Presentation on theme: "JS 113: Organic and Inorganic Analyses"— Presentation transcript:
1 JS 113: Organic and Inorganic Analyses AnnouncementsSchedule and AssignmentsReturn and Review Exam 1Learning Objectives- Organic AnalysesDefine- Elements vs.. CompoundsDifference between solid, liquid or gas and define phaseDistinguish Organic vs.. Inorganic compoundsDistinguish between qualitative and quantitative analysisExplain equilibrium and Henry’s lawDescribe chromatography, gas chromatography (GC) and retention timeDefine Rf and electrophoresisReview spectrophotometryDescribe Mass Spec and GC-MSLearning Objectives- Inorganic AnalysesDescribe the usefulness of trace elements in comparisons of phys. evidenceDistinguish continuous and line emission spectraDescribe the following instruments/techniques and how they are used :Emission spectrographInductively Coupled Plasma Emission SpectrometryAtomic absorption spectrophotometerNeutron activation analysisX-ray DiffractionDefine proton, neutrons and electrons, mass and charge relationship atomic number and atomic mass, orbital energy levels, isotope, radioactivityExplain how atoms absorb a definite amount of energy and release energy in the form of light
2 Announcements and Assignments Read chapters 5 and 6Read Chapters 9 and 10 on Drugs and ToxicologyStudy for the Quiz – Chapters 5, 6, 9 and 10Guest LecturesTom AbercrombieSandra SachsReturn and review exams
3 Elements and Compounds Element- simplest substances known providing building blocks for all matter109 known elements – 89 natural, others createdPeriodic Table- elements listed by name and symbol arranged in rows with similar chemical properties. e.g. carbon (C )Atom- smallest particle of an element that can exist and retain its identityCompound: when 2 or more elements are combined to form a new substance different in physical and chemical properties from its elemental constituents e.g. CO2
5 Physical StatesSolid, liquid, and gas – different forms or states of matterSolid- definite shape and volumeLiquid- definite volume and takes shape of containerGas- neither definite shape nor volumeSubstances can change from one form to anotherFreezing- Water to Ice (0C) or Vaporizing- water to steam (100C)Sublimation - solid gasNo new chemical substance is being formed. Attractive forces changePhases- substances can be distinguished by a visible boundaryFor example- Oil and Vinegar or Sugar in Water
6 Organic vs.. Inorganic substances Organic v. InorganicOrganic: contains carbon ( C ) combined w/: H, O, N, S, P, Cl, BrInorganic substance: all other known (no C )Qualitative vs. Quantitative determinationsQualitativeresults in the identity of the materialRequires determination of numerous propertiesFor example- powder reveals presence of heroin and quinineQuantitativeresult in percentage combination of components of a mixturePrecise measurement of a single property of the materialFor example – powder contains 10% heroin and 90% quinineAnalytical techniques for identification of organic compoundsSpectrophotometry- study of absorption of light by chemical substances usually requires material to be in pure statesChromatography- separating and identifying components of a mixture
7 Chromatography Principles (1) Useful to separate mixtures into componentsWilliam Henry (1803):Henry’s Law - When a volatile chemical compound is dissolved in a liquid and is brought to equilibrium with air, there is a fixed ratio between the concentration of the volatile compound in air and its concentration in the liquid and this ratio remains constant for a given temperatureDistribution or partitioning determined by solubility of the gas in the liquid. The higher the solubility the greater the tendency to remain in the liquid phase
8 Chromatography Principles (2) One phase moves continuously in one directionAir is forced to move continuously over the water and since B (clear) has greater % in moving gas, its molecules will travel over the liquid faster than A (Dark)Race between chemical compounds.Substances are first mixedMaterials with preference for moving phase slowly pull aheadAt end, all substances separated crossing the finish line at different timesGas Chromatography- GC, High Performance Liquid chromatography- HPLC, Thin Layer chromatography- TLC.
