Module 02 - Instrumentation - Forensic Analysis PDF

20-09-2024 Module- II Syllabus Chemical microscope, Spectroscopy – basics –...

20-09-2024 Module- II Syllabus Chemical microscope, Spectroscopy – basics – Fourier Transform-Infrared (FT-IR), Ultraviolet-Visible (UV-Vis), Raman, mass spectrometry. Elemental analysis – X-ray fluorescence (XRF), powder X-ray techniques (PXRD). Gas Chromatography (GC), and High Performance Liquid Chromatography (HPLC) – SEM- EDAX. Tutorial: Instrumentation analysis of forensic samples By Dr. Manoj Acharya Copy Right @ VIT Bhopal Light travels in straight line but this concept could not explain Various forms of electromagnetic radiations are Infra-red, Ultra-violet, phenomenon like Interference, Refraction, Diffraction etc. X-rays, Radio waves, Microwaves etc. To explain these phenomenon light is suppose to travel in waves. Characteristics of Electromagnetic radiations Electromagnetic consist of discrete packages of energy which are All the properties of light can be explained by corpuscular theory called as photons. Photons consist of oscillating electrical and and wave theory. magnetic field. Radiant energy is the energy transmitted from one body to Electromagnetic radiations are another in the form of radiations. produced by the oscillation of electric and magnetic field residing on the Radiant energy has wave nature and associated with electric as atom and they are perpendicular to well as magnetic fields, these radiations are called each other. electromagnetic radiation. Electromagnetic radiations are characterised by wavelength or frequency or wave number. Radiant energy is emitted by fluctuation of magnetic and electric field. Dr Manoj Acharya 1  20-09-2024 Frequency (ν): – It is defined as the number of times electrical field When an atom/molecule undergoes transition from lower energy radiation oscillates in one second. The unit for frequency is Hertz (Hz). level to higher energy level with the absorption of photon results in 1 Hz = 1 cycle per second. absorption spectrum. Wavelength (λ): – It is the distance between two nearest parts of the When an atom/molecule falls from excited energy level to ground wave in the same phase i.e. distance between two nearest crest or state with the emission of photon results in emission spectrum. troughs. Spectroscopy is a branch of science which deals with the Energy of electromagnetic radiation study of interaction of matter with light or it deals with is directly proportional to its interaction of electromagnetic radiation with matter with frequency and no medium is required for their propagation. They light emission and absorption spectra. can travel in vacuum. When different types of electromagnetic radiations The relationship between wavelength & frequency can be written as: interact with matter it give different types of ν=c/λ spectroscopies. Like UV-Visible Spectroscopy, IR As photon is subjected to energy, so E=hν=hc/λ Spectroscopy, X-Ray Diffraction, Mass Spectroscopy, Atomic Absorption Spectroscopy, NMR Spectroscopy. Electromagnetic Spectrum Ultraviolet-Visible Spectroscopy or Electronic Spectroscopy Ultraviolet and visible (UV-Vis) absorption spectroscopy is the measurement of the attenuation (reduction in the strength of signal) of a beam of light after it passes through a sample or after reflection from a sample surface. Absorption measurements can be at a single wavelength or over an extended spectral range. Ultraviolet and visible (UV-Vis) spectroscopy is useful to measure the number of conjugated double bonds and aromatic conjugation within various molecules. It also distinguishes between conjugated and non-conjugated system, homoannular and hetroannular conjugated dienes etc. Dr Manoj Acharya 2  20-09-2024 Ultraviolet and visible (UV-Vis) spectroscopy is helpful in UV spectroscopy is not useful below 200nm because in this region  Detection of functional group absorption occurs by Nitrogen & Oxygen. To study below 200nm the  Detection of impurities whole path length is evacuated, hence the region is known as  Qualitative Analysis Vacuum UV region.  