Modern Analytical Techniques

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IB OPTION A SL & HL

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A1 Analytical techniques

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A2 Principles of spectroscopy

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The Electromagnetic Spectrum:

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Absorption spectra have parts ‘missing’. In
the diagram below the troughs indicate
energy absorbed by the molecules

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Observing with a hand held spectroscope gives
you evidence for line emission spectra similar to
the one shown below

Here light is being emitted as the excited electrons drop

down to lower ‘allowed’ energy levels.
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9
 The Infra-red Spectrophotometer

• a beam of infra red radiation is passed through the sample

• a similar beam is passed through the reference cell
• the frequency of radiation is varied
• bonds vibrating with a similar frequency absorb the radiation
• the amount of radiation absorbed by the sample is compared with the reference
• the results are collected, stored and plotted

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BENDING AND STRETCHING IN WATER MOLECULES

SYMMETRIC STRETCHING

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BENDING AND STRETCHING IN WATER MOLECULES

ASYMMETRIC STRETCHING

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BENDING AND STRETCHING IN WATER MOLECULES

BENDING

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 INFRA RED SPECTRA - INTERPRETATION

Vertical axis Absorbance the stronger the absorbance the larger the peak

Horizontal axis Frequency wavenumber (waves per centimetre) / cm-1

Wavelength microns (m); 1 micron = 1000 nanometres
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 FINGERPRINT REGION

• organic molecules have a lot of C-C and C-H bonds within their structure
• spectra obtained will have peaks in the 1400 cm-1 to 800 cm-1 range
• this is referred to as the “fingerprint” region
• the pattern obtained is characteristic of a particular compound the frequency
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of any absorption is also affected by adjoining atoms or groups.
 IR SPECTRUM OF A CARBONYL COMPOUND

• carbonyl compounds show a sharp, strong absorption between 1700 and 1760 cm -1
• this is due to the presence of the C=O bond

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 IR SPECTRUM OF AN ALCOHOL

• alcohols show a broad absorption between 3200 and 3600 cm-1

• this is due to the presence of the O-H bond

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 WHAT IS IT!
One can tell the difference between alcohols, aldehydes and
carboxylic acids by comparison of their spectra.

O-H STRETCH ALCOHOL

C=O STRETCH ALDEHYDE

O-H STRETCH

AND
CARBOXYLIC
ACID
C=O STRETCH
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A4 Mass spectrometry

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An outline of what happens in a
mass spectrometer

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Peaks appear due to characteristic fragments (e.g. 29 due to C 2H5+) and
differences between two peaks also indicates the loss of certain units (e.g.
18 for H2O, 28 for CO and 44 for CO2). Many of the fragments do not
show up themselves on the spectrum because they are not ions.

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FRAGMENTATION PATTERNS OF FUNCTIONAL
GROUPS
Interpretation of thousands of spectra has shown that many
classes of organic compound show characteristic
fragmentation patterns due to their functional groups. It is often
possible to identify the type of compound from its spectrum by
looking at the ...
• position of peaks
• differences between major peaks.

Alkanes The mass spectra of these simple hydrocarbons have

peaks at m/z values corresponding to the ions produced by
breaking C-C bonds.

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2,6-dimethyloctane

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A5 Nuclear magnetic resonance
(NMR) spectroscopy

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INTERPRETATION OF SPECTRA
• NMR spectra provide information about
the structure of organic molecules from the
• number of different signals in the spectrum
• position of the signals (chemical shift)
• splitting pattern of the signals(Higher level)
• intensity of the signals

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 LOW RESOLUTION - HIGH RESOLUTION

• low resolution nmr gives 1 peak for each environmentally different group of protons
• high resolution gives more complex signals - doublets, triplets, quartets, multiplets
• the signal produced indicates the number of protons on adjacent carbon atoms

LOW RESOLUTION SPECTRUM OF 1-BROMOPROPANE

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 How do you identify the different H
environments in a molecule?
• For example CH3.CH2.OH
• There are 3 environments
• Three H’s associated with CH3
• Two H’s associated with CH2
• And 1 H associated with OH

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 INTEGRATION

• the area under a signal is proportional to the number of hydrogen atoms present
• an integration device scans the area under the peaks
• lines on the spectrum show the relative abundance of each hydrogen type

By measuring the distances between the integration lines one can

work out the simple ratio between the various types of hydrogen.

before integration after integration

NOTICE THAT THE O-H SIGNAL IS ONLY A SINGLET

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 INTEGRATION

Measure the
distance between
the top and
bottom lines.

