Spectrophotometer Selection and Troubleshooting

Spectrophotometers play huge roles in life science laboratories. They can be used to determine the current growth state of a bacterial culture, as well as quantitate the total amount of protein in a crude cell lysate or the concentration of an already purified protein. Spectrophotometers give glimpses into the lives of bacteria or tell how successful a protein purification or sample preparation was.

But when it comes to choosing the right spectrophotometer, scientists often wish that the selection process was as easy as choosing the right wavelength on the instrument. There are so many types of spectrophotometers out there and so many features to choose from that it can be quite overwhelming to pick the right one.

This article focuses on some features to consider when buying a new spectrophotometer as well as what to do when the reading does not go as planned.

Identifying the right spectrophotometer for experimental needs

Before diving into the selection process, Jeffrey Lai from Arise Biotech, suggests referring to publications for similar applications first. “This way [the scientist] can get a general idea which type of instrument can be used for the application.” Otherwise, it helps to define the experimental measurements to choose the right spectrophotometer. These will provide limitations on which spectrophotometer can be required.

For example, “different types of spectrophotometers will offer different measurable wavelength ranges. Knowing these limitations can help narrow down from a wide field of spectrophotometers to a few different options,” explains Gilbert Vial from Shimadzu Scientific Instruments. To provide a broad dynamic wavelength range, scientists should select an instrument with multiple light sources.

Another option to consider is fluorescence as a complimentary method to absorbance. Knowing that they will need this feature reduces the list of potential spectrophotometers to choose from.

Sample properties play a huge role

For many researchers, sample volume is very important. For general applications, in which sample volumes are within the mL or sub-mL range, most spectrophotometers use 1 cm path length cuvettes. However, more and more molecular biology applications rely on sample volumes within the μL range. These require specialized instruments that use micro-volume cuvettes for samples below 1 μL. Other instruments even work without a cuvette; the scientist just drops a sample of less than 1 μL on the optical sample pad. As such, microvolume spectrophotometers also increase the dynamic range over which samples can be measured. However, these instruments are more expensive, hard to operate, and not easy to clean.

If a scientist also “needs to compare several samples at the same time, a microtiter-plate spectrophotometer or a spectrophotometer that can accept multiple cuvettes would be good,” explains Lai. On the other hand, Andrew Jones from Denovix says that if they “run kinetic assays where samples need to be heated and measured over a period of time then they will likely want to do this in cuvettes”. The same is true for inhomogeneous samples when measuring cell density or where stirring is needed.

Instrument calibration should also be a consideration for labs looking to buy a spectrophotometer. Some microvolume instruments or specialized instruments will require periodic calibration checks or even recalibration by engineers. This additional expense should be factored into the buying decision.

Lastly, instrument handiness and data access are important to keep in mind. The instrument should be fully integrated rather than being controlled by a PC. This saves lab space and simplifies machine handling. Additionally, researchers want easy access to their data or an export function via email or network folders. In this case, they should make sure that the spectrophotometer can connect to the Internet or an email provider to avoid looking for USB drives all the time.

Troubleshooting tips for a spectrophotometer

Most spectrophotometers are robust and stable. However, problems come up every once in a while leading to unreliable results. Generally, spectrophotometry errors fall into one of the three categories: sample, cuvette, or instrument.

“The most common sources of error come from sample preparation. If the sample is too diluted or concentrated, then it will obscure the photometric readings,” Vial explains. Also, it should be taken into account that the spectrophotometer does not discriminate contaminants from the sample of interest. These additional compounds might absorb at the same wavelength as the sample thus altering the reading results. So, samples should always be kept pure by using fresh pipettes and clean cuvettes.

Cuvette errors are less common but easier to fix. Generally, it is important that the cuvette carrying the sample is clean and free from scratches. Also, the cuvette needs to be inserted in the proper orientation within the cuvette holder and instrument. Plus, a common mistake among scientists is that they forget to use a quartz cuvette when measuring wavelengths within the UV spectrum.

When it comes to instrument errors, it can be rather difficult to get to the bottom of them. Each spectrophotometric instrument has its own specific instructions and reasons why it could misbehave. Hence, it is always good to check the instrument’s manual when a new error crops up.

“Follow the instructions of the operation manual to use the instrument, keep regular maintenance and the instrument will provide many years of service without any trouble,” Lai notes. Lastly, it should not be forgotten to calibrate the baseline regularly and replace the lamps, filters, and vacuum pumps when they reach their ends of life. These basic maintenance tasks will ensure that a spectrophotometer works properly for years.