Chem2404 Notes Entire Semester

1 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Neutron Neut ron Activat Acti vation ion Analysi Anal ysis s General Principle: Incident neutron hits target nucleus, which creates an intermediate radionuclide, radionuclide, which then decays producing radiation. By using the constituent radiation and decay products we can find out composition of sample. Explained: *An incident neutron hits the target nucleus. - prompt γ  ray emitted * Compound nucleus formed, then intermediate radionuclide formed - β particle emitted - delayed γ  ray emitted *The constituents of the sample are identified by the decay products, i.e. the γ  rays emitted – of the new compound isotope. NAA creates an elemental fingerprint fingerprint of the sample.

Advantages of NAA: Simultaneous detection for most elements Highly sensitive for most elements Little to none sample manipulation manipulation required, sample intact (nondestructive) Highly selective as many parameters can be changed Fast and efficient Disadvantages Disadvantages of NAA: Neutron source required Not all elements can be detected Unknown chemical structure only elemental elemental makeup known (up to 20 at a time). Parameters Parameters of NAA: Irradiation Irradiation time, decay time, counting counting time. Procedure: Sample is typically in mg to g scale. Inside polyethylene or quartz container. Radiated in groups in a reactor. Various parameters can be set such as

- type of radiation, energy, neutron flux – hence all are performed with respect to a standard. - sample is then removed, allowed to cool and spectrum is then recorded. Decay can be detected by ionisation detectors, or scintillation counters. γ  rays interact with matter in various ways – the photoelectric effect and Compton scattering.

The Photoelectron Effect

2 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Incident γ  ray annihilated, and e - is ejected. The highest probability of the electron being ejected is when γ  is low energy (< MeV) and the atom is heavy.

Compton Scattering Only some of the γ  energy is lost with interaction with the electron, the products formed are low energy γ  and e-. Highest probability is when γ  is medium energy and the atom is heavy.

Detector Efficiency Only a fraction of emitted radiation is actually detected. With higher resolution it is possible to discriminate between similar energies of radiation.

Ionisation Detectors *Chamber with electrons and filled with gas at 0.1 – 1 atm *Detects the presence of  α,β radiation and X-rays, not effective with γ 

3 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

*Ions are produced when the gas inside is ionised by the incoming radiation, and these ions move to electrodes, which generates a current. *Voltage at 200-300V so as to collect all ion pairs, but low enough to avoid secondary ionisation *Magnitude of current ≡ energy of radiation Scintillation Detectors *Incident αβγ  radiation causes ionisation and atom excitation. *De-excitation *De-excitation produces emission of light *e.g. band gap of pure NaI is large hence p(electron transition) transition) is low, and any hv is not visible *It is then doped with Ti, providing accessible energy levels levels Requirements: -Material reacts to αβγ  radiation -High light output and linear energy response -Short delay between excitation and emission -Easy machining Suitability: -X-rays, γ  and high energy electrons -γ  rays have good efficiencies efficiencies because, crystals are high density, contain a high charge element and large crystals can be grown -NOT good for fast counting, long recovery time (230ns), hence low resolution See LN for more types of scintillation scintillation detectors – notably Ge, Li, Si.

Activation Processes i-NAA ii-Charged Particle Activation Analysis iii-Instrumental iii-Instrumental Photon Activation Analysis (IPAA)

4 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA NAA – Classification of Neutron Activation Analysis ! Origin of  γ  rays *PGNAA (Prompt Gamma Ray NAA), measures γ  emitted by the intermediate nuclide *NAA, measures the delayed γ  emitted by final nuclide PGNAA - γ  detected as they’re produced. That is the γ  emitted from the intermediate intermediate radionuclide. *This is more difficult than NAA as it must be done on site. Used to distinguish prompt γ  from other (e.g. background) radiation. *Used for elements with high neutron capture -elements that decay rapidly -elements that produce STABLE isotopes -elements with weak γ  decay intensity ! Separation Process *Is there any chemical separation ? *If NO – INAA *If YES -Before irradiation : Chemical Neutron Activation Analysis (CNAA) -After irradiation : Radiochemical Neutron Activation Analysis (RNAA) CNAA – example : Vanadium in Marine Animals *Sample – wet ash/HNO 3 at 65C – cation exchange column – final sample –NAA RNAA – example: see LN Further classification; neutron energies. *Thermal (~0.025 eV) *Epithermal (~0.1 – 1eV), resonance neutrons (1 – 1000ev) *Fast (>0.5 meV) *14 MeV fast (14-MeV INAA, no chemical separation) CPAA - Charged Particle Activation Analysis Analyte activated by charged charged particles, particles, e.g. e.g. protons, protons, deuterons, deuterons, α particles w/ energies energies in MeV Less sources of these particles, hence used less often than NAA. Prompt γ rays detected by PIGE, PIGE, PIPPS, PIPPS, GRALE Particle Induced Gamma Ray Emission Analysis Particle Induced Prompt Photon Spectroscopy Gamma Ray Analysis of Light Elements IPAA - Instrumental Photon Activation Analysis

5 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Interference in Activation Analysis – NAA Effectiveness can be decreased by Interference reactions -Primary, -Primary, Secondary, 2 nd order interference, interference, γ-ray spectral interference. interference. Primary

See LN for other sources of interference, and explanation.