Compare the
heights from
each signal and
make them into a
simple ratio.

HOW TO WORK OUT THE SIMPLE RATIOS

• Measure how much each integration line rises as it goes of a set of signals
• Compare the relative values and work out the simple ratio between them
• In the above spectrum the rises are in the ratio... 1:2:3

IMPORTANT: It doesn’t provide the actual number of H’s in each environment, just the ratio
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 MRI
• In 2003 two scientists, Paul C Lauterbur (1929-
2007) and Peter Mansfield (1933- ) won the
Nobel Prize in Physiology or Medicine for their
work n developing the use of NMR in diagnostic
medicine. Because the protons in water, lipids,
carbohydrates, etc give different signals,
depending upon their environment, an image of
the whole body can be built up by placing the
patient inside the magnet of a large NMR
machine. This is known as magnetic resonance
imaging
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How NMR is used in body scanners

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A6 Atomic absorption (AA)
spectroscopy

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This is what an Atomic Absorption
spectroscopic machine looks like

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This is the basic set up for AAS

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Schematically

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Schematically

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UV-vis Spectroscopy and Beer’s
Law
• UV-vis spectroscopy provides useful qualitative and
quantitative information about a chemical sample. Since
electromagnetic radiation in the ultraviolet and visible
range causes electrons to be promoted between energy
levels, the shape and wavelength of maximum
absorbance of a UV-vis spectrum can help chemists
identify a particular chemical. UV-vis spectra can also
provide quantitative information about a particular
chemical through a relationship called Beer’s Law.
Beer’s Law states that the amount of light absorbed by a
chemical is directly related to the concentration of the
chemical in a solution. See equation 1.
A = ε bc
Equation 1

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 A = ε bc

In this equation, the absorbance (A) of the solution is

directly proportional to the concentration (c) in moles/Liter,
the pathlength (b) in cm-1 and the molar absorptivity (e ) in
Lcm-1mol-1. Under the conditions of this experiment, the
molar absorptivity and the path length are constant. Thus
the absorbance of a solution is directly proportional to the
concentration of solution. This concentration is expressed
in molarity or moles/Liter.

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This equation and relationship between absorbance and concentration
can be used to of several samples of a particular chemical on the x-
axis and the corresponding maximum absorbance on the y-axis, a
Beer’s Law Plot is obtained.

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The Plot can then be used to determine an unknown concentration of
the same chemical. At dilute concentrations, many chemical solutions
obey Beer’s Law, resulting in a straight line plot with the general
equation for a straight line y = mx + b.

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To find the unknown concentration of a solution, use the equation for a straight
line and solve for x.
y = mx + b

Sample calculation: Using the Beer's Law Plot above, determine the
unknown concentration of a solution with an absorbance reading of .600.

In this example, the slope (m) is 6.414 and the intercept ( b) is -7.99 x 10 -3.
Solving for x,

.600 = 6.414 x - 0.00799

x = 0.095 M

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A7 Chromatography

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What is Chromatography?
Chromatography is a technique for
separating mixtures into their components
in order to analyze, identify, purify,
and/or quantify the mixture or
components.

• Analyze
Separate • Identify
• Purify

Mixture Components
• Quantify

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Uses for Chromatography

Chromatography is used by scientists to:

• Analyze – examine a mixture, its components,

and their relations to one another
• Identify – determine the identity of a mixture or
components based on known components
• Purify – separate components in order to isolate
one of interest for further study
• Quantify – determine the amount of the a mixture
and/or the components present in the sample

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Definition of Chromatography
Detailed Definition:
Chromatography is a laboratory technique that
separates components within a mixture by using the
differential affinities of the components for a mobile
medium and for a stationary adsorbing medium through
which they pass.

