Spectrophotometry is a method used to

measure how much a chemical substance

absorbs light by measuring the intensity
of light as a beam of light passes through
sample solution. The basic principle is that
each compound absorbs or transmits light
over a certain range of wavelength. This
measurement can also be used to
measure the amount of a known chemical
substance. Spectrophotometry is one of
Every chemical compound absorbs,
transmits, or reflects light
(electromagnetic radiation) over a certain
range of wavelength. Spectrophotometry
is a measurement of how much a
chemical substance absorbs or transmits.
Spectrophotometry is widely used for
quantitative analysis in various areas
(e.g., chemistry, physics, biology,
biochemistry, material and chemical
In biochemistry, for example, it is used to
determine enzyme-catalyzed reactions. In
clinical applications, it is used to examine
blood or tissues for clinical diagnosis.
There are also several variations of the
spectrophotometry such as atomic
absorption spectrophotometry and atomic
emission spectrophotometry.
A spectrophotometer is an instrument
that measures the amount of photons
(the intensity of light) absorbed after it
passes through sample solution. With
the spectrophotometer, the amount of a
known chemical substance
(concentrations) can also be determined
by measuring the intensity of light
detected. Depending on the range of
wavelength of light source, it can be
UV-visible spectrophotometer: uses
light over the ultraviolet range (185 -
400 nm) and visible range (400 - 700
nm) of electromagnetic radiation
spectrum.
IR spectrophotometer: uses light over
the infrared range (700 - 15000 nm) of
electromagnetic radiation spectrum.
In visible spectrophotometry, the absorption or
the transmission of a certain substance can be
determined by the observed color. For
instance, a solution sample that absorbs light
over all visible ranges (i.e., transmits none of
visible wavelengths) appears black in theory.
On the other hand, if all visible wavelengths
are transmitted (i.e., absorbs nothing), the
solution sample appears white. If a solution
sample absorbs red light (~700 nm), it appears
green because green is the complementary
 Devices and
mechanism
Figure 1 below illustrates the basic
structure of spectrophotometers. It
consists of a light source, a collimator, a
monochromator, a wavelength selector,
a cuvette for sample solution, a
photoelectric detector, and a digital
display or a meter. Detailed mechanism
is described below. Figure 2 shows a
A spectrophotometer, in general, consists of two
devices; a spectrometer and a photometer. A
spectrometer is a device that produces, typically
disperses and measures light. A photometer indicates
the photoelectric detector that measures the intensity
of light.
Spectrometer: It produces a desired range of
wavelength of light. First a collimator (lens) transmits a
straight beam of light (photons) that passes through a
monochromator (prism) to split it into several
component wavelengths (spectrum). Then a
wavelength selector (slit) transmits only the desired
wavelengths, as shown in Figure 1.
You need a
spectrometer to
produce a variety of
wavelengths
because different
compounds absorb
best at different
wavelengths. For
example, p-
nitrophenol (acid
form) has the
Looking at the graph
that measures
absorbance and
wavelength, an
isosbestic point can
also be observed.
An isosbestic point is
the wavelength in
which the absorbance
of two or more species
are the same. The
appearance of an
Referring back to Figure 1 (and Figure 5), the
amount of photons that goes through the cuvette
and into the detector is dependent on the length
of the cuvette and the concentration of the
sample. Once you know the intensity of light after
it passes through the cuvette, you can relate it to
transmittance (T). Transmittance is the fraction of
light that passes through the sample. This can be
calculated using the equation:
transmittance(T)=It/Io
Where It is the light intensity after the beam of light
passes through the cuvette and Io is the light intensity
Transmittance is related to absorption by the
expression:
Asorbance(A)= -log(T)= -log(It/Io)

Where absorbance stands for the amount of photons

that is absorbed. With the amount of absorbance known
from the above equation, you can determine the
unknown concentration of the sample by using Beer-
Lambert Law. Figure 5 illustrates transmittance of light
through a sample. The length l is used for Beer-Lambert
Law described below.
Beer-Lambert Law
Beer-Lambert Law (also known as Beer's Law)
states that there is a linear relationship between
the absorbance and the concentration of a
sample. For this reason, Beer's Law can only be
applied when there is a linear relationship. Beer's
Law is written as:
A=£lC
Where
A is the measure of absorbance (no units),
ϵ is the molar absorptivity (or absorption coefficient),
l is the path length, and
The molar extinction coefficient is given
as a constant and varies for each
molecule. Since absorbance does not
carry any units, the units for ϵ must
cancel out the units of length and
concentration. As a result, ϵ has the
units: L·mol-1·cm-1. The path length is
measured in centimeters. Because a
standard spectrometer uses a cuvette
that is 1 cm in width, l is always
Guanosine has a maximum absorbance of 275
nm. ϵ275=8400−1 and the path length is 1 cm.
Using a spectrophotometer, you find the
that A275=0.70. What is the concentration of
guanosine?

Solution
To solve this problem, you must use Beer's Law.
A=ϵ⁢
0.70 = (8400 M-1 cm-1)(1 cm)(c)
Next, divide both side by [(8400 M-1 cm-1)(1 cm)]
c = 8.33x10-5 mol/L