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
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
Sandra Sachs
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, 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 ( C) 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 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 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
Sample placed between two carbon electrodes- direct current heat vaporizes and exices sample’s atoms
Emitted light collected by a lense focused onto a prism dispersing component frequencies
directed toward a photographic plate to record

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
Unlike 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 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

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

51. Excitation at the Atomic Level: Atomic Absorption Spectrophotometry
Elements are selective in the frequencies of light they absorb
Selectivity is determined by the electron energy levels in each element
Atomic Absorption Spectrophotometer, a photon of light will interact with an electron causing it to jump into a higher orbital
Energy must correspond to the energy difference between the two orbitals
E=hf E=energy difference, h-frequency of absorbed light and h – Planks constant
Any energy value more or less will not affect the transition
Like 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 level
As it falls back it releases energy
Emission spectrum - energy loss comes about in the form of light emission
Each element has its own unique set of energy levels each emits a unique set of frequencies
Emission spectrum is a picture of the energy levels surrounding the nucleus of each element
Atomic Absorption spectroscopy measures the value and amount of light energy going into the atom
Emission 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 energy
New tool for identifying and quantitating elements
Atoms of single elements must have protons= electrons. Not so with neutrons
Total number of protons and neutrons = atomic mass
Isotopes 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 decay
Radioactivity is the emission of radiation accompanying decay of unstable nuclei
Alpha: helium atoms minus electrons
Beta: electrons
Gamma: 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 neutrons
When a neutron is captured by the nucleus of an atom a new isotope is formed activated and many decompose by emitting radioactivity
To 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 elements
Sensitive to one-billionth of a gram (1ng)
Multiplex capable- simultaneously analyze elements
Limitation is cost
Metals, drugs, paint, soil, gunpowder residue and hair
Example 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 Diffraction
ES, AA and NAA reveal presence of elements not how combined into compounds
Focusing a beam of X-rays at a crystal and studying how the atoms in the substance interact is called X ray diffraction
95% of all inorganic compounds are crystalline in nature
Limitation- lack of sensitivity- fails to detect those present at 5% level in mixtures (ES, AA and NAA more sensitive)

58. Summary 1
Inorganic 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 comparison
Emission spectroscopy, inductively coupled plasma and atomic absorption spectrophotometry are techniques used by forensic scientists to determine elemental composition of materials
In 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 2
In 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)