, in some part, have not been characterized in some way by UV/VIS/NIR technologies.”

Though spectrophotometers have been lab fixtures for decades, their technology has continued to evolve. “Spectrophotometers have seen steady improvements over the past 60 years, particularly in the areas of source and detector technology,” says Travis Burt, UV-VIS-NIR, fluorescence product marketing manager at Agilent Technologies. “Supported by rapid advances in electronics and firmware, they have become better, more flexible and more adaptable than their predecessors.” If you are in the market for one, here are some considerations that may help you choose the spectrophotometer that’s right for you.

What sample volumes will you use? A look at small-volume spectrophotometry.

Traditional sample vessels are spectrophotometer cuvettes, but the drive toward high-throughput screening, as well as the need to conserve time and money, have encouraged the development of spectrophotometers that run samples in microplates. The use of more than one type of spectrophotometer vessel in a lab is becoming more common, with an expanding range of labware available. Using sample vessels that vary in size and shape, however, can affect the path length, or the amount of sample that light passes through. BioTek’s Eon™ Microplate Spectrophotometer, controlled by its Gen5™ Data Analysis Software, automatically corrects sample path length. “Direct nucleic acid and protein quantification in microplates requires normalization to a 1-cm path length; this also allows results from different instruments or cuvette spectrophotometers to be compared,” says Lenore Buehrer, product manager at BioTek. “For those preferring a fixed path length, or who are transitioning into a higher-throughput microplate format, an integrated, standard cuvette port is available on Eon.”

Additionally, the Eon can be used with BioTek's Take3™ Micro-Volume Plate to measure 2-µL samples, and cuvettes and may be integrated into robotic systems for automated processing. The ability to measure very low volumes is becoming increasingly important in standard and microplate-based spectrophotometers. By using BioTek's Take3 Micro-Volume Plates, the Eon can read up to 48 2-µL samples for direct nucleic acid and protein quantification without dilution, thereby conserving precious samples.

Thermo Scientific’s NanoDrop specializes in micro-volume instrumentation. The company’s UV/VIS spectrophotometers are designed to work without containment devices like cuvettes, microplates and capillaries. “This has several advantages over traditional methods, including quick and easy sample placement and removal, as well as the ability to use sample volumes as low as 1 µl,” says Andrew Page, applications manager for Thermo Scientific NanoDrop products. “The patented sample-retention system combines fiber optic technology and the natural liquid surface tension to capture and hold a 1- to 2-µl sample between two optical surfaces during measurement. By eliminating traditional containment devices, the volume of the required sample is greatly reduced, which conserves most of the sample for downstream analysis, allowing scientists to quickly and easily quantify and assess purity of samples such as proteins and nucleic acids.”

Another advantage to eliminating sample vessels is that path length can be varied during measurement. “By measuring at several different path lengths and automatically picking the best one, a Thermo Scientific NanoDrop 2000 or 2000c instrument can measure samples approximately 200x more concentrated than is possible with a traditional cuvette-based system,” says Page. “In addition, to address the demands of higher-throughput environments, the Thermo Scientific NanoDrop 8000 spectrophotometer has been designed to perform up to eight 1- to 2-µL measurements simultaneously.”

GE Healthcare Life Science’s NanoVue Plus is another UV/Visible spectrophotometer designed for quick and accurate quantification of nucleic acids and protein samples using small samples (0.5 to 5.0 µl). To load the NanoVue, you drop a sample onto the sample plate without cuvettes, capillaries or microplates. The gold color of the sample plate is compatible with most chemicals used in samples, and its hydrophobic coating reduces sample contamination during repeated use.

What sample vessels will you use? A look at microplate spectrophotometers.

Buehrer says researchers who previously used conventional spectrophotometers that relied on sample cuvettes for endpoint, kinetic, scanning and temperature-controlled experiments can benefit now from the higher throughput afforded by 96- and 384-well (or in some cases, 1536-well) plates. “BioTek's Eon Microplate Spectrophotometer combines multiple functions in a single instrument so that users can reach beyond standard UV/VIS measurements into very low-volume measurements and expand application choice with multiple shake modes and natural convection heating,” says Buehrer. “Eon uses powerful, proven monochromator-based optics and accommodates many different labware vessels, including high-density microplates, to increase flexibility in assay choice and among multiple users.”

