JS 113: Organic and Inorganic Analyses

1. JS 113: Organic and Inorganic Analyses • Announcements • Schedule and Assignments • Return and Review Exam 1 • Learning Objectives- Organic Analyses • Define- Elements vs.. Compounds • Difference between solid, liquid or gas and define phase • Distinguish Organic vs.. Inorganic compounds • Distinguish between qualitative and quantitative analysis • Explain equilibrium and Henry’s law • Describe chromatography, gas chromatography (GC) and retention time • Define Rf and electrophoresis • Review spectrophotometry • Describe Mass Spec and GC-MS • Learning Objectives- Inorganic Analyses • Describe the usefulness of trace elements in comparisons of phys. evidence • Distinguish continuous and line emission spectra • Describe the following instruments/techniques and how they are used : • Emission spectrograph • Inductively Coupled Plasma Emission Spectrometry • Atomic absorption spectrophotometer • Neutron activation analysis • X-ray Diffraction • Define proton, neutrons and electrons, mass and charge relationship atomic number and atomic mass, orbital energy levels, isotope, radioactivity • Explain how atoms absorb a definite amount of energy and release energy in the form of light

2. Announcements and Assignments • Assignments: • Read chapters 5 and 6 • Read Chapters 9 and 10 on Drugs and Toxicology • Study for the Quiz – Chapters 5, 6, 9 and 10 • Guest Lectures • Tom Abercrombie- 100807 • Sandra Sachs 101007 • Return and review exams

3. Elements and Compounds • Element- simplest substances known providing building blocks for all matter • 109 known elements – 89 natural, others created • Periodic 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 identity • Compound: 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

4. The Periodic Table

5. Physical States • Solid, liquid, and gas – different forms or states of matter • Solid- definite shape and volume • Liquid- definite volume and takes shape of container • Gas- neither definite shape nor volume • Substances can change from one form to another • Freezing- Water to Ice (0C) or Vaporizing- water to steam (100C) • Sublimation - solid gas • No new chemical substance is being formed. Attractive forces change • Phases- substances can be distinguished by a visible boundary • For example- Oil and Vinegar or Sugar in Water

6. Organic vs.. Inorganic substances • Organic v. Inorganic • Organic: contains carbon ( C ) combined w/: H, O, N, S, P, Cl, Br • Inorganic substance: all other known (no C ) • Qualitative vs. Quantitative determinations • Qualitative • results in the identity of the material • Requires determination of numerous properties • For example- powder reveals presence of heroin and quinine • Quantitative • result in percentage combination of components of a mixture • Precise measurement of a single property of the material • For example – powder contains 10% heroin and 90% quinine • Analytical techniques for identification of organic compounds • Spectrophotometry- study of absorption of light by chemical substances usually requires material to be in pure states • Chromatography- separating and identifying components of a mixture

7. Chromatography Principles (1) • Useful to separate mixtures into components • William 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 temperature • Distribution 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 direction • Air 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 mixed • Materials with preference for moving phase slowly pull ahead • At end, all substances separated crossing the finish line at different times • Gas Chromatography- GC, High Performance Liquid chromatography- HPLC, Thin Layer chromatography- TLC.

9. Gas Chromatography (1) • Separates mixtures – stationary liquid and moving gas • Stationary liquid is in columns • Packed columns contain liquid fixed on particles are 2-6m in length and 3mm diameter • Capillary columns composed of glass, 15-60 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 faster • Once 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 state • As components emerge they enter the detector • Flame ionizes substance generating an electric signal • Recorded on a strip chart recorder as a function of time = chromatogram • Recorder response v time • Retention time- • Time required for a component to emerge • Provides a useful identifying characteristic of a material • Not 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 recorded • Pyrolysis GC • Important extension of GC • Many forms of physical evidence, paint, fibers,plastics, can be dissolved in a solvent by heating or pyrolysis to high temps (500-1000C) for injection into the GC • Pyrolyzers 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 particles • As liquid carries the sample, different components are slowed to different degrees depending on their interaction with the stationary phase • Major advantage over GC is it takes place at room temperature • GC- 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 phase • TLC Procedure • Sample is dissolved in a solvent • Spotted onto the lower edge of the plate • The plate is placed into a closed chamber with liquid • The liquid slowly rises up by capillary action. Separation occurs as the components with the greatest affinity for the moving phase migrate faster • Visualized UV fluorescence or developed with a chemical reagent spray  color spots

14. TLC -2 Q K • Questioned 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 same • ID cannot be considered definitive as other materials may have similar migration • Distance 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 = 0.8 • Rapid and sensitive down to 100ug • Principal 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 towel • Spot 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 depth • Slowly drop your plate into the solvent • Permit the front to move up at least 3 inches • Remove the plate and let air dry • Answer 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. Electrophoresis • Separation of materials according to migration rates on a stationary solid phase • Uses electric potential across the stationary medium • Medium may include starch or agarose coated on a glass plate of polymer in a capillary • Substances possessing an electric charge migrate. The speed depends on size and charge • Principal applications are the separation of mixtures of proteins and DNA

