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analytical chemistry
Learning outcomes
You should be able to:
explain and use the terms Rf value in thin-layer predict the number of peaks in a carbon-13 NMR
chromatography and retention time in gas spectrum for a given molecule
liquid chromatography, and interpret gasliquid analyse and interpret a proton NMR spectrum of a
chromatograms to find the percentage composition simple molecule to deduce the dierent types of
of a mixture proton present, the relative numbers of each type
use a mass spectrum to deduce the molecular of proton present, the number of non-equivalent
mass of an organic molecule, the number of carbon protons adjacent to a given proton and the possible
atoms in a compound using the M + 1 peak, and structures for the molecule
the presence of bromine and chlorine atoms in a predict the chemical shifts and splitting patterns of
compound using the M + 2 peak the protons in a given molecule
suggest the identity of molecules formed by simple in obtaining an NMR spectrum, describe the use of
fragmentation in a given mass spectrum tetramethylsilane, TMS, as the standard for chemical
analyse a carbon-13 NMR spectrum of a simple shift measurements, and the need for deuterated
molecule to deduce the dierent environments of the solvents, e.g. CDCl3
carbon atoms present and the possible structures for describe the identification of O H and N H protons
the molecule by proton exchange using D2O.
Cambridge International A Level Chemistry
Introduction
We can detect and identify any chemical
substance using a range of different techniques.
Specialised instruments have been developed
to carry out tests, such as gas-liquid
chromatography, nuclear magnetic resonance
(NMR) spectroscopy and mass spectrometry. The
instruments used are often very sensitive, so
chemical substances can be detected at very
low concentrations.
a b
solvent front
supporting rod
paper clip
paper
glass tank
solvent
sample to be
separated
pure reference pure reference mixture of solutes
compounds compounds to be identified
Figure 29.2 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 (see Figure29.3) and comparing these values with those in
tables of data.
Chapter 29: Analytical chemistry
overlapping
1 Look at this
paper chromatogram:
rotate paper
through 90
mixture of original three solutes now
three solutes mixture completely separated
A B C
Figure 29.4 Two-way paper chromatography to separate
solutes with similar Rf values in a solvent. The technique
can also be used with thin-layer chromatography (see
page 436). a The solvent used was ethanol. Which sample of
ink, A, B or C, has the greatest relative solubility
in ethanol?
b Work out the Rf value of the ink whose partition
coefficient in ethanol and water lies between the
values of the other two inks.
Cambridge International A Level Chemistry
thin layer of
SiO2 or Al2O3
coated onto a High-performance liquid
glass or plastic chromatography
surface
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,
mixture of bonded onto a solid support, e.g. small particles of silica.
solutes
This is packed tightly into a column. The solvent chosen
solvent 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
Figure 29.7 Thin-layer chromatography. (Figure29.8).
The tiny solid particles in the column have a very large
surface area over which partitioning can occur, resulting
Polar molecules have a greater attraction for a polar solid in excellent separation. The more polar components in
used as the stationary phase, and they are adsorbed more the mixture have a greater relative solubility in the polar
strongly onto its surface. Therefore they travel more slowly solvent. Therefore they are carried through the column
up the thin layer of alumina or silica, and separation faster than components whose molecules are more non-
occurs. Solutes are located on the chromatogram and polar (which dissolve better in the non-polar stationary
identified by comparing with standard known substances phase in the column). The detector records retention
or by calculating Rf values. times, i.e. how long it takes each component to pass
Chapter 29: Analytical chemistry
packed column
I ICI IBI IAI
separated materials
from the column
mobile
phase
detectors
solvent
delivery injector
system
waste collected
through the column. The area under each peak recorded HPLC is used:
is proportional to the amount of solute emerging from the in medical research to separate peptides and proteins
column (Figure29.9). to analyse urine samples from athletes for banned
substances such as steroids or stimulants
for monitoring pollutants in the atmosphere and in rivers,
Recorder response
437
e.g. measuring levels of pesticides
by food standards agencies to check the accuracy of the
data on food labels.