9 Gas Chromatography (1)Separates mixtures – stationary liquid and moving gasStationary liquid is in columnsPacked columns contain liquid fixed on particles are 2-6m in length and 3mm diameterCapillary columns composed of glass, m and 0.25 to 0.75mm diameter. Stationary liquid phase is a thin film on column inner wall.Carrier gas (N) flows thru column carrying components of a mixture. Those with a greater affinity for gas are fasterOnce traversing the column, emerge separated into its components
10 Gas Chromatography (2)Sample injected into a heated port with a heated column sample in vapor stateAs components emerge they enter the detectorFlame ionizes substance generating an electric signalRecorded on a strip chart recorder as a function of time = chromatogramRecorder response v timeRetention time-Time required for a component to emergeProvides a useful identifying characteristic of a materialNot considered absolute ID as other materials may have similar RT
11 Gas Chromatography (3)GC is extremely sensitive and quantitative (down to ng – how small is that?)Amount of substance is proportional to the peak area recordedPyrolysis GCImportant extension of GCMany forms of physical evidence, paint, fibers,plastics, can be dissolved in a solvent by heating or pyrolysis to high temps ( C) for injection into the GCPyrolyzers permit the gaseous products to enter the carrier gas stream where they flow thru the GC column and the material produces a pyrogram – fingerprint of the material with many points of comparison
12 High-Performance Liquid Chromatography (HPLC) Moving phase is liquid and stationary phase are coated solid particlesAs liquid carries the sample, different components are slowed to different degrees depending on their interaction with the stationary phaseMajor advantage over GC is it takes place at room temperatureGC- needs to heat material. Any temperature sensitive material may be destroyed. Explosives are generally heat sensitive and therefore are more readily separated by HPLC
13 Thin Layer Chromatography (TLC -1) Moving liquid phase, solid stationary phaseTLC ProcedureSample is dissolved in a solventSpotted onto the lower edge of the plateThe plate is placed into a closed chamber with liquidThe liquid slowly rises up by capillary action. Separation occurs as the components with the greatest affinity for the moving phase migrate fasterVisualized UV fluorescence or developed with a chemical reagent spray color spots
14 TLC -2Q KQuestioned sample (Q) must be developed alongside a standard or known (K) sample. If Q and K travel the same distance up the plate from the origins then they can be tentatively identified as the sameID cannot be considered definitive as other materials may have similar migrationDistance traveled up can be assigned an Rf value = distance traveled by the component divided by the distance traveled by the liquid phase. For example if the moving phase travels 10cm and spot 8cm then Rf = 8cm/10cm = 0.8Rapid and sensitive down to 100ugPrincipal application is detection and identification of components in a complex mixture
15 Pen Ink TLC Hands on exercise Draw a straight line with pencil 1 inch from the bottom of your “plate” = paper towelSpot at least 8 different inks across the plate at ½ inch intervals- Label your plate with team name and pen ink (eg. red expo marker)Pour your solvent in to approximately ¼ inch depthSlowly drop your plate into the solventPermit the front to move up at least 3 inchesRemove the plate and let air dryAnswer the following:1) Are there differences in migration?2) Do you see any evidence of separation of dyes?3) Are there any inks that do not migrate?4) Based on your observations, which inks have the most affinity for the mobile phase? For the stationary phase?