17. Spectrophotometry Review • Theory of Light- White light = ROYGBIV • Light is a wave - wavelength is inversely proportional to frequency- Visible light is only a small part of the electromagnetic spectrum • Color = visual indication of an objects ability to absorb some and reflect visible light components • Different materials have different absorptions • Absorption 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. Spectrophotometer • Instrument used to measure and record the absorption spectrum of a chemical substance • Components- 1. Radiation source • 1- Radiation source (UV, vis, IR) • 2. Monochromator or frequency selector • 3. Sample holder • 4. 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 frequency • UV spec of heroin has max absorption at 278nm providing materials probable identity • Will 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 spectra • Each IR spectra is equivalent to a “fingerprint” of that substance and no other • Fourier transform infrared spectrophotometer FT-IR • Considered 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 fragment • Fragments 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

22. Mass Spectrometry (2)

23. Mass Spectrometry (3) • Sample first injected into a heated inlet port and carrier gas sweeps it into the GC column • GC separates the mixture into its components • Ion source filiment wire emits electrons striking the sample molecules causing them to fragment according to mass • Detector 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 1 • Organic substances contain C. Inorganic ones comprise all others • Choice of analytical techniques depends on substance category (organic vs inorganic) and the need for qualitative vs. quantitative determinations • Qualitative relates just to the identity of the material whereas quantitative relates to the percent composition of components in a mixture • Chromatography, spectrophotometry and mass spec are used by forensic scientists to identify or compare organic materials • Chromatography is a means of separating and tentatively identifying the components of a mixture. • Spectrophotometry is the study of the absorption of light by chemical substances • Mass spectrometry characterizes by observing a substance’s fragmentation patterns after collision with high energy electrons

25. Summary 2 • GC 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 chromatogram • A 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 explosives • TLC uses a solid stationary phase and mobile liquid phase • Electrophoresis uses electric potential to separate proteins and DNA of different size and charge on a gel-coated plate or polymer filled capillary • Most 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 Silicon • 99% 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 scrapings • Examples 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 arsenic • Complete the tests  results identical to tests previously recorded for knowns to be a valid conclusion as to the chemical identity of evidence • Comparison to ascertain common origin- • Example: Brass pipe found on the suspect compared to a broken pipe at a crime scene • Condition of the pipes may not allow fitting of broken edges • Pipes 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 crust • Purification is not 100% and cannot exclude all minor impurities • Manufactured products and natural materials contain small quantities of elements in trace amounts (< 1%) • Trace elements provide additional points of comparison • See Table 6.2 for Brass example • Soil, fibers, glass and metallic objects- Kennedy

29. Brass Pipe Trace Elements

30. Evidence in the Kennedy Assassination • Did Lee Harvey Oswald act alone? • Warren Commission concluded he was alone assassin • Oswald fired 3 shots from behind in the Texas School Book Depository • President hit by 2 bullets, 1 missing the limo • 1 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 stretcher • Second 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 cases • Oswald seen there in the am • Critics of the Warren commission cite • eyewitness accounts and acoustical data contending someone else fired from a region in front of the limo • One 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 20-1200ppm 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 indistinguishable • Q2, 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 derived • There 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 silver • Both bullets have a composition highly consistent with WCC/MC bullet lead although other sources cannot entirely be ruled out • Bullet 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 bullet • None of these can totally verify the Warren Commission’s reconstruction but results are consistent • Analysis was conducted by Neutron Activation analysis

35. Emission Spectrum of Elements • Elements selectively absorb and emit light • Techniques used to determine elemental composition of materials • Emission spectroscopy • Inductively Coupled Plasma Emission Spectrometry and • Atomic Absorption Spectrophotometry

36. Emission Spectra • Light 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 colors • Example- 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 composition • Vaporized and excited by high temp each element  light of select frequencies characteristic of this element • Line 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 light • Separate light into component frequencies • Record resultant spectra

40. Emission spectra of evidence • Contains numerous elements hence numerous lines • Identification by comparison to a standard chart showing position of principal spectral lines of all elements • More 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 atoms • Inductively Coupled Plasma is caused by a chain reaction of colliding electrons • high voltage spark releases electrons from argon gas • Acceleration in magnetic field more collisions and more release • Discharge sustained by RF energy

42. Inductively Coupled Plasma Emission Spectrometry (ICP) is Hot, very hot • ICP discharge acts like a very intense continuous flame-7000-10,000 oC • Sample introduced into hot plasma collides with argon electrons  charged particles (ions) emit light of characteristic wavelengths corresponding to identity of elements • Applications- mutilated bullets and glass fragments. • Bullets not suitable for comparison to test fired bullets. • copper, arsenic, silver, antimony, bismuth, cadmium and tin • Class characteristic as currently no way (no database) of providing statistical significance • Accepted in NJ Supreme Court – State v Noel. 1999

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 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 • For example if one wanted to determine the presence • of antimony, the discharge lamp would be constructed • with 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 sample • Concentration of absorbing element will be directly proportional to the quantity of light absorbed. • Sensitive to trace levels • Limitation is that only one element at a time can be measured • Modification 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 sun • Nucleus 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 structure • Each element contains a different number of protons= Atomic number • The periodic table represent the atomic number = number(s) of protons • Element is a collection of atoms all having the same number of protons.

48. The Periodic Table

49. Atomic structure of elements • Electrons move around the nucleus confined to a path of flight = electron orbital • Each orbital is associated with a definite amount of energy = energy level • Each 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 orbitals • When an atom absorbs energy or light its electrons are pushed into higher energy orbitals = excited state • Because energy levels have fixed values only definite amounts can be absorbed