Quantitative Analysis  Single compound without chromophore Principle of Ultraviolet-Visible Spectroscopy  Drugs with chromophoric reagent  Detection of conjugation of the compound When an atom/molecule abosrbs UV radiation then the electrons in outermost shell (ground state) are promoted to higher energy state. UV radiation region extends from 150nm to 400nm & visible region In ground state the spin of electrons in molecule is paired (Opposite). extends from 400nm to 800nm. In excited energy state if the spin of electrons is paired then its called UV region can be further subdivided into Near UV region & Far UV excited singlet state. On the other hand if the spin of electrons is region. parallel then its called excited triplet state. The near UV region extends from 200nm to 400 nm and the region Excited triplet state is lower in energy than excited singlet state, so below 200nm is known as Far UV region or Vacuum UV region. its more stable. In UV spectroscopy the absorption of energy produces changes in the Possible Electronic Transitions valence electron of molecule and different types of electronic transitions are possible. S.No. Electronic Transition 1 σ → σ* transition Electronic Transitions 2 π → π* transition 3 n → σ* transition 4 n → π* transition 5 σ → π* transition 6 π → σ* transition σ → σ* transition σ electron from orbital is excited to corresponding anti-bonding orbital σ* and energy required for such type of transition is large. For example- In saturated hydrocarbon (like Methane, Ethane, Propane) σ → σ* transition required higher energy and shows absorbance maxima at 125 nm. Dr Manoj Acharya 3  20-09-2024 π → π* transition n → π* transition π electron in a bonding orbital is excited to corresponding anti- An electron from non-bonding orbital is promoted to anti-bonding π* bonding orbital π*. Compounds containing multiple bonds like orbital. Compounds containing double bond involving hetero atoms alkenes, alkynes, carbonyl, nitriles, cyanide, azo, aromatic compounds (RCHO, RCOR, C=O, C≡N, N=O) undergo such transitions and required etc. undergo π → π* transitions. less energy. For example- Alkenes generally absorb in the region 170 to 205 nm. n → π* transitions require minimum energy and show absorption at longer wavelength around 300 nm. n → σ* transition This type of transition takes place in saturated compounds σ → π* transition & π → σ* transition containing one hetroatom with lone pair of electrons like O, N, S and halogens are capable of n → σ* transition. These transitions usually σ → π* transition & π → σ* transition electronic transitions are requires less energy than σ → σ* transitions. forbidden transitions & are only theoretically possible. Thus, n → π* For example- Saturated Halide, Alcohols, Ether, Aldehyde, Ketone & π → π* electronic transitions show absorption in region above 200 and Amines. The number of organic functional groups with n → σ* nm which is accessible to UV-visible spectrophotometer. peaks in UV region is small (150 – 250 nm). Terms used in UV-Visible Spectroscopy Absorption & Intensity shift Chromophore Bathochromic or Red Shift The part of a molecule responsible for imparting colour, are called as chromospheres or the functional groups containing multiple bonds When absorption maxima (λmax) of a capable of absorbing radiations above 200 nm due to n → π* & π → compound shifts to longer π* transitions. wavelength, it is known as For example- Ethylenes, Acetylenes, NO2, N=O, C=O, C=N, C≡N, C=C, bathochromic shift or red shift. The C=S, etc effect is due to presence of an auxochrome or by the change of Auxochrome solvent. The functional groups attached to a chromophore which modifies the ability of the chromophore to absorb light , altering the wavelength or intensity of absorption. For example- An auxochrome group like –OH, -OCH3 causes The functional group with non-bonding electrons that does not absorption of compound at longer wavelength. absorb radiation in near UV region but when attached to a chromophore alters the wavelength & intensity of absorption. Dr Manoj Acharya 4  20-09-2024 Hypsochromic Shift or Blue Shift When absorption maxima (λmax) of a compound shifts to shorter wavelength, it is known as hypsochromic shift or blue shift. The effect is due to presence of an group causes removal of conjugation or by the change of solvent. Hyperchromic Shift When absorption intensity (ε) of a compound is increased, it is known as hyperchromic shift. The introduction of auxochrome increases the intensity of absorption. Hypochromic Shift When absorption intensity (ε) of a compound is decreased, it is known as hypochromic shift. Application of UV Spectroscopy UV/Vis spectroscopy is useful tool for single component analysis of sample with known or suspected composition such as Qualitative & Quantitative Analysis: pharmaceuticals. – It is used for characterizing aromatic compounds and conjugated olefins. UV spectroscopy identify a class or group of compounds in sample. – It can be used to find out molar concentration of the solute under Many drug groups produce characteristic UV spectra. study. Detection of impurities: – It is one of the important method to detect UV spectroscopy is used to identify coloured inks and fibres. impurities in organic solvents. Detection of isomers are possible. Determination of molecular weight using Beer’s law. UV/Vis spectroscopy is routinely used in analytical chemistry for the quantitative determination of different analytes, such as transition metal ions, conjugated organic