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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).

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  • 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.