Unit 2: Practical Scientific Procedures and Techniques
Unit 2C: Undertake Chromatographic techniques to identify components in
mixtures
Vocational Scenario:
I am a newly appointed technical assistant at a large chemical plant, Chemicalequip. As part of my
induction period and to progress in my role, I have to demonstrate skills in a range of practical
procedures and techniques. The company is often required to identify substances. I need to
demonstrate that I can carry out different chromatographic techniques to separate and identify
components in mixtures. In a report of the techniques I have used, I will need to explain the
techniques, analyse and evaluate my results and suggest improvements.
Chromatography:
Chromatography is an analytical separation technique that is used to separate compounds and
mixtures and to recognise the components or substances that make up the mixture/compound. This
allows the identification of the purity of the chemical substance. Summarising the concept of
chromatography is that it is a simple concept that involves the movement of either a gas or liquid
over a fixed paper or powder.
In more depth, overall, in all types of chromatography, substances are broken down into individual
components as they move in a mobile phase, which travels across a stationary phase. The mobile
phase refers to a substance, which can either be liquid or gas, but is usually liquid, carrying the
components of the mixture along the stationary phase. The stationary phase is the material, usually
solid, that remains in a fixed position during the chromatography process, soaking up the mixture as
it passes through. One thing to note is that substances will all move at varying speeds, some faster
than others and some slower than others in a certain period.
There are multiple types of chromatographic techniques and these techniques have been produced
and developed each with a distinctive purpose and suitability for a particular compound. These
techniques have also been produced by the modification of the mobile and stationary phase. Here
are a few types of chromatographic techniques: Gas chromatography, Liquid chromatography, Ion
exchange chromatography, Thin layer chromatography and Paper chromatography. Out of these
techniques listed I will be performing and analysing Thin layer chromatography and Paper
chromatography.
But I will briefly discuss other types of chromatography and their techniques:
In Gas chromatography, compounds are separated by a process that uses gas travelling down a glass
or metal cylinder with consideration for both solubility and reactions with the stationary phase. Ion
exchange chromatography relies on the connection between charged molecules and stationary
phases that have contrasting charges to achieve separation.
Theory:
,Mobile phase- The mobile phase refers to a substance which can either be liquid or gas but is usually
liquid that carries the components of the mixture along the stationary phase.
Stationary phase- The stationary phase is the material usually solid that stays in a fixed position
during the process of chromatography and soaks up the mixture as it passes through.
Adsorption- Adsorption refers to the process by which a substance's molecules attach themselves to
the face of another molecule. This process is reversible and temporary.
Paper chromatography and thin layer chromatography:
A method used for isolating low-molecular-mass substances according to how they are divided
during the stationary phase and mobile phase is paper chromatography. In paper chromatography,
the stationary phase is paper, and the mobile phase is either an aqueous liquid or an organic solvent
that is non aqueous. The separation of each chemical in the mixture is determined by the degree of
attraction towards the mobile and stationary phases. Thus, chemicals with a higher attraction to the
paper will remain closer to the stationary phase, usually at the beginning, while chemicals with a
higher attraction to the solvent/liquid will stay closer to the mobile phase, resulting in their higher
travel up the paper. This characteristic is unique to each chemical and helps with the differentiation
when in a mixture.
Thin layer chromatography or TLC is like paper chromatography however the difference is the
migration of the chemicals, materials used and adsorption. In thin-layer chromatography, a glass,
metal, or plastic plate is covered with a thin layer of a neutral unreactive compound such as silica or
aluminium oxide. This plate which is referred to as TLC plate is the stationary phase. A capillary tube
is used to deposit a little amount of mixture/sample near the bottom of the TLC plate. Once this is
done the TLC plate is inserted upright and into a sealed beaker with an organic solvent. The organic
solvent is the mobile phase. Each element in the mixture is absorbed onto the solid surface due to
their variety in solubility and if the sample attaches to the solid surface differently based on their
solubility and the adsorption strength to the TLC plate. Adsorption refers to the process by which a
substance's molecules attach themselves to the face of another molecule. This process is reversible
and temporary. Some elements will be higher up on the plate compared to other elements. Once
the solvent has reached the top of the plate, the plate is removed from the beaker and dried, once
dried, analysis begins. In TLC, each element in the mixture will separate according to how much it is
attracted to the stationary and mobile phases. As a result, elements that are more attracted to the
adsorbent material coated on the plate will stay closer to the stationary phase, typically at the
beginning of the plate, while elements that are more attracted to the solvent will stay closer to the
mobile phase and ascend up the plate further. The sample is gradually carried up the plate by the
mobile phase. Depending on how much they are attracted to the plate or the solvent, different
portions of the sample travel over different distances. The distinct components within the mixture
can be easily identified and therefore separated due to the different movements around the plate.