16 ElectrophoresisSeparation of materials according to migration rates on a stationary solid phaseUses electric potential across the stationary mediumMedium may include starch or agarose coated on a glass plate of polymer in a capillarySubstances possessing an electric charge migrate. The speed depends on size and chargePrincipal applications are the separation of mixtures of proteins and DNA
17 Spectrophotometry Review Theory of Light- White light = ROYGBIVLight is a wave - wavelength is inversely proportional to frequency- Visible light is only a small part of the electromagnetic spectrumColor = visual indication of an objects ability to absorb some and reflect visible light componentsDifferent materials have different absorptionsAbsorption of UV, visible and IR are particularly applicable for identification of organic substances. How much? - Beer’s Law- A=kc , A= absorption c=concentration k=proportionality
18 SpectrophotometerInstrument used to measure and record the absorption spectrum of a chemical substanceComponents- 1. Radiation source1- Radiation source (UV, vis, IR)2. Monochromator or frequency selector3. Sample holder4. Detection to convert electromagnetic radiation into an electric signal (digitizer)5. Recorder
19 UV and Visible Spectrophotometry Measures the absorbance of UV and visible light as a function of wavelength or frequencyUV spec of heroin has max absorption at 278nm providing materials probable identityWill not provide definitive result - other material may have a similar UV absorption
20 IR Spectrum IR specs provide far more complex patterns Different materials always have distinctively different IR spectraEach IR spectra is equivalent to a “fingerprint” of that substance and no otherFourier transform infrared spectrophotometer FT-IRConsidered specific in itself for identification
21 Mass Spectrometry (1)GC coupled to a MS overcomes limitation of GC (cannot produce specific identification alone)Material emerging from GC, enters a vacuum where they are bombarded by high energy electrons causing them to lose electrons and acquire a positive charge (ions).These ions are unstable and fragmentFragments pass through an electric field where they are separated according to their masses.No two substances produce the same fragmentation pattern under carefully controlled conditions.Very sensitive – one millionth of a gram
23 Mass Spectrometry (3)Sample first injected into a heated inlet port and carrier gas sweeps it into the GC columnGC separates the mixture into its componentsIon source filiment wire emits electrons striking the sample molecules causing them to fragment according to massDetector counts the fragments passing thru the quadrupole Signal is small and must be amplified.Measures abundance of each fragment displaying the mass spectrum
24 Summary 1Organic substances contain C. Inorganic ones comprise all othersChoice of analytical techniques depends on substance category (organic vs inorganic) and the need for qualitative vs. quantitative determinationsQualitative relates just to the identity of the material whereas quantitative relates to the percent composition of components in a mixtureChromatography, spectrophotometry and mass spec are used by forensic scientists to identify or compare organic materialsChromatography is a means of separating and tentatively identifying the components of a mixture.Spectrophotometry is the study of the absorption of light by chemical substancesMass spectrometry characterizes by observing a substance’s fragmentation patterns after collision with high energy electrons
25 Summary 2GC separates components of a mixture on the basis of their distribution between a moving (carrier) gas and a stationary phase which is a thin film of liquid contained in a column. The record of the separation is a chromatogramA direct connection between GC and MS allows components to flow into the MS (GC-MS). Fragmentation of each component produces a fingerprint pattern of the substance.HPLC separates compounds in a stationary phase and mobile liquid phase with temp sensitive compounds like explosivesTLC uses a solid stationary phase and mobile liquid phaseElectrophoresis uses electric potential to separate proteins and DNA of different size and charge on a gel-coated plate or polymer filled capillaryMost labs use UV and IR spec to characterize chemical compounds. UV spec produces simple vs. IR complex spectra and distinctive spectra providing a fingerprint of the substance
26 Most Abundant Elements 75% of the earth’s crust is compose of 2 elements: Oxygen and Silicon99% made up of only 10 elements with carbon comprising less than 0.1%!Expect non-carbon containing elements to be present in physical evidence- e.g. iron, steel, copper, aluminum- tools, coins, weapons, metal scrapingsExamples include- inorganic chemicals such as pigments in paints and dyes and in explosives or poisons such as mercury, lead or arsenic
