Infrared Spectrometry

Infrared Spectrocopy is mainly used to identify functional

group present in particular molecules or samples

(qualitative).
Each

IR spectrum possessed its own fingerprint region.

For

example, two sample with similar functional group can

only be distinguish using this fingerprint region .
Among

the analytical instrument that utilize such

spectrocopy is Fourier Transform Infra-Red (FTIR).

Basic Principal of FTIR

It is measured based upon interferogram of samples signal which is

obtained from interferometer.

Interferometer is main component of FTIR consist of 2 mirror ( movable

mirror and fixed mirror) in which perpendicular to each other and a beam
splitter.

Beam splitter will split the IR source into two equal beam.

The first beam will shone upon the movable mirror and the rest will go to
fixed mirror.

Both of this beam will be reflected back and converge at the beam splitter
and interference will occur among them and resulting interferogram.

Schematic Diagram of
Interferometer

Interferogram contains all the infrared frequencies.

This interferogram will be shone upon the sample and certain frequency
will be detect by a detector and the computer will interpret based upon
Fourier Transform to produce infra-red spectrum.

Block Diagram of FTIR

Interferometer

IR source

Sample

Interferogram

Detector

IR spectrum

Data
Interpretion

Theory

In electromagnetic radiation, the wavenumber (v = 1/v) of IR is within

12800 cm -1 until 10 cm -1 . In analytical chemistry, the main region used
was 4000 cm -1 until 400 cm -1.

The energy in IR radiation is not enough to give excitation or emission just

like in UV region, however IR radiation may cause vibration and rotation
towards the covalent bonds of molecules.

Each type of bond have different profiles of vibration.

C
C

Symmetric Stretch

Asymmetric Stretch

Bending

Theory Cont..

When a radiated molecule possessed an equal frequency with its vibration

frequency, the molecule will successfully absorbed the energy hence
causing the bonds of the molecule to vibrate and rotate. The frequency at
which the molecule vibrates differs with different types of bonds. From
this each functional group present in the molecule can be indentified,
since different functional group have different types of bonds.

Examples:

Types of bond

Frequency
Range,cm-1

C-H

2850-2970

C-N

1180-1360

C-O

1050-1300

Theory Cont..

However, not every IR radiation is absorbed. Only

molecules that undergo net change dipole moment.

Example
H

A dipole moment
O

No dipole moment

Quantitative Analysis

IR spectroscopy was also bound for quantitative

analysis.

Such technique can be done by the aid of Beers Law

A = log10 (Io/I)

Io = transmittance of light before entering sample

I = transmittance of light after entering sample

Sample Preparation

Usually for IR spectroscopy, solid and liquid sample is analysed.

Solid Sample

KBr Pelleting

Solid sample and KBr(Potassium Bromide) was grinded and the mixture was pressed using
hydraulic pressure to formed a thin layered pellet. KBr was placed on top of flat salts plates

Nujol Mull

Grinded solid sample is added with few drops of Nujol oils to formed mulls. Then, this mull
is placed in between flat salt plates

Usage inorganic solvent such as CH2Cl, CH3CCl, and CCl4 and placed in flat salt plates

Liquid Sample

Simply few drops of sample in between of flat salt plate

Flat salt plates

Flat salt plates

Sample in the
middle

Spectrum Interpretation

Examples of real IR
chromatogram of
HDPE

Instrument

Chromatography

Chromatography is a technique developed to separate

components of a sample by utilizing mobile phase and
stationary phase.

Mobile phase: a phase or medium that moves upon certain

time.

Stationary phase : a phase or medium that stationary upon

certain time.

Principal of Chromatography

Using the principal like dissolve like.

Sample will be eluted together with mobile phase.

If component of sample is more soluble in stationary

phase, it will be delayed or retain, thus leaving the
other component of sample and mobile phase moving
ahead. Hence, the component is separated.

Stationary
phase

Chromatogram
Signal

Time
tRM

tRA

tRB

Chromatogram

Retention Time,tR

It is time required for a component to be detected by detector.

This time also useful for qualitative information.

Dead Time, tRm

It is time for unretained species such as mobile phase.

Selectivity Factor,

It is an indicator to show how well the components are separated

= (tR)B (tR)M
(tR)B (tR)M

Peak under area,A

It is useful for quantitative information

Types of Chromatography

Gas Chromatography

Chromatography

HPLC Chromatography

Gas Chromatography

It is a technique used isolate sample that is volatile and

thermally stable .

Samples are separated by the inert gas (mobile phase)

and columns (stationary phase).

Basic Principles and

Operation

Schematic Diagram of GC:

Recorder

Injection Port

Detector

Pressure Control

Oven
Column
Carrier Gas

Basic Principles and Operation

GC consists of :

Carrier Gas: H2,N2

Separation column

Injection port

Detector

Recorder

Usually the sample is injected to the system through septum in which

located on top of injection port. The temperature at injection port,
must be higher than the boiling point of the sample to make sure it is
volatile and can be carried into the column by carrier gas.

In separation column, component of sample which is more volatile will

be eluted first and followed by less volatile

It is suitable for both qualitative and quantitative analysis.

Temperature Programming

To resolve components completely using a technique known as

temperature programming.

The technique is applied by maintaining a low temperature for a

short period of time, and increasing the temperature to help force
out the longer-sticking compounds.

Types of GC column
Two

types of column

Features

Open Tubular

Packed

Length,m

1.5 - 10

1-6

Diameter,mm

0.25 0.75

2-4

Chemical Inertness

Best

Poor

Sample size,ng

10-1000

10 106

Speed

Fast

Slow

Efficiency, Theoretical 600-4000

plates/m
(more efficient)

500-1000
(less efficient)

Sample Preparation

Usually sample is diluted in volatile sample such as

ethanol and directly injected to GC provided the sample
is clean.

