Introduction to
Spectrophotometry
Why Spectrophotometry?
Imagine you are to make a 1μM
solution of a specific protein that you
believe could have anti-carcinogenic
properties.
 Unfortunately if the protein is 100X
more concentrated it has serious side
effects.
 How could you be sure of the
concentration?

Why Spectrophotometry?
A spectrophotometer or “spec” could
be used to quantify the concentration
of a solution.
 A spec shines a beam a light on a
sample.
 The molecules in the sample either
absorb light energy, reflect the light or
the light transmits between the atoms
and molecules of the sample.

Spectrophotometry Overview
The spec measures the amount of light
transmitted through the sample or
transmittance.
 Although the spec measures the percent
of light transmitted (%T), by using an
equation it can convert the transmittance
data to absorbance.
 Absorbance or transmittance data can
then be compared to known standard
curves to quantify a sample.

Spectrophotometry
Overview
Spectrophotometers shine either UV
light, visible light or both on a sample.
 Depending on the sample, different
parts of the EM spectrum are used.

Physics Review
Why do blue objects look blue?
 The object absorbs all other
wavelengths of visible light except blue
which is reflected.
 Visible, or white, light ranges from blues
at 350 nm to red at 700 nm
 The different wavelengths of light also
contain different amounts of energy.
 The spec detects the differences in
energy

Electromagnetic Spectrum
How a Spec Works
Features

UV/VIS Specs contain two lamps
Tungsten lamp – 350-700 nm
 Deuterium lamp – 200-350 nm


Wide variety of other options

Scanning
• Time or wavelength
Digital / graphic output
 One dial

How a Spec Works
Features

Common features (Spec 20)


Our spec (Spec 20)


Lamp, prism or grating, sample holder,
display, detector, and knob
Knob rotates a prism which directs a
different color through the sample
Fancy specs have an automatic feature
that scans for the maximum absorbance
which then indicates the ideal wavelength
for that sample
How a Spec Works
The wavelengths of light from
tungsten lamp shine on a sample.
 The molecules in the sample either
absorb or transmit the light energy of
one wavelength or another.
 The detector measures the amount of
light being transmitted by the sample.

How a Spec Works
The spec reports the value directly as
(% transmittance) or converts it to
the amount of light absorb in
absorbance units (au).
 To convert between transmittance and
au the spec uses an equation based
on Beer’s Law.
 A=2-log10(%T)

How a Spec Works
Beer's law: the absorbance is directly
proportional to the concentration of a
solution.
 So, as absorbance of a sample increases,
the transmittance decreases and vice
versa.
 If molecules are not absorbing all the
energy from a light beam, then the
remaining light penetrates (transmits)
through the sample.

Beer’s Law
Beer’s law is a relationship between the light
absorbance of a substance and its concentration It
is often written:
A = a×b×c where:
A = Absorbance of solution
a = absorptivity, or how well the substance absorbs light
b = path length, length of sample holder
c = concentration of solution
Beer’s Law
Often a and b will not change in an experiment, we
can combine them into a new constant, k. Thus: A =
kc. Note that this is the equation of a straight line y
= mx + b where y = A, x= c, m = k and b = 0.
(Remember that m is the slope of the line, and b is
the y-intercept.)
Beer’s law then states that a plot of absorbance vs.
concentration will give a straight line passing
through the origin. Such a graph is labeled a Beer s
Law Plot . The slope of the line is characteristic of
and depends upon the solution used.
How a Spec Works

To be detected by a visible light spec,
molecules must be colored or have a
colored indicator added.
starch & iodine
 protein and biuret’s solution


Colorless molecules are not detected
by a VIS spec since the do not absorb
any wavelengths within the white light.
How a Spec Works:
A blue example
Consider a blue solution
 When the white light shines on a blue
molecule, all wavelength are
absorbed except blue.
 The blue wavelengths are transmitted
or reflected off the molecules.
 Some of these blue wavelengths hit
the detector and blue light has a
certain amount of energy.

How a Spec Works:
An example
If the color of the solution is unknown,
then all of the wavelengths could be
measured and graphed.
 An absorbance spectrum is a graph
that shows a samples at different
wavelengths.
 The peaks on the spectrum would
indicate the wavelength at which a
molecule absorbs the most light is
called lambamax.

How a Spec Works:
A “clear” example



Molecules such as proteins and nucleic
acids, DNA & RNA, are often the most
important in biotech labs.
When proteins and DNA are in solution they
are “colorless” or do not absorb light in the
visible light spectrum.
In order to assay, measure, these solutions
either a UV light needs to be used or an
indicator is added to the solution.
How a Spec Works:
A “clear” example
Proteins can be made visible by
adding Bradford reagent or Biuret
reagent.
 Diphenylamine (DPA) or ethidium
bromide (EtBr) make nucleic acids
visible.
 DPA turns DNA blue and RNA green
 EtBr interacts with DNA and UV light
by glowing orange

How a Spec Works
Identification
Certain molecules have known values
for lambamax.
 This can be used to identify unknowns
in a solution.
 Hemoglobin absorbs the most light at
395 nm, so any solution with a peak at
395 may indicate the possible
presence of hemoglobin.

How a Spec Works
Concentration
The concentration of a solution also
affects the solution’s absorbance.
 If there are more molecules in one
solution then another, then there are
more molecules to absorb light.
 The amount of light that a sample
absorbs indicates how many
molecules (the concentration) are
present.

How a Spec Works
Concentration



If there are twice as many molecules in a solution,
there would be twice as much absorbance.
Likewise, half as many molecules absorb half as
much light.
On a plot of absorbance versus concentration, the
resulting graph yields a straight line. The equation
for the straight line (termed regression line) can be
used to determine the concentration of an
unknown solution once the %T has measured.
How a Spec Works
Concentration
Some specs can monitor changes in
samples over time.
 Enzyme studies are conducted by
monitoring the change in a colored
product over time.

Review Questions
What is measured in a spectrophotometer?
 What is the difference in a UV and VIS spec?
 What happens to the absorbance of a sample
as the concentration of a sample increases or
decreases?
 What color of light has a wavelength of 530
nm?
 If a molecule absorbs light at 530 nm, what
color would it be?
 What color do we no it is not?
