In a basic biology lab class, a spectrophotometer appears pretty robust and easy to use. That’s still the case for the most part, despite the increasing sophistication of the instruments—some covering wavelengths from the near-infrared (NIR) through ultraviolet (UV). To get the most accurate results, scientists need to start with the best spectrophotometer for the application. Then, some expert tips provide the best ways to troubleshoot any problems that arise. In this article we will explore ways to avoid problems all together—and get high-accuracy results—when using a spectrophotometer. Despite the breadth in spectrophotometers, many best-use practices apply across all of them.
For the key features in selecting a spectrophotometer, Jennifer Firestine, division director of physical sciences and professor of chemistry at Lindenwood University, gives a great overview:
“The correct choice of spectrophotometer depends on the wavelengths of interest and the amount of accuracy needed. Lower-end instruments are robust and user-friendly but usually only allow for readings at one wavelength at a time. A simple manual adjustment is required for measurement at various wavelengths. These units are easily moved and used in various locations. A computer-controlled higher-end instrument gives the ability to scan over a range of wavelengths, therefore providing a larger range of accurate data in one analysis.”
Building on Firestine’s efficient summary, let’s explore some of the details.
Start with a list
To pick the right spectrophotometer and any necessary accessories, Sudhir Dahal, molecular spectroscopy product manager at Shimadzu Scientific Instruments, recommends “outlining all the requirements needed to carry out your application.”
Here are some important features to consider:
- wavelength range
- spectral bandwidth
- absorbance or transmittance measurement requirements based on the highest absorbing sample if the samples cannot be diluted
- sample volume
- required accessories: such as special holders for solid, film, or powder samples; reflectance-measurement accessories; temperature-controlled holders; and autosamplers.
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For liquid samples, the volume plays a fundamental role in the instrument choice. “If you are looking at milliliter-range sample measurements, choose a cuvette-base instrument,” says Jeffrey Lai, product supervisor at Blue-Ray Biotech. “For microliter-range samples, a non-cuvette micro-volume spectrophotometer would be the better choice.”
The ongoing theme here will be: The characteristics of a sample will determine the best choice in a spectrophotometer.
Features worth considering
In addition to a basic list, other features impact the performance of a spectrophotometer. How the sample interacts with the light, for example, determines the required sensitivity of the device. “Measuring very high absorbance, or very low transmission, is best achieved with a research-grade spectrophotometer that has been designed for these more challenging measurements,” says Ursula Tems, business manager for laboratory analysis at Agilent.
The required sensitivity creates a decision point. As Dahal puts it: “Higher prices provide spectrophotometers with better specs; however, going beyond the needed specs of your applications is unnecessary, unless you think you may need it for your future applications.”
Fluorescent capabilities can also be added. “Fluorescence is an important option to be considered, offering great sensitivity and specificity,” says Andrew Jones, market development manager at DeNovix. “Fluorescence is also considered highly complementary to absorbance for many applications.”
User features also matter. Jones notes that, “in addition to pre-defined applications, the ability to accommodate new assays and applications is important.” He asks: “Can custom methods and new assays requiring standard curves be easily created and saved?” Any ease of use really matters with a spectrophotometer that will be used “by a wide range of researchers with different levels of experience,” Jones notes. “In a multi-user lab, the ability to define user accounts that allow data and protocols to be handled individually may be useful.”
Avoiding errors
With any spectrophotometer, scientists need to look out for errors. Tems points out three common sources of errors:
- sampling errors
- cuvette errors
- instrument errors
To avoid sampling errors, a sample must not be so concentrated that it’s absorbance is too high, and the sample must be placed accurately in the instrument so that light is passing through it, Tems notes. As another source of sample error, she asks: “If measuring over a long time or where environment plays a role, is the sample evaporating, does it need stirring or temperature control to remain consistent?”
Avoiding cuvette errors starts with the right cuvette. The characteristics of samples determine the best choice. “Samples that absorb UV light—for example, liquid biomolecules—require quartz cuvettes that transmit UV light, whereas visible light–absorbing samples can be measured with optical glass or polystyrene disposable cuvettes,” Tems explains. The cuvette must also be clean. That’s worth repeating. As Firestine emphasizes: “Clean, correctly aligned cuvettes are very important.”
Instrument errors can come from leaving the lid open during measurements, room light, or not getting a proper baseline. “Another common error is failure to allow the instrument lamps to fully warm up,” Firestine says. “This can cause inconsistency in the beam.”
Some of these points about avoiding errors really get to one basic idea in science: Don’t rush the work.
Keep it consistent
For some of the best error prevention, Lai says, “Stick to the instructions in the manual for proper operation and maintain periodic calibration, and you can get rid of most mechanical or electronic errors.”
Like all instruments, spectrophotometers work best when properly maintained and used as directed. Then, be methodical, work consistently and take the time needed to properly prepare and place a sample. Make sure that the spectrophotometer’s lamp is up to temperature, and the device is in proper working order. By following those steps, scientists will get the best results.
Image: The Agilent Cary 7000 universal measurement spectrophotometer (UMS).