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Chapter 18 Chromatography Page 1

Chapter 18 Chromatography
Topics A7 and A10 from the IB HL Chemistry Curriculum

A7 Chromatography (2 Hours)
Assessment Statement Obj Teachers Notes
A.7.1 State the reasons for using chromatography.

1 The quantitative and quantitative aspects of chromatography should be
outlined.
A.7.2 Explain that all chromatographic techniques
involve adsorption on a stationary phase and
partition between a stationary phase and a mobile
phase.

3 Components in a mixture have different tendencies to adsorb onto a
surface or dissolve in a solvent. This provides a means of separating the
components of a mixture.
A.7.3 Outline the use of paper chromatography, thin-
layer chromatography (TLC) and column
chromatography.
2 An outline of the operation for each technique will be assessed. This
should include an understanding and calculation of R
f
values were relevant.
Students should be aware that, in some instances, paper chromatograms
may need to be developed, for example, in the separation of sugars.

A10 Chromatography (2 Hours)
Assessment Statement Obj Teachers Notes
A.10.1 Describe the techniques of gas-liquid
chromatography (GLC) and high-performance
liquid chromatography (HPLC).

2 An outline of the operation for each technique will be assessed. This
should include an understanding of R
f
value and its dependence on other
factors where relevant.
A.10.2 Deduce which chromatographic technique is most
appropriate for separating the components in a
particular mixture.

3 Aim 8: HPLC can identify compounds that are temperature sensitive. Uses
include: analysis of oil; alcoholic beverages; antioxidants, sugars, and
vitamins in foods; pharmaceuticals; polymers; biochemical and
biotechnology research; and quality control of insecticides and herbicides.
GLC can identify compounds that can vaporize without decomposing. Uses
include: analysis of urine samples from athletes for drugs, underground
mine gases, and blood alcohol levels.


Chromatography

Chromatography is a technique for separating and identifying the components of a mixture. Many different forms of
chromatography are used but they all work on the same principle. The components of the mixture have different
affinities for two phases: a stationary phase and a mobile phase and so are separated as the mobile phase moves
through the stationary phase. A component which has a strong attraction for the mobile phase will move quickly,
whereas a component with a strong attraction for the stationary phase will be held back.

If the stationary and mobile phases are carefully chosen, the different components will move at different speeds and so
be separated effectively. Polar compounds, for example, are more likely to move quickly when the mobile phase is a
polar solvent.

Two main types of chromatography

There are two main types of chromatography: partition and adsorption chromatography.
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Partition chromatography

Chromatography using a non-volatile liquid stationary phase held on an inert solid surface is known as partition
chromatography. The components distribute themselves between the two phases according to their relative solubility.
Paper chromatography and gas liquid chromatography are examples. The more soluble or volatile the component, the
faster it will move.

Adsorption chromatography

Chromatography which uses a solid stationary phase and a mobile liquid or gas phase is known as adsorption
chromatography. Some components of the mixture are attracted to the solid surface and the other components which
are less strongly bonded travel faster with the mobile phase. Thin-layer chromatography is an example. As the
stationary phase is generally a polar solid, the more polar solutes are more readily adsorbed than the less polar solutes.

Exercise:

Distinguish between adsorption and partition chromatography by stating the states of matter used in the stationary and
mobile phases.

Paper chromatography

You probably first used this method of partition chromatography to separate the different colors in black ink on a piece
of filter paper. The different colors in the ink move at different rates over the paper and so separate.

(a) Small spots of solutions containing the samples tested are
placed on the base line. The paper is suspended in a
closed container to ensure that the paper is saturated.
(b) The different components have different affinities for the
water in the paper and the solvent and so separate as
solvent moves up the paper.
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This simple form of partition chromatography is used mainly for qualitative analysis. Paper contains about 10% water
and this forms the stationary phase. Water is adsorbed by forming hydrogen bonds with the OH groups in the cellulose
of the paper. The mobile phase is a liquid solvent, such as water or ethanol. It moves up the paper by capillary action
and, as it does so, dissolves the spots to be analyzed at different rates, depending on their relative solubility in the
stationary and mobile phases.

When some of the solvent has almost reached the top of the paper, the level is marked as the solvent front. The paper
is then removed from the solvent and dried. The resulting chromatogram is treated with a dye, or ultraviolet light if the
different components are not visible. The organic dye ninhydrin, for example, is used to identify the different amino
acids produced when a protein molecule has been hydrolysed. Exposure to iodine vapor for 5-10 min can also be used
in some procedures. The different components appear as brown spots.