Projectile and Gunshot Residues

6 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Locards Principle: No “clean contact”. There is always exchange of contaminants and materials when two bodies interact. Small Arms Round Schematic

Shotgun Cartridge

NB: On discharge, propellant explodes, the projectile is forced down a barrel. -

Barrel rifled to add spin to bullet If bullet larger than diamter of barrell, then creates, scrateches, etc. Fragments clips off 

Residues Powder from bullet residues from propellant deposited onto shooter Used to analyse whether death was suicide, identify shooter, identify bullet fragments  Typical propellant propellant is black black poweder. 15C: 15C: 75KNO 3: 10S Primer also has composition, explosive (lead styphnate), oxidiser(BaNO 3), fuel (Sb 2S3 )

Projectile and Gunshot Residues Detection: Testing for nitrates. Nitrate chemical assay for most propellants (nitro-cellulose).

7 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Spot test

Bullet Leads: Also have unique and identifyable composition. -Soft; 99% Pb, 1ppm < [Sb] < 1500 ppm -Hard; 95-99% Pb, 0.4% < [Sb] < 4% -Metal Jacket; Cu-Zn Cu-Zn alloy. 90% Cu/10% Zn Zn or 95%Cu/ 5% Zn Spot test for Pb; area swabbed with soln of Sodium Rhodizonate. Acid wash to extract insoluble Pb. If present free particles turn reddish-purple. reddish-purple. Firearms Discharge Residue (DFR) Gunshot Residue (GS) Cartridge Discharge Resuidue (CDR)

Spectroscopy General Principle Principle AAS: Atomic absorption, electrons of the atoms of the sample are promoted to higher energy states, by absorbing a characteristic characteristic λ of energy, energy, i.e. specific λ. Specific to a particular particular electron transition in a particular element. Absorption of sample measured over 100s of λ. Dimensionless Dimensionless value of absorbance at λ corresponds linearly with prevalence of element in sample.

8 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

General Principle Principle AES: Atoms in sample are excited by flame and/or atomization. Each element in sample emits emits excess excess energy at a characteris characteristic tic λ which falls falls in visible visible the spectrum (i.e. 400-800nm). Each element has characteristic emission spectrum which may have many many λ. Intensity of emission corresponds linearly with [element]. [element].

Beer-Lambert Law: Transmittance T = I / I 0 (where 0 < T < 1)

9 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Absorbance A = log 10 (I / I0 ) = -log 10 (T)

Deviations from Beer-Lambert: -Applies well in dilute solutions, derivation may be due to chemical/ instrumental instrumental error or fault. Chemical -Interations between solute and solvent -Refractive -Refractive index of solvent, (i.e. ε changes) -Concentration -Concentration of certain species dependant on chemical equilibrum e.g. acid dissociation -Complex equilibria of metal ions, ligand dependant -The presence presence of a dimer/ dimer/ monomer equilibrium will cause cause ε and absorbption absorbption spectra spectra to change -The presence of intefering species in sample

Instrumental -Less of a problem, generally. -Radiation is not entirely monochromatic - spread of wavelengths is used, okay at ABS max -Can be a problem problem on the side of an absorption – ε changes rapidly with λ, hard to accurately accurately determine [sample] -Stray radiation, mismatched cells, air bubbles in solution (affects path length) – all affect result. -Better to calibrate against standard standard samples samples rather rather than rely rely on ε values. Interpolate, instead of extrapolating. extrapolating.

X-Ray Fluorescence:

-

Sensitive to all most all elements (>13 Al), rapid analysis, several elements can analysed together, equipment relatively cheap and portable, sample can be µg to g range.

10 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -

However, no speciation information, no compounds identified only elements.

-

Electr Electrons ons in in shells shells,, princ principl iple e quantu quantum m number number (1), (1), inco incomin ming g energy energy (α, β, γ) is absorbed and knocks out core (K) shell electron. Ejected electron goes into continuum, atom ionised.

-

-

As electron is lost from core and not valence shell (L, M), resulting ion is an excited state. Excited ion relaxes relaxes to ground state as electrons in valence band band fill hole in core.  The excess energy energy of a L or M shell electron electron is lost lost as x-ray radiation energy. energy.

Auger Effect: -  The emitted x-ray x-ray as a result result of above, above, may not escape escape the atom and and instead an an outer shell electron is ejected. - Auger spectroscopy characterizes elements by energies of ejected electrons -

X-Ray Fluorescence: NB:

11 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

- X-ray fluorescence; resonance transition. Electron on continuum has a wave function defined by De Broglie λ = h/mv - Transition probability maximised when phase and frequency of bound and free electrons are the same.  The intensity of each emission emission is dependent dependent on; Probabilty of atom ionisation Probability that core shell will be filled Probability that flourescence flourescence photon will escape or be absorbed by ion (Auger Effect) -

XRF Sources: -Most common source of incident photons is x-ray tube. -Cathode is heated to emit electrons which are accelerated towards anode -Continuous x-ray spectrum emitted as electron trajectories bent by W (tungsten) nucleus.