Types samples that is usually anaylsed using GC such as

essential oils, steroids, aromatics and alkaloids.

Types of Detector

Gas Chromatography detectors:

Flame Ionization (FID) for hydrocarbon samples.

Thermal Conductivity for universal detector

Electron Capture(ECD) for halogenated compound

Mass spectrometry suitable for any species

Instrumentation
Detector port

Injection port

Separation
column

GC oven

GC-MS

Gas Chromatography-Mass Spectrometry,

GC-MS is an analytical instrument to
analyze quantitatively.
It is two different analytical instrument
that are combined together.
It is used for identification of thousands
components that are present in natural
and biological systems by means of its
molecular weight.

GC-MS operational principle

The process in the separation column similar with

ordinary GC. The process differs as soon as it enters
mass spectrometry detector.

The compound separated will be exposed to electron

bombardment that cause the compound break into
small fragments.

Each fragments are in ion form with certain mass and

denoted as m/z

Basic Principle of Mass

Spectrometry

Where the
molecules is
defragmented
into smaller
components

GC-MS operational principle

All of the fragments will be recorded in mass spectrum in terms of

%relative abundance.

M+ ion
Parent ion

GC-MS Chromatogram

Instrumentation

High Performance Liquid

Chromatography (HPLC)

It is used to analyse less volatile compound and

thermally unstable.

This technique is very accurate yet sensitive.

The mobile phase is in liquid form and the stationary

phase is short column that is polar and sometimes no
polar.

HPLC Schematic Diagram

Basic Principles of HPLC

The sample usually is diluted into with solvent that is

miscible with mobile phase.

The sample is injected through the injection port

simultaneously with the movement of mobile phase.

The more attracted component of sample towards

stationary phase will be delayed/retained, and the rest
will be eluted.

Basic Principles of HPLC

There are two modes of separation:

Reversed phase: stationary phase- non polar compound

(hydrocarbon)
mobile phase polar solvent such as alcohol

During analysis, the polar component is eluted first since it is

more retain
attracted towards mobile phase as compared to stationary phase.

Normal phase: stationary phase polar compound (silica SiO 2)

mobile phase non polar solvent : isopropanol

The non polar component is eluted first since it is more retain

attracted towards mobile phase as compared to stationary phase.

Elution

During analysis the types of elution can be vary by

means of eluant composition upon time.

Isocratic elution: Eluant composition is pumped constantly

through out the analysis

Gradient elution: Eluant composition and flow rate is

changed within some time through out the analysis.

The significance of this technique to ensure complete

separation of peaks as well as the shortening of
retention time.

Qualitative and
Quantitative measure

Both measurement is similar towards GC process.

The retention time obtained will be usually compared

with standard retention time to obtain qualitative
result.

The peak under area will result the quantitative

measure.

Chromatogram
The HPLC
chromatogram of
patulin

Detectors

UV-visible absorption detector

Fluorescence detector

Mass Spectrometer detector

Gel Permeation
Chromatography

It is suitable for sample that have high molecular-mass

species such as proteins and polymers

It is also known as size exclusion chromatography.

GPC-theory

The stationary phase consists of uniform packing which

sizes around 10m silica or polymer particles.

This packing will have uniform pores to allow the diffusion

of solute and solvent.

Generally, the extraction is depend on the sizes of solute.

Components having smaller diameter than the pores will be

eluted last since it will passed through inside in the column
and entrapped for the greatest time.

As for larger components, it cannot passed through the

packing hence will be eluted directly. This component will
be detected first.

As for gel permeation, hydrophobic packing was used which

means it is especially design for insoluble water molecules
such as polymers

< 10m particles will get trapped and takes time to be eluted

Hydrophobic
packing

> 10m particles will be excluded and will be eluted first

Chromatogram

Instrument

The gel column

Thermal Gravimetric Analysis

Thermal analysis measurement towards the changes of a

particular properties in a material upon control heating
or cooling.

In TGA, mass loss upon heating is measured.

Basic Principal of TGA

The mass of sample is continuously recorded as a

function of time as the temperature increased in
certain range.

In the end, the result obtain is the percentage of

decompose sample and the temperature range of
sample decompose.

Thermogram

% mass

Ti

Tf

Ti = Initial temperature of decomposition

Tf = final temperature of decomposition

Temperature
(K)

Thermogram

Example
for TGA
result

Thermogram

The sample thermogram obtained will be compared

against the standard thermogram for qualitative
analysis.

Temperature profile can be obtained from this analysis

Sample preparation

Types of sample suitable for analysis are plastic, rubber,

and nylon.

Sample is weighed in the range of 0.1mg 10 mg

The sample is purge with either oxygen or nitrogen gas

at 40 50 cm3 min-1.

Temperature range is set between 30oC 900oC.

Heating rate is 5 20oC/min.

TGA instrument

X-Ray Diffraction
Spectrometry

X-ray are short wavelength electromagnetic radiation

however produced high energy electron.

It is used to determine the chemical composition and

crystallography of a particular material without the
destruction of material.

Basic Principle of XRD

High energy beam such as electrons is focused on

sample.

This will cause the transition of electron from high

energy level to low energy level will emit X-ray
radiation.

The X-rays energy is differ based upon the energy

difference gap between high level and low level

Basic Principles of XRD

The transition of electron from high energy level to lower will e

high energy in terms of x-ray radiation

Atoms

After high
energy
bombardment

Schematic Diagram XDS

Electron beam
Computer

Detector

sample

Instrument

Sample preparation

Sample must be grinded and sieves until < 250 mesh.

Sample is placed in container holder and ready for

analysis.