Indicator
– Quickest method.
 Indicator solutions change colors when
a molecule of interest is present.
 Allows scientist to detect colorless
molecules in a solution.
 Examples: Bradford protein reagent
Diphenylamine (DPA) or
Biuret (NaOH+CuSO4).
What kind of data is this?
Spectrophotometer
(Spec)
 An instrument that measures the
amount of light that passes through
(is transmitted through) a sample.
Uses
a type of light to detect
molecules in a solution
Light is a type of energy, and
the energy is reported as
wavelengths, in nanometers
(nm).
Ultraviolet
(UV)
Spectrophotometers.
 Uses
ultraviolet light of wave lengths
from 200 nm to 350 nm.
Visible
(VIS) Light Spectrum
Spectrophotometers.
 Uses
visible light (white light) of wave
lengths from 350 nm to 700 nm.
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Shines
a beam of light on a sample.
The molecules in the sample interact
with the light waves in of 3 ways:
 Absorb the energy
 Reflect the energy
 Transmit the energy between and
through the atoms and molecules of
the sample.
Blue molecules are blue because they
reflect blue light.
Blue molecules absorb the other
colors of visible light.
Consider
blue molecules, all the
wavelengths of light are absorbed,
except for the blue ones.
The blue wavelengths are
transmitted or reflected off the
molecules. If these blue
wavelengths hit a detector (such as
in the spectrophotometer or the
nerve cells in your eye), they appear
blue.
Molecules
are whatever color of
light that they do not absorb.
Green molecules appear green
because they absorb most
wavelengths of visible light,
except the green wavelengths.
The
spectrophotometer
measures the amount of light
transmitted through the sample
(Transmittance).
By using an equation (Beers law),
it converts the transmittance
data to an absorbance value.
What kind of data is this?
The
concentration of an unknown
sample can be determined by
comparing the absorbance data to
standards of known concentration.
The data generated with the set of
known standards is called a
standard curve.
Inner
parts
 Lamp
 Prism
or grating that direct
light of a specific wavelength.
Visible
spectrophotometer
 Contains
a tungsten lamp that
produces white light.
Ultraviolet
spectrophotometer
 Contains
a deuterium lamp
that produces light in the UV
light part of the spectrum.
Outer
parts:
Display
Wavelength
Selection
Sample Holder
Knobs or buttons used to calibrate the
spec to measure the designated molecule.
Visible
 White
Spectrophotometer
light hits the prism or
grating, it is split into the
colors of the rainbow (Visible
Spectrum).
 The wavelength knob rotates
the prism/grating, directing
different color of light toward
the sample.
The
wavelength of light
produced by the tungsten lamp
range from about 350 nm (Violet
light) to 700 nm (red light).
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 and
reports that value directly (%
transmittance) or converts it to
the amount of light absorbed in
absorbance units (au) using
Beers Law.
A = 2 – log10%T
After
collecting data for your
concentration an absorption
spectrum graph is created.
These
can be used when
attempting to identify unknown
substances (e.g. CSI)
The absorbance spectrum is a
graph of a sample’s absorbance
at different wavelengths.
The spectrophotometer can
measure the amount of
absorbance
or
lack of absorbance of different
colored light for a given molecule.
The
concentration of molecules
in a solution affects the
solution’s absorbance.
Remember
[ ] is a ratio – when we
change one number – it affects the
ratio
 If
there are more molecules in one
solution than in another, than there
are more molecules to absorb the
light.
Determines
the presence and
concentrations of samples.
Determines the purity of a
sample.
Look at the change of
samples over time.
A. Measure the absorbance of standards
containing known concentrations of the
analyte
B. Plot a standard curve with absorbance
on the X axis and analyte concentration on
the Y axis
C. Measure the absorbance of the
unknown(s)
D. Determine the concentration of material
of interest in the unknowns based on the
standard curve
http://web.umr.edu/~gbert/color/AAcolor.html
http://www.chm.davidson.edu/java/spec/spec.html