Troubleshooting in UV/Vis Spectrophotometry

Tanja Musiol, Ph.D., Global Product Manager Detection, Eppendorf AG, Germany

Spectrophotometry is a widely used analytical technique in a broad range of industries, providing quantitative measurement of the reflection or transmission properties of a material as a function of the amount of light it absorbs. The most commonly used spectrophotometers operate in the UV/Vis range.

Challenges in spectrophotometric measurement vary, depending on the volume and concentration of the samples. In bioresearch applications, UV/Vis spectrophotometers are used to measure analytes such as nucleic acids, proteins and cells from biological samples. These samples are often limited in volume or highly concentrated, which presents unique challenges. We have recently even seen growth in applications measuring mere drops, or microvolumes, of sample. Such samples are typically high-concentration, and researchers look for ways to avoid sample dilution when obtaining measurements.

Most problems in the application of UV/Vis spectrophotometry result from the user choosing the wrong method or the wrong cuvette for the sample at hand. The second leading cause of problems is using the wrong purification strategy. Where possible, users should invest in a spectrophotometer that is optimized for use in the modern life-science lab—and follow the tips below.

Low volume vs. low concentration

To determine the best method for spectrophotometric measurement, the analyst must correctly identify the characteristics of the sample. Users often confuse low volume with low concentration, resulting in questionable data. The understanding and application of the Beer-Lambert law is useful in setting up a spectrophotometric analysis and is especially important when working with biomolecules, many of which absorb light. The Beer-Lambert law relates the absorption of light to the properties of the material through which the light is traveling. The concentration of a substance is directly proportional to the amount of light absorbed by the sample itself and inversely proportional to the logarithm of the transmitted light. The formula is: A=Ɛ x c x d (where Ɛ=sample specific factor extinction coefficient, c=concentration of solution and d=path length). The absorbance between samples varies linearly with concentration.

Sample purity

Sample purity is important, not just in spectrophotometric measurement but also in quantification and other downstream applications. Contaminants in the sample may include proteins, buffer components or even cells, depending on the matrix and the analyte of interest. The sample-preparation strategy, whether it uses spin columns or automated kit solutions with vacuum manifolds, should be optimized for your sample amount and concentration. Sample-preparation kit suppliers provide guidance and manuals on selecting the best chemistry and procedure for a particular sample, in addition to tips on preventing overload of the column filters or other difficulties that could lead to impure sample eluates.

Choosing the right cuvette

Cuvettes are optically transparent cells that hold the analyte in solution and are used to introduce samples into the light path. There are two types of cuvettes—UV- and non-UV-transparent. Some cuvettes can only be used in the visible range. Be sure to assess the condition of the cuvette. If it is scratched or contaminated, if it carries fingerprints or condensation, inaccurate measurements may result. Spectrophotometers vary in flexibility, and some can accommodate a wider range of cuvettes than others. In addition to the variety of standard cuvettes, special microliter measuring cells, which can be used in the quantification of minute volumes, are available for some instruments. Users also can find microvolume cuvettes, which are specially designed for use with small volumes of highly concentrated biomolecules, such as proteins and nucleic acids, without sample dilution. Or small sample volumes of biological samples can be quantified by pipetting the sample directly into the measuring window of a spectrophotometer designed specifically for microvolume analysis.

Software requirements

No one wants to spend time on instrument training. This can be particularly challenging in academia, where students are running analyses. Method development and data management can be very difficult, and programming a method from scratch wastes valuable lab time. Many of today’s sophisticated instruments come with software that has been designed for ease of use; some can be put into use right out of the box, with no training on the programming.

Assessing the experience level of your staff and your tolerance for dedicated training and programming time is an important step when selecting an instrument. Data management and storage are also important aspects to consider. In many cases, a power outage or system malfunction can result in lost data. Technology developed over the last decade enables the direct and easy connection to a PC, eliminating the need for additional software or storage devices.

Spectrophotometry will continue to be a workhorse in the modern lab. Just as recent technology advances have yielded instruments optimized for biological-science applications, we expect continued evolution to meet changing needs. We also expect the instruments to get easier and easier to use.