Chapter 5 and 6

Organic and Inorganic

Chemical Analysis

Phase Changes: (physical state changes)

• Melting: from solid directly into liquid

• Freezing: from liquid directly into solid

• Vaporization: from liquid directly into gas

• Condensation: from gas directly into liquid

• Sublimation: from solid directly into gas

• Deposition: from gas directly into solid

Phase Diagrams:

Matter

Uniform Composition?

Heterogeneous

Can be separatedby physical methods

Pure Substance

Can it be broken down further ?

Compound Element

Yes No

No NoYes Yes

Homogeneous(solution)

Mixture

Selecting an Analytical Technique

•  Organic: a substance composed of carbon (often contain hydrogen and smaller amounts of oxygen, nitrogen, chlorine, or phosphorus)

•  Inorganic: a chemical compound not based on carbon

Questions to consider in choosing an analytical (chemical) method:

• Quantitative or qualitative required• Sample size and sample preparation requirements• What level of analysis is required (ex. ± 1.0% or ± 0.001%)• Detection levels• Destructive or non-destructive• Availability of instrumentation• Admissibility

What Is Chromatography?

• Laboratory technique for separating mixtures into their component compounds

• Uses some version of a technique in which two phases (one mobile, one stationary) flow past one another

• Chromatographic systems have a stationary phase and a mobile phase (usually liquid or gas)

• The mixture to be separated is placed on the stationary phase

• The mobile phase then “pushes” the components of the mixture through the system

• Each component adsorbs on the stationary phase with a different strength (stronger means moves more slowly through the system)

• Each component comes out the end of the system at a different time (retention time)

• When the molecules reach the far end of the surface, they are detected or measured one at a time as they emerge

Chromatography Review

• Chromatography is non-destructive

• Separation of components depends on both their solubility in the mobile phase and their differential affinity to the stationary phase.

Amphetamine

Methamphetamine and MDMA

Hydrocodone

Cocaine

Oxycodone

RT: 2.80 - 7.43 SM: 9G

3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0Time (min)

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

1000

20

40

60

80

100 NL:7.91E6m/z= 43.50-44.50 F: MS level4

NL:1.18E7m/z= 57.50-58.50 F: MS level4

NL:5.92E5m/z= 298.50-299.50 F: MS level4

NL:2.17E6m/z= 81.50-82.50 F: MS level4

NL:2.40E5m/z= 314.50-315.50 F: MS level4

Street Drugs in Real Time

Types of Chromatography• Paper Chromatography• Thin-Layer Chromatography (TLC)• Liquid Chromatography (HPLC)• Gas Chromatography (GC)

Paper Chromatography• Stationary phase

a sheet or strip of paper• mobile phase

a liquid solvent

• Sample mixture spotted onto the paper

• Capillary action moves mobile phase

• Components appear as separate spots spread

out on the paper after drying

Thin Layer Chromatography (TLC) • Stationary Phase

a thin layer of adsorbent coating on a sheet of plastic or glass

• Mobile Phase a liquid solvent

• Sample mixture spotted onto the adsorbent - Solids must first be dissolved - Liquids can be directly applied

• Some components bind to the adsorbent strongly; some weakly

• Components appear as separate spots after development

TLC

Retention Factor (Rf)

• An indication of how far a compound travels in a particular solvent

• Good gauge of whether an unknown and a known compound are similar

• Rf = distance the solute (D1) moves divided by the distance traveled by the solvent front (D2)

• Rf = D1

D2

Gas Chromatography• Stationary phase

a solid or very “syrupy” liquid in a tube (column)• Mobile phase

an inert gas (carrier gas)• Usually nitrogen or helium

Analysis Using the GC

• Retention time can be used as an identifying characteristic of a substance retention times may not be unique

• An extremely sensitive technique area under a peak is proportional to the quantity of

substance presentallows quantitation of sample

Retention time: time between when the sample is injected and when it exits the column reaching the detector

Retention Time

Tm is the time it takes for the mobile phase to pass through the column

High Performance Liquid Chromatography (HPLC)• Stationary phase

fine solid particles which are chemically treated• Mobile phase

a liquid

• Advantage: takes place at room temperatureUsed for organic explosives that are heat sensitive as well as heat sensitive drugs

• Atomic Mass Scale - based upon 12C isotope. This isotope is assigned a mass of exactly 12 atomic mass units (amu) and the masses of all other atoms are given relative to this standard.