Terminology:
• Differential – showing a difference, distinctive
• Affinity – natural attraction or force between things
• Mobile Medium – gas or liquid that carries the components
(mobile phase)
• Stationary Medium – the part of the apparatus that does
not move with the sample (stationary phase) 46
Uses for Chromatography
Real-life examples of uses for
chromatography:
• Pharmaceutical Company – determine amount of
each chemical found in new product
• Hospital – detect blood or alcohol levels in a
patient’s blood stream
• Law Enforcement – to compare a sample found at
a crime scene to samples from suspects
• Environmental Agency – determine the level of
pollutants in the water supply
• Manufacturing Plant – to purify a chemical
needed to make a product
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Definition of Chromatography
Simplified Definition:
Chromatography separates the components of
a mixture by their distinctive attraction to the
mobile phase and the stationary phase.

Explanation:
• Compound is placed on stationary phase
• Mobile phase passes through the stationary phase
• Mobile phase solubilizes the components
• Mobile phase carries the individual components a
certain distance through the stationary phase,
depending on their attraction to both of the
phases 48
 Types
Types of
of Chromatography
Chromatography
• Liquid Chromatography – separates liquid samples
with a liquid solvent (mobile phase) and a column
composed of solid beads (stationary phase)

• Gas Chromatography – separates vaporized samples

with a carrier gas (mobile phase) and a column
composed of a liquid or of solid beads (stationary phase)

• Paper Chromatography – separates dried liquid

samples with a liquid solvent (mobile phase) and a paper
strip (stationary phase)

• Thin-Layer Chromatography – separates dried liquid

samples with a liquid solvent (mobile phase) and a glass
plate covered with a thin layer of alumina or silica gel
(stationary phase)
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 Principles of Paper
Chromatography
• Capillary Action – the movement of liquid within the spaces of a
porous material due to the forces of adhesion, cohesion, and
surface tension. The liquid is able to move up the filter paper
because its attraction to itself is stronger than the force of gravity.

• Solubility – the degree to which a material (solute) dissolves into

a solvent. Solutes dissolve into solvents that have similar
properties. (Like dissolves like) This allows different solutes to be
separated by different combinations of solvents.

Separation of components depends on both their solubility in the

mobile phase and their differential affinity to the mobile phase and
the stationary phase.

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Developing the Chromatograms

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 Observing the
Chromatograms

0% 20% 50% 70% 100%

Concentration of Isopropanol 52
 Kinds of Chromatography

1. Paper Chromatography

2. Column Chromatography

3. Thin-layer Chromatography

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 LIQUID COLUMN CHROMATOGRAPHY

A sample mixture is passed through a column

packed with solid particles which may or may not be
coated with another liquid.
With the proper solvents, packing conditions, some
components in the sample will travel the column
more slowly than others resulting in the desired
separation.

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Column Chromatography

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• The blue compound is obviously more polar than the
yellow one - it perhaps even has the ability to hydrogen
bond. You can tell this because the blue compound
doesn't travel through the column very quickly. That
means that it must adsorb more strongly to the silica gel
or alumina than the yellow one. The less polar yellow
one spends more of its time in the solvent and therefore
washes through the column much faster.
• The process of washing a compound through a column
using a solvent is known as elution. The solvent is
sometimes known as the eluent.