X-Ray Fluorescence: Choice of emission

12 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

AAS: Advantages : - High sensitivity, ppm levels of analyte usually, ppb sometimes possible - Rapid analysis - Simple anayses

13 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Disadvantages: -Not as accurate as many wet chemical methds -Instumentation and equipment is expensive -No information given about speciation (i.e. molecular and chemical structure is unknown) Flame Emission AAS – Flame Photometry:

 The sample is is aspirated aspirated into a flame, flame, and them atomised atomised at flame flame temperature, temperature, the atoms then; - Absorb energy from the flame, to reach excited atomic electronic state. state. From this state, they may decay from this excited state which results in a spectrum of lines, (i.e. a characteristic characteristic EMISSION spectrum.) This is then recorded, and is known as flame emission spectrometry. -OR, they may absorb energy from a specific appropriate cathode light source with a specific energy energy and λ, to reach the same excited excited state. The The output radiation radiation is measured measured and the difference (i.e. loss in intensity) is proportional to the amount of sample. This is the Atomic ABSORPTION spectrum. i.e. similar techniques -FE has no light source, atomization / excitation all done by flame. Emission spectrum measured – measurement from excited state atoms decaying to ground -AAS has a light source, atomization by flame, excitation by light, measurement is from ground state to excited state atoms. Difference between the two techniques is the relative population of atoms in ground and excited states. Sensitivity Sensitivity of AAS is prop to number of ground state atoms.

Flame Photometer : Relatively inexpensive inexpensive instrumentation, uses photoelectric detector and filters, NOT a monochromator, and detects only the presence of radiation and not wavelength specific. Used to determine presence of Na, K, Li, Ca, Ba etc etc Filters can be used as λ selectors – -Interference -Interference filters are used if there are a limited number of known λ

14 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -Energy throuput is more important than λ resolution -If cost is more important than flexibility NB : Note that, different fuel mixtures and composition result in different temperatures.

Atomic Absorption Spectroscopy:

-Sample is aspirated into flame, but most atoms are in ground electronic state. Some are thermally excited -Ground state state atoms atoms absorb absorb characteristic characteristic λ energy from from radiation radiation source source or cathode lamp lamp made of element -loss of intensity of incident light is measured. Atomic Absorption Spectroscopy: -Width of absoprtion line very narrow, for high res you need a very sharp line, use hollow cathode lamp

15 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

-Ne or Ar gas ionised by high voltage. Positive Ne+ or Ar+ ions accelerated towards cathode -Metal ions “sputtered” into gas phase in excited electronic states, emit radiation to return to ground state. -Emits MONOCHROMATIC MONOCHROMATIC light, De Beer’s law applies, hence [atomic vapour] ∝ [analyte]

Background correction, there is a chopper which periodically blocks any radiation reaching the sample from the lamp. The difference in the lamp + flame signals and flame only signals allows the flame only signal to be subtracted.

Atomic Absorption Spectroscopy: Other forms of AAS Graphite Furnace: -Sample placed in electrically heated (300K) graphite tube in light path. All of sample introduced to light path -Higher sensitivity, flameless, responsible responsible for decline of NAA

16 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -Comparable sensitivity sensitivity to NAA, lower instrumental and operating costs

Flameless AAS: See lecture notes Analytical Methods: - Construct a standard curve using known concentrations of analyte -OR -Add known quantities of desired element to analyte and measure the corresponding increase in signal, then extrapolate back to concentration of unknown. (This is STANDARD ADDITION) Emission Spectroscopy: Spectroscopy : -Similar to AAS but there is NO light source. Flame promotes atoms into excited state, then decay to ground state produces radiation which recorded. This technique relies on promotion of atoms to excited state. 99% of the atoms are still in ground state, and only 1% are excited, therefore only a small % of analyte gives an emission signal. Normal AAS has superior detection limits due to more % of atoms being excited, at elements that absorb at low λ.

