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inform users how to select the most appropriate analysis technique for specific proteins or mixture thereof.
Spectrophotometric Methods
Almost all biological macromolecules are characterised by their optical properties
via use of spectroscopic measurements. The technique employs electromagnetic
radiation (200–800 nm) to interact with matter relative to a standard, or using an
assigned extinction coefficient. The functional groups or regions, such as basic
groups, aromatic groups, peptide bonds or aggregated proteins within the protein
absorb light in the ultraviolet or visible range of the electromagnetic spectrum
(200–800 nm). This absorbance is read by spectrophotometer and used for calculating protein concentration by comparing it with reference standard or known absorptivity for specific protein (Maehre et al. 2018). Depending on properties of
electromagnetic radiation and its interaction with matter, different spectroscopic
methods are used for analysis of proteins in sample. A wide range of spectrophotometric methods are available for accurate determination of protein concentration
essential for quantitative biochemical, biophysical, molecular, and structural biology studies.
UV Visible Light Spectroscopy
These techniques are most frequently used for analytical and research work. In this
type of spectroscopic technique proteins can be quantified by their intrinsic chromophores such as peptide bonds, aromatic amino acids (tyrosine, tryptophan), and certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem). In
UV-Vis region absorption of light by chromophores arises due to electronic transitions within atoms or molecules from a bonding or non bonding to an anti-bonding/
orbital. The amount of excitation energy depends on energy difference between
ground and exited state.
Among the following possible transitions (σ-π*, π-π*, π-σ*, σ-σ *), conjugated
π systems exhibit π-π* transition absorb in region between 200 and 800 nm while
other type require light in the vacuum UV region. Such molecules find application
in colorimetric methods. For a given concentration, greater the degree of conjugation in molecule smaller will be difference in energy between the ground and excited
state and hence greater will be intensity of absorbance. UV Visible spectroscopy can
be divided into two classes, absorbance and fluorescence, based on sample- radiation interactions.
Recent Advances in Analysis of Food Proteins
inform users how to select the most appropriate analysis technique for specific proteins or mixture thereof.
Spectrophotometric Methods
Almost all biological macromolecules are characterised by their optical properties
via use of spectroscopic measurements. The technique employs electromagnetic
radiation (200–800 nm) to interact with matter relative to a standard, or using an
assigned extinction coefficient. The functional groups or regions, such as basic
groups, aromatic groups, peptide bonds or aggregated proteins within the protein
absorb light in the ultraviolet or visible range of the electromagnetic spectrum
(200–800 nm). This absorbance is read by spectrophotometer and used for calculating protein concentration by comparing it with reference standard or known absorptivity for specific protein (Maehre et al. 2018). Depending on properties of
electromagnetic radiation and its interaction with matter, different spectroscopic
methods are used for analysis of proteins in sample. A wide range of spectrophotometric methods are available for accurate determination of protein concentration
essential for quantitative biochemical, biophysical, molecular, and structural biology studies.
UV Visible Light Spectroscopy
These techniques are most frequently used for analytical and research work. In this
type of spectroscopic technique proteins can be quantified by their intrinsic chromophores such as peptide bonds, aromatic amino acids (tyrosine, tryptophan), and certain prosthetic groups and coenzymes (e.g. porphyrine groups such as in haem). In
UV-Vis region absorption of light by chromophores arises due to electronic transitions within atoms or molecules from a bonding or non bonding to an anti-bonding/
orbital. The amount of excitation energy depends on energy difference between
ground and exited state.
Among the following possible transitions (σ-π*, π-π*, π-σ*, σ-σ *), conjugated
π systems exhibit π-π* transition absorb in region between 200 and 800 nm while
other type require light in the vacuum UV region. Such molecules find application
in colorimetric methods. For a given concentration, greater the degree of conjugation in molecule smaller will be difference in energy between the ground and excited
state and hence greater will be intensity of absorbance. UV Visible spectroscopy can
be divided into two classes, absorbance and fluorescence, based on sample- radiation interactions.
Recent Advances in Analysis of Food Proteins