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A8HL Visible and ultraviolet (UV-Vis)
spectroscopy

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 COLOURED IONS
A characteristic of transition metals is their ability to form coloured compounds

Theory ions with a d10 (full) or d0 (empty) configuration are colourless

ions with partially filled d-orbitals tend to be coloured
it is caused by the ease of transition of electrons between energy levels
energy is absorbed when an electron is promoted to a higher level
the frequency of light is proportional to the energy difference

ions with d10 (full) Cu+,Ag+ Zn2+

or d0 (empty) Sc3+ configuration are colourless
e.g. titanium(IV) oxide TiO2 is white

colour depends on ... transition element

oxidation state
ligand
coordination number
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 SPLITTING OF 3d ORBITALS

Placing ligands around a central ion causes the energies of the d orbitals to change
Some of the d orbitals gain energy and some lose energy
In an octahedral complex, two (z2 and x2-y2) go higher and three go lower
In a tetrahedral complex, three (xy, xz and yz) go higher and two go lower

OCTAHEDRAL TETRAHEDRAL

3d 3d

Degree of splitting depends on the CENTRAL ION and the LIGAND

The energy difference between the levels affects how much energy is absorbed when
an electron is promoted. The amount of energy governs the colour of light absorbed.
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 COLOURED IONS
The observed colour of a solution depends on the wavelengths absorbed
Copper sulphate solution appears blue because the energy absorbed corresponds to
red and yellow wavelengths. Wavelengths corresponding to blue light aren’t absorbed.

WHITE LIGHT SOLUTION

GOES IN APPEARS BLUE

ENERGY CORRESPONDING TO
THESE COLOURS IS ABSORBED

Absorbed colour nm Observed colour nm

VIOLET 400 GREEN-YELLOW 560
BLUE 450 YELLOW 600
BLUE-GREEN 490 RED 620
YELLOW-GREEN 570 VIOLET 410
YELLOW 580 DARK BLUE 430
ORANGE 600 BLUE 450
RED 650 GREEN 520
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 COLOURED IONS
a solution of copper(II)sulphate is blue because
red and yellow wavelengths are absorbed

blue and green

white light not absorbed

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 COLOURED IONS
a solution of copper(II)sulphate is blue because
red and yellow wavelengths are absorbed

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 Ultraviolet (UV) Spectroscopy – Use and Analysis
Of all the forms of radiation that go to make up the electromagnetic spectrum UV is
probably the most familiar to the general public (after the radiation associated with visible
light which is, for the most part, taken for granted).
UV radiation is widely known as something to be aware of in hot weather in having a
satisfactory effect of tanning the skin but which also has the capacity to damage skin cells
to the extent that skin cancer is a direct consequence of overexposure to UV radiation. This
damage is associated with the high energy of UV radiation which is directly related to its
high frequency and its low wavelength (see the equations below).
This is designed to introduce you to UV spectroscopy and give you enough information to
ensure that you understand how you can use the technique as a quantitative as appose to a
qualitative method..

E = energy; c = speed of light;  = wavelength;

 = frequency; h = Planck’s constant

c =  E = h E = (hc)/  E  1/ 

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 Ultraviolet (UV) Spectroscopy – Use and Analysis
This slide is part automatically animated – if animation does not occur click left hand mouse button.
When continuous wave radiation is passed through a When continuous wave radiation passes through a
prism a diffraction pattern is produced (called a transparent material (solid or liquid) some of the
spectrum) made up of all the wavelengths associated radiation might be absorbed by that material.
with the incident radiation.
Spectrum with ‘gaps’ in it

Spectrum

Transparent material that

Diffraction prism
absorbs some radiation
Radiation source

If, having passed through the material, the beam is diffracted by passing through a prism it will produce a light
spectrum that has gaps in it (caused by the absorption of radiation by the transparent material through which is
passed).
The effect of absorption of radiation on the transparent material is to change is from a low energy state (called the
ground state) to a higher energy state (called the excited state).
The difference between all the spectroscopic techniques is that they use different wavelength radiation that has
different associated energy which can cause different modes of excitation in a molecule.
For instance, with infra red spectroscopy the low energy radiation simply causes bonds to bend and stretch when a
molecule absorbs the radiation. With high energy UV radiation the absorption of energy causes transition of bonding
electrons from a low energy orbital to a higher energy orbital.
The energy of the ‘missing’ parts of the spectrum corresponds exactly to the energy difference between the orbitals
involved in the transition. 65
 Ultraviolet (UV) Spectroscopy – Use and Analysis
The bonding orbitals with which you are familiar are the -bonding orbitals typified
* by simple alkanes. These are low energy (that is, stable).
Unoccupied
Next (in terms of increasing energy) are the -bonding orbitals present in all
Energy Levels
* functional groups that contain double and triple bonds (e.g. carbonyl groups and

