291
proteins (Imafidon and Sosulski 1990) requiring different conversion factors. Also
use of concentrated sulphuric acid at high temperature and catalysts poses a serious
threat. This method, however, is modified to measure micro quantities of proteins
such as micro and macro Kjeldhal analysis, automated Kjeldhal analysis and colorimetric Kjeldhal analysis of nitrogen (Owusu-Apenten 2002). In spite of introduction of sophisticated instrumental methods e.g., spectrophotometric, potentiometric
with ion selective electrode, FIA, ion chromatography, chemiluminiscence, and others, modification of commercial sample digestion by Kjeldahl microwave digestion
decreases the digestion time and improves precision and is thus considered as a
worthy alternative to overcome the drawbacks of classical Kjeldhal method (SáezPlaza et al. 2013).
UV Absorption Method
With the advent of UV-Vis spectrometer, there has been an improvement in the
analytical techniques of proteins. This method is based on the principle that most of
proteins in food contain fair amount of aromatic amino acids such as tryptophan and
tyrosine that have strong absorbance in ultraviolet region of electromagnetic spectrum at 280 nm. The absorbance can be used for measurement of protein concentration using Beer-Lambert’s law given as:
A
I I E cl
m
log
/
0
(4)
where, A = absorbance of solution, I 0 = intensity of light incident on sample cell, I
= intensity of light exiting the sample cell, c = molar concentration of absorbing
species, l = length of sample cell (cm), E m = molar absorptivity.
It is thus clear from the Beer-Lambert law that greater the number of molecules
capable of absorbing light of a given wavelength, the greater the extent of light
absorption.
Since proportions of these aromatic amino acids in proteins vary, extinction coefficients (E 280 ) or molar absorptivity (E m ), which for individual proteins lies in the
range 0.4–1.5, must be determined for estimation of protein content. Moreover,
presence of nonprotein chromatophores such as nucleic acid contaminations that
have 10 times more absorbance at this wavelength (280 nm) obscures the quantitation of protein in crude sample. This interference can be, to some degree, eliminated
by measuring absorbance at 260 and 280 nm and then protein concentration (Pc)
can be calculated using following equation (Warburg and Christian 1942;
Layne 1957):
Pc mg ml
A
A
nm
nm
/
.
.
1 55
076
280
260
(5)
Since nucleic acids have identical absorbance at 280 and 235 nm, interference by
nucleic acids in protein quantification can be neglected and protein concentration
can be calculated by following formula (Whitaker and Granum 1980):
Recent Advances in Analysis of Food Proteins
proteins (Imafidon and Sosulski 1990) requiring different conversion factors. Also
use of concentrated sulphuric acid at high temperature and catalysts poses a serious
threat. This method, however, is modified to measure micro quantities of proteins
such as micro and macro Kjeldhal analysis, automated Kjeldhal analysis and colorimetric Kjeldhal analysis of nitrogen (Owusu-Apenten 2002). In spite of introduction of sophisticated instrumental methods e.g., spectrophotometric, potentiometric
with ion selective electrode, FIA, ion chromatography, chemiluminiscence, and others, modification of commercial sample digestion by Kjeldahl microwave digestion
decreases the digestion time and improves precision and is thus considered as a
worthy alternative to overcome the drawbacks of classical Kjeldhal method (SáezPlaza et al. 2013).
UV Absorption Method
With the advent of UV-Vis spectrometer, there has been an improvement in the
analytical techniques of proteins. This method is based on the principle that most of
proteins in food contain fair amount of aromatic amino acids such as tryptophan and
tyrosine that have strong absorbance in ultraviolet region of electromagnetic spectrum at 280 nm. The absorbance can be used for measurement of protein concentration using Beer-Lambert’s law given as:
A
I I E cl
m
log
/
0
(4)
where, A = absorbance of solution, I 0 = intensity of light incident on sample cell, I
= intensity of light exiting the sample cell, c = molar concentration of absorbing
species, l = length of sample cell (cm), E m = molar absorptivity.
It is thus clear from the Beer-Lambert law that greater the number of molecules
capable of absorbing light of a given wavelength, the greater the extent of light
absorption.
Since proportions of these aromatic amino acids in proteins vary, extinction coefficients (E 280 ) or molar absorptivity (E m ), which for individual proteins lies in the
range 0.4–1.5, must be determined for estimation of protein content. Moreover,
presence of nonprotein chromatophores such as nucleic acid contaminations that
have 10 times more absorbance at this wavelength (280 nm) obscures the quantitation of protein in crude sample. This interference can be, to some degree, eliminated
by measuring absorbance at 260 and 280 nm and then protein concentration (Pc)
can be calculated using following equation (Warburg and Christian 1942;
Layne 1957):
Pc mg ml
A
A
nm
nm
/
.
.
1 55
076
280
260
(5)
Since nucleic acids have identical absorbance at 280 and 235 nm, interference by
nucleic acids in protein quantification can be neglected and protein concentration
can be calculated by following formula (Whitaker and Granum 1980):
Recent Advances in Analysis of Food Proteins