27 Identification vs. Comparison Review Identification of inorganic evidence –Examples: Explosive formulation suspected of containing potassium chlorate or a powder suspected to contain arsenicComplete the tests results identical to tests previously recorded for knowns to be a valid conclusion as to the chemical identity of evidenceComparison to ascertain common origin-Example: Brass pipe found on the suspect compared to a broken pipe at a crime sceneCondition of the pipes may not allow fitting of broken edgesPipes are alike because they are brass (alloy of copper and zinc) but hundreds of thousands of brass pipes known to exist.Distinguishing these pipes requires comparison using chemical analyses on trace elements providing meaningful criterion to increase probability the two pipes originated from the same source
28 Dirt is Good! Or Trace with trace elements! Raw materials originate from earth’s crustPurification is not 100% and cannot exclude all minor impuritiesManufactured products and natural materials contain small quantities of elements in trace amounts (< 1%)Trace elements provide additional points of comparisonSee Table 6.2 for Brass exampleSoil, fibers, glass and metallic objects- Kennedy
30 Evidence in the Kennedy Assassination Did Lee Harvey Oswald act alone?Warren Commission concluded he was alone assassinOswald fired 3 shots from behind in the Texas School Book DepositoryPresident hit by 2 bullets, 1 missing the limo1 bullet hit the president in the back, exited his throat and then struck Governor Connelly then exited his chest, struck his right wrist and then lodged in his left thigh. Bullet later found in the governors stretcherSecond bullet in the skull fatally wounded Kennedy
31 Evidence in the Kennedy Assassination In the Texas book repository room, a 6.5mm Mannlicher Carcano military rifle was found with Oswald’s palm print and3 spent 6.5mm Western Cartridge Co. Mannlicher-Carcano (WCC/MC) cartridge casesOswald seen there in the amCritics of the Warren commission citeeyewitness accounts and acoustical data contending someone else fired from a region in front of the limoOne bullet caused both president and Connelly's back wound? If so the bullet would be mutilated and deformed. Instead no deformity some flattening and only 1 % weight loss
32 Evidence in the Kennedy Assassination 1977 US House of Representative Select Committee on Assassinations requested the bullets and bullet fragments recovered from the car and various wound areas be examined for trace element levels.Lead alloys are used in manufacture of bullets. Antimony added to lead as a hardening agent; copper, bismuth and silver commonly found. Antimony and Silver were compared Previous studies showed these have probative value for WCC/MC bullets. Ranges of antimony ppm and AG 5-15 ppm
33 Evidence in the Kennedy Assassination Results indicate Q1 and Q9 (bullet from Connelly's stretcher and Connelly's wrist) were indistinguishableQ2, Q4,5 and Q14, Large fragment from the car, fragments from Kennedy’s brain and small fragments found in the car were also indistinguishable.
34 Evidence in the Kennedy Assassination Conclusions derivedThere is evidence of only two bullets- one of composition of 815 ppm antimony and 9.3 silver, the other of composition 622 ppm antimony and 8.1 ppm silverBoth bullets have a composition highly consistent with WCC/MC bullet lead although other sources cannot entirely be ruled outBullet from Connelly stretcher also damaged Connelly's wrist. Absence of bullet fragments from the back wounds of Kennedy and Connelly prevented any effort at linking these wounds to the stretcher bulletNone of these can totally verify the Warren Commission’s reconstruction but results are consistentAnalysis was conducted by Neutron Activation analysis
35 Emission Spectrum of Elements Elements selectively absorb and emit lightTechniques used to determine elemental composition of materialsEmission spectroscopyInductively Coupled Plasma Emission Spectrometry andAtomic Absorption Spectrophotometry
36 Emission SpectraLight emitted from a bulb or any other light source is passed through a prism, separating it into component colors or frequencies = Emission Spectrum- the resulting display of colorsExample- sunlight passing through a prism yielding rainbow colors. This is called a continuous spectrum as all colors merge or blend into one another to form a continuous band
37 Continuous vs. Line Spectrum Unlike white light from the sun continuous spectrum, other light sources such as sodium, neon or mercury arc lamps when passed through a prism result in several individual colored lines separated by dark spaces. Each line represents a definite wavelength or frequency of light called a line spectrum
38 Continuous vs. Line Spectrum Solid or liquid heated continuous spectrum-not very indicative of compositionVaporized and excited by high temp each element light of select frequencies characteristic of this elementLine spectra produced are in essence a fingerprint of an element and a practical method of identification.
