Absorbance measurement in Pharma analysis (DNA/RNA)

1.Overview of Absorbance 

Absorbance is a measure of the quantity of light absorbed by a sample. When light hits something that takes some of the light, it is defined as absorbance. If all light passes through a sample, none was absorbed, so the absorbance is zero  and the light that is absorbed is not lost; it is transformed to heat or chemical energy as the absorbing molecule gets excited. Absorbance is a physical process we come across every day as we see colors: White light such as sunlight contains all colors of visible light. When it shines on the green grass, the grass absorbs all colors but the green light is reemitted and this is what gives the grass its color. The property is measured using spectroscopy, particularly for quantitative analysis. Typical units of absorbance are called "absorbance units," which have the abbreviation AU and are dimensionless. 

In biology and chemistry, the principle of absorbance is used to quantify absorbing molecules in solution. Many biomolecules are absorbing at specific wavelengths themselves. Nucleic acids and proteins absorb UV light, chlorophyll absorbs light of blue and orange/red and hemoglobin absorbs yellow-green light. These analytes can be quantified without further procession, at least if they are found in a solution with low background absorbance. However, absorbance quantification is not limited to a handful of absorbing molecules. Chemical reactions assist in producing absorbing molecules for quantification. These reactions depend on a specific substrate or on an enzyme. Hence, color development (and absorbance) is more intense, the more of the compound/enzyme is found in the sample.

2. Absorbance measurement in nucleic acids concentration

Absorption spectroscopy of nucleic acids (DNA and RNA) is a very useful tool in molecular biology. Nucleic acids (DNA and RNA) and their components show strong absorption in the region 240-270 nm. At neutral pH the absorption maxima for guanosine shifts to 253 nm and for cytidine to 271 nm. This is the reason why Nucleic acid absorption peak is around 260 nm. However, depending on the base composition and environment, the peak can shift from 255 to 265 nm.

Figure 1: Absorption by DNA at 260 nm

There are two approaches to determine DNA/RNA concentration, namely Fluoresence and Absorbance measurement but Absorbance is more and more popular when without the need of any sample preparation or staining procedure absorbance measurements at this wavelength can be exploited fast and easy. By taking additional measurements at other wavelengths such as 230 nm, 280 nm or 340 nm, nucleic acid purity can be evaluated on top. The nucleic acid quantification based on UV absorbance is quick, easy, and robust, if you disregard the impact pH value, differing in water vs. buffer, can have on results. UV measurements with a spectrophotometer allow quantifying DNA in up to highly concentrated samples with several µg/µL

To observe the UV response and absorbance linearity, we measured the absorbance of various concentration levels of DNA samples in the different environments. With a high absorbance linearity spectrometer, users can measure a wide range of sample concentration levels accurately and with minimal sample preparation between measurements. This can be critical for applications where very precise measurement accuracy is desired or where you have a limited amount of sample to work with.  

3. Applications by using spectrometers

The SR6 from Ocean Optics is a multipurpose spectrometer, suitable as a setup for the lab or as a customized system integrated into high-volume industrial and OEM applications. The spectrometer is available in models covering wavelength ranges within ~180-1100 nm, and connects to light sources, optical fibers and sampling optics to optimize spectral setups for various applications.

One of the distinguishing features of the SR6 is its response in the UV region, especially the range from 180-280 nm. The UV response of SR6 is primarily a function of the detector and can best be taken advantage of by pairing it with optical bench components – gratings and the like – designed for the UV. Adding a UV light source and sampling optics that transmit UV light provides the optimum configuration for the application.

Figure 2: SR6 Absorbance for Oligo DNA Concentrations 0.5 - 100 µg/mL DNA (30 concentrations)

With the spectroscopy software: Ocean Direct, which permit to write custom software solutions for spectrometer to optimize the performance, the SNR (signal to noise ratio) can be improved. As a result, in a given period of time the SR6 will perform significantly more spectral averages and yield a far superior SNR per unit time – as high as 3500:1.

Our setup comprised the SR6 spectrometer, a deuterium-tungsten halogen lamp (using only the deuterium bulb for UV illumination), a cuvette holder with 1 cm quartz cuvette, and solarization-resistant optical fibers. The spectrometer and light source were warmed up for 30 minutes prior to use, and a fiber optic variable attenuator was used, as needed, to control the light from the deuterium source to the spectrometer.