Transcript JS 113: Organic Analysis

JS 113: Organic and Inorganic Analyses
I.
II.
Announcements
A.
Schedule and Assignments
B.
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
A.
Describe the usefulness of trace elements in comparisons of phys. evidence
B.
Distinguish continuous and line emission spectra
C.
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
D.
Define proton, neutrons and electrons, mass and charge relationship atomic number and atomic
mass, orbital energy levels, isotope, radioactivity
E.
Explain how atoms absorb a definite amount of energy and release energy in the form of light
A.
B.
C.
D.
E.
F.
G.
H.
I.
III.
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
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
The Periodic Table
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
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
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
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.
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
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
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 (5001000C) 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
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
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
TLC -2
• 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
Q K
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?
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
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 LawA=kc , A= absorption c=concentration k=proportionality
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
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
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
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
Mass Spectrometry (2)
Mass Spectrometry (3)
1.
2.
3.
4.
5.
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
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
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
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
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
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
Brass Pipe Trace Elements
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
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
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
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.
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
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
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
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
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.
Emission Spectrometer
• Main components:
– Vaporizes and excites
atoms to emit light
– Separate light into
component frequencies
– Record resultant
spectra
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
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
Inductively Coupled Plasma Emission
Spectrometry (ICP) is Hot, very hot
• ICP discharge acts like a very intense continuous flame7000-10,000 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
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
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?
Fundamentals of the Atom
• Subatomic particles: proton, electron and
neutron- basic structural units of the atom
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
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.
The Periodic Table
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
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
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!
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
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
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
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
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 20-30 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
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)
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
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 20-30 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)