Gasliquid chromatography
5 10 15 20 min Gasliquid chromatography, which is referred to as
Time / min GLC, is similar to HPLC but a gaseous sample enters the
Figure 29.9 The chromatogram from a vitamin E HPLC column. The column contains the stationary phase and
analysis carried out by a food scientist investigating the sample is moved through by an inert carrier gas. This
chilli peppers. method is used with gases, liquids and volatile solids (as
they must be in the form of a vapour). The apparatus is
shown in Figure29.10.
carrier gas
computer
(mobile
or recorder
phase) in
sample injected
through silicone
rubber septum
exit
injector detector Figure 29.10 Gasliquid
port
detector chromatography. The
injector
oven oven maintains a constant
oven
temperature, higher
column (15 m long, than the boiling point of
36 mm diameter) the components in the
column oven mixture to be analysed.
Cambridge International A Level Chemistry
pentan-2-one
all the peaks. For example, for a mixture of three esters A,
ethoxyethane
B and C:
heptane
Recorder response
(approx.) % of ester A
hex-1-ene
= ___________________________________
100
sum of the areas (or heights) of A, B and C
Energy
Nuclear magnetic resonance (NMR) spectroscopy is a
before field
widely used analytical technique for organic compounds. is applied with
NMR is based on the fact that the nucleus of each after field is applied
hydrogen atom in an organic molecule behaves like a tiny
magnet. The nucleus of a hydrogen atom consists of a Figure 29.14 Hydrogen (1H) nuclei will absorb energy in the
single proton. This proton can spin. The spinning motion radiowave range when they flip from the lower energy level,
of the positively charged proton causes a very small lining up with the applied magnetic field, to the higher energy
level, lining up against it.
magnetic field to be set up.
In NMR we put the sample to be analysed in a
magnetic field. The hydrogen nuclei (protons) either line molecular environments flip at different field strengths.
up with the field or, by spinning in the opposite direction, The different field strengths are measured relative to a
line up against it (Figure29.13). reference compound, which is given a value of zero. The
There is a tiny difference in energy between the standard compound chosen is tetramethylsilane (TMS).
oppositely spinning 1H nuclei. This difference corresponds TMS is an inert, volatile liquid that mixes well with
to the energy carried by waves in the radiowave range most organic compounds. Its formula is Si(CH3)4, so all
of the electromagnetic radiation spectrum. In NMR its H atoms are equivalent (i.e. they are all in the same
spectroscopy the nuclei flip between the two energy molecular environment). TMS only gives one, sharp
levels (Figure29.14). Only atoms whose mass number is an absorption, called a peak, and this peak is at a higher
odd number, e.g. 1H or 13C, absorb energy in the range of frequency than most other protons (Figure29.15). All
other absorptions are measured by their shift away from 439
frequencies that are analysed.
The size of the gap between the nuclear energy levels the TMS line on the NMR spectrum. This is called the
varies slightly, depending on the other atoms in the chemical shift (), and is measured in units of parts per
molecule (the molecular environment). Therefore, NMR million (ppm).
can be used to identify 1H atoms in different parts of
a molecule. This is easier to visualise by looking at an single peak
example. If we look at a molecule of methanol, CH3OH, we of TMS
can see that there are 1H atoms in two different molecular (which we use
as a standard)
environments. We have the 1H atoms in the CH3 group
and the 1H atom in the OH group. The energy absorbed
by the CH3 1H atoms is different from the energy
absorbed by the 1H atoms in OH. 0 chemical
shift ()
In NMR spectroscopy, we vary the magnetic field as
that is easier than varying the wavelength of radiowaves. Figure 29.15 The standard TMS peak used as a reference on
As the magnetic field is varied, the 1H nuclei in different NMR spectra.