Increasing energy
alkenes).
Higher energy still are the non-bonding orbitals present on atoms that have lone
pair(s) of electrons (oxygen, nitrogen, sulfur and halogen containing compounds).
n All of the above 3 kinds of orbitals may be occupied in the ground state.

Two other sort of orbitals, called antibonding orbitals, can only be occupied by an
Occupied electron in an excited state (having absorbed UV for instance). These are the * and

Energy * orbitals (the * denotes antibonding). Although you are not too familiar with the
concept of an antibonding orbital just remember the following – whilst electron
Levels density in a bonding orbital is a stabilising influence it is a destabilising influence
 (bond weakening) in an antibonding orbital.
Antibonding orbitals are unoccupied in the ground state

A transition of an electron from occupied to an unoccupied energy level can be

UV

caused by UV radiation. Not all transitions are allowed but the definition of which
are and which are not are beyond the scope of this tutorial. For the time being be
aware that commonly seen transitions are  to * which correctly implies that UV is
useful with compounds containing double bonds.
A schematic of the transition of an electron from  to * is shown on the left.

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 Ultraviolet (UV) Spectroscopy – The Output
The output from a UV scanning spectrometer is not the most informative looking piece of data!! It looks like a series
of broad humps of varying height. An example is shown below.

*Absorbance has no units

– it is actually the
logarithm of the ratio of

Increasing absorbance *
light intensity incident on
the sample divided by the
Beer Lambert Law light intensity leaving the
sample.

A = .c.l

Decreasing wavelength in nm

There are two particular strengths of UV (i) it is very sensitive (ii) it is very useful in determining the quantity of
a known compound in a solution of unknown concentration. It is not so useful in determining structure although
it has been used in this way in the past.
The concentration of a sample is related to the absorbance according to the Beer Lambert Law which is described
above.
A = absorbance; c = concentration in moles l-1; l = pathlength in cm ;  = molar absorptivity (also known as
extinction coefficient) which has units of moles-1 L cm -1. 67
 Ultraviolet (UV) Spectroscopy – Analysing the Output
Absorbance
Beer Lambert Handling samples of known concentration
1.0
If you know the structure of your compound X and you
Law wish to acquire UV data you would do the following.
A = .c.l Prepare a known concentration solution of your sample.
Run a UV spectrum (typically from 500 down to 220 nm).
0.5 From the spectrum read off the wavelength values for each
of the maxima of the spectra (see left)
Read off the absorbance values of each of the maxima (see
left).
0.0 Then using the known concentration (in moles L -1 ) and the
350 400 450
known pathlength (1 cm) calculate the molar absorptivity ()
wavelength (nm)
for each of the maxima.
Determining concentration of samples with Finally quote the data as follows (for instance for the largest
known molar absorptivity (). peak in the spectrum to the left and assuming a concentration
Having used the calculation in the yellow box to of 0.0001 moles L-1 ).
work out the molar absorptivity of a compound you max = 487nm A= 0.75
can now use UV to determine the concentration of  = 0.75 /(0.001 x 1.0) = 7500 moles-1 L cm -1
compound X in other samples (provided that these
sample only contain pure X).
Simply run the UV of the unknown and take the absorbance reading at the maxima for which you have a known value
of . In the case above this is at the peak with the highest wavelength (see above).
Having found the absorbance value and knowing  and l you can calculate c.
This is the the principle used in many experiments to determine the concentration of a known compound in a
particular test sample – for instance monitoring of drug metabolites in the urine of drug takers; monitoring
biomolecules produced in the body during particular disease states
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Organic molecules that contain a double
bond can absorb UV

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phenolphthalein

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Retinol

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Other conjugated systems that
absorb in the visible UV