Carbon Monoxide Poisoning: -Between 1979 and 1988 average of 1100 Carbon Monoxide (CO) deaths per year. -Estimated 10,000 people in the US every year seek medical help due to CO exposure -Acute CO exposure accounts for 3500 deaths in the US per year Symptoms : -Blood and tissue becom deep red -Colour of skin being dark and inflamed, pinkish discolouration is a result of  carboxyhemoglobin

17 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Detection : CO released from blood by treatment with 85% H3PO 4 CO catalytically converted to methane CO + 3H 2 -> CH4 + H2O Methane detected by gas chromatography

Cyanide Poisoning : -Long history see Lecture Notes -Qualitative -Qualitative tests for CN (Cyanide)

Cyanide Poisoning : Qualitative Tests

18 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Cyanide Poisoning : Quantitative Quantitative Tests

Electrophoresis : General Principle:

19 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -Charged specieies specieies sperated on the basis of different rates of migration through a conductive medim under the inlfuence of DC electric field -This is due to relative differences in the analytes’ mass to charge ratio -Sample in electrolyte (aqueous buffer) is loaded as narrow band across slab of the stationary phase -Layer of porous material, e.g. paper, gel or agarose / polycrylamid polycrylamid supported on glass plate

-Both the solute (analyte) and buffer migrate in electric field -Electroosmotic flow : all charged species (anions, cations, neutral) molecules swept with buffer -Electrophoretic -Electrophoretic flow: solute also migrates in response to electric field -Cation mobility added to buffer -Anion mobility against that of buffer -Neutral species migrate along with buffer Slab Gel Electrophoresis : -Two ends of stationary phase suspended in reservoirs of buffer (electrolyte) -Each reservoir contains electrode connected to DC power -Positively charged species migrate towards a negative electrode, negative species migrate towards positive electrode -Ions with a high charge to volume ratio (i.e highly charged ions and/or a combination of  small ions) move fastest – this is the basis of seperation - Neutral species migrate slowly, and all analytes are identifed visually, staining or UV fluorescence Resolution : Can be improved by maintaining pH gradient along gel (isoelectric foucssing) foucssing) Migrating proteins reach pH regions equal to isoelectric point, are neutralized hence stop. Seperated proteins become focussed into narrow bands of gel Dis(continous) electrophoresis electrophoresis involves two different buffers at different pH Proteins migrating between pH domains are concentrated in sharp bands

SDS –PAGE Electrophoresis : -Rate of migration dependant on charge/volume ratio -Molecules of similar size but different charge can migrate at different rates -If charge is equalized, migration rate is dependant on molecular size (i.e. mass) -Use polyacrlamide gel (PAGE) and a detergent sodum dodecyl sulfate (SDS) -SDS dissociates proteins into polypeptide units

20 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -Electrostatic -Electrostatic binding of SDS to polypeptides produces aggregates aggregates of similar charge -These migrate depending on their mol wt and then compare migration rates with known mol wts DRAWBACKS : slab gel techniques useful for qualitiative analysis analysis but not so useful for quantitative analysis

Best seperation at high voltages, but high voltages lead to unwanted heating which limits the potentials in gel electrophoresis to 500V. This puts a limit of seperation efficiency. efficiency.

Capillary Electrophoresis : Lower power dissipation, highly efficient cooling means much higher potentials 20,00 – 60,000 V Improvements in sensitivity and resolution and analysis time Sample sizes 0.1 to 10 nL quantitative analysis analysis

-Electroosmotic flow – very small sample cross section, hence capillary walls influence flow -Si-O-H groups on campillary wall ionized by buffer, cations associated with Si-O - groups form immobile layer. -Second layer of loosely held cations, attracted to cathode. They migrate with associated solvating molecules, solution moves towards cathode -At pH less than 2, few Si-OH ionized, electroosmotic flow eliminated -Seperation of cations, anions and neutral solutes on basis of differing electrophoretic electrophoretic mobilities -Soluutes detected, identified and quantified as passed over detector Elution order : 1 Fastest moving moving (small, highly highly charged charged cations), 2 slower slower cations, cations, 3 neutral species, species, 4 slowest anion, 5 fastest anions : Capillary Electrophoresis : Electroosmotic flow has a flat profile across capillary, bands of analyte intrinsically narrower than

21 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

-To analyse anions of interest – reverse flow direction by making capillary wall cationic, then incorporate cationic surfactant eg, cetyltrimethylammonium bromide into buffer -Adsorption of surfactant on capillary surface and hydrophobic interactions between surfactant molecules make wall positively charged, hence flow toward anode

-Electroosmotic flow can be eliminated by derivatising capillar walls, -No electroosmotic flow, so only cations migrate to cathode, neutral species stationary anions go to anode (i.e. source reservoir). Use excess R3SiCl – nHCl Sample introduced by electrokinetic electrokinetic injection: -One end of capillary and corresponding electrode removed from buffer and added to smal chamber with sample. -Apply potential and sample enters capillary by ionic migration and electroosmotic flow -Capillary -Capillary and electrode returned to buffer. -Alternative -Alternative is pressure injection using a pressure differential to drive sample into capillary. capillary. Capillary Electrophoresis :

22 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

-Detection by absorbance, fluorescence, electrochemistry, electrochemistry, mass spec etc -Beer Lambert law applicable, detector designs to maximise path length as [analyte] is low Variations Variations : look this shit up -Capillary zone electrophoresis (CZE) -Capillary Gel Electrophoresis (CGE) -Capillary Electrochromatography Electrochromatography (CEC) -Micellar Electrokinetic Capillary Chromatography (MEKC) X-Ray Diffraction: -One of many diffraction techiques, others being electron and neutron diffraction. -Crystals make up the analyte and repeat themselves Unit cell: Contains the minimum contents, which if repeated through space, are sufficient enough to specify the structure of whole crystal. In a 3D lattice, unit cell is a volume element and is defined as the parallepapied which produces macroscopic crystal when repeated in 3D Atoms of a crystal are repeated regularly in 3D space and form a grid of a plane of atoms. If X-ray energies are chosen chosen such that the λ of the X-rays X-rays = the distance between between atoms atoms in a crystal, then the X-rays visualize visualize a series of planes within the crystal, it acting as a “diffraction grating”.