The different components are identified by their R
f
value (retention factor) which compares the distance they have
moved relative to the maximum distance moved by the solvent, the solvent front. Use this equation:

R
f
= distance moved by component above the base line distance moved by the solvent front

Components with a high R
f
value are attracted to the solvent and so move quickly through the system. Components
with a low R
f
value are more strongly bonded to the water adsorbed in the paper and so do not move as far.

Exercise:

A student wanted to investigate the green color in some leaves by paper
chromatography using the organic solvent ethanol. The results are shown
on the right.

(a) Suggest why the student used ethanol and not water in her
investigation.


(b) State and explain the conclusion which the student can make about the coloring matter in the leaves.


(c) Explain why some of the colored material had not moved from the original spot.


(d) Explain why a pencil and not a pen is used to draw the base line.


(e) Suggest why repeating the experiment with a different solvent may give more information.


(f) Identify the mobile and stationary phase in the separation technique.


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Thin-layer chromatography (TLC)

Thin-layer chromatography is an example of adsorption chromatography. It follows the same procedure as paper
chromatography, with small spots of the test solutions placed on the base line using a capillary tube. The stationary
phase is a thin layer of absorbent particles of alumina or silica supported on a glass or thin plastic plate and the mobile
phase is a liquid solvent. The different components separate and can be identified. The technique is used in qualitative
analysis to determine whether a substance is pure. TLC has four advantages over paper chromatography.

It is about three times quicker than paper chromatography.
It is more efficient it works on very small samples and the separated components can be easily recovered in a pure
form.
The results are more easily reproduced.
A range of mixtures can be separated by changing the mobile and stationary phases.

Exercise:

Three compounds were separated using thin-layer chromatography on a silica gel stationary phase.

Compound Distance travelled (cm)
A 2.5
B 7.5
C 10.0
solvent 15.0


Calculate the R
f
values and comment on the relative polarity of the components.










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Column chromatography

This technique is essentially TLC on a large scale, with a column filled with an adsorbent material such as silica or
alumina. The tap at the bottom is first closed so that the column can be saturated with the solvent. The sample
to be separated is dissolved in a minimum volume of solvent and added to the column from the top. Fresh solvent is
added in the same way to wash the sample down the stationary phase and the tap is opened. The different components
are separated as they pass through the column at different rates and are collected at the bottom as different fractions.
In the laboratory, this method is used to obtain small quantities of pure compounds. In industry, columns several
meters high are used to obtain larger quantities of materials.




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Gas-liquid chromatography (GLC)

GLC is used to separate and identify small samples of gases and volatile liquids. In this technique, the sample to be
analyzed is injected through a self-sealing cap into an oven where it is vaporized. The vapor is then carried by a
unreactive gas, the mobile phase, over a non-volatile liquid stationary phase. Nitrogen and helium are typical carrier
gases; long chain alkanes of high boiling point supported on a silicon dioxide surface act as the stationary phase.



The components of the sample are partitioned between the stationary and mobile phases, depending on their relative
boiling points and relative solubilities in the two phases. They pass through the column at different rates, each
component leaving after a characteristic interval known as the retention time. Volatile components with low solubility
in the liquid stationary phase emerge first from the column (they have shorter retention times). An unknown compound
can be identified by comparing the retention time with
that of known compounds measured under the same
conditions. The area under each peak is a measure of the
amount of each substance present. It is important that the
temperature of the oven is carefully controlled as it affects
the rate at which molecules move through the apparatus.
The figure on the right shows a chromatogram of a mixture
of primary alcohols. Methanol has the lowest boiling point
and so has the shortest retention time. The less volatile
pentan-1-ol has the longest retention time.

The technique can be made more precise, if the
chromatogram is calibrated. A substance of known
concentration is passed through the column under the
same operating conditions as those used for the test
sample. For example, a standard solution of propan-1-ol is
used when testing for blood alcohol levels. As the ratios of
the areas under the peaks are proportional to their relative
concentrations, the concentration of ethanol can be
accurately determined.
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Exercise:

(a) The chromatogram on the right was obtained
when a mixture containing the aldehydes butanal,
ethanal, pentanal, and propanal was analyzed.
Suggest which peak corresponds to which
compound.

(b) Deduce the percentage composition of the
mixture.



(c) Explain why it is important that the temperature of the oven is carefully controlled during GLC.


(d) Explain why hydrogen is unsuitable as a carrier gas
GLC is used to identify components that can vaporize without decomposition. It is used, for example, to test urine
samples for illegal substances such as steroids and stimulants, to test to blood alcohol levels, and to analyze
underground mine gases.