3H
Absorption of energy
2H
1H
O O O
RCH2 C 2.02.9
H3C C R2CH C
N CH3 CH R
N 2
CHR
N 2 2.32.9
O CH 3
CH R
O 2
CHR
O 2 3.34.3
Br or Cl CH3 Br or Cl CH2R Br or Cl CHR2 3.04.2
OH 4.510.0(a)
CH CH 45.6.0
O O
C C 5.012.0(a)
NH2 NH
441
H 6.58.0
O
C 9.010
H
O
C 11.012.0(a)
H
Table 29.1 1H NMR chemical shifts relative to TMS. Chemical shifts are typical values that can vary slightly depending on the
solvent, concentration and substituents. (a)OH and NH chemical shifts are very variable (sometimes outside these limits and are
often broad. Signals are not usually seen as split peaks).
This interference is called spinspin coupling. The exact Table29.2 shows the relative intensities and distribution of the
splitting pattern of a peak depends on the number of splitting patterns you are likely to meet.
hydrogen atoms on the adjacent carbon atom or atoms. Figure29.19 shows another high-resolution NMR
spectrum. You should try to interpret it by following
The number of signals a peak splits into equals n + 1 these steps:
where n is the number of 1H atoms on the adjacent Step 1 Use values to identify the environment of the
carbon atom.
equivalent protons (1H atoms) present at each
peak (remembering the peak at zero is the TMS
The high-resolution NMR spectrum of ethanol illustrates this standard reference peak).
n + 1 rule used to interpret splitting patterns (Figure29.18). Step 2 Look at the relative areas under each peak
The CH3 peak is split into three because there are two 1H to determine how many of each type of non-
atoms on the adjacent CH2 group. n + 1 = 3 (as n= 2); this is equivalent protons (1H atoms) are present.
called a triplet.
Step 3 Apply the n + 1 rule to the splitting patterns to see
The CH2 peak is split into four because there are three
1H atoms on the adjacent CH group. n + 1 = 4 (as n = 3);
which protons (1H atoms) are on adjacent carbon
3 atoms in the unknown molecule.
this is called a quartet.
The OH peak is not usually split as its 1H atom is Step 4 Put all this information together to identify the
constantly being exchanged with the 1H atoms of other unknown molecule.
ethanol molecules and any water present. This results in
one average peak being produced.
3H
1H
Absorption of energy
442
2H
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0
/ ppm
Figure 29.18 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.
Number of Using the n + 1 rule, the Relative intensities in the Observed on the NMR spectrum as
1
adjacent H atoms peak will be split into splitting pattern
2H
Absorption of energy
6 5 4 3 2 1 0 1H
Chemical shift, / ppm
2H 2H
Figure 29.19 The high-resolution NMR spectrum of
an unknown ester in a glue. 1H
spectrum. By checking against the peaks in the original NMR The solvent used to prepare samples for 13C NMR
spectrum, without D2O, we can tell if the OH or NH analysis is CDCl3. This accounts for the small signal near
groups are present in the sample. The 1H atom in the OH or 80ppm that can be ignored when interpreting a spectrum,
NH group is referred to as a labile proton. as it is caused by the atoms of 13C in the solvent molecules.
Chapter 29: Analytical chemistry
Figure 29.21 shows the 13C NMR spectrum for propanone, Figure 29.22 shows another example of a carbon-13 NMR
(CH3)2CO. spectrum, that of ethylbenzene, C6H5CH2CH3.
Note that there are only two peaks: one for the carbon The carbon atoms in the benzene ring are almost
atom in the carbonyl group, C O, and the other for the equivalent but will be affected to slightly different extents
carbon atoms in the methyl groups, CH3. Although by the presence of the ethyl group in the molecule. Hence
there are two CH3 groups in propanone, they are both the series of lines clustered near 125ppm.
equivalent and so appear as only a single peak (just as
equivalent H atoms do in proton NMR).
C6H5CH2CH3
H3C
C O
H3C
Figure 29.21 The carbon-13 NMR spectrum of propanone. Figure 29.22 The carbon-13 NMR spectrum of ethylbenzene.