39 Emission Spectrometer Main components:Vaporizes and excites atoms to emit lightSeparate light into component frequenciesRecord resultant spectraSample placed between two carbon electrodes- direct current heat vaporizes and exices sample’s atomsEmitted light collected by a lense focused onto a prism dispersing component frequenciesdirected toward a photographic plate to record
40 Emission spectra of evidence Contains numerous elements hence numerous linesIdentification by comparison to a standard chart showing position of principal spectral lines of all elementsMore commonly in forensic analysis is the simple comparison of two or more specimen line-by-line
41 Inductively Coupled Plasma Emission Spectrometry (ICP) Identifies and measures elements through light energy emitted by excited atomsInductively Coupled Plasma is caused by a chain reaction of colliding electronshigh voltage spark releases electrons from argon gasAcceleration in magnetic field more collisions and more releaseDischarge sustained by RF energyUnlike ES in that instead of electrical arc, uses hot plasma torch 3 concentric quartz tubes through which argon gas flows. Radio frequency coil caries a current wrapped around the tubes. RF current causes the creation of an intense magnetic field.
42 Inductively Coupled Plasma Emission Spectrometry (ICP) is Hot, very hot ICP discharge acts like a very intense continuous flame ,000 oCSample introduced into hot plasma collides with argon electrons charged particles (ions) emit light of characteristic wavelengths corresponding to identity of elementsApplications- mutilated bullets and glass fragments.Bullets not suitable for comparison to test fired bullets.copper, arsenic, silver, antimony, bismuth, cadmium and tinClass characteristic as currently no way (no database) of providing statistical significanceAccepted in NJ Supreme Court – State v Noel
43 Atomic Absorption Spectrophotometry When atoms are vaporized they absorb the same frequencies of light that are emitted when excited.First the sample is partially vaporized (acetylene flame) leaving a substantial number unexcited.Second it is exposed to radiation sourceThis source, the discharge lamp is chosen to emit only frequencies of light putatively present in the emission spectrum of the element in questionFor example if one wanted to determine the presenceof antimony, the discharge lamp would be constructedwith antimony. The sample will absorb light only when it contains antimony
44 Atomic Absorption Spectrophotometry Application is the accurate determination of an element’s concentration in a sampleConcentration of absorbing element will be directly proportional to the quantity of light absorbed.Sensitive to trace levelsLimitation is that only one element at a time can be measuredModification by substituting heated graphic furnace or heated strip of metal (tantalum) for the flame more efficient volatilization resulting in 1 trillionth of a gram sensitivity!How does this work at the atomic level?
45 Fundamentals of the Atom Subatomic particles: proton, electron and neutron- basic structural units of the atom
46 Fundamentals of the Atom Electrons (-) orbiting around a central nucleus analogous to the solar system where the planets revolve around the sunNucleus contains protons (+) and neutrons (neutral)Atoms have no net electrical charge therefore # protons= # electrons
47 Atomic structure of elements Behavior of elements is related to the differences in the atomic structureEach element contains a different number of protons= Atomic numberThe periodic table represent the atomic number = number(s) of protonsElement is a collection of atoms all having the same number of protons.
49 Atomic structure of elements Electrons move around the nucleus confined to a path of flight = electron orbitalEach orbital is associated with a definite amount of energy = energy levelEach element has its own characteristic energy levels located at varying distances from the nucleus- some are full, some empty
50 Excitation at the Atomic Level Atoms in stable states have electrons positioned in their lowest possible orbitalsWhen an atom absorbs energy or light its electrons are pushed into higher energy orbitals = excited stateBecause energy levels have fixed values only definite amounts can be absorbed
51 Excitation at the Atomic Level: Atomic Absorption Spectrophotometry Elements are selective in the frequencies of light they absorbSelectivity is determined by the electron energy levels in each elementAtomic Absorption Spectrophotometer, a photon of light will interact with an electron causing it to jump into a higher orbitalEnergy must correspond to the energy difference between the two orbitalsE=hf E=energy difference, h-frequency of absorbed light and h – Planks constantAny energy value more or less will not affect the transitionLike playing pool – too little force you wont make it in, too much it might bounce out!