compounds, and biological macromolecules. Spectroscopic analysis is commonly carried out in solutions but solids and gases may also be studied. Dr Manoj Acharya 5  20-09-2024 Instrumentation Infrared Spectroscopy The ordinary IR region extends from 2.5 to 15. The region from 0.8 to 2.5  is called Near IR region and from 15  to 200  is called Infrared spectrum gives information about the structure of a Far IR region. compound. The IR spectroscopy is employed to identify the two compounds or to The absorption of IR radiation causes bonds in a molecule to stretch determine the structure of a new compound. This technique is also and bend with respect to one another. useful to predict the presence of certain functional group which absorbs definite frequencies. IR radiation lies between the visible and microwave portion of electromagnetic spectrum. Principle of Infrared Spectroscopy IR spectroscopy is concerned with the study of absorption of infrared Absorption in the Infrared region is due to the changes in the radiations, which causes vibrational transition in the molecule. Hence vibrational and rotational levels. it also known as Vibrational Spectroscopy. When radiations with frequency range less than 100 cm-1 are The IR spectra which is obtained from IR spectroscopy provide raw absorbed then molecular rotation takes place in the substance. When data and its non-conclusive so, mathematical algorithm Fourier more energetic radiations in the region 102 to 104 cm-1 are passed transformation is used which converts raw data into actual spectrum. through the sample then molecular vibrations also takes place. Dr Manoj Acharya 6  20-09-2024 In IR spectroscopy the absorbed radiations bring changes in the Stretching vibrations are of two types: vibrational energy which depends upon:- (a) Symmetric Stretching (b) Asymmetric Stretching Masses of atom present in the molecule Symmetric Stretching: The movement of atom with respect to a particular atom in a molecule is in same direction. Strength of bond Asymmetric Stretching: The movement of atom with respect to a The arrangement of atoms within the molecule particular atom in a molecule is in opposite direction. When IR radiation is passed through the sample then vibrational & Bending rotational energies of the molecule is increased and two types of vibrations are found:- In this type of vibration the position of the atom changes with respect to the original bond axis. Bending vibrations of four type:-  Stretching  Bending Scissoring:- The two atoms approaches each other Stretching Twisting :- One atom moves up and other moves down the plane Stretching: The distance between atoms increases or decreases but with respect to the central atom. the atoms remain in the same bond axis. Rocking :- The movement of two atoms takes place in the same Finger Print Region in IR Spectrum direction The important function of IR spectroscopy is to determine the Wagging :- The two atoms moves up & below the plane with respect structure of compounds. to the central atom. The possibility that two compounds having the same IR spectrum is least so IR spectrum is called “Finger Print” of a molecule. Two identical compounds may have same spectra when run in the same medium under the similar conditions. The region below 1500 cm-1 is rich bending vibrations and stretching vibrations. Bending vibrations are more than stretching vibrations as each compound has its own absorption pattern in the region. This region is called “Finger Print Region”. Dr Manoj Acharya 7  20-09-2024 Instrumentation IR Spectroscopy exploits the property that alkali halides become plastic when subjected to pressure and form a sheet that is transparent in the infrared region. Potassium bromide (KBr) is the commonest alkali halide used in the pellets. Potassium bromide is transparent from the near ultraviolet to long- wave infrared wavelengths (0.25-25 µm) and has no significant optical absorption lines in its high transmission region. In infrared spectroscopy, samples are analysed by grinding with powdered potassium bromide and pressing into a disc. To obtain an IR spectrum of a solid, a sample is combined with Nujol (paraffin based solution/mineral oils) in a mortar and pestle or some other device to make a mull (a very thick suspension), and is usually sandwiched between potassium- or sodium chloride plates before being placed in the spectrometer. The liquid membrane method involves dripping several drops of the Infrared has been used to measure blood alcohol content; to analyse “neat” sample onto an NaCl or KBr aperture plate and sandwiching it drug, fibre and paint samples; and to visualize wounds such as bruises under another aperture plate, such that no gas bubbles are trapped. or bite marks on tissue. The thickness is adjusted according to the sample absorbance by