Exercise:

Blood samples can be analyzed for ethanol levels by dissolving the sample in a solvent. A known amount of propan-1-ol
is then added to calibrate the chromatogram. The GLC traces of two blood samples are shown below. Each shows two
peaks corresponding to ethanol and a standard solution of propan-1-ol.



(a) Identify which of the peaks corresponds to ethanol and which of peaks corresponds to propan-1-ol.


(b) State which of the samples contains the higher blood alcohol level.

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(c) Suggest how an increase in temperature changes the retention time of ethanol and propan-1-ol.


(d) Explain why it is necessary to use a reference when different blood samples are analysed.



The separated components leaving the column are typically detected using flame ionization. The gas ionizes as it passes
through a hydrogen flame; and the resulting current gives a measure of the amount of each component present.
Alternatively, the emerging gas can be analyzed with a mass spectrometer (GCMS), which allows each sample to be
identified directly. This powerful combination of techniques is particularly useful in forensic science, and in food and
drug testing.

Worked Example:

Describe how a combination of GLC and mass spectrometry can be used to test blood samples qualitatively and
quantitatively for the presence of illegal drugs.

Solution:

Blood sample is dissolved in a solvent containing a reference and is injected into the apparatus. The vaporized sample is
carried through the column by an inert carrier gas at constant temperature. The different components of the blood
leave the column at different retention times and are identified by mass spectroscopy. The different components are
ionized, accelerated by an electric field and deflected by a magnetic field. The mass spectrum produced is compared
with a data base of known illegal drugs.




High-performance liquid chromatography (HPLC)

GLC cannot be used for non-volatile substances or for substances which decompose at temperatures near their boiling
points. Instead an improved form of column chromatography known as high-performance liquid chromatography
(HPLC) is used. High pressure, rather than gravity, is used to force the mixture to be analyzed through a column tightly
packed with very fine solid particles. The method can be used to separate components which are very similar to each
other. The chromatogram produced is similar to that of GLC, although ultraviolet light is now used to detect the
different components. The technique can also be improved in combination with mass spectrometry.


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Exercise:

The amount of caffeine (C
8
H
10
N
4
O
2
) added to paracetamol (C
8
H
9
NO
2
) tablets must be carefully controlled. Small
amounts of caffeine can increase the pain-relieving properties of the paracetamol, but large amounts of caffeine in
combination with paracetamol can lead to liver damage. A paracetamol tablet was analyzed using HPLC and the
following chromatogram produced.



(a) Explain why HPLC is used to analyze the tablet.



(b) Identify which of the peaks corresponds to caffeine and explain your choice.



(c) Explain the relative retention times of the two substances.




Choosing a chromatographic technique

The choice of which chromatographic technique to use in any given situation depends on whether a quantitative or
qualitative method is needed and on the nature of the sample. Column chromatography can be used with large
amounts, but GLC is very sensitive and so it can be used with smaller samples. HPLC is the preferred method when the
sample is non-volatile or decomposes near its boiling point.

GLC is used in the analysis of urine samples from athletes for illegal drugs; blood samples for ethanol levels and
underground mine gases.

HPLC is used in the analysis of oil pollutants; alcoholic beverages; and antioxidants, sugars and vitamins in foods. It is
used in the pharmaceutical and polymer industries, for quality control of insecticides and herbicides, and in
biochemical and biotechnology research.
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Worked example:

Suggest which chromatographic technique could be used in the following situations.

(a) The separation and analysis of proteins.

(b) The detection of illegal drugs from hair samples.

(c) The monitoring of trace atmospheric gases such as CFCs in the atmosphere.

(d) The analysis of ions in solution.

(e) The separation of liquid hydrocarbons.


Solution

(a) HPLC as the molecules have high molecule mass and are non-volatile.
(b) GLC as it is very sensitive and detects very small amounts.
(c) GLC as the gases are present in small amounts.
(d) HPLC as the ions are non-volatile.
(e) GLC as all the liquids are volatile.

Exercise:

(a) Suggest, with a reason, which chromatographic technique could be used to separate a mixture of liquid
hydrocarbons from crude oil.





(b) Explain why HPLC is used in preference to other chromatographic methods in drug production.





(c) Identify, giving a reason whether GLC or HPLC should be used to determine the composition of a mixture of sugars.



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(a) Describe a chromatographic technique used to identify the amino acids formed when a protein is hydrolysed. (4)







(b) Suggest a chromatographic technique that could be used to detect the alcohol concentration in a sample of blood.
Outline the essential features of this technique.

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