Flashcards in Lecture 13 - Theory of spectroscopy Deck (10):
Spectra of organic molecules (7)
Often multiple peaks
o Wavelength - λ (greek lambda)
o Wavelength of peak absorbtion- λ max
o Measurements usually made at λ max because of
– Maximum sensitivity
– Minimum slope
• T = I/I0
• where I0 is the intensity of the incident light and I is the intensity of the transmitted light
• In essence transmittance = light out / light in
• Often given as %.
• A = log10 ( I0/I )
• = - log10 (T)
• Sometimes also called “optical density (OD)”
• This is used rather than transmission as absorbance is additive.
• Linear relationship to concentration
• Absorbance of a sample depends on the wavelength. This must be clearly specified. Often this is written as a superscript e.g.
• Absorbance also depends on the path length- as a longer time means more absorbance as more time to do so. This may be written as a subscript
• However, the default path length is always assumed to be 1cm, unless it is stated otherwise.
Beer-Lambert law (
• Absorbance of a solution
• A = ε x l x c
• Where l is the path length in cm
• c is the concentration, and
• ε (epsilon) is the extinction coefficient depending on the substance being studied and the wavelength (unique to every substance)
• Absorbance is proportional to length e.g. 1 cm = 0.12 so 3 cm = 0.36.
• Applies for a pure solution
• In principle for dilute solutions (is used to talk about dilute solutions when using this equation).
• Absorbance is proportional to concentration for dilute solutions
• A = ε x l x c
• If l = 1cm, and
• ε is the molar extinction coefficient, then
• Molar concentration (mol/l) = A/ ε
Molar extinction coefficient (MEC):
• The MEC is the theoretical absorbance of 1cm of a one molar solution of the substance being studied
• Units: M-1cm-1
Practical considerations (5)
• Most spectrophotometers are accurate in the absorbance range 0.1-1.5
• Solution must be homogeneous
• Solution must be clear- no turbidity
Suspended particles, dust, droplets etc will scatter light and give a false reading
• Need to use a “blank” cuvette with solvent
– To allow for any absorbance of the cuvette or solvent
• Need to zero using blank after changing wavelength
– Because output of light source and sensitivity of detector vary with wavelength.
Need to know if DNA is pure.
Peak at 260nm – depends on GC vs AT bonds
Spectrophotometry is commonly used for quantitation
Absorbance of nucleic acids can vary with the base composition (number of A,C,G,T)
But for an “average” DNA sample we assume an A260 of 1.0 ≡ 50μg/ml
• Spectrophotometry is also used to check the purity of the DNA sample
• In pure DNA the ratio A260 / A280 is ~1.8-2.0
• A lower value implies the sample is contaminated (most likely with protein)
• The absorption peak at 280nm can be used to quantitate protein, but not very sensitive
• Better to use Bradford reagent
• Develops blue colour in the presence of protein
• The Bradford assay is not measuring the absorbance of a pure substance
• It is measuring the product of a reaction
• Beer Lambert law does not apply
• Necessary to construct a standard curve
Measuring growth of bacteria (2)
• Because bacteria scatter light, and stop it passing through the cuvette, you can measure the concentration of bacteria in a spectrophotometer.