inserting spacers between the aperture plates or by appropriately It also used to detect blood and explosives. Near-IR and Fourier tightening the screws (without breaking the aperture plates). This type transform IR have been tapped for pharmaceutical forensics as well as ink and fibre analysis. of cell is called a "liquid cell." IR spectroscopy can be used to identify the paint left from car in an Application of Infrared Spectroscopy accident. Investigators can find out the composition of paint and IR can be used in many forensic applications, such as the identification figure out which vehicle have that specific paint. of various inks, sweat prints, hair, and other fibres, and toxic industrial materials and chemicals Moisture- and protein-sensitive diffuse reflectance near-infrared spectroscopy is capable to establish the time of death from skeletal remains & it works on the principle that at death, a person’s bones start to lose water, and the proteins begin to decompose Dr Manoj Acharya 8  20-09-2024 Raman Spectroscopy: A Scattering Technique Raman effect Raman spectroscopy was discovered by C. V. Raman in 1928 If any substance gaseous, liquid or even solid is exposed to radiation of definite frequency then light scattered at right angle contains It is a spectroscopic technique used to observe vibration , rotational, frequency different from the incident radiation and it is the and other low-frequency modes in a system and uses laser light source characteristic of the substance under examination. Thus the to irradiate the sample & generates infinitely small amount of Raman phenomenon due to which the scattering light has a slightly different scattered light. frequency from that of incident radiation and there is change in the Raman spectroscopy is commonly used in chemistry to provide a atomic oscillations within the molecule is called Raman effect. fingerprint by which molecules can be identified. Principle of Raman Spectroscopy Raman spectroscopy (RS) is a versatile method for analysis of a wide range of forensic samples. It can be used for both qualitative as well A Raman spectrum is presented as an intensity-versus-wavelength as quantitative purpose. shift. Raman spectra can be recorded over a range of 4000–10 cm−1. Qualitative analysis can be performed by measuring the frequency of In Raman spectroscopy, sample is illuminated with a monochromatic scattered radiations while quantitative analysis can be performed by measuring the intensity of scattered radiations. laser beam which interacts with the molecules of sample and originates a scattered light. Dr Manoj Acharya 9  20-09-2024 Raman spectra arise due to inelastic collision (Reduction in Kinetic Energy) between incident monochromatic radiation and molecules of The scattered light having a frequency different from that of incident sample. When a monochromatic radiation strikes at sample, it scatters light (inelastic scattering) is used to construct a Raman spectrum. in all directions after its interaction with sample molecules. If v1 and v2 are the frequencies of incident and scattered Much of this scattered radiation has a frequency which is equal to radiation/light respectively then the difference v = v1 - v2 is known frequency of incident radiation and constitutes Rayleigh scattering. as Raman frequency. Only a small fraction of scattered radiation has a frequency different from frequency of incident radiation and constitutes Raman scattering. In Raman spectra, raman lines When the frequency of incident radiation is higher than frequency of appears on either side of the line scattered radiation i.e. vi>vs, Stokes lines appear in Raman spectrum. of incident radiation with higher as well as lower frequencies. The Raman line on lower frequency When the frequency of incident radiation is lower than frequency of side are called Strokes lines (i.e. vi scattered radiation i.e. vi vs ) and those on the higher spectrum. Scattered radiation is usually measured at right angle to frequency side are termed as anti incident radiation. strokes lines (i.e. vs < vi ) Stokes shifted Raman bands involve Raman scattering arises from molecular vibration causing a change in the transitions from lower to higher polarizability. This means that intense Raman scattering occurs from energy vibrational levels and symmetric vibrations which induce a large distortion of the electron therefore, Stokes bands are more cloud around the molecule. intense than anti-Stokes bands and hence are measured in conventional A peak appearing in the Raman spectrum will be derived from a Raman spectroscopy. specific molecular vibration or lattice vibration. Peak position shows the specific vibrational mode of each molecular functional group Anti-Stokes bands are measured with fluorescing samples because included in the material. fluorescence