BMG Labtech’s UV/VIS microplate (and cuvette) spectrometer uses technology that is able to capture an entire absorbance spectrum per well, rather than just a single wavelength. “The SPECTROstar Nano can capture a full UV-to-visible spectrum [220 to 1,000 nm] in less than one second per well, at 1- to 10-nm resolutions,” says E.J. Dell, business development and applications scientist at BMG Labtech. “Most other absorbance microplate readers use monochromators or filters. When a monochromator-based microplate reader is used to generate a spectrum at a 10-nm resolution from 220 to 1,000nm, it is more than 60 times slower and it has a 10-fold lower resolution than a spectrometer. In other words, by the time a monochromator-based instrument would generate a spectrum for one well of a 96-well plate, the SPECTROstar Nano would have generated spectra for all 96 wells at 1nm resolution.”

Michael Fejtl, international sales and marketing specialist at BMG Labtech, notes that when using the SPECTROstar Nano, “ELISA, protein quantitation, reporter gene assays, DNA 260/280 quantitation, microbial growth and protein aggregation can be read in one reading step, because the spectrometer can measure a whole spectrum or up to eight individual wavelengths,” says Fejtl. “It reads at a rate of less than one second per well, using sample volumes as low as 2 µL with the LVis plate.” The SPECTROstar Nano is also flexible in that it can read absorbance measurements from microplates or cuvettes, in endpoint or kinetic assays.

Using a spectrometer for absorbance assays with a microplate reader improves one’s flexibility, speed and dynamic range, according to Fejtl. “For example, in ELISA assays customers usually read [the assay] at a given wavelength provided by the kit manufacturer,” he explains. “However, without spectral information the customer cannot be certain that this wavelength is the optimal one to choose in the absorbance spectrum. This information is easily derived using a spectrometer. Here, the full spectrum shows the ideal peak, allowing customers to check if the reported wavelength is the best one to use. By using full spectrum analysis, customers will greatly benefit from an increased dynamic range in their assays.” Furthermore, the SPECTROstar Nano captures all wavelengths in one single measurement, obviating the need to read your plates multiple times when multiple wavelengths are needed, such as for DNA.

Molecular Devices recently introduced the SpectraMax® Paradigm microplate reader, which Anita Kant, application scientist at Molecular Devices, describes as future-ready. “An end user can spend just two minutes to insert a cartridge to introduce a new read mode or a new technology,” says Kant. “The cartridge contains the light source and detection accessories. This year we introduced the TUNE cartridge for fluorescence intensity detection with wavelength optimization function. It is unique, as it combines flexibility of monochromators in selecting wavelength and sensitivity of filters.” Kant recommends that when choosing a microplate spectrophotometer, one should “make sure that sensitivity for individual modes is not compensated if choosing a multimode microplate reader.” She also says to keep flexibility in mind, especially with regard to different modes, the various microplates that can be used and possible wavelength selections.

GE Healthcare Life Sciences recently launched a new line of dual-beam Ultrospec instruments in three models that differ in bandwidth optics and performance requirements. The easy-to-use 7000 model, with a 2-nm bandwidth, is ideal for most common lab applications. The 8000 model features a 1-nm bandwidth and is compatible with the European Pharmacopoeia. The 9000 model has a variable bandwidth and supports method development and complex calculations using GE Healthcare Life Sciences’ upgraded Datrys software. All models can be run from a PC or as stand-alone instruments using a color, touch-screen interface. You also have a choice of data-management options: export to a PC, store to internal memory, download to a USB stick drive or output via the built-in printer.

“Consider the applications first, then consider how you want to handle the data—does it print out, does it export to a PC [or] do you save it to a USB memory stick? Often this will depend on the lab environment,” says Sandra Lloyd-Lewis, product manager-spectrophotometry at GE Healthcare Life Sciences. “Also think about ease of use, ongoing running costs and level of experience of the users.” She also notes the importance of sample volume. “The low-volume instruments brought with them not only the ability to use small, 2-µl or less sample volumes but also a real advancement in ease of use,” says Lloyd-Lewis. “GE Healthcare’s [low-volume spectrophotometer] NanoVue’s tag line is ‘drop, measure, done.’ You literally can just drop your sample in it, drop the sample head down and measure it, and you’re done. You wipe away the sample, and you don’t have to worry about cuvettes.”

How do you want to light the sample?

