BASIC INSTRUMENTAL

ANALYSIS

INTRODUCTION TO
SPECTROSCOPIC METHOD OF
ANALYSIS

CHAPTER 7: Spectrochemical Analysis

7.1 Principle of Spectroscopy
7.2 UV-Vis & Infrared Absorption Spectrocopy
7.3 Nuclear Magnetic Resonance
CHAPTER 8: Chromatographic Analysis
8.1 Chromatography Overview
8.2 Column Chromatography
8.3 Gas Chromatography 8.4 High-Performance Liquid Chromatography

UV/VIS Photometers and Spectrophotometer

Specrometer
Spectroscopic instrument that uses a monochromator or a
polychromator in conjunction with a transducer to
convert radiant intensities into electrical signal
Spectrophotometers
Spectrometers that allows measurement of the ratio of the
radiant powers of to beams, a requirement to measure
absorbance
Photometers
Use a filter for wavelength selection in conjunction with a
suitable radiation transucer

 UV-VISIBLE
SPECTROPHOTOMETER

2.4.1 Molecular UV/Visible Spectrophotometers

Absorption of ultraviolet and
visible radiation by
molecules generally occurs
in one or more electronic
absorption bands, made up
of many discrete lines:
Electronic energy state
Vibrational energy state
Rotational energy state

a) gases
b) non-polar
c) polar

2.5 Infrared Absorption Spectroscopy (FTIR)

2.5.1 Molecular Species that Absorb IR
All molecular species absorb IR radiation, except for a
few homonuclear species molecules such as O2, N2 and
Cl2
Each has a unique IR absorption spectrum
IR spectroscopy is a less satisfactory tool for quantitative
analyses than UV/Vis spectroscopy due to:
Lower sensitivity
Frequent deviations from Beers Law
Less precise

2.5.2 Infrared Spectrum

Why infrared spectra/spectrum exhibit
narrow, closely spaced absorption
bands?
Due to transitions among the various
vibrational quantum levels
Rotational level is often hindered or
prevented, and the effects of small
energy differences are not detected.
Therefore, a typical IR spectrum for a
liquid consists of a series of
vibrational bands

2.5.4 Qualitative Applications

2.5.4 Qualitative Applications

2.5.5 Quantitative Analysis and Applications

2.5.6 Sample Handling Techniques

2.5.6 Sample Handling Techniques

FTIR FIGURES

The Electromagnetic Spectrum

NMR, MRI
EPR/ESR

What is NMR?

NMR is an experiment in which the resonance frequencies of nuclear

magnetic systems are investigated.

NMR always employs some form of magnetic field (usually a strong

externally applied field B0)

NMR is a form of both absorption and emission spectroscopy, in which

resonant radiation is absorbed by an ensemble of nuclei in a sample, a
process causing detectable emissions via a magnetically induced
electromotive force.

A. Abragam, The Principles of Nuclear Magnetism, 1961, Oxford: Clarendon Press.

Things that can be learned from NMR data

Covalent chemical structure (2D structure)

Which atoms/functional groups are present in a molecule

How the atoms are connected (covalently bonded)

3D Structure

Conformation
Stereochemistry

Molecular motion
Chemical dynamics and exchange
Diffusion rate
3D Distribution of NMR spins in a medium an image!

(Better known as MRI)

Plus many more things of interest to chemists

Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
Nuclear magnetic resonance spectroscopy is a powerful
analytical technique used to characterize organic molecules
by identifying carbon-hydrogen frameworks within molecules.
Two common types of NMR spectroscopy are used to
characterize organic structure: 1H NMR is used to determine
the type and number of H atoms in a molecule; 13C NMR is
used to determine the type of carbon atoms in the molecule.
The source of energy in NMR is radio waves which have long
wavelengths, and thus low energy and frequency.
When low-energy radio waves interact with a molecule, they
can change the nuclear spins of some elements, including 1H
and 13C.
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Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
When a charged particle such as a proton spins on its axis, it creates
a magnetic field. Thus, the nucleus can be considered to be a tiny bar
magnet.
Normally, these tiny bar magnets are randomly oriented in space.
However, in the presence of a magnetic field B0, they are oriented with
or against this applied field. More nuclei are oriented with the applied
field because this arrangement is lower in energy.
The energy difference between these two states is very small (<0.1
cal).

18

Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
In a magnetic field, there are now two energy states for a
proton: a lower energy state with the nucleus aligned in the
same direction as B0, and a higher energy state in which the
nucleus aligned against B0.
When an external energy source (h) that matches the
energy difference (E) between these two states is applied,
energy is absorbed, causing the nucleus to spin flip from
one orientation to another.
The energy difference between these two nuclear spin states
corresponds to the low frequency RF region of the
electromagnetic spectrum.
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Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
Thus, two variables characterize NMR: an applied magnetic
field B0, the strength of which is measured in tesla (T), and
the frequency of radiation used for resonance, measured in
hertz (Hz), or megahertz (MHz)(1 MHz = 106 Hz).