causes interference with Stokes bands. The same vibrational modes for each functional group will show a The magnitude of Raman shifts does not depend on wavelength of shift in peak position due to the nearby environment surrounding the incident radiation but Raman scattering depends on intensity of functional group, thus the Raman spectrum shows the "molecular incident radiation. fingerprint" of the target. A change in polarizability (distortion in electron cloud) during molecular vibration is an essential requirement to obtain Raman spectrum of sample. Dr Manoj Acharya 10  20-09-2024 Application of Raman Spectroscopy Fourier transform-Raman and infrared spectroscopy is used to characterize benzodiazepines (A class of psychoactive drugs, used for Raman spectroscopy is useful in the forensic analysis of different the treatment of anxiety and panic, seizures (fits) and insomnia or types of inks in questioned documents. trouble sleeping) (delorazepam, fludiazepam, flurazepam, Characterizing trace amounts of body fluids using Raman tetrazepam). spectroscopy. Raman spectroscopy is used for the rapid identification of drug Infrared and Raman spectroscopy can be used for the identification particles on nail surface and under a coating of nail varnish without of explosives. any interference from nail or nail varnish and clothing. Raman spectrophotometric method is used to analysed eight barbiturates (Sedatives, Sleep inducing drugs) (phenobarbital, pentobarbital, barbital, secobarbital, ambarbital, hexobarbital, butabarbital, mephobarbital) and three sodium salt analogs. Raman spectroscopy is used for the determination of drug content of street sample of cocaine, amphetamine, ecstasy, ketamine (Psychomotor stimulants) and deposited in latent fingerprints. Mass Spectrometry (MS) Mass spectrometry is a powerful analytical technique used to quantify known materials, to identify unknown compounds within a Mass Number sample, and to elucidate the structure and chemical properties of different molecules. The mass number (A) also known as atomic mass number or nucleon number is total number of protons and neutrons in the atomic nucleus. A mass spectrometer generates multiple ions from the sample under Difference between Mass Number & Atomic Weight investigation, it then separates them according to their specific mass- to-charge ratio (m/z), and then records the relative abundance of Mass number is the number of protons and neutrons in an atom, and each ion type. it tells us about the mass of the atom in atomic mass units (amu). Atomic weight is the average mass of all the isotopes of a certain type. It is a weighted average that takes into account the abundances This technique basically studies the effect of ionizing energy on of all of the different isotopes. molecules. It depends upon chemical reactions in the gas phase in which sample molecules are consumed during the formation of ionic and neutral species. Dr Manoj Acharya 11  20-09-2024 Principle A mass spectrometer generates multiple ions from the sample under investigation, it then separates them according to their specific mass- to-charge ratio (m/z), and then records the relative abundance of each ion type. The first step in the mass spectrometric analysis of compounds is the production of gas phase ions of the compound, basically by electron ionization. This molecular ion undergoes fragmentation i.e. A sample, which may be solid, liquid, or gas, is ionized by bombarding it with electrons. This may cause some of the sample's molecules to break into charged fragments. Each primary product ion derived from the molecular ion, in turn, undergoes fragmentation, and so on. These ions are then separated according to their mass-to-charge ratio, typically by accelerating them and subjecting them to an electric or magnetic field: ions of the same mass-to-charge ratio will undergo the same amount of deflection and are detected in proportion to their abundance. Mass spectra is a plot of ion abundance versus mass-to-charge ratio. Ions provide information concerning the nature and the structure of their precursor (ancestor) molecule. In the spectrum of a pure compound, the molecular ion, if present, appears at the highest value of m/z (followed by ions containing heavier isotopes) and gives the molecular mass of the compound. Vaporization-Ionization-Acceleration-Deflection-Detection Dr Manoj Acharya 12  20-09-2024 Application of Mass Spectrometry Arson Investigation: An arson investigator might be able to identify the use of an accelerant through burn patterns or lingering odours, a Isotope dating and tracing: Isotope ratios are used to determine the mass spectrometer can break down any residue and provide an age of materials. For example as in Carbon Dating. accurate report of its molecular makeup. This