Similarities and differences:
Similarities Differences
Paper chromatography Thin layer chromatography Paper Thin layer
chromatography chromatography
, Uses a stationary Uses a stationary phase for Specialised paper as Thin layer of
phase for separation separation the stationary phase unreactive
substance on a flat
surface as
stationary phase
Uses a mobile phase Uses a mobile phase for Uses a liquid or a Uses a liquid or a
for separation separation solvent for mobile gas for mobile
phase phase
Sample is applied as Sample is applied as spots The separation The separation
spots near the bottom near the bottom of mechanism depends mechanism
of stationary phase stationary phase on different depends on
attraction to the different attraction
paper and solvent to the stationary
and mobile phase
Components are seen Components are seen as Less even movement More even
as spots spots through the movement through
stationary phase and stationary phase
longer separation and faster
time separation time
Used in various Used in various scientific Less sensitive due to More sensitive due
scientific fields fields thicker stationary to thinner
phase stationary phase
Rely on separation of Rely on separation of Limited range of Variety of
components components absorbencies for the absorbencies for
stationary phase the stationary
phase
Uses of chromatography:
Chromatography is used in a variety of ways depending on the technique used as I’ve stated a few
types of chromatography earlier on but there are 14 types of chromatography, and each type has a
different purpose, but they all are used in the scientific industries whether it be pharmaceutical,
clinical labs, food and beverage safety, forensics, chemical or biochemical. I will be discussing the
uses of paper chromatography and thin layer chromatography.
Paper chromatography's main use is in the pharmaceutical industry, and its task is to figure out the
number of chemicals present in a product that has been produced, as well as finding out how pure
said product is. Before items are sent to the public, it is also used in the food and beverage industry
to check for contaminants and determine whether any chemical substances are present. Identifying
any materials discovered at a crime scene is an additional use for it in forensic science. It is also used
in chemical reactions to see reaction mixtures and in biochemical reactions to separate amino acids
and other biological elements.
Many scientific industries employ thin layer chromatography. It is employed in the pharmaceutical
business to assess the purity of substances produced, much like paper chromatography. Due to its
ability to detect purity, it is used in clinical laboratories to identify drugs in a patient's system and, if
the patient has overdosed it helps with identifying the drugs in the patients system. This information
, is important as it helps with the decision of the best course of action and treatment for the patient.
Additionally, the food and beverage business it to protect the public and look for contaminants. It is
also used in herb analysis to ensure that the herbs used in certain herbal medicines are safe and
authentic and not harmful.
Factors affecting chromatography:
There are a variety of factors that affect chromatography majority of which are individual to the type
of chromatography. I will discuss factors that affect thin layer chromatography and paper
chromatography as these are the types of chromatography we will be investigating and carrying out.
Thin layer chromatography:
TLC plate
Temperature
Application technique
Paper chromatography:
The paper used
Choice of solvent
Amount travelled by solvent
Characteristics of the components
Principle and theory of chromatography:
Polarity:
The structure of a molecule and the distinction in electronegativity within molecules decides
whether it is polar or non-polar. In polar molecules, there is an uneven distribution of electrons
which leads toa positive charge and a negative charge on different ends. Non-polar molecules lack
this polarity and opposing charges and have an even distribution of electrons meaning there is
usually no charge. This difference influences their behaviour specifically with solubility and
compatibility of other substances. Polar molecules interact with polar molecules as they can form
hydrogen bonds and non-polar molecules prefer to interact with other non-polar molecules.
It is important to know the polarity of your solvent as it will help you predict how quickly the
components will travel up the stationary phase and separate. Usually if the solvent is polar the
components will move much faster than a non-polar solvent. Also, the polarity of molecules affects
how soluble they are in the mobile phase. Polar molecules will dissolve better in polar solvents and
non-polar molecules will dissolve better in non-polar solvents. This connection between polarity and
solubility is important for chromatographic separations to work efficiently and effectively.
Molecule size and solubility:
Tiny molecules dissolve much quicker in a solvent since they can move around gaps that are within
the larger solvent molecules. Temperature affects solid solubility, because when it increases, the
bonds that are between the solute and solvent starts to become weaker because of an increase of
kinetic energy in the solvent which increases solubility. Also, molecules that resemble the mobile
