Biomedical Engineering Reference
In-Depth Information
It should also be kept in mind that the Beer-Lambert law
often is not valid at higher concentrations, since there occur inter-
actions between chromophores and other molecules
6
.Thiseffect
is observed especially at reading of proteins in the UV. The solvent
may influence the absorbance too, because, for example, some of
the aromatic amino acid residues are buried within hydrophobic
core of the molecule and become exposed during unfolding of the
protein when the composition of the solvent is changed or the
protein is denaturated by dilution.
If you are sure that the Beer-Lambert law is fulfilled and a lin-
ear correlation between signal and amount is approximately given,
the amount of analyte may be calculated by a simple equation:
M
U
−M
B
·
Q
S
M
S
−M
B
=
Q
U
Q: quantity or concentration; M: analytical signal; U: unknown
(sample);S:standard;B:blank.
Since most of the analytical methods are influenced by an un-
Controls
known set of circumstances, it is recommended to run each test
with appropriate controls. The most important control is the blank,
i.e., the assay containing all components with the exception of the
analyte. It is also recommended to run each sample in triplicate to
get a rational mean and to detect false values.
6
Galla H-F (1988) Spektroskopische Methoden in der Biochemie. Thie-
me, Stuttgart
