BIO-360 BIOCHEMISTRY LABORATORY II
• II. WEEK
2nd WEEK
SPECTROPHOTOMETRICAL ANALYSIS
COLORIMETRIC ANALYSIS
These analyzes are used to
determine the concentrations of
substances with unknown
concentrations. According to
Beer-Lambert's law, the
concentration in the sample can
be determined according to the
absorbance values.
A=ε x l x c
A= absorbance
ε= molar absorption coefficient I= the lenght passing through by light
Each substance has its own ε value.
If the absorbance is outside the
scale, the sample must be
diluted.
The exact value of the molar
absorbance coefficient must be
known in order to determine the
concentration by the absorbance
value. Ɛ values should be
recalculated instead of using the
molar absorbance coefficient
values defined in the literature.
To determine the molar
absorption coefficients at certain
wavelengths, dilution is made
from the stocks prepared for the
substance and a series of
suspensions is prepared. The
standard graph is drawn with the
absorbance values obtained from
these suspensions.
CLASSIFICATION OF
SPECTROPHOTOMETRICAL ANALYSIS
Emission
Emission spectrophotometry
Flame spectrophotometry
Absorbtion Colorimetry
Visible region or UV spectrophotometry IR (infrared) spectrophotometry
Atomic absorbtion
Modern analysis
NMR spectrophotometry Mass spektrophotometry
X light reflection spectrophotometry
Beer-Lambert Law
The amount of light passing through a medium is inversely proportional to the path and ambient concentration of light in the medium.
If the UV-visible region lights pass through a solution containing the substance, the solution absorbs some of these rays (absorption), while others absorb very little or undergo transmission (transmission).
http://www.brighthub.com/environment/renewable- energy/articles/79089.aspx
The light intensity (I) from a colored solution in a cuvette is less than the light intensity (Io) entering the solution.
http://life.nthu.edu.tw/~labcjw/BioPhyChem/Spectroscopy/beerslaw.htm