can help identify any Toxicology Analysis: Mass spectrometry is useful in cases involving unique or exotic compounds that may be present. Discovering a poison/toxins. Tissue or bodily fluids samples are collected and the similar mix used at multiple crime scenes may be useful for presence of toxic substance is determined, and if present, in what identifying the work of a serial arsonist. concentration. Analysis of Explosives: When a bomb detonates, it may not leave Trace Evidence: Mass spectrometry is also useful in analysing trace behind much in the way of physical evidence, only small fragments evidence. Investigators at a crime scene may find microscopic and chemical residues can be retrieved from the crime scene. materials like carpet fiber, glass splinters or paint flakes. Commercial explosive manufacturers each utilize their own unique mix of chemicals and a spectrometer can analyse this residue to A mass spectrometer can determine the precise mix of dyes used in identify the particular makeup of the explosive involved. carpet fibres, the makeup of materials that went into any particular Even in cases where a bomber used a homemade mix, the analysis glass fragment and the precise set of polymers present in any paint may identify the type of materials used and give investigators a push sample. in the right direction to identify the source. X-ray fluorescence XRF (X-ray fluorescence) is a non-destructive analytical technique used to determine the elemental composition of materials. XRF analysers determine the chemistry of a sample by measuring the fluorescent or secondary X-ray emitted from a sample when it is excited by a primary X-ray source. Each of the elements present in a sample produces a set of characteristic fluorescent X-rays ("a fingerprint") that is unique for that specific element, which is why XRF spectroscopy is an excellent technology for qualitative and quantitative analysis of material composition. Principle of X-ray fluorescence (XRF) When an element is placed in a beam of x-rays, the x-rays are absorbed. The absorbing atoms become ionized (e.g. due to the x-ray beam ejects the electron in the inner shell). Dr Manoj Acharya 13  20-09-2024 An electron from higher energy shell (e.g., the L shell) then fall into the position vacated by dislodged inner electron and emit x-rays or characteristic wavelength. This process is called x-ray fluorescence. The wavelength of fluorescence is characteristic of the element being excited, measurement of this wavelength enable us to identify the fluorescing element. The measurement of the fluorescence intensity makes possible the quantitative determination of an element. Most atoms have several electron orbitals (K shell, L shell, M shell). When x-ray energy causes electrons to transfer in and out of these shell levels, XRF peaks with varying intensities are created and will be present in the spectrum. A graphical representation of X-ray intensity peaks as a function of Determination of Chloride, Strontium in blood serum and bone tissue. energy peaks. The peak energy identifies the element, and the peak height/intensity is indicative of its concentration. Elemental analysis of tissues, bone and body fluids. Application of X-ray fluorescence (XRF) Used in the determination of pesticides on fruits and herbal drugs. X-ray fluorescence spectroscopy (XRF) is a method for measuring the Determination of trace elements in plants and foods. thickness of coatings and for analysing materials. Measurement of heavy metals in soil, sediments, water & aerosols. It can be used for the qualitative and quantitative determination of the elemental composition of a material sample as well as for Powder X-ray techniques (PXRD) measuring coatings, coating systems and analysis of lead based paints. Powder diffraction is a scientific technique using X-Ray, neutron, or An X-ray fluorescence (XRF) is used for non-destructive chemical electron diffraction on powder or microcrystalline samples for analyses of rocks, minerals, sediments, ceramics and fluids. structural characterization of materials. X-ray fluorescence (XRF) indicates protein distribution & provides a An instrument dedicated to performing such powder measurements is diagnostic link for the medical practitioner. called a powder diffractometer. X-rays are electromagnetic radiations with wavelength in the range Study of paintings and sculptures etc. 0.1-100 ºA (0.01nm – 10nm) Dr Manoj Acharya 14  20-09-2024 Max von Laue, in 1912, discovered X-ray diffraction i.e. crystalline Crystal monochrometers, is required to produce monochromatic X- substances act as three-dimensional diffraction gratings for X-ray rays needed for diffraction. These X-rays are collimated (made parallel wavelengths / set at particular angle) and directed onto the sample. As the sample and