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Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
The frequency needed for resonance and the applied
magnetic field strength are proportionally related:

NMR spectrometers are referred to as 300 MHz instruments,

500 MHz instruments, and so forth, depending on the
frequency of the RF radiation used for resonance.
These spectrometers use very powerful magnets to create a
small but measurable energy difference between two
possible spin states.
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Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
Figure 14.1
Schematic of an NMR spectrometer

22

Nuclear Magnetic Resonance Spectroscopy

Introduction to NMR Spectroscopy
Protons in different environments absorb at slightly different
frequencies, so they are distinguishable by NMR.
The frequency at which a particular proton absorbs is
determined by its electronic environment.
The size of the magnetic field generated by the electrons
around a proton determines where it absorbs.
Modern NMR spectrometers use a constant magnetic field
strength B0, and then a narrow range of frequencies is
applied to achieve the resonance of all protons.
Only nuclei that contain odd mass numbers (such as 1H, 13C,
19
F and 31P) or odd atomic numbers (such as 2H and 14N) give
rise to NMR signals.
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CHROMATOGRAPHY SEPARATION

4.1 A General Description of Chromatography

Separation of Methods
Method
Mechanical phase
separation
Precipitation and
filtration
Distillation
Extraction
Ion exchange

Basis of Method
Difference in solubility of compounds
formed
Difference in volatility of compounds
Difference in solubility in two immiscible
liquids
Difference in interaction of reactants
with ion-exchange resin

Chromatography

Difference in rate of movement of a

solute through a stationary phase

Electrophoresis

Difference in migration rate of charged

species in an electric field

Field-flow fractionation

Difference in interaction with a field or

gradient applied perpendicular to
transport direction

4.1 A General Description of Chromatography

Chromatography
A technique in which the components of a mixture are
separated based on differences in the rates at which they
are carried through a fixed or stationary phase by a
gaseous or liquid mobile phase
Stationary phase
A solid or an immobilized liquid on which analyte species are
partitioned during passage of a mobile phase
Mobile phase
A liquid or a gas that carries analytes through a liquid or solid
stationary phase

4.1.1 Classification of chromatography

The stationary phase:
narrow tube
Mobile phase: forced
through tube under
pressure or by gravity

stationary phase:
flat plate or pores
of paper

Chromatography
Methods:

Colum
Chromatography

Mobile phase: by
capillary action or
influence by gravity

Planar
Chromatography

Which one is which?

4.1.1 Classification of chromatography

Classification of Column Chromatography Methods

4.5 Application of Chromatography

Gas Chromatography
High Liquid Performance Chromatography

GAS LIQUID
CHROMATOGRAPHY
Chromatography used for separation and analysis
of volatile compounds

The GC Process
Sample is injected through septum
The sample is vaporized and injected onto the
chromatographic column
The sample is transported through the column
by the flow of gaseous mobile phase

GC PRINCIPLE
uses a gas as the mobile phase and either a liquid or solid as the
stationary phase
The analytes are adsorbed (or dissolved) in the stationary phase
due to an equilibrium based on the vapor pressure and other
additional interactive forces
The mobile phase in GC is referred to as the carrier gas (because
there is little interaction between the analyte and the gas phase)

Gas-solid chromatography (GSC) uses a solid stationary phase

Gas-liquid chromatography (GLC) uses a liquid stationary phase that
is bonded or coated onto a solid support

5.1 Apparatus

5.3 Applications of Gas-Liquid Chromatography

Gas-liquid chromatography is applicable to species
that are appreciably volatile and thermally stable at
temperature up to a few hundred degrees C.
Consequently, GC has been widely applied to the
separation and determination of the components in a
variety of sample types

Applications of GC
Petrochemical (Volatiles, adulterants)
Environmental (Organic pollutants, PCBs)
Forensic (Arson, explosives, poisonous gas)
Pharmaceutical (Solvents, components)
Oleochemicals (Components, volatiles)
Cosmetics (Essential oils, perfume components,
formulations)
Food, polymer, textile, etc

HIGH PERFORMANCE
LIQUID
CHROMATOGRAPHY
Performance means enhanced efficiency and
resolution by the use of small diameter (2-5m)
stationary phase particles
The liquid is pressurized (to several hundred psi) for
efficient flow rates

6.1 Scope of LC
The types of HPLC are often classified by
separation mechanism or by the type of
stationary phase:

Partition / Liquid-liquid chromatography

Adsorption / liquid solid chromatography
Ion exchange
Size-exclusion
Affinity chromatography
Chiral chromatography

HPLC is the most widely used because of

its sensitivity
accurate quantitative determinations
suitability for separating nonvolatile species or
thermally fragile ones
widespread applicability to substances that are
prime interest to industry. (eg. Proteins, drug,
antibiotics, etc.)

Application of Liquid Chromatography

6.2 Apparatus

Schematics of The HPLC

System