This experiment is from the official website of Wuhan University School of Pharmacy

Operation method

Qualitative scanning and quantitative analysis of tryptophan by ultraviolet absorption spectroscopy

Principle

Ultraviolet-visible spectroscopy is the electronic spectra of substances with ultraviolet-visible light, which studies the molecular absorption spectra of substances in the ultraviolet-visible region arising from the leaps of valence electrons between electronic energy levels. When monochromatic light of different wavelengths passes through the analyzed substance, the absorbance or transmittance at different wavelengths can be measured, and the absorption spectral curve of the substance can be obtained by graphing the wavelength λ with ABS as the vertical coordinate and the horizontal coordinate. Generally, the UV region is 190 ~ 400 nm, and the visible region is 400 ~ 800 nm. The qualitative analysis of UV absorption spectra provides an information basis for the qualitative analysis of compounds. Although the molecular structures are different, as long as they have the same chromophore, their maximum absorption wavelength values are the same. Therefore, by comparing the scanning spectra and maximum absorption wavelength values of unknown compounds with the standard spectrograms of known compounds in the same solvent and measurement conditions, a basic identification can be obtained. The 20 amino acids involved in the composition of proteins have no light absorption in the visible region; only tyrosine, phenylalanine and tryptophan have light absorption in the ultraviolet region, among which tryptophan has the strongest ability to absorb ultraviolet light, and the maximum ultraviolet absorption peaks of tryptophan and tyrosine are at 280 nm. Therefore, according to the characteristics of their ultraviolet absorption spectra, they can be measured by spectroscopic scanning in the qualitative measurement mode of the UV-visible spectral analyzer. Their absorbance-wavelength profiles are measured by spectral scanning for accurate and reliable qualitative identification of it. The characteristic maximum absorption peak at 280 nm for proteins is caused by the tryptophan and tyrosine it contains. This property can be utilized to determine the protein content.

Materials and Instruments

Tryptophan standard solution UV-Vis spectrometer

Move

1. Configure 20 μg-mL-1 tryptophan standard solution.

2. Turn on the UV-visible spectrometer, initialize the instrument and warm up for 5min.

3. Qualitative scanning: Select the qualitative analysis mode in the application menu, and set up the scanning range of 200 ~ 400nm for the horizontal coordinate (wavelength value) and 0 ~ 1ABS for the vertical coordinate (ABS or %T value) as well as the scanning in the configuration menu.

4. Quantitative analysis: fixed wavelength scanning. Set the wavelength, change the concentration of the analyte, you can get different ABS values, according to which the purpose of quantitative determination can be achieved.

5. Refer to different instructions for different instrument models.

Caveat

1. Determine the maximum wavelength peak of tryptophan at different wavelengths.

2. Fixed wavelength scanning, plotting the working curve for quantitative measurement and calculating the unknown concentration.

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