Unit 4: analytical chemistry

Alauddin sir
A & O level Chemistry Teacher
Edexcel & Cambridge syllabus
+8801720991977

Unit 4: analytical chemistry

Chromatography

Paper chromatography

You will be familiar with the technique of paper chromatography. It is used to separate mixtures as a solvent
moves up a piece of absorbent paper. We call the solvent the mobile phase, and water trapped between the
cellulose fibers of the paper is the stationary phase. The substances in the mixture will have different
affinities for the solvent and for the water, and so they move at different rates over the paper.

Figure: a Paper chromatography. b The chromatogram produced. Components of the mixture can be
identified by comparison with pure reference compounds or by calculating Rf values and comparing these
values with those in tables of data.

The Rf values (retardation factors) of substances are calculated as shown in Figure. The conditions must be
identical to those quoted in the Rf data table, e.g. the same temperature and the same solvent used.

Colored substances can be seen directly on the paper but others are sprayed with a chemical that forms
colored compounds on the chromatogram. For example, amino acids can be revealed as bluish spots by
ninhydrin spray.

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Calculation of Rf

distance traveled by sample ( x )

Rf=
distance traveled by solvent ( y)

Calculated Rf values, are compared with reference values obtained under identical conditions.

Thin-layer chromatography

In thin-layer chromatography, referred to as TLC, the stationary phase is a solid that adsorbs solute
molecules onto its surface.

The solid stationary phase is usually alumina (Al 2O3) or silica (SiO2), which is made into slurry with water
and spread onto a microscope slide. This is then put into an oven, where it dries out into a solid white
coating on the glass. A chromatogram is then made in a similar way to paper chromatography.

Making a thin-layer chromatogram

Thin-layer chromatography.

Polar molecules have a greater attraction for a polar solid used as the stationary phase, and they are adsorbed
more strongly onto its surface. Therefore they travel more slowly up the thin layer of alumina or silica, and
separation occurs. Solutes are located on the chromatogram and identified by comparing with standard
known substances or by calculating Rf values.

TLC is quicker than paper chromatography and can be used on smaller samples, making it useful in forensic
science, where it can be used to identify drugs and explosive residues. For example, TLC is used for the
analysis of a substance that is suspected to be cannabis. The stationary phase is silica sprayed with silver
nitrate solution, which is then dried. The mobile phase is methylbenzene.

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High-performance liquid chromatography

High-performance liquid chromatography, referred to as HPLC, uses partitioning to separate and identify the
components in a mixture. The stationary phase is a non-volatile liquid, such as a long-chain hydrocarbon
liquid, bonded onto a solid support, e.g. small particles of silica. This is packed tightly into a column. The
solvent chosen for the mobile phase is usually polar, e.g. a methanol/water solvent. This has to be forced
under pressure through the densely packed column where separation occurs.

The tiny solid particles in the column have a very large surface area over which partitioning can occur,
resulting in excellent separation. The more polar components in the mixture have a greater relative solubility
in the polar solvent. Therefore they are carried through the column faster than components whose molecules
are more non-polar (which dissolve better in the non-polar stationary phase in the column). The detector
records retention times, i.e. how long it takes each component to pass through the column. The area under
each peak recorded is proportional to the amount of solute emerging from the column.

Figure: High-performance liquid chromatography.

The chromatogram from a vitamin E HPLC

analysis carried out by a food scientist
investigating chilli peppers.
HPLC is used:

 in medical research to separate peptides

and proteins
 to analyze urine samples from athletes for banned substances such as steroids or stimulants
 for monitoring pollutants in the atmosphere and in rivers, e.g. measuring levels of pesticides
 by food standards agencies to check the accuracy of the data on food labels.

Gas–liquid chromatography

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Gas–liquid chromatography, which is referred to as GLC, is similar to HPLC but a gaseous sample enters
the column. The column contains the stationary phase and the sample is moved through by an inert carrier
gas. This method is used with gases, liquids and volatile solids (as they must be in the form of a vapor). The
apparatus is shown in Figure

The chromatogram must be obtained using the same carrier gas, flow rate, stationary phase and temperature
that were used when the standard data was obtained.

Figure shows a chromatogram obtained using GLC.

A gas chromatogram from a mixture of volatile organic compounds

Analysis by gas–liquid chromatography does have some limitations. For example, similar compounds will
have similar retention times and if a newly discovered compound is detected it will not have a match in the
computer’s database of retention times.

Determination of the percentage composition of a mixture by GLC

For quantitative analysis, the component peaks are first identified and then the area of each is measured. The
peaks are roughly triangular in shape so their area is approximately:

The amount of each component in a mixture is found by expressing it as a percentage of the sum of the areas
under all the peaks. For example, for a mixture of three esters A, B and C:
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 peak area ( ¿ height ) of A
( ¿ ) % of ester A= × 100
∑ of theareas ( ¿ height ) of A , B ,∧C
GLC is used in

 Testing for steroids in competing athletes

 Testing the fuels used in Formula One motor racing.
 It is also used for medical diagnosis in analyzing blood samples.

With GLC it is possible to determine the percentages of dissolved oxygen, nitrogen, carbon dioxide and
carbon monoxide in blood samples as small as 1.0 cm 3. GLC is often combined with mass spectrometry to
separate then rapidly identify the components of a mixture.

Proton (1H) nuclear magnetic resonance

How NMR works

Nuclear magnetic resonance (NMR) spectroscopy is a widely used analytical technique for organic
compounds. NMR is based on the fact that the nucleus of each hydrogen atom in an organic molecule
behaves like a tiny magnet. The nucleus of a hydrogen atom consists of a single proton. This proton can
spin. The spinning motion of the positively charged proton causes a very small magnetic field to be set up.

