Detecting and Quantifying Biological Molecules Using Spectrophotometry

Detecting and Quantifying Biological
Molecules Using Spectrophotometry
Dr Sue Whittle and Dr Sue Bickerdike

University of Leeds
Detecting and Quantifying Biological Molecules Using Spectrophotometry, created by Dr Sue Whittle and Dr Sue
Bickerdike, University of Leeds, 2010, is licensed under the Creative Commons Attribution-Non-Commercial-Share
Alike 2.0 UK: England & Wales License. To view a copy of this license, visit http://creativecommons.org/licenses/by-
nc-sa/2.0/uk/ or send a letter to Creative Commons, 171 Second Street, Suite 300, San Francisco, California 94105,
USA.
Detecting and Quantifying Biological
Molecules Using Spectrophotometry
The
Background
spectrophotometer
Theory video
Determination of
Calibration Peak
Curves Wavelength
Background Theory
Many coloured solutions absorb light in proportion to the amount of

coloured material present. The concentration of a coloured substance
can therefore be determined by measuring the amount of light a
solution absorbs.
Light In Light Out
As many substances of interest in Biochemistry are coloured (or more precisely, absorb light, even
though it may be in the ultraviolet region of the spectrum when they do not appear to have a
colour) this provides a very important method of biochemical analysis.
In principle, one could make up a set of standard solutions of

known concentration and compare them visually against a white
background with the unknown. It is more precise, however, to use
an instrument, a colorimeter or a spectrophotometer, in which a
photoelectric cell measures the amount of light passing through
the solution.
Spectrophotometer
Colorimeter
In a colorimeter, light from a tungsten filament lamp
passes through a suitable filter which transmits light
Light Source
of a fairly wide band of wavelengths.
In a spectrophotometer, a monochromator replaces

the filter and produces light of a very narrow band of
Filter
wavelengths, a few nm at most (1 nm (nanometer) =
10-9 m = 10 Å). A prism or a diffraction grating gives a Prism
spectrum, from which a slit selects the chosen
wavelength.
This has 3 main advantages:
1. A spectrophotometer can be used to measure

an absorption spectrum. This is a plot of
absorption against wavelength.
2. The simple theory of light absorption applies only to monochromatic light. Therefore the use of a
broad band of wavelengths often involves more laborious standardisation procedures.
3. Where absorption peaks are sharp, a spectrophotometer can give much greater
sensitivity than a colorimeter, as well as increased selectivity between different absorbing
substances.
Spectrophotometers are widely used, in biochemical research and clinical practice, for
determination of the concentrations of substances in solution. Often a few micrograms of material
can be estimated conveniently and rapidly.
With the simpler instruments only visible light can be used; therefore test
compounds must be coloured. More elaborate spectrophotometers operate
also in the ultraviolet and infra red regions. The UV is particularly useful for
the study of proteins and nucleotides.
Many substances without specific absorption bands can be chemically (or

enzymically) converted into coloured derivatives, and hence estimated
colorimetrically.
In all work with colorimeters and spectrophotometers, the relationship between absorbance and
concentration must be carefully studied, using standard solutions of the substance to be estimated. It
is often necessary to measure the absorbance of "blank" solutions containing all the components
except the substance in question, because these other components may contribute to the measured
absorbance.
Beer-Lambert Law
The absorbance of the solution (sometimes called optical density or

extinction) is measured by the spectrophotometer. It is equal to log10
Io/I, where Io is the intensity of the incident light, and I is the intensity
of the light transmitted through the solution.
I0 I
Note: Since absorbance is derived from a ratio it has no units.
Lambert's Law states that for a given solution the absorbance is proportional to
the path length (l) of the light through the cell.
Beer's Law states that the absorbance is proportional to the concentration (c) of the
solution.
Beer-Lambert Law
The Beer-Lambert Law combines the two and states that:
Absorbance = εcl
Where:
c is the concentration
l is the path length
ε is a constant.
If c is the molar concentration and l is in cm, then ε is called the molar absorption coefficient (also
called the molar extinction coefficient). This is the absorbance of a molar solution of the compound, in
a 1 cm cell, at the specified wavelength. Its units are molar-1 cm-1 since absorbance has no dimensions.
The scales of colorimeters and spectrophotometers are often calibrated both in absorbance units (=
optical density = extinction) and in percentage transmission. The absorbance scale is used when
measuring the concentration of solutions. The Beer-Lambert Law is normally obeyed, provided the light is
monochromatic. Departures usually indicate a change in molecular constitution with changing
concentration.
The specific wavelengths at which compounds absorb (i.e. their absorption spectra) will vary
depending on the specific compound.
If we want to use the absorption of light to determine concentration we need to measure this
absorbance where it is highest, i.e. at the peak wavelength for that compound.
Since these peaks are often very sharp, a small change in the wavelength used will cause a large
change in the absorbance measured.
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Detecting and Quantifying Biological

Molecules Using Spectrophotometry

Dr Sue Whittle and Dr Sue Bickerdike

Many coloured solutions absorb light in proportion to the amount of

In principle, one could make up a set of standard solutions of

In a spectrophotometer, a monochromator replaces

1. A spectrophotometer can be used to measure

Many substances without specific absorption bands can be chemically (or

The absorbance of the solution (sometimes called optical density or

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