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.

B IV

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.

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.

Outer parts:
Display

Wavelength
Selection

Sample Holder

Knobs or buttons used to calibrate the

spec to measure the designated
molecule.

Visible spectrophotometer
Contains

a tungsten lamp that

produces white light.
ght

Ultraviolet
spectrophotometer
Contains

a deuterium lamp
that produces light in the UV
light part of the spectrum.

Visible Spectrophotometer
White

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.

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 samples 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
solutions absorbance.

If

Remember [ ] is a ratio when we

change one number it affects the
ratio

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