Cambridge International A Level Chemistry
Mass spectrometry
You have already seen how a mass spectrometer
works (see pageX). The mass spectrum of an element
can be used to measure relative isotopic masses and
446 Figure 29.24 The fragmentation of propanone: +CH3 causes
their relative abundances. This information is used to
the peak at 15 and CH3C+O causes the peak at 43.
calculate relative atomic masses. However, the main
use of mass spectrometry is in the identification of
The electron bombardment has caused the C C single
organic compounds. As in other forms of spectroscopy,
bonds in the propanone molecules to break. This has
a substance can be identified by matching its spectrum
resulted in the fragments at m/e 15 and 43 that are
against the spectra of known substances stored in a
observed in Figure29.22. The breaking of single bonds,
database. This technique is known as fingerprinting.
such as C C, C O or C N, is the most common cause
In a mass spectrometer the sample is first vaporised.
of fragmentation.
When vapour from the sample enters the machine it
is bombarded by high-energy electrons. This knocks
electrons from the molecules and breaks covalent bonds, que io
fragmenting the molecule. Figure29.23 shows the mass
spectrum produced by propanone. 9 Look at Figure29.25 on page 447, which shows the
mass spectrum of ethanol, C2H5OH. A structural
100 isomer of ethanol is methoxymethane, an ether with
43
the formula CH3OCH3.
Relative abundance (%)
80
a Predict the mass-to-charge ratio of a fragment
60 that would appear on the mass spectrum of
58 methoxymethane but does not appear on
40 ethanols mass spectrum.
b Give the formula of the ion responsible for the
20 15 peak in your answer to part a.
0
0 20 40 60 80 100
Mass-to-charge ratio (m/e)
80 45 [C2H5]+ [C2H5O]+
40
[C2H3]+ [C2H5OH]+
60 15 20
60 [M + 1]
40
0
20 10 20 30 40 50
Mass-to-charge ratio, m/e
Identify the fragments with mass-to-charge In any organic compound there will be 1.10% carbon-13.
ratios of: We can use this fact to work out the number of carbon
i 15 iii 45 atoms (n) in a molecule. We apply the equation:
ii 43 iv 60.
abundance of [M + 1]+ ion
100 ______________________
n = ___
1.1 abundance of M+ ion
37Cl 25
35 56
79Br 50 0
30 50 70 90 110
81Br 50 Mass-to-charge ratio, m/e
Table 29.6 Naturally occurring isotopes of chlorine Figure 29.26 The mass spectrum of chlorobenzene, showing
and bromine. the [M + 2] peak. (Note that there are also tiny [M + 1] and
[M + 3] peaks corresponding to 13C in the molecule.)
One Cl or Br atom per molecule
The M, [M + 2] and [M + 4] peaks also occur in
Imagine a sample of chloromethane, CH3Cl. We will have
dibromomethane but the relative heights of peaks are
molecules of CH335Cl (75%) and molecules of CH337Cl
easier to work out. Because the ratio 79Br:81Br is 1:1, the
(25%). The molecular ion will be CH335Cl+, and two units
M:[M + 2]:[M + 4] height ratio is 1:2:1.
beyond that on the mass spectrum will be the peak for
CH337Cl+. The peak for CH337Cl+ will be one-third the
448 height of the molecular ion. This is the [M+ 2] peak. que io
In the mass spectrum of bromomethane, CH3Br, we
will have two molecular ion peaks of approximately the 11 a List the ions responsible for the M, [M + 2] and
same height one for CH379Br+ and the other for CH381Br+ [M + 4] peaks in a mass spectrum of dibromomethane.
(the [M + 2] peak). b What would be the mass-to-charge ratio and
You should look out for the relative heights mentioned relative abundances of the major peaks with the
highest charge-to-mass ratios in the mass spectrum
here when interpreting mass spectra.
of chloroethane?
if the [M + 2] peak is one-third the height of the M peak, this c How many peaks would you see beyond the
suggests the presence of one chlorine atom per molecule molecular ion peak in 1,1-dibromoethane? What
if the [M + 2] peak is the same as the height of the would be their mass-to-charge ratios and
M peak, this suggests the presence of one bromine abundances relative to the molecular ion? (Ignore
atom per molecule. peaks due to 13C.)
An example of the [M + 2] peak is shown on the mass
spectrum of chlorobenzene (Figure29.26).