Bragg Equation:  These are two parallel parallel incident rays, rays, with angle angle θ, in two parallel parallel planes. planes. The two rays rays will be in phase with with the path difference difference of the two rays is some integer multiple of λ. Path length length difference = 2x.

23 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Generation of X-rays X-rays : e- accelerated accelerated b electric field field to hit a metal (Cu, Mo) target. target. e- are slowed by collisions and X-ray white radiation is emmitted. Incident electrons of appropriate energy can knock out inner K-shell electrons. e- from L, M shells fill vacancy in K shell. As this occurs, monochromatic X-ray radiation K α is emitted. Extraneous λ removed by filter (monochromator), and almost monochromatic K a is used in diffraction. Diffraction Experiement Experiement : X-rays directed directed upon ½ gram powdered powdered sample. i) ii)

Elec Electr tron onic ic det detec ecto torr move moves s in arc arc aro aroun und d samp sample le,, meas measur urin ing g inte intens nsit ity y of dif diffr frac acte ted d x-rays as a function of 2 θ Detect Detector or rema remains ins consta constant, nt, and and samp sample le is is rota rotate te – inte intensi nsity ty of xra xrays ys plotte plotted d as as a function of 2θ

24 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA C-14 Dating: Developed by William libby in 1946, Nobel Prize Chemistry 1960. Amount of C14 in atmosphere assumed to be relatively constant. Carbon incorporated into living tissue as equilibrium through photosynthesis. Upon death no new C14 only decays to a declining amount of N14 Formerly, β particles counted in gas proportional proportional counter counter or liquid scintillation counter. Large amounts of sample needed, this is burned to produced CO 2 trapped as CaCO 3 and measured. Accelerator Accelerator mass spec provides accurate counts of C 14 and C12. Minute samples (mg).

Assume that levels of C 14 production have not changed since sample died. Then current rate of C14 decay is 15.3 disintegrations/min/gm disintegrations/min/gm of sample. This is proportional to A0. Let current rate of disintegration of C 14 be R, then

C-14 Dating Example: Charcoal sample had 14C disintegration rate of 9.65/min/g. Disintegration Disintegration in living matter is 15.3. This is assumed to be constant since charcoal sample was formed. Calcualte Calcualte age of  charcoal.

25 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA  Thus, Δt = 8268 ln(15.3/9.65) = 3810 years old. Isotope Ratios :

-Sr and Rb are “locked in” to rock once they are formed. Sr isotope gives age and can be characteristic characteristic of location -Different rocks are formed at different time periods. Rock weathers, becomes soil and Sr, Rb are absorbed by plants and enter food chain. -Sr and Rb isotopes of humans and animals will match their diets. Sr deposited in teeth enamel and bones, ratio of teeth characteristic characteristic location in childhood. -Difference in Sr isotope ratio in teeth and bones indicates migration. See iceman death. Constancy of Atmospheric CO2 : -Libby assumed atmospheric 14C is constant, produced by neutrons from cosmic rays. -Cosmic rays deflected by magnetic fields of sun and earth which are variable. Cosmic flux influenced by periodic 11 year sunspot cycles. -Hence, atmospheric 14C from N(np) varies with time, depending on Earth’s natural CO2 reservoirs. -Redistribution -Redistribution of global CO2 between reservoirs varies with climate change, and natural levels are variable.

Constancy of Atmospheric CO2 :

26 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA -Carbon isotope ratios determined by accurate mass spectroscopy -Original reference for δ13C was Cretaceous marine fossil, -Chosen as it has one of the the highest 13C/12C ratios ratios of all natural sources. sources. Standard assigned a δ13C value of zero almost all naturally-occurring naturally-occurring samples have negative δ 13C values.

Examples of C4 products include sugar cane and maize

Largest stable stable carbon isotope isotope fractionation fractionation involves atmospheric atmospheric CH4, δ13C values of ~ -50 % recorded. recorded. Atmospheric CO2 has has a δ13C values values of ~ -7 %.  The δ13C value used used routinely by plant physiologists physiologists to distinguish distinguish between the the C3 and C4 plant groups for applications applications such as determining drought resistance in plants, monitoring attempts to breed in drought resistance, and in detecting adulteration of foods. C4 plants have have higher δ13C values ranging ranging from –17 % to –9 %; mean of –13 % relative relative to PDB, C3 plants show show δ13C values ranging ranging from –32 % to –20 %; average average value value of –27 %.

