Spectrophotometry

Spectrophotometry is a method to measure how much a chemical substance

absorbs light (at one or more wavelengths) by measuring the intensity of light as the
beam of light passes through a sample solution. This depends on the concentration
of that chemical substance. The basic principle is that each compound absorbs light
over a certain range of wavelength.
Spectrophotometer is an instrument that measures the intensity of light after it
passes through a sample solution.

Figure 1. Spectrophotometer device

Depending on the range of wave length of the light source,

spectrophotometers can be classified into two different types (see Fig. 2):
 UV range spectrophotometer: Uses light over the ultraviolet range, and wave
length ranges between 185 - 400 nm.
 Visible range spectrophotometer: Uses a tungsten light range and wave
length ranges between 400 - 700 nm.
Note: Notation of wavelength is Lambda (λ)
Nanometer: It is the unit of measuring wavelength in nano-meter (nm = 1*10-9 m).

Figure 2. Range of wave lengths of spectrophotometers

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 The need for a spectrometer is to produce various wavelengths since the
absorbance depends on the compatibility of components with different wavelengths.
For example, the highest absorption of p-nitrophenol (acid form) occurs at
approximately 320 nm, while p-nitrophenolate (basic form) takes place at 400 nm.
See Fig.3.

Figure 3. Peak absorbances of p-Nitrophenol (acid form) and p-Nitrophenolate (basic form)

Principles of Spectrophotometry
Spectrophotometry depends on 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 the substance in the
sample solution.
The equation below represents the origin of absorbance:

𝑨 =a b c
where
 𝑨 :indicates the measure of absorbance (without unit).
 𝒂 :is the molar extinction coefficient or molar absorptivity (or absorption
coefficient).
 𝒃 :is the path length
 𝒄 :represents the concentration.

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Device Contents
It consists of:
1. Light source: It is usually a tungsten lamp, where the light emits in the visible
range only.
2. Monochromator: It is a coloured glass filter that absorbs most of the light and
allows only light of the complementary colour with a sufficiently narrow
wavelength. It should be noted that the selection of filters depends on the colour
of the solution under test.
3. Wavelength selector.
4. Cuvette for sample solution: These are glass tubes of usually 1-cm diameter and
uniform thickness in which absorbance is measured.
5. Photoelectric detector: Either a photocell or a phototube may be used to convert
the transmitted light into electrical energy.
6. Digital Display unit.

Figure 4. The components of the spectrophotometer

Table1. Complementary colours of the filters used in a spectrophotometer

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 The intensity of incident light is symbolized as (I0) which is the original light or
“total light”. When the light passes through a blank solution, it is only minimally
absorbed and is symbolized as transmitted light (It). There are important values
which are Transmittance (T) Absorbance (A).
𝐈𝐭
𝐓=
𝐈𝟎
𝐀 = − 𝐥𝐨𝐠 𝟏𝟎 𝐓
𝐈𝐭 = the intensity of light after passing through the cuvette.
𝐈𝟎= the intensity of light before passing through the cuvette.

In spectrophotometry, we need to measure the intensity of light that crossed a

blank solution, and then measure the intensity of light that crossed or passed through
a sample.
Calculation of Transmittance and Absorbance
The number of photons transferred or absorbed totally is dependent on the
concentration of the sample and the length of the cuvette .
The transmittance and absorption relation is:
Absorbance (A) = −log (T)

–log (T)= - log (lt/l0)

The absorbance of an unknown sample can be calculated using the formula

given below.

A (Test) − A (Blank)
𝐶 (𝑇𝑒𝑠𝑡 ) = ∗ 𝐶 (𝑆𝑡𝑎𝑛𝑑𝑎𝑟𝑑)
A (Standard) − A (Blank)
A (T)−A(B)
Or by symbols: 𝐶 (𝑇 ) = ∗ 𝐶(𝑆𝑇)
A (S)−A(B)

where :
C (T) or C (Test) = the concentration of the sample
C (ST) or C ( Standard) = the concentration of the standard
A ( T ) or A ( Test) = absorbance of the test
A (ST) or A ( standard ) = absorbance of the standard
A (B) or A ( Blank) = absorbance of the blank

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 In an experiment, three different solutions are prepared in three tubes and are
referred to as ‘Blank’, ‘Test’, and ‘Standard’ and marked by the first capital letter of
each word as B, T, and S, respectively.
Sample ( test ): is made from the biological fluid being analyzed and subjected to
all steps of the analysis to determine the concentration of an analytic in it.
Standard solution (s) (ST): are defined as the solutions that contain a known
accurate amount (s) (i.e. concentration) of a substance or element. These solutions
are used to compare a known concentration of a substance with the unknown
concentration of the same substance in the sample.
Blank solution (B): Is a solution that contains all of the reagents needed in analysis
of a substance except the substance under test or the standard.

Prepared by: Ali Abdulrasool Hussein

Reviewer: Abdulkareem H. Issa
Oct 2022