detector are rotated, the intensity of the reflected X-rays is X-ray powder diffraction (XRD) is a rapid analytical technique recorded. primarily used for phase identification of a crystalline material and When the geometry of the incident X-rays impinging (imposing) the can provide information on unit cell dimensions. The analysed sample satisfies the Bragg Equation Bragg’s Law (nλ = 2d sin θ), material is finely ground, homogenized, and average bulk composition constructive interference occurs and a peak in intensity occurs. is determined. The geometry of an X-ray X-rays are generated by a cathode ray tube by heating the filament to diffractometer is such that the produce electrons, accelerating the electron towards the target by sample rotates in the path of the applying the voltage & bombarding the target material with electron. collimated X-ray beam at an angle θ while the X-ray detector is mounted on an arm to collect the When electrons have sufficient energy to dislodge inner shell electrons diffracted X-rays and rotates at an of the target material, characteristic X-ray spectra are produced. angle of 2θ. Where n = an integer (Order of reflection),  = Wavelength of the Application of Powder X-ray Diffraction incident X-ray, d = inter-planar spacing of the crystal,  = Angle of incidence. X-ray powder diffraction is most widely used for the identification of unknown crystalline materials (e.g. minerals, inorganic compounds). Bragg’s law relates the wavelength of electromagnetic radiation to the Determination of unknown solids is critical to studies in geology, diffraction angle and the lattice spacing in a crystalline sample. environmental science, material science, engineering and biology, forensic science. The diffracted X-rays are then detected, processed and counted. By scanning the sample through a range of 2θ angles, all possible Characterization of crystalline materials diffraction directions of the lattice should be attained due to the random orientation of the powdered material. Identification of fine-grained minerals such as clays and mixed layer Conversion of the diffraction peaks to Determination of unit cell dimensions d-spacings allows identification of the Measurement of sample purity mineral because each mineral has a set of unique d-spacings. This is achieved Determining the thickness, roughness and density of the film using by comparison of d-spacings with glancing incidence X-ray reflectivity measurements. standard reference patterns. Dr Manoj Acharya 15  20-09-2024 Make textural measurements, such as the orientation of grains, in a The liquid stationary phase is coated onto an inert powdered or polycrystalline sample. granular solid support which is either spread on a supporting sheet in Chromatography the form of a thin layer or packed into a column. ‘Chromatography’ is an analytical technique for identification, During chromatographic separation the solute molecules are separation and purification of components of a mixture on the basis of continuously moving from mobile phase to stationary phase. The rate difference in their affinity for stationary and mobile phase. of migration depends on the time solute spends in mobile phase. The Chromatography involves two mutually immiscible phase (Stationary & process transferring of solute from mobile phase to stationary phase is Mobile phase) which are brought into contact. A sample to be known as “Sorption”. analysed is introduced into a mobile phase. Partition Ratio is the concentration of solute molecules in stationary The sample is then carried along through a stationary phase packed in phase to the concentration of solute molecules in mobile phase. the form of column or thin layer. Different constituents of the sample undergo repeated partition Retention Factor (Rf)= between mobile phase and stationary phase. This result in gradual Distance travelled by the constituent substance on stationary phase separation of constituents of the sample into bands in the mobile Distance travelled by the solvent on stationary phase phase. Rf value is the good indicator of whether an unknown compound and Gas Chromatography (GC) known compound are similar, if not identical. When any separation process involving a moving gaseous phase for Types of Chromatography separation of compounds from mixture is known as Gas Chromatography. Paper Chromatography Thin Layer Chromatography Principle Liquid Column Chromatography Size Exclusion Chromatography The mixture is introduced into the moving carrier gas (H2, He, N2, Ar, Ion Exchange Chromatography Affinity Chromatography CO) and its allowed to pass through column (Stationary Phase - Liquid High Performance Liquid Chromatography / Solid) so that the component of mixture distributes themselves between two phases. Gas Chromatography Distribution is