Chapter 6 – Chromatography
Chromatography is a technique that is used to separate the substances present in a mixture. It is also
widely used to determine the identity of a substance. Chromatography can be both qualitative and
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How it works?
All methods of chromatography have a stationary phase and a moving or mobile phase. Components
in the mixture move because they are dissolved in a solvent or mixed with a carrier gas (gas
chromatography) that is travelling over a particular type of stationary phase. The components undergo
a continual process of adsorption onto the solid stationary phase, followed by desorption back into the
liquid mobile phase.
The rate of movement of each component depends mainly upon:
1. How strongly it adsorbs onto the stationary phase.
2. How readily it dissolves in the mobile phase.
Since each component undergoes these two processes to a different degree, the components separate.
The simplest forms of chromatography are Thin Layer and Paper chromatography. In paper
chromatography a piece of high-quality absorbent paper, similar to filter paper, is used as the stationary
phase. Thin-layer chromatography (TLC) is very similar to paper chromatography. In this case the
stationary phase is a thin layer of a fine powder such as alumina (aluminium oxide) spread on a glass or
plastic plate. Paper and thin-layer chromatography are useful for qualitative analysis.
Similar procedures are used in both techniques.
1. In thin-layer chromatography a solution of the sample to be analysed is made up with suitable
solvent.
2. A small a spot as possible of this solution is placed onto one end of the chromatography plate
and allowed to dry. (Sometimes a hair dryer is used to accelerate the process). The position of
this spot is called the origin.
3. The plate is then placed in a container with the edge of the plate below the spot submerged in a
solvent. It is important to have the sample spot, the origin, above the level of the solvent so that
it can be transported up the plate and not dissolve into the liquid in the beaker.
4. Allow the solvent to rise up the plate until it almost reaches the top, mark this spot, this is called
the solvent front.
5. Calculate the Rf. Values of the components. If it is possible run some standards with your sample
to aid identification.
E.g.
A mixture of three chemicals, A, B and C, are spotted on to TLC plate at the origin. Chemical A is very
soluble in the mobile phase but has little affinity for the stationary phase. Chemical B is only slightly
soluble in the mobile phase but has a strong attraction for the stationary phase, chemical C lies
somewhere in between these two extremes.
Sketch what the resulting chromatogram may look like.
Complete Key Question 1 page 64
Column Chromatography, High Performance and Gas
Chromatography
Another chromatographic technique, column chromatography, can be used to separate the components
in a mixture. The stationary phase is a solid, or a solid that has been thinly coated in a viscous liquid,
and packed into a glass column. The sample is applied carefully to the top of the packing and a solvent,
which acts as the mobile phase, is dripped slowly onto the column from a reservoir above. A tap at the
bottom of the column allows the solvent, which is called the eluent, to leave the column at the same rate
as it enters it at the other end.
Two instrumental chromatographic techniques based on column chromatography, high performance
liquid chromatography (HPLC) and gas chromatography, are commonly used for the separation and
identification of very complex mixtures of similar compounds, such as drugs in blood and hydrocarbons
in oil samples.
High performance liquid chromatography
High performance, or high pressure, liquid chromatography (HPLC) makes possible extremely sensitive
analysis of a wide range of compounds. It is now used routinely for pharmaceutical and industrial
analyses. For example, it can be used to detect barbiturates (sleeping tablets) in the blood, a procedure
that is useful in hospitals in cases where it is suspected that a patient’s medical condition is caused by
overdose.
There are many ways in which this technique differs from traditional chromatography.
in HPLC:
 the size of the particles in the solid used in the column is often 10–20 times smaller than in a
column chromatography
 the very small size of the solid particles allows for more frequent adsorption and desorption of
the components, giving much better separation of similar compounds
 the small particle size creates a considerable resistance to the flow of the mobile phase and so the
solvent is pumped through under high pressure—up to about 14 000 kPa
 a range of solids is available for use in HPLC columns, some with chemicals specially bonded to
their surfaces to improve the separation of particular classes of compounds.
In HPLC, the components are usually detected by passing the eluent stream through a beam of UV light.
Many organic compounds absorb UV light, so when an organic compound passes in front of the beam of
light, a reduced signal is picked up by a detector. The amount of light received by the detector is
recorded on a chart that moves slowly at a constant speed. The resulting trace is called a chromatogram.
The time taken for a component to pass through the column is called the retention time, Rt, and is
characteristic of the compound for the conditions of the experiment. It is analogous to the Rf value in
paper and thin-layer chromatography. The retention times are used to identify the components associated
with the peaks on a chromatogram. The relative amounts of each component in a mixture may be
determined by comparing the areas under each peak with areas under peaks for standard samples.
Draw Diagram here
Gas chromatography
The most sensitive of the chromatographic techniques is gas chromatography (GC). It is capable of
detecting as little as 10−12 g of a compound. However, it is limited to compounds that can be readily
vaporised without decomposing. Such compounds usually have relative molecular masses less than 300.
High performance liquid chromatography, on the other hand, can separate compounds with relative
molecular masses of 1000 or more. The extreme sensitivity of gas chromatography makes it ideal for the
analysis of trace contaminants in samples or for the detection of tiny amounts of very potent compounds.
For example, urine samples are routinely taken from athletes competing in major events to ensure that
the athletes are not benefiting from the use of illegal, performance-enhancing drugs. These samples are
analysed by GC. There are two types of gas chromatography, gas liquid chromatography (GLC) and gas
solid chromatography (GSC). Both gas chromatographic techniques operate in a similar way as outlined
below.
Another Diagram
Gas chromatography has the following features:
The mobile phase is a gas, generally nitrogen, called the carrier gas.
Procedure:
1. A small amount of sample is injected into the top of the column through
an injection port.
2. The injection port is heated to a temperature sufficient to instantly vaporise the sample, which is
then swept into the column by the carrier gas.
3. The column is a loop, or series of loops, of glass that has an internal diameter of about 4 mm and
is 2–3 m long in total. In gas–liquid chromatography, the column is packed with a porous solid
that has been coated with a liquid hydrocarbon or ester with a high boiling point. This liquid acts
as a liquid stationary phase. In gas–solid chromatography, the column is packed with an
adsorbant solid such as silica gel or alumina. The solid acts as a solid stationary phase.The
column is mounted in an oven and heated.
4. The components of the sample repeatedly pass into and out of solution with the stationary phase.
The least soluble are swept out first by the gas into the detector.
5. One of the most useful detectors is the fl ame ionisation detector, invented by an Australian, Ian
McWilliam. In this detector, organic compounds leaving the column are burnt in a hydrogen–
oxygen flame. Ions produced in the flame are attracted to electrodes and cause a current to flow.
This current is used to indicate the presence of the compound.
Complete Key Question 2 page 71
Complete selection of Questions from pages72-75
Chromatography
Mobile phase
Stationary phase
Solutes
Affinity
Dissolves
Adsorbed
Condenses
Vaporizes
Chromatogram
Rf value
Retention Time
Peak Height
Area of peak
Standards