52 Emission at the Atomic Level: Energy is a two way street Electrons will not remain in high energy state and quickly fall back to its original energy levelAs it falls back it releases energyEmission spectrum - energy loss comes about in the form of light emissionEach element has its own unique set of energy levels each emits a unique set of frequenciesEmission spectrum is a picture of the energy levels surrounding the nucleus of each elementAtomic Absorption spectroscopy measures the value and amount of light energy going into the atomEmission spectroscopy collects and measures the various light energies given off.Either method- Atom are identified by the existence of characteristic energy levels
53 Neutron Activation Analysis Changing the number of subatomic particles nuclear energyNew tool for identifying and quantitating elementsAtoms of single elements must have protons= electrons. Not so with neutronsTotal number of protons and neutrons = atomic massIsotopes are atoms having the same no. of protons but different numbers of neutrons
54 Neutron Activation Analysis Most elements have many isotopes. Some are stable others are not and decompose with time by radioactive decayRadioactivity is the emission of radiation accompanying decay of unstable nucleiAlpha: helium atoms minus electronsBeta: electronsGamma: high energy form of electromagnetic radiation emitted by a radioactive element
55 Neutron Activation Analysis Neutron Activation Analysis is the technique of bombarding specimen with neutrons and measuring the resultant gamma-ray radioactivity.Scientists create radioactive isotopes by bombarding atoms with neutronsWhen a neutron is captured by the nucleus of an atom a new isotope is formed activated and many decompose by emitting radioactivityTo identify the activated isotope one measures the gamma irradiation. Gamma rays of each element is associated with characteristic energy values. Once identified the amount can be measured by the intensity of the gamma ray radiation
56 Neutron Activation Analysis Advantage – non destructive method for identifying and quantifying trace elementsSensitive to one-billionth of a gram (1ng)Multiplex capable- simultaneously analyze elementsLimitation is costMetals, drugs, paint, soil, gunpowder residue and hairExample from NAA comparison of stolen copper telegraphic wires – 4 wires at scene of theft compared to B seized at a scrap yard and suspected of being stolenA1 and B matched
57 X ray DiffractionES, AA and NAA reveal presence of elements not how combined into compoundsFocusing a beam of X-rays at a crystal and studying how the atoms in the substance interact is called X ray diffraction95% of all inorganic compounds are crystalline in natureLimitation- lack of sensitivity- fails to detect those present at 5% level in mixtures (ES, AA and NAA more sensitive)
58 Summary 1Inorganic substances are present in tools, explosives, poisons and metals as well as in paints and dyes. Trace elements are useful because they provide “invisible’ markers that may be used to establish source of material or for additional points of comparisonEmission spectroscopy, inductively coupled plasma and atomic absorption spectrophotometry are techniques used by forensic scientists to determine elemental composition of materialsIn ES a sample is vaporized and atoms achieve an excited state. Excited atoms emit light separated into its components in a line spectrum. Each element can be identified by its characteristic line frequencies.In ISP, the sample in the form of an aerosol is introduced in a hot plasma creating charged particle that emit light of characteristic wavelengths corresponding to identity of the elements
59 Summary 2In AAS, the sample is partially vaporized (acetylene flame). Second it is exposed to radiation source This source, the discharge lamp is chosen to emit only frequencies of light putatively present in the emission spectrum of the element in question. Finally if the element is present a portion of the light will be absorbed. Many elements can be detected at the level of one-trillionth of a gram.NAA measures the gamma-ray frequencies of specimens that have been bombarded with neutrons. Highly sensitive and non-destructive method for simultaneously identifying and quantifying trace elements. However, it requires a nuclear reactor and is expensive.X-ray diffraction is used to study crystalline materials. As X-rays penetrate crystals a portion of the beam is reflected and the reflected beams from the crystal’s planes combine to form a series of light and dark bands known as a diffraction pattern. Each compound is known to produce its own unique diffraction pattern giving a means for fingerprinting inorganic compounds. This works on organic compounds too (e.g. DNA)