-
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 FTIRIR
sourceInterferometerInterferogramSampleDetectorData InterpretionIR
spectrum
-
TheoryIn 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.
CHSymmetric StretchAsymmetric StretchBendingHHHHHCC
-
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 bondFrequency
Range,cm-1C-H2850-2970C-N1180-1360C-O1050-1300
-
Theory Cont..However, not every IR radiation is absorbed. Only
molecules that undergo net change dipole moment.Example
CHOOA dipole momentNo dipole moment
-
Quantitative AnalysisIR 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
sampleI = transmittance of light after entering sample
-
Sample PreparationUsually for IR spectroscopy, solid and liquid
sample is analysed. Solid SampleKBr PelletingSolid 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 MullGrinded 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 platesLiquid SampleSimply few drops of
sample in between of flat salt plate
-
Flat salt platesFlat salt platesSample in the middle
-
Spectrum InterpretationExamples of real IR chromatogram of
HDPE
-
Instrument
-
ChromatographyChromatography 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 ChromatographyUsing 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
-
ChromatogramSignalTimetRAtRBtRMA
-
ChromatogramRetention Time,tRIt is time required for a component
to be detected by detector.This time also useful for qualitative
information.Dead Time, tRmIt 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,AIt is useful for quantitative information
-
Types of ChromatographyChromatographyGas ChromatographyHPLC
Chromatography
-
Gas ChromatographyIt 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 OperationSchematic Diagram of GC:Carrier
GasInjection PortPressure ControlDetectorOvenRecorderColumn
-
Basic Principles and OperationGC consists of :Carrier Gas:
H2,N2Separation columnInjection portDetectorRecorder
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 volatileIt
is suitable for both qualitative and quantitative analysis.
-
Temperature ProgrammingTo 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 columnTwo types of column
FeaturesOpen TubularPackedLength,m1.5 - 101-6Diameter,mm0.25
0.752-4Chemical InertnessBestPoorSample size,ng10-100010
106SpeedFastSlowEfficiency, Theoretical plates/m600-4000(more
efficient)500-1000(less efficient)
-
Sample PreparationUsually 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 DetectorGas Chromatography detectors:Flame Ionization
(FID) for hydrocarbon samples.Thermal Conductivity for universal
detectorElectron Capture(ECD) for halogenated compoundMass
spectrometry suitable for any species
-
InstrumentationGC ovenSeparation columnInjection portDetector
port
-
GC-MSGas 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 principleThe 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 SpectrometryWhere the molecules is
defragmented into smaller components
-
GC-MS operational principleAll of the fragments will be recorded
in mass spectrum in terms of %relative abundance.Parent ionM+
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 HPLCThe 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 HPLCThere are two modes of
separation:Reversed phase: stationary phase- non polar compound
(hydrocarbon) mobile phase polar solvent such as alcoholDuring
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 SiO2)
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.
-
ElutionDuring 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 analysisGradient
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 measureBoth 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.
-
ChromatogramThe HPLC chromatogram of patulin
-
DetectorsUV-visible absorption detectorFluorescence detectorMass
Spectrometer detector
-
Gel Permeation ChromatographyIt is suitable for sample that have
high molecular-mass species such as proteins and polymersIt is also
known as size exclusion chromatography.
-
GPC-theoryThe 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
> 10m particles will be excluded and will be eluted first
Hydrophobic packing
-
Chromatogram
-
InstrumentThe gel column
-
Thermal Gravimetric AnalysisThermal 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 TGAThe 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 Temperature (K)Ti TfTi = Initial temperature of
decompositionTf = final temperature of decomposition
-
ThermogramExamplefor TGA result
-
ThermogramThe sample thermogram obtained will be compared
against the standard thermogram for qualitative
analysis.Temperature profile can be obtained from this analysis
-
Sample preparationTypes 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 SpectrometryX-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 XRDHigh 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 XRDAfter high energy bombardmentThe
transition of electron from high energy level to lower will emit
high energy in terms of x-ray radiationAtoms
-
Schematic Diagram XDSDetectorComputersampleElectron beam
-
Instrument
-
Sample preparationSample must be grinded and sieves until <
250 mesh.Sample is placed in container holder and ready for
analysis.