Spectrophotometry is a widely used analytical technique that provides quantitative measurement of the reflection or transmission properties of a material as a function of the amount of light it absorbs. Most commonly, spectrophotometers operate in the UV/Visible wavelength range.
Challenges in spectrophotometric measurement can vary depending on the volume and concentration of the samples. In biological research applications, UV/Vis spectrophotometry is 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 of sample. Such samples are usually highly concentrated, thereby leading researchers to look for ways to avoid sample dilution when obtaining measurements.
Most problems in the application of UV/Vis spectrophotometry result from user error: choosing the wrong method or the wrong cuvette to contain the sample. The second leading cause of problems is using the wrong purification strategy. Whenever possible, users should invest in a spectrophotometer that is optimized for use in the modern life-science laboratory.

Low Volume versus Low Concentration: Remember the Beer-Lambert Law

To determine the best method for spectrophotometric measurement, the sample characteristics must initially be correctly identified. There is much confusion about differences between low volume and low concentration, resulting in questionable data. The Beer-Lambert law states 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). Remembering the Beer-Lambert law is particularly useful when preparing to run a spectrophotometric analysis and is especially important when working with biomolecules, many of which absorb light. The absorbance between samples varies linearly with concentration.

Sample Purity

Sample purity is imperative—not just for ensuring ideal spectrophotometric measurement, but also for quantification and other downstream applications and analyses. 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 utilizing 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. They also provide useful tips on preventing overload of the column filters or other difficulties that may lead to potentially impure sample eluates.

The Container: Choosing the Right Cuvette

Cuvettes are optically transparent cells that hold the analyte in solution while the sample is exposed to light. There are two cuvette types: UV- and non-UV-transparent. Some cuvettes can only be used in the visible range; it is critical to choose the correct type of cuvette with the appropriate wavelength being measured. It is also important to observe the physical condition of the cuvette being used for analysis. If it is scratched or contaminated, or carries fingerprints or condensation, inaccurate measurements may result. It is highly advisable to wipe the external surface of the cuvette with a laboratory tissue immersed in alcohol to retain maximum transparency for analysis. Using disposable cuvettes can alleviate suthc problems.
Spectrophotometers vary in flexibility. In addition to the variety of standard cuvettes, there are special microliter measuring cells available for some instruments that can be used in the quantification of minute volumes. Another type of cuvette—the microvolume cuvette—is specially designed for use of small volumes of highly concentrated biomolecules, such as proteins and nucleic acids, without sample dilution.

UV/Vis Instrument Software

Method development and data management can be very difficult, and programming a method from scratch wastes valuable lab time. However, many of today’s instruments come with software that has been designed for ease of use simply “out of the box,” making them accessible to a wide variety of training levels and users.Assessing the experience level of your staff, and allotting for important training time is an important step when selecting an instrument. Data management and storage are also important aspects to consider because a power outage or system malfunction can result in loss of valuable data. However, technology developed over the last decade has enabled the direct and easy connection to a computer, eliminating the need for additional software or storage devices.