UV/VIS Spectroscopy for Determination of Nucleic Acids (DNA and RNA)

UV/VIS Spectroscopy for Determination of Nucleic Acids (DNA and RNA)


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  • Date: 01 Aug,2021

UV/VIS Spectroscopy is a very powerful and popular method in life science. It has become a standard method that is used on a daily basis in many laboratories. This is mainly due to its simplicity; it does not require complex sample preparation, it is easy to perform and results are obtained quickly, usually within seconds. A typical mea­surement requires only a small amount of sample, and as it is a non-destructive method, samples can be used for subsequent analyses. The field of life sciences typically applies UV/VIS spectroscopy in the analysis of nucleic acids, proteins and bacterial cell cultures. The most common applications are the concentration determination of nucleic acids – DNA and RNA – the purity of nucleic acids, the concentration determination of proteins by direct measurement or colorimetric assays, the study of enzymatic reactions, and monitoring growth curves of bacterial cell suspensions.

For an in-depth introduction to UV/VIS spectroscopy we recommend the “UV/VIS Spectrophotometry – Fundamentals and Applications” guide, which is available here: www.mt.com/uv-vis

 Beer-Lambert Law:

Many compounds absorb ultraviolet or visible light, a behavior that is described by the Beer-Lambert law. It states that the absorbance of a sample is proportional to its concentration. This is the principle used in simple concen­tration determinations of samples such as DNA and proteins. To be more precise, the Beer-Lambert law states that the absorbance is proportional to the extinction coefficient Σ(epsilon), the path length d and the concentration c.

A= Σ ∙c∙d

A: photometric absorbance

Σ: extinction coefficient

c: concentration of sample

d: path length

Spectrophotometer Instrument Types

There are essentially two different instrument categories in life sciences; the cuvette based spectrophotometer and the micro-volume spectrophotometer. Hybrid instruments come with an additional cuvette shaft and can be used for both micro-volume and cuvette measurements. However, their accessory range is limited, which also restricts the scope of applications.


Cuvette-Based Spectrophotometers

Conventional UV/VIS spectrophotometers with a large sample area for cuvettes can be used for various applications. They can accommodate many different cuvette types with a wide volume range and allow the usage of many optional accessories such as changers and thermostat holders.

Micro-Volume Spectrophotometers

Micro-volume instruments are dedicated to those applications where only a small amount of sample is available or when the sample is highly concentrated. The sample volume required for a measurement is typically between 1 and 3 μL. The direct measurement of highly concentrated samples eliminates the tedious dilution step and avoids pipetting errors. It is also convenient that the sample is loaded directly onto the measurement surface, without the need for cuvettes.

Most micro-volume instruments cover a wide concentration range by changing the optical path length of the measurement platform. For example, decreasing the path length by the factor 10 has the same effect as diluting the sample by a factor of 10. This is because reducing the path length also reduces the photometric absorbance.This way very high concentrations can be measured at moderate photometric absorbance levels. In other words, the shorter the path length, the higher the concentrations that can be measured. To achieve accurate results with this approach it is key that the exact path length is known.

METTLER TOLEDO Solutions for Life Science

METTLER TOLEDO offers two dedicated UV/VIS spectrophotometers for life science applications:

UV5Bio Spectrophotometer for Cuvette Measurements

The UV5Bio spectrophotometer is based on the FastTrack™ technology, which allows fast and accurate measurements. The open sample area on the top makes loading and unloading cuvettes and other accessories uncomplicated. Running an analysis is self-explanatory with the aid of the OneClick™ color touchscreen user interface. Its clear structure and intuitive smartphone-type functions means there is no need for extensive training. Users are expertly guided through each step of their work. The software includes a comprehensive package of life science methods ready to use.

 UV5Nano Spectrophotometer for Micro-Volume Measurements

The UV5Nano spectrophotometer combines innovative technology with intuitive usability. It can be used for small volumes of down to 1 μL, saving time for dilutions and sparing precious samples. The additional cuvette slot allows measurements of large volumes of up to 3 mL. The micro-volume measurements are based on the innovative LockPath™ technology ensuring high accuracy and repeatability. Two independent measurements at two precisely defined path lengths are taken so that a wide concentration range can be covered. The LockPath technology secures the path length at a fixed position, thus excluding path length drift over time. However, some laboratories need documented verification of their procedures. In this case, where utmost certainty is required, a calibration procedure for the path length can be performed.


METTLER TOLEDO is a leading global manufacturer of precision instruments. The Company is the world’s largest manufacturer and marketer of weighing instruments for use in laboratory, industrial and food retailing applications. The Company also holds top-three market positions for several related analytical instruments and is a leading provider of automated chemistry systems used in drug and chemical compound discovery and development. In addition, the Company is the world’s largest manufacturer and marketer of metal detection systems used in production and packaging. Additional information about METTLER TOLEDO is available at www.mt.com.

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