Arson -Generally -Generally initiated with an accelerant, accelerant, supplying large amounts of heat within a short time -Often at a specific location as multiple points of origin are suspicious. -Available -Available O2 consumed quickly, leavning excess of fuel. Pyrolysis products may be detected.

27 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Proof: proof of arson is difficult to establish. It must be proven the fire was deliberatley lit with intent to cause damage. Much of the evidence is damaged or destroyed or contaminated by fire. Common Accelerants : Petroleum based products eg gasoline, kerosene. Solid accelerants accelerants include oxidisers eg fertilizer or oil. Early Attempts 1940 – Arson debris in chamber, tube connected to vaccum in between two cold traps 1952 – Distillation with alcohol and water. Compare volume, flammability, boiling range etc with common accelerants. 1957 – Purge and trap. Similar to 1940 method, but extraction with volatile solvent solvent (CCl4). 1977 – Carbon Wire absorption/desorption. absorption/desorption. Volatile components adsorbed onto carbon. Carbon removed heated, destroyed and vapours analysed. Chromatography 1905 – Ramsey seperates mixtures of gases / vapours with solid adsorbents 1906 – Tswett obtains discrete discrete coloured bands bands of plant pigments on column of adsorbent adsorbent ‘chromatography’. 1941 – Partition chromatography chromatography demonstrated. Martin and Synge win Nobel Prize. -Martin and Synge achieved seperation of mixture between two liquid phases : a

stationary phase and mobile phase -One liquid was bound to granulated solid packed in glass tube with second liquid immiscible with first percolated through -Filter papers was stationary phase, other mobile phase was CHCl3 Elution Gas Chromatography Demonstrated : Long column (generaly coiled) packed with an inert solid support. A non-volatile solvent (stationary (stationary phase) coats the support.  The mixture to to be analysed analysed is volatilised volatilised and carried carried through the column by an inert inert carrier carrier gas. The compounds in the analyte are selectively retarded by the stationary phase to an extent determined by the differing affinities of the compounds in the analyte for the stationary phase.  The analyte successively distills from from and redissolves redissolves in the stationary phase phase as it travels travels along the column in the mobile mobile phase, giving a highly highly efficient separation. separation.  The oven temperature temperature can be be varied during during the analysis analysis to allow allow slow moving components components to move along the column.  The components of the the mixture are are analysed analysed by a detector detector

Chiral Stationary Phases Requirements Requirements for chiral stationary phases : -Highly viscous even at elevated temperatures. -Have proper surface tension to wet capillary wall completely. -Must form rapid and reversible diastereomeric associate complexes -H-bonding, dispersion, dipole-dipole and steric interactions must be present -High temperature stability and no racemization racemization at elevated temperatures temperatures  They can either either be : chiral chiral stationary stationary phase which which exists as a viscous liquid. Or a chiral molecule dissolved in a liquid which is stationary.

28 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Cyclodextrins {(C6H10O5)n} : Crystalline oligosaccharides produced by enzymic hydrolysis of starch. Five optically active sites per monomer. Hydrophobic cavity with –CH2OH groups inside one side and –OH groups lining opposite hydrophilic side. Cyclodextrins now generally generally used as derivatised derivatised (acetylated, permethylated, permethylated, etc.) compounds. Derivatised cyclodextrins cyclodextrins often liquids (mixture of compounds, depending on extent and position of derivatisation). The either form a viscous liquid that can be used as the stationary phase or have increased solubility in viscous liquid. Can be chemically anchored to a liquid. Solute classes : I Low polarity : saturated hydrocarbons, aromatic hydrocarbons, hydrocarbons, sulfides, mercaptans II Intermediate : Ethers, ketones, aldehydes, esters, nitriles III Polar : Alcohols, carboxylic acids, phenols, nitro compounds IV Very Polar : Amino alcohols, hydroxy acids, polyphenols, polyhydroxyalcohols. polyhydroxyalcohols. Capillary (Open Tubular) Columns Dramatic improvements in resolution. Lengths of capillary tubing, inside is coated with a thin film of the stationary phase. -Very high specifc gas permeability due to ‘openness’. Long columns, very high number of  theoretical plates per m. Carrier gas flow uniform due to low resistance -Very small amounts of stationary phase required due to capacity of column is reduced and seperation efficiency is improved.

Capillary (Open Tubular) Columns

Wall coated open tubular (WCOT): Liquid phase deposited directly on the glass surface (which may have been pretreated). Porous layer open tubular (PLOT): Wall surface extended during fabrication by macro elongated crystal crystal deposits or addition addition of fumed slica during during column drawing. Support coated open tubular (SCOT) : Column wall coated with mixture of finely divided solid support, e.g., alumina or silica.