controlled by three physical transport phenomena:  Flow  Diffusion  Partition of the solute between the mobile phase and stationary phase Dr Manoj Acharya 16  20-09-2024 The process of distribution of the solute between two phases continues till dynamic equilibrium is established. At this stage the concentration of molecules of each solute in the stationary & mobile phase is constant. Partition Ratio = Concentration of solute molecules in stationary phase Concentration of solute molecules in mobile phase Partition Ratio depends upon:  Nature of solute (Volatile / Non Volatile)  Nature of solvent (Polar / Non Polar)  Concentration of Liquid Phase (In case of GLC)  Temperature Application of Gas Chromatography Scanning Electron Microscopy-Energy Dispersive X-ray GC is widely used for analysis of body fluids for the presence of illegal Analysis (SEM-EDAX) substances, to testing of fibre and blood from a crime scene, and to Scanning Electron Microscopy (SEM) is used to determine the detect residue from explosives. morphological aspects of sample (shape, size of particles), with EDAX GC is used to determine which fluids and compounds are present we can determine information on the chemical composition of inside a human body after death. This is vital in determining whether sample. The instrument is the same for both analysis, so the or the person was intoxicated either from alcohol or drug abuse at the information can be complementary. The choice of one or another time of death, or indeed whether there is any poison or other harmful depends on need. substance present in their body. EDAX develops the best solutions for micro- and nano- Gas chromatography can also be used to test samples found at a crime characterization, where elemental and/or structural information is scene, whether these be blood samples or fibre samples from clothing required, making analysis easier and more accurate. or other materials. Gas chromatography−differen al mobility spectrometry (GC−DMS) The scanning electron microscope (SEM) uses a focused beam of high- was investigated as a tool for analysis of ignitable liquids from fire energy electrons to generate a variety of signals at the surface of solid debris. specimens. Dr Manoj Acharya 17  20-09-2024 The signals that derive from electron-sample interactions reveal Principle- (SEM-EDAX) information about the sample including external morphology Accelerated electrons in an SEM carry significant amounts of kinetic (texture), chemical composition, and crystalline structure and energy, and this energy is dissipated as a variety of signals produced orientation of materials making up the sample. by electron-sample interactions when the incident electrons are In most applications, data are collected over a selected area of the decelerated in the solid sample. surface of the sample, and a 2-dimensional image is generated that These signals include secondary electrons (that produce SEM displays spatial variations in these properties. images), backscattered electrons (BSE), diffracted backscattered Areas ranging from approximately 1 cm to 5 microns in width can be electrons (EBSD is used to determine crystal structures and imaged in a scanning mode using conventional SEM techniques orientations of minerals), photons (Characteristic X-ray that are used (magnification ranging from 20X to approximately 30,000X, spatial for elemental analysis and continuum X-rays), visible light resolution of 50 to 100 nm). (Cathodoluminescence –CL) and heat. X-ray generation is produced by inelastic collisions of the incident The SEM is also capable of performing analyses of selected point electrons with electrons in discrete orbitals (shells) of atoms in the locations on the sample; this approach is especially useful in sample. As the excited electrons return to lower energy states, they qualitatively or semi-quantitatively determining chemical yield X-rays that are of a fixed wavelength (i.e. related to the compositions (using EDAX), crystalline structure, and crystal difference in energy levels of electrons in different shells for a given orientations (using Electron backscattered Diffraction “EBSD”). element). Application of SEM-EDAX Typical Applications  Characterization of material structures  Assessment of reaction interfaces, service environment and degradation mechanisms  Characterization of surface defects, stains and residues on metals, glasses, ceramics and polymers  Measurement of the thickness of layered structures, metallised layers, oxide films, composite materials using cross sectional imaging  Particulate and contaminant analysis on and within materials. Typical Industries using SEM / EDX  Aerospace, Automotive  Materials, Minerals  Glass, Ceramics and Refractories  Healthcare, Medical Devices  Semiconductors, Electronics. Dr Manoj Acharya 18