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Typical absorbtions

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 A9HL Nuclear magnetic
resonance (NMR) spectroscopy

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 TETRAMETHYLSILANE - TMS
PROVIDES THE REFERENCE SIGNAL

• non-toxic liquid - SAFE TO USE

• inert - DOESN’T REACT WITH COMPOUND BEING ANALYSED
• has a low boiling point - CAN BE DISTILLED OFF AND USED AGAIN
• all the hydrogen atoms are chemically equivalent - PRODUCES A SINGLE PEAK
• twelve hydrogens so it produces an intense peak - DON’T NEED TO USE MUCH
• signal is outside the range shown by most protons - WON’T OBSCURE MAIN SIGNALS
• given the chemical shift of  = 0
• the position of all other signals is measured relative to TMS

The molecule contains four

methyl groups attached to a
silicon atom in a tetrahedral
arrangement. All the hydrogen
atoms are chemically equivalent.

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76
Multiplicity & splitting

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 MULTIPLICITY (Spin-spin splitting)

• low resolution nmr gives 1 peak for each environmentally different group of protons
• high resolution gives more complex signals - doublets, triplets, quartets, multiplets
• the signal produced indicates the number of protons on adjacent carbon atoms

Number of peaks = number of chemically different H’s on adjacent atoms + 1

1 neighbouring H 2 peaks “doublet” 1:1

2 neighbouring H’s 3 peaks “triplet” 1:2:1

3 neighbouring H’s 4 peaks “quartet” 1:3:3:1

4 neighbouring H’s 5 peaks “quintet” 1:4:6:4:1

Signals for the H in an O-H bond are unaffected by hydrogens on adjacent atoms - get a singlet
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 LOW RESOLUTION - HIGH RESOLUTION

• low resolution nmr gives 1 peak for each environmentally different group of protons
• high resolution gives more complex signals - doublets, triplets, quartets, multiplets
• the signal produced indicates the number of protons on adjacent carbon atoms

HIGH RESOLUTION SPECTRUM OF 1-BROMOPROPANE

The broad
peaks are split
into sharper
signals

The splitting pattern depends on the number of hydrogen atoms on adjacent atoms

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 INTEGRATION

Measure the
distance between
the top and
bottom lines.

Compare the
heights from
each signal and
make them into a
simple ratio.

HOW TO WORK OUT THE SIMPLE RATIOS

• Measure how much each integration line rises as it goes of a set of signals
• Compare the relative values and work out the simple ratio between them
• In the above spectrum the rises are in the ratio... 1:2:3

IMPORTANT: It doesn’t provide the actual number of H’s in each environment, just the ratio
80
 NMR SPECTROSCOPY

“CENSUS”
“CENSUS”QUESTIONS
QUESTIONS
- -describe
describewhere
whereeach
eachhydrogen
hydrogenlives
lives 1 2 3
- -say
sayhow
howmany
manyhydrogens
hydrogenslive
liveon
onthat
thatatom
atom
- -say
sayhow
howmany
manychemically
chemicallydifferent
differenthydrogen
hydrogen
atoms live on adjacent atoms
atoms live on adjacent atoms 1-BROMOPROPANE

UNIQUE DESCRIPTION OF THE POSITION H’S ON THE CHEMICALLY DIFFERENT SIGNAL

ATOM
OF THE HYDROGEN ATOMS ATOM H’S ON ADJACENT ATOMS SPLIT INTO

On an end carbon, two away

1 from the carbon with the 3 2 2+1 = 3
bromine atom on it
On a carbon atom second from
2 the end and one away from the 2 3+2 = 5 5+1 = 6
carbon with the bromine atom

On an end carbon atom which

3 also has the bromine atom on it 2 2 2+1 = 3

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CONTENTS
CONTENTS
A10HL Chromatography

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This is what a HPLC machine looks like

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What does a HLPC chromatogram look like?

Response

5 10 15 20 25

Retention Time

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Chromatogram of Orange Juice Compounds

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