29 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Column design : Packed columns are glass or stainless steel coil of 1-5 m total length and 5 mm inner diameter filled with stationary phase Capillary columns columns : thin fused silica capillary are typically typically 10 –100m in length with 250 um inner diameter. Stationary phase coated on inner surface. Dual column assemblies : used for pesticide analyses and provide rapid throughput. Use two different columns permits discrimination discrimination between difficult to separate components -faster than running the same sample twice Injection : sample introduced to column in a single aliquot. Direct (on column, splitless) injector : carrier gas travels between heated block and liner.  Travels alogn alogn outside of liner liner before entering entering liner to mix with analyte analyte gases. gases. Ensures that carrier gas is heated to temperature of analyte before mixing.

Injection: Injection:

30 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA

Oven: Oven: Temperature programming. Can run isothermally (at 25C). Can vary temperature during analysis. Assists in separation and resolution of anayltes. Can be programmed to change temperature in any number of intervals. Detection: Detection: Many types of detection. Flame Ionization (FID), Thermal Condctivity (TCD), Electron Capture (ECD), mass spec.

Flame Ionization Detection: Detection: Eluate mixed with H2 and air burned inside detector. Organic Organic carbon atoms form CH radical. These radicals react with oxygen atoms to form CHO+ ions and e-. CHO+ ions carry current to cathodic collector above flame. CH radicals are not produced by CO or COO. Approximatley in in 100,000 carbon atoms produces an ion. This is proportional to the number of susceptible (i.e. NON CO) carbon

31 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA atoms entereing flame. Differing substacnes such as alkanes or chlorinated alkanes require require calibration. No special requirements on carrier gas. N2 or He acceptable. FID insensitive to inorganics. Thermal Conductivity Detector: Eluent gas passed over tungsten-rhenium tungsten-rhenium filament. Electrical resistance resistance of filament increases increases with filament temp. Carrier fas only flowing over filament, filament temp is constant. Solute in gas stream cools filament and signal recorded Electron Capture Detector: Invented in 1957. Similar level of sensitivity to FID. Limited dynamic range. Useful in analysis of halogenated hydrocarbons. hydrocarbons.

Atomic Emission Detector: Expensive process. Eluent atomised by microwave plasma. Atoms in eluent emit a characteristic light. Emissions of all types of atoms in sample are split by diffraction grating. Emissions of individual atoms measured by photodiode. Computer produces chromatograms of each element. Photoionization Detector: Often used in combination with another detector to detect specific species in a mixture. Can be used with another detector as PID is NON-destructive. UV light ionises the analyte, the ions are collected at electrodes.

PIDs contain a single lamp which is changed to match ionization potential of analyte. Constant light source and pressure – Intensity is proportional to concentration of analyte even though only a fraction is analysed.

Chemiluminescence Detector: This is a MOLECULAR emission spectra. Analyte excited to higher electronic, vibrational vibrational or rotational states. Excitation NOT by lamp or laser but a CHEMICAL reaction with a strong oxidant.

32 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA  Two decay paths paths present : chemiluminescence chemiluminescence where light is produced produced or molecular molecular collisions where not light is produced. To avoid the latter – performed under low pressure ~1 torr. Emission spectrum detected on decay back to ground state. Emitted light detected by photomultiplier tube (PMT). Important that : light is generated, easily detectable at low levels, levels, selective for target analyte. Response is linear. I.e. light intensity is proportional to [analyte]. Flame Photometric Detector:

Mass Selective Detector:Output Detector:Output from column fed into mass spectrometer. spectrometer. Column at 1atm, mass spec at 10^-6 atm. Technically difficult for packed column. Simple for low flow rate of capillary column. Output from capillary column fed directly to MS vacuum chamber. Vacuum increases flow rate through the column. There is some loss of chromatographic efficiency. Compensate Compensate with a longer column. -Analyte ionized by mass spec -Ionized molecule breaks into reproducible fragment ions -Fragment ions filtered according to mass/charge (m/z) ratios. -Ions generate current at detector which is proportional to [analyte]

33 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Presented as : total ion chromatogram (TIC), mass spectrum or selected ion monitoring.

-For each TIC peak plot the abundance of each ion vs m/z -Reproducible -Reproducible breakdown of analyte gives a unique ‘fingerprint’. Structure can be deduced from: -A unique breakdown pattern -Comparsion with a known compound from a library of spectra

Selected Ion Monitoring: Abundance and relative intensities of characteristic characteristic ions are monitored to extreme precision. Ratios and retention times are matched against known compounds. Useful in determining trace amounts of analyte is a complex sample. Extremely sensitive sensitive – detection accurate in 10^-12 gram range

Mass Spectroscopy: Is the seperation of matter on the basis of atomic or molecular mass. 1898: Beam of positive ions deflected by electric and magnetic fields 1897: Electron discovered 1912: Demonstrated of two neon isotopes via magnetic deflection 1919: First mass spec built Ionization techniques: electron impact, chemical ionization, fast atom bombardment (FAB), secondary ion mass spec (SIMS), electrospray electrospray ionization (ESI), matrix assisted laser (MALDI)

34 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Electron Impact: Analyte is volatilised and passed into ionization chamber. Analyte is passed through beam of e- generated from heated filament. Electrons are energetic enough (~70eV) to ionize analyte. M + e-  M+o + 2e- here M+o is the molecular ion.

