INTRODUCTION TO SPECTROSCOPIC METHOD OF ANALYSISBASIC INSTRUMENTAL ANALYSIS

CHAPTER 7: Spectrochemical Analysis

7.1 Principle of Spectroscopy7.2 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 SpectrophotometerSpecrometer Spectroscopic instrument that uses a monochromator or a polychromator in conjunction with a transducer to convert radiant intensities into electrical signal

SpectrophotometersSpectrometers that allows measurement of the ratio of the radiant powers of to beams, a requirement to measure absorbance

PhotometersUse a filter for wavelength selection in conjunction with a suitable radiation transucer

UV-VISIBLE SPECTROPHOTOMETER

2.4.1 Molecular UV/Visible SpectrophotometersAbsorption of ultraviolet and visible radiation by molecules generally occurs in one or more electronic absorption bands, made up of many discrete lines:

Electronic energy stateVibrational energy stateRotational energy state

a) gasesb) non-polarc) polar

2.5 Infrared Absorption Spectroscopy (FTIR)2.5.1 Molecular Species that Absorb IRAll molecular species absorb IR radiation, except for a few homonuclear species molecules such as O2, N2 and Cl2Each has a unique IR absorption spectrumIR spectroscopy is a less satisfactory tool for quantitative analyses than UV/Vis spectroscopy due to:Lower sensitivityFrequent deviations from Beers LawLess precise

2.5.2 Infrared SpectrumWhy 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 SpectrumNMR, MRIEPR/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 dataCovalent chemical structure (2D structure)Which atoms/functional groups are present in a moleculeHow the atoms are connected (covalently bonded)3D StructureConformationStereochemistryMolecular motion Chemical dynamics and exchangeDiffusion rate3D Distribution of NMR spins in a medium an image!(Better known as MRI)Plus many more things of interest to chemists

*Nuclear Magnetic Resonance SpectroscopyNuclear 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.Introduction to NMR Spectroscopy

*Nuclear Magnetic Resonance SpectroscopyWhen 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 (

*Nuclear Magnetic Resonance SpectroscopyIn 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.Introduction to NMR Spectroscopy

*Nuclear Magnetic Resonance SpectroscopyThus, 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).Introduction to NMR Spectroscopy

*Nuclear Magnetic Resonance SpectroscopyThe 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.Introduction to NMR Spectroscopy

*Nuclear Magnetic Resonance SpectroscopyIntroduction to NMR SpectroscopyFigure 14.1Schematic of an NMR spectrometer

*Nuclear Magnetic Resonance SpectroscopyProtons 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, 19F and 31P) or odd atomic numbers (such as 2H and 14N) give rise to NMR signals.Introduction to NMR Spectroscopy

CHROMATOGRAPHY SEPARATION

4.1 A General Description of ChromatographySeparation of Methods

MethodBasis of MethodMechanical phase separation Precipitation and filtration Distillation Extraction Ion exchangeDifference in solubility of compounds formedDifference in volatility of compoundsDifference in solubility in two immiscible liquidsDifference in interaction of reactants with ion-exchange resinChromatographyDifference in rate of movement of a solute through a stationary phaseElectrophoresisDifference in migration rate of charged species in an electric fieldField-flow fractionationDifference in interaction with a field or gradient applied perpendicular to transport direction

4.1 A General Description of ChromatographyChromatographyA 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 phaseA solid or an immobilized liquid on which analyte species are partitioned during passage of a mobile phase

Mobile phaseA liquid or a gas that carries analytes through a liquid or solid stationary phase

4.1.1 Classification of chromatographyThe stationary phase: narrow tube

Mobile phase: forced through tube under pressure or by gravity stationary phase: flat plate or pores of paper

Mobile phase: by capillary action or influence by gravity

Which one is which?

4.1.1 Classification of chromatographyClassification of Column Chromatography Methods

4.5 Application of ChromatographyGas ChromatographyHigh Liquid Performance Chromatography

GAS LIQUID CHROMATOGRAPHY

Chromatography used for separation and analysisof volatile compounds

The GC ProcessSample 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 PRINCIPLEuses a gas as the mobile phase and either a liquid or solid as the stationary phaseThe analytes are adsorbed (or dissolved) in the stationary phase due to an equilibrium based on the vapor pressure and other additional interactive forcesThe 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 phaseGas-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 ChromatographyGas-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 GCPetrochemical (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 particlesThe liquid is pressurized (to several hundred psi) for efficient flow rates

6.1 Scope of LCThe 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 chromatographyChiral chromatography

HPLC is the most widely used because ofits sensitivityaccurate quantitative determinationssuitability for separating nonvolatile species or thermally fragile oneswidespread 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

*The visible region of the spectrum comprises photon energies of 36 to 72 kcal/mole. The near UV region (to 200 nm) extends this energy range to 143 kcal/mole.