Assignment : Chemical perspective

Topics : Chromatography + Spectrophotometry

Group : 01

Group leader : Danish Liaquat

Group members : Zain Ahmad

Sheraz Rafie
Hamza Faiz
Shehryar Ayub

Submitted To: Sir Kashif Iqbal

Class: Bs Chemistry 3rd Semester

Institute: Khawaja Fareed Collage RYK

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 classified into two
different types:

• 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.

Spectrophotometry is a method 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 the most useful methods of quantitative analysis in
various fields such as chemistry, physics, biochemistry, material and chemical
engineering and clinical applications.

Explanation
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 engineering,
clinical applications, industrial applications, etc.). Any application that deals with
chemical substances or materials can use this technique. 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.
Figure 1: Basic structure of spectrophotometers (CC BY-4.0; Heesung Shim via LibreTexts)

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.
• Photometer: After the desired range of wavelength of light passes through the
solution of a sample in cuvette, the photometer detects the amount of photons
that is absorbed and then sends a signal to a galvanometer or a digital display,
as illustrated in Figure 1.

Figure 2: A single wavelength spectrophotometer

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 maximum absorbance at approximately 320 nm and p-
nitrophenolate (basic form) absorb best at 400nm, as shown in Figure 3.

Figure 3: Absorbance of two different compounds

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
isosbestic point in a reaction demonstrates that an intermediate is NOT required
to form a product from a reactant. Figure 4 shows an example of an isosbestic
point.

Figure 4: An example of isosbestic point (CC BY-4.0; Heesung Shim via Libre Texts
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 before the beam of light passes through the cuvette.
Transmittance is related to absorption by the expression:
Absorbance(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.

Figure 5: Transmittance (CC BY-4.0; Heesung Shim via LibreTexts)

Chromatography
What is it?
Some materials appear homogenous, but are actually
a combination of substances. For example, green plants contain a mixture
of different pigments. In addition, the black ink in the pens that are used in
this experiment is a mixture of different colored materials. In many
instances, we can separate these materials by dissolving them in an
appropriate liquid and allowing them to move through an absorbent
matrix, like paper. Chromatography is a method used by scientists for
separating organic and inorganic compounds so that they can be analyzed
and studied. By analyzing a compound, a scientist can figure out what
makes up that compound. Chromatography is a great physical method for
observing mixtures and solvents. The word chromatography means "color
writing" which is a way that a chemist can test liquid mixtures. While
studying the coloring materials in plant life, a Russian botanist invented
chromatography in 1903. His name was M.S. Tswett. Chromatography is
such an important technique that two nobel prizes have been awarded to
chromatographers. Over 60% of chemical analysis worldwide is currently
done with chromatography or a variation thereon. Chromatography is used
in many different ways. Some people use chromatography to find out what
is in a solid or a liquid. It is also used to determine what unknown
substances are. The Police, F.B.I., and other detectives use
chromatography when trying to solve a crime. It is also used to determine
the presence of cocaine in urine, alcohol in blood, PCB's in fish, and lead in
water. Chromatography is used by many different people in many different
ways. Chromatography is based on differential migration. The solutes in a
mobile phase go through a stationary phase. Solutes with a greater affinity
for the mobile phase will spend more time in this phase than the solutes
that prefer the stationary phase. As the solutes move through the
stationary phase they separate. This is called chromatographic
development.
How it works

In all chromatography there is a mobile phase and a stationary phase. The

stationary phase is the phase that doesn't move and the mobile phase is
the phase that does move. The mobile phase moves through the stationary
phase picking up the compounds to be tested. As the mobile phase
continues to travel through the stationary phase it takes the compounds
with it. At different points in the stationary phase the different
components of the compound are going to be absorbed and are going to
stop moving with the mobile phase. This is how the results of any
chromatography are gotten, from the point at which the different
components of the compound stop moving and separate from the other
components. In paper and thin-layer chromatography the mobile phase is
the solvent. The stationary phase in paper chromatography is the strip or
piece of paper that is placed in the solvent. In thin-layer chromatography
the stationary phase is the thin-layer cell. Both these kinds of
chromatography use capillary action to move the solvent through the
stationary phase. What is the Retention Factor, Rf ? The retention factor,
Rf, is a quantitative indication of how far a particular compound travels in a
particular solvent. The Rf value is a good indicator of whether an unknown
compound and a known compound are similar, if not identical. If the Rf
value for the unknown compound is close or the same as the Rf value for
the known compound then the two compounds are most likely similar or
identical. The retention factor, Rf, is defined as

Rf = distance the solute (D1) moves divided by the

distance traveled by the solvent front (D2) Rf = D1 / D2 where D1 =
distance that color traveled, measured from center of the band of color to
the point where the food color was applied D2 = total distance that solvent
traveled
The Different Types of Chromatography

There are four main types of chromatography. These are Liquid

Chromatography, Gas Chromatography, Thin-Layer
Chromatography and Paper Chromatography.

Liquid Chromatography
is used in the world to test water samples to look for
pollution in lakes and rivers. It is used to analyze metal ions and
organic compounds in solutions. Liquid chromatography uses
liquids which may incorporate hydrophilic, insoluble molecules.

Gas Chromatography
is used in airports to detect bombs and isused is
forensics in many different ways. It is used to analyze fibers on a
persons body and also analyze blood found at a crime scene. In gas
chromatography helium is used to move a gaseous mixture
through a column of absorbent material.
Thin-layer Chromatography uses an absorbent material on flat
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Chromatography the Real World
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Paper Chromatography is one of the most common types of

chromatography. It uses a strip of paper as the stationary phase.
Capillary action is used to pull the solvents up through the paper
and separate the solutes.

The table below summarizes the information from

above.
Liquid test water Used to analyze metal
Type of Applications in Why and What is it
Chromatograph samplesto look ions and organic
y for pollution, compounds in
solutions.It uses
liquids which may
incorporate
hydrophilic, insoluble
molecules.
Gas detect bombs in Used to analyze
Chromatograph airports, identify volatilegases. Helium
y and quantify such is used tomove the
drugs as alcohol, gaseous mixture
used in forensics through a column of
tocompare fibers absorbent material.
found on a victim
Thin-Layer detecting pesticide Uses an absorbent
Chromatograph or insecticide material on flat glass
y residues in food, plates. This is a
also used in simpleand rapid
forensics to method to check the
analyzethe dye purity of theorganic
composition compound.
offibers
Paper separating The most common
Chromatograph amino acids and typeof
y anions,RNA chromatography. The
fingerprinting, paper is the stationary
separating and phase. This uses
testing capillary action to pull
histamines, the solutes up through
antibiotics the paper and separate
the solutes.