User Manual

SECTION 3 – Theory and Practice of Spectroscopy
Measurements
3.1 THEORY OF SPECTROSCOPY MEASUREMENT
UV-visible spectroscopy is the measurement of the absorbance of light at a specific wavelength in a
sample. This is used to identify the presence and concentration of molecular entities within the sample.
The Beer-Lambert law is used to relate the absorption of light to the properties of the sample through
which the light is travelling through. The Beer-Lambert law states that:
A is the absorbance
is the molar absorption coefficient (l mol
-1
cm
-1
)
c is the concentration (mol l
-1
)
l is the path length (cm)
This law shows that absorbance is linear to concentration but this is only true for low concentrations. For
absorbance levels above 3 the concentration starts to move away from the linear relationship.
Transmittance is the proportion of the light which passes through the sample:
Therefore: T = I
t
I
o
Absorbance is inversely related to transmittance:
A = log 1
T
3.2 NUCLEIC ACID DETERMINATION
DNA, RNA and oligonucleotides can be measured directly in aqueous solutions in a diluted or undiluted
form. Aqueous buffers with low ion concentrations (e.g. TE buffer) are ideal for this method. The
concentration is commonly determined by measuring at 260nm against a blank and then evaluating
against a factor.
The Genova Plus has pre-defined methods installed which assume that absorption of 1 OD (A) is equivalent
to approximately: 50µg/ml dsDNA, 37µg/ml ssDNA, 40µg/ml RNA and 30µg/ml for oligonucleotides.
DNA interference by contaminants can be assessed by the calculation of an absorption ratio. The ratios
A260/A280 and A260/A230 are used to estimate the purity of nucleic acids, since proteins absorb at
280nm and substances such as peptides, phenols, aromatic compounds or carbohydrates absorb at
230nm. Pure DNA should have an A260/A280 ratio of approximately 1.8 and pure RNA 2.0. In pure
nucleic acid samples the A260/A230 ratio should be approximately 2.2.
Where:
I
o
is the incident light
l
t
is the transmitted light
L is the path length
14
I
o
I
t
L