Extinction Coefficient Measurement Method

The extinction coefficient is a measure of the intensity of light absorbed by a specific wavelength of a chemical substance. The molar extinction coefficient of proteins at 280nm is almost entirely dependent on the number of aromatic residues, especially tryptophan, and can be predicted by the amino acid sequence. If the molar extinction coefficient is known, it can be used to determine the concentration of protein in the solution. Spectrophotometry is a common method used to determine the extinction coefficient and is the method most frequently used in the determination of the extinction coefficient of most bioproducts.

 

According to the Lambert-Beer law, when a monochromatic light passes through a flow cell, if the flowing phase does not absorb light, the absorbance A is proportional to the concentration C of the absorbing component and the optical path length L of the flow cell. Therefore, the extinction coefficient of the protein can be calculated by the formula ℇ=A/cl, provided the absolute content of the protein and the extinction value of the test substance at the UV 280 nm wavelength under a 1 cm light path are measured.

 

The following are some commonly used methods for determining extinction coefficient:

 

1. Spectrophotometry

This method uses a spectrophotometer to measure the absorbance of light of different wavelengths passing through a specific sample. In the experiment, a solution of a certain concentration is placed in a cuvette, which is then placed in the light path of the spectrophotometer. The spectrophotometer measures the difference in light intensity passing through the sample and a reference sample (usually pure solvent) to determine the sample's absorbance of a specific wavelength of light. Through the Beer-Lambert law, the extinction coefficient can be calculated. This law states that absorbance (A) is proportional to the concentration of the substance (c), the path length of the solution (l), and the extinction coefficient of the substance (ε): A = ε l c.

 

2. Transmission Spectrometry

Transmission spectrometry is an analytical technique that determines the extinction coefficient of a substance by measuring the transmittance or absorbance characteristics of a sample to a series of different wavelengths of light. This usually involves the use of a spectrometer, which can provide detailed information about how a substance interacts with different wavelengths of light. By analyzing the transmission spectrum, the extinction coefficient at a specific wavelength can be calculated.

 

3. Reflection Spectrometry

When the sample is a solid or has poor transmittance to light, reflection spectrometry can be used to measure the extinction coefficient. This method measures the intensity of light reflected back after being irradiated onto the sample surface. By analyzing the reflectance spectrum, the extinction coefficient can be indirectly calculated.

 

4. Infrared Spectrometry

For certain specific types of samples, such as organic compounds containing specific functional groups, infrared spectrometry is a very useful method. This method relies on the absorbance characteristics of substances to infrared light, which are related to the molecular structure of the substance. By measuring the infrared spectrum, the extinction coefficient of specific chemical bonds can be determined.

 

When determining the extinction coefficient, it is important to ensure the use of appropriate experimental conditions and calibration methods to obtain accurate and reproducible results. Additionally, factors such as sample processing, solvent purity, and the control of temperature and pressure can also affect the measurement results.