Stray light from the environment can interfere with spectrophotometric measurements, especially across the UV-VIS wavelength range, but new technology is working around this issue. Agilent’s new Cary 60 UV-VIS spectrophotometer takes advantage of Agilent’s xenon flash lamp technology for greater measurement flexibility and ease of use. “The intense, focused, pulsed xenon lamp ensures that measurements taken in open-air conditions are not affected by the presence of room light,” says Burt. “Fiber optic-based probes are used to exploit this capability by channeling the light directly to the sample, rather than having to place the sample inside the instrument. Immersing the fiber optic probe head into a solution, in any vessel, affords for easy sampling, eliminates any need for expensive UV transmissive cuvettes and can avoid interferences such as condensation on the vessel when samples are taken straight out of the fridge.” The Cary 60 system can analyze samples down to 0.54 µL with a high degree of accuracy and reproducibility and can scan the entire wavelength range (190 to 1,100 nm) in less than three seconds.

Does it have the potential to meet future needs?

As with the purchase of other lab instruments, it is wise to select a spectrophotometer according to your current needs as well as potential future needs. “Applications and/or samples may change, and the ability to utilize different accessories to address these changes is important,” says Mark Talbott, molecular spectroscopy product manager at Shimadzu. Shimadzu recently released two UV-VIS spectrophotometers, the UV-2600 and UV-2700. “With its double-monochromator design and Lo-Ray-Light diffraction gratings, the UV-2700 achieves ultra-low stray light of 0.00005 %T at 220 nm. Its photometric performance range has been expanded to 8 Abs, with a transmittance value of 0.000001%. This eliminates the need to dilute samples and allows the measurement of low-transmittance samples,” Talbott says.

Agilent, too, recommends buying with the future in mind. “It’s critical that an instrument meets the purchaser’s performance requirements and measurement needs today, with low maintenance costs, and [has] the capacity to adapt to meet the needs of tomorrow,” says Burt. For example, according to Burt, the Cary 60 system’s lamp warranty is longer than the warranty on most complete systems. “Users typically find ongoing maintenance costs are zero, [because] they never have to change a lamp throughout the life of the instrument. This [is] not the case with traditional deuterium-based UV systems, where lamp life can be measured in hours of use. The broad range of sampling accessories and room-light immunity ensure that the system can be applied to almost any educational activity or measurement need. In fact, the USB connectivity and 12V supply mean that the instrument can even be moved out into the field if the sample can’t be brought into the lab.”

Lynch agrees that taking a longer-range view of your spectrophotometric needs is the way to go. “A person looking for a new system needs to consider not only . . . current [usage], but also longer-term applications that may be coming,” says Lynch. “A platform that enables expandability should be a critical part of any purchase decision.”

Do you need infrared capabilities?

The longer wavelengths and lower frequencies of infrared or near-infrared wavelengths in spectrophotometry can enable you to analyze the transmittance or reflectance of denser samples better than in the UV/VIS range. Shimadzu’s UV-3600 UV-VIS-NIR spectrophotometer allows a measurement wavelength range of 185 to 3300 nm. It also includes three detectors to cover the wide wavelength range: a photomultiplier tube for the UV/VIS ranges and InGaAs and PbS detectors for the NIR range. Positioning the InGaAs detector between the other two ensures that the sensitivity remains high in the middle region, where sensitivity otherwise might decline in the transition between the photomultiplier tube and the PbS detector. Similarly, PerkinElmer’s Lambda 950 UV/VIS/NIR spectrophotometer is compatible with wavelengths of up to 3,300 nm.

Shimadzu’s newest spectrophotometer, the UV-2600, measures wavelengths up to 1,400 nm, using the ISR-2600Plus two-detector integrating sphere. “This wide range permits measurements in the near-infrared region,” says Talbott. “One area still to improve upon is the smoother, more seamless integration between UV-VIS-NIR or NIR-MIR-FAR (near-mid-far infrared) regions. Doing this will increase functionality and ease of use, thereby improving productivity and efficiency in the lab. In addition, the introduction of a new detector to reduce noise and increase the signal-to-noise ratio will be a huge enhancement.”

Despite the range of features available to the spectrophotometry customer today, there are always improvements in the pipeline. Expect progress in sensitivity via new detector designs, improvements in light separation and the use of tunable lasers, says Lynch. “The basic optical bench we use today is still on the cutting edge of performance capabilities,” he adds, “and we expect it to be around for years to come.”

The image at the top of the page is Shimadzu's UV-2600, UV-2700 UV-VIS spectrophotometer.

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