In NMR we put the sample to be analyzed in a magnetic field. The hydrogen nuclei (protons) either line up
with the field or, by spinning in the opposite direction, line up against it (Figure 29.13).

There is a tiny difference in energy between the oppositely spinning 1H nuclei. This difference corresponds
to the energy carried by waves in the radiowave range of the electromagnetic radiation spectrum. In NMR
spectroscopy the nuclei ‘flip’ between the two energy levels (Figure 29.14). Only atoms whose mass number
is an odd number, e.g. 1H or 13C, absorb energy in the range of frequencies that are analyzed.

The size of the gap between the nuclear energy levels varies slightly, depending on the other atoms in the
molecule (the molecular environment). Therefore, NMR can be used to identify 1H atoms in different parts
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of a molecule. This is easier to visualize by looking at an example. If we look at a molecule of methanol,
CH3OH, we can see that there are 1H atoms in two different molecular environments. We have the 1H atoms
in the −CH3 group and the 1H atom in the −OH group. The energy absorbed by the −CH 3 1H atoms is
different from the energy absorbed by the 1H atoms in −OH.

As the magnetic field is varied, the 1H nuclei in different molecular environments flip at different field
strengths. The different field strengths are measured relative to a reference compound, which is given a
value of zero. The standard compound chosen is tetramethylsilane (TMS). TMS is an inert, volatile liquid
that mixes well with most organic compounds. Its formula is Si(CH 3)4, so all its H atoms are equivalent.
TMS only gives one, sharp absorption, called a peak, and this peak is at a higher frequency than most other
protons. All other absorptions are measured by their shift away from the TMS line on the NMR spectrum.
This is called the chemical shift (δ), and is measured in units of parts per million (ppm).

Low-resolution NMR

A low-resolution NMR spectrum shows a single peak for each non-equivalent hydrogen atom; an example is
shown in the following Figure.

The low-resolution NMR spectrum of ethanol, H3CH2OH.

There are three peaks on ethanol’s low-resolution NMR spectrum. These correspond to the 1H atoms in
−OH, −CH2 and −CH3. Note how the heights of the peaks vary. The area under each peak tells us the relative
number of equivalent 1H atoms responsible for that particular chemical shift. The largest peak will be from
the −CH3 hydrogen atoms, the middle peak from the −CH 2− hydrogen atoms and the smallest peak from the
−OH hydrogen. The relative areas under the peaks are shown on the NMR spectrum by the labels 1H, 2H
and 3H.

High-resolution NMR

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High-resolution NMR gives us more information to interpret. Peaks that appear as one ‘signal’ on a low-
resolution NMR spectrum are often revealed to be made up of a cluster of closely grouped peaks. This is
because the magnetic fields generated by spinning nuclei interfere slightly with those of neighboring nuclei.

This interference is called spin–spin coupling. The exact splitting pattern of a peak depends on the number
of hydrogen atoms on the adjacent carbon atom or atoms.

The high-resolution NMR spectrum of ethanol illustrates this n + 1 rule used to interpret splitting patterns

 The −CH3 peak is split into three because there are two 1H atoms on the adjacent CH 2 group. n + 1 =
3 (as n = 2); this is called a triplet.
 The −CH2− peak is split into four because there are three 1H atoms on the adjacent −CH3 group.
n + 1 = 4 (as n = 3); this is called a quartet.
 The −OH peak is not usually split as its 1H atom is constantly being exchanged with the 1H atoms of
other ethanol molecules and any water present. This results in one average peak being produced.

The high-resolution NMR spectrum of ethanol, showing the splitting pattern in two of the peaks. The area under each
series of peaks still represents the number of equivalent 1H atoms in the molecule, as in low-resolution NMR.

Splitting patterns in high-resolution NMR spectra.

Infra-red spectroscopy

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Use infrared spectra, or data from infrared spectra, to deduce functional groups present in organic
compounds and predict infrared absorptions, given wavenumber data, due to familiar functional groups. This
will be limited to:

i) C–H stretching absorptions in alkanes, alkenes and aldehydes

ii) O–H stretching absorptions in alcohols and carboxylic acids

iii) N–H stretching absorption in amines

iv) C=O stretching absorptions in aldehydes and ketones

v) C–X stretching absorption in halogenoalkanes

vi) As an analytical tool to show the change in functional groups during the oxidation of an alcohol to a
carbonyl

Demonstrate an understanding that only molecules which change their polarity as they vibrate can absorb
infrared radiation

Molecules that change their polarity as they vibrate (due to the movement of the dipoles in the polar bonds)
absorb infrared radiation.

Demonstrate an understanding that H2O, CO2, CH4 and NO molecules absorb IR radiation and are
greenhouse gases, but O2 and N2 are not.

Oxygen and nitrogen do not change their polarity. So they do not absorb IR radiation unlike water, carbon
dioxide, methane and nitrogen monoxide and therefore they are not greenhouse gases.

 In infrared (lR) spectroscopy, a beam of lR radiation goes through a sample of a chemical.

 The lR energy is absorbed by the covalent bonds in the molecules, increasing their vibrational
energy.
 The bending or stretching vibrations of different bonds result in absorption at different frequencies.
 Bonds between different atoms absorb different frequencies of lR radiation. Bonds in different
places in a molecule absorb different frequencies too - so the O-H group in an alcohol and the O-H
in a carboxylic acid absorb different frequencies.

Vibrational frequencies of some common covalent bonds are shown below:

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