100 200
180
80
Relative abundance (%)
160
60 140
120
40
e
100
m/
80
20
60
0 40
8.8 8.9 9.0 9.1
Time / min
Figure 29.27 The x-axis shows retention time, the y-axis the amounts and the z-axis is the charge/mass ratio of the mass spectra.
These 3-D data show the peaks on a mass spectrum for one component in a gasliquid chromatogram.
Cambridge International A Level Chemistry
100
MH+ que io
13 Look at Figure29.28.
Abundance (%)
Summary
Chromatography separates mixtures of substances Protons in different chemical environments
for identification. In chromatography, the mobile produce signals at different chemical shifts. The
phase moves the components of a mixture through chemical shift provides information about the
450 or over the stationary phase. Separation occurs by protons environment.
the transfer of the components to the stationary Protons on neighbouring carbon atoms cause signals
phase either by: to be split. The splitting pattern establishes which
partition between two liquids (due to the groups of protons are on adjacent carbon atoms. The
different solubility of solutes in the mobile phase n + 1 rule predicts the splitting pattern.
and stationary phase) Protons on OH and NH can be identified
partition between a gas and a liquid by the addition of D2O to the NMR sample, which
adsorption on a solid surface. collapses the peak due to an OH or an NH
The stationary phase may be solid or liquid; the proton.
mobile phase may be liquid or gas. Carbon-13 NMR can also help to determine the
In paper and thin-layer chromatography (TLC) the structure of organic molecules.
components of a mixture are identified by their The mass spectrum of a compound enables
Rf values. the relative molecular mass of the compound
In gasliquid chromatography (GLC) and high- to be determined using the molecular ion peak.
performance liquid chromatography (HPLC), the The molecular ion peak, M, is the peak produced
components of a mixture are identified by their by the loss of one electron from a molecule of
retention times; the amount of each component is the compound.
found by measuring the area of each peak (estimates We can deduce the number of carbon atoms in a
can be made from peak heights). compound using the [M + 1] peak and the presence
The proton NMR spectrum of a compound provides of a single bromine or chlorine atom using the
detailed information about the structure of the [M + 2] peak (and two Cl or Br atoms by the [M + 4]
compound. In particular, the spectrum for the peak as well).
protons, 1H, in a compound can provide a complete We can also use mass spectroscopy to identify
determination of the compounds structure. unknown organic compounds by fingerprinting
Chapter 29: Analytical chemistry
(matching the spectrum to other known spectra). times but can be fingerprinted by their unique
The fragmentation peaks give us clues as to the mass spectra). It is used in airport security checks,
structure of the original molecule. food industries and in forensic, environmental and
Gasliquid chromatography/mass spectrometry medical testing.
(GLCMS) provides a more powerful tool for A combination of techniques (such as infra-red, NMR
identifying the components in a mixture than GLC and mass spectroscopy) must be used to confirm the
alone (compounds can have similar retention structure of newly discovered compounds.
End-of-chapter questions
1 a Identify the fragments that would cause peaks in the mass spectrum of HOCH2COCH3 with the following
m/e values:
i m/e = 15 [1]
ii m/e = 17 [1]
iii m/e = 31 [1]
iv m/e = 43 [1]
v m/e = 57 [1]
vi m/e = 59 [1]
b At what value for m/e would you find the molecular ion peak? [1]
Total = 7
2 The gasliquid chromatogram for a mixture of organic compounds is shown below.
90
pentane
80
70
60 octane
pentan-1-ol
50
40
A
30
20
10 B
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
C
a Give the correct labels for a, B and C. [3]
b What percentage of the mixture is pentan-1-ol? [6]
c Give an explanation for the different retention times. [3]
d i How would the chromatogram change if the liquid in the stationary phase was much more polar? [1]
ii Explain your answer. [2]
e Why is gasliquid chromatography useful in testing for anabolic steroids in the blood of athletes? [2]
f Explain why the use of gasliquid chromatography linked to a mass spectrometer is so useful. [2]
g Why is it difficult to separate dyes using gasliquid chromatography? [2]
Total = 21