70eV e- generally provide enough energy s.t. molecular ion undergoes further fragmentation. fragmentation. These fragmented ions may undergo further fragmentation to produce ‘daughter ions’. Fragmentation Fragmentation pattern (m/z value of ions, relative intensities, intensities, isotope patters) create characteristic fingerprint. “Double focussing” may be carried out. This is an electrostatic electrostatic analyser (‘energy monochromator’) which focusses ions and provides higher resolution through more accurate determination determination of m/z values. Very accurate measurement is possible. E.g., CF3+(68.9952) easily distinguished from C4H5O +(69.0263) or C5H9+(69.0705) all m/z 69.

Chemical Ionization: Volatile analyte analyte is admitted to source with large excess of CH4. Electron bombardment as in above. Electron bombardment is relatively inefficient, inefficient, only 1% of molecules are ionized.

35 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Almost all ionization involves excess methane, analyte is not fragmented. fragmented. Small transfer of  energy to M. Very little fragmentation. Soft ionization, limited value in structural determination. Fast Atom Bombardment Mass Spec: Analyte in a viscous solvent introduced to high vacuum ion source in mass spec.  The sample (analyte (analyte + solvent) solvent) is bombarded bombarded with highenergy highenergy (8 – 10keV) 10keV) atoms like Ar, Xe.  The ions generated generated from the impact are analysed by a mass spec. Several types types of ions are are possible : ionization of atomic/molecular atomic/molecular species in sample, ejection of existing ions, atom/ion transfer to analyte. Common solvents are glycerol, 1-thioglycerol, triethanolamine triethanolamine etc. These are chosen as they are viscous, have a low BP, and keeps the analyte in a liquid state in a vacuum. Solvent absorbs most of the energy and minimizes damage to analyte. Minimal fragmentation of  analyte. ‘Soft’. Secondary Ion Mass Spec: Surface analysis, only used for solid samples. Surface of  material bombarded with energetic ions (1-30keV) these are primary ions eg Cs +, O2+, Ar+ etc. Positive, negative charged ions ejected from surface of sample and into mass spec. Analyse single surface layer or conduct ‘depth profiling’.

Depth profile is obtained via count rate of element vs. time. Measure depth of crater to convert element conc as a function of time to element concentration as a function of  distance from surface. Synchotron: Electromagnetic Electromagnetic radiation is generated when charged particles travelling near c, change direction (velocity). Depending on extent of change, synchotron radiation can cover a wide range of λ. Synchotrons produce hv from infrared to visible to UV to X-rays. Synchotron light is emitted in a narrow cone pointed in direction of particle travel. travel. Charged particles (electrons (electrons or positrons) are accelerated accelerated close to c. Particles change direction under influence of magnets. This direction change results in emission of stream of  photons tangent to orbit. Charged particles held in circular orbit by array of magnets constantly emitting photons as they change direction.

36 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Photons are are polychromatic, polychromatic, with with λ ranging from infrared infrared to short x-rays. x-rays. Characteris Characteristics tics of  these photons : high flux and extrememly bright, millions of times brighter than sun, highly polarized and can travel in nano-second pulsed, rays are highly collimated (converge). Electrons constrained constrained in circular path by magnets. Synchotron radiation produced by magnets. Insertion devices such as undulators and wigglers are rows of small magnets with alternating alternating polarity. Bending magnet light is available at all sources. These move eperpendicular perpendicular to travel direction.

Major forensic uses include : non-destructive non-destructive elemental analysis eg toxic heavy metals. High spatial resoultion in imaging, diffraction identification and quantification of crystalline crystalline materials. X-ray fluorescence analysis. Trace element analysis. IR spectroscopy. X-ray diffraction. Reasons for use: Intensity of radiation (many photons in small area). Highly collimated beam, wide energy range.

Fingerprinting: We have many sebaceous fluids, fats and oils on the surface of our skin. E.g amino acids, stearic acids, lactic acids, squalane. Superglue Fuming : Cyanoacrylate monomer contacts fingerprint residue, and the polymer grows only along fingerprint ridge trail. Polymer morphology can influence ease of  visualization. Iodine Fuming: Good for sebaceous deposits on both porous and non-porous materials. Non destructive and can be used with other techniques. Best for fresh prints. Image is not permanent, nonpolar I2 insoluble in water soluble in hydrocarbons (e.g. squalane, oleic acid).

37 of 37 – SEMESTER 1 – CHEM 2403 – A.GUPTA Ninhydrin: Ninhydrin reacts with amino acids from skin to form intense blue/purple colour and fingerprint image. Spraying the sample with ZnCl2 solution produces fluorescent complex. This enables detection of very weak fingerprints. ZnL2 produces more strongly fluorescent complex.

Luminol: It is possible to detect blood through the use of luminol which fluoresces.