• Most elements in nature exist as mixtures of isotopes (atoms of an element that have different numbers of neutrons but same number of protons).

Mass ScaleAtomic and Molecular Weights

Creates charged particles (ions) from gas phase molecules.

The Mass Spec analyzes ions to provide information about the molecular weight of the compound and its chemical structure.

Mass Spectrometry (MS or mass spec)Basic Ideas

Mass Spectrometer (MS)

• As the sample leaves the GC or HPLC, it enters the Mass spec.

• Within the MS, a beam of electrons is shot at the substance breaking it down into fragments

• These fragments pass through an electric field which separates them by their masses

• The fragment masses are then recorded• Each substance breaks down into its own

characteristic pattern

Mass SpectrometerAtomic Spectra

mass number (amu)

Int.

Mass Spectrum

Cl

mass number (amu)

Int.

Mass Spectrum

C

mass number (amu)

Int.

Mass Spectrum

P35

37

35Cl: 75% abundant37Cl: 24% abundant

31

12Cl: 98.9% abundant13Cl: 1.11% abundant

31P: 100% abundant

12

13

• The unique feature of mass spectrometry is that under carefully controlled conditions, no two substances produce the same fragmentation pattern

• Allows for identifying chemical substance

• Each mass spectrum is unique to each drug and so serves as specific test for identifying the substance

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Mass SpectrometerUnknown white powdery substance ingested by unconscious patient. What do you do? Is it Heroin, Cocaine, Caffeine?

Mass Spectrum of Unknown Compound

Mass Spectrometer

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Heroin other peaks at 327 and 369

43

94 146

204215

268

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Caffeine

42

55

67

82

109

194

MS of Unknown

MS Library Heroin

Mass Spectrometer

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

42

82

122 150

182

272

303

Cocaine

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Caffeine

42

55

67

82

109

194

MS of Unknown

MS Library

Cocaine

Mass Spectrometer

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Caffeine

42

55

67

82

109

194

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Caffeine

42

55

67

82

109

194

MS of Unknown

MS Library Caffeine

Mass Spectrometer

N

N N

N

CH3

CH3

H3C

O

OUnknown white powdery substance ingested by unconscious patient. What do you do?

Mass Spectrum

Mol. Wgt = 194

Caffeine

25 50 75 100 125 150 175 200 225 250 275 300

Inte

nsity

Mass

Electrophoresis

• Separates materials based on their migration rates on a stationary solid phase

• Passes an electrical current through the medium and allows for classification of proteins (DNA)

Most useful applications of Electrophoresis

• Characterization of proteins and DNA in dried blood

• Proteins migrate at speeds that vary according to their electrical charge and size resulting in characteristic band patterns

Spectroscopy and Spectrophotometry

• Study of absorption of light by chemical substances• Used for identification of various organic materials

or for presence of trace elements• Electromagnetic spectrum – entire range of “light

waves”• Colors – absorption or reflection of various

wavelengths of visible light• Ultraviolet or infrared radiation (either side of visible

region)• X-ray – high energy, short wavelength

• Incoherent light – light comprised of waves that are out of step with each other

                                                         

• Coherent or laser light – light whose waves are pulsating in unison– Laser (light amplification by the simulated

emission of radiation)

• Photon – small packet of electromagnetic radiation energy.

• Each photon contains a unit of energyE = hν

E = energy of photonν = frequency of radiationh = Plank’s constant (6.626 X 10-34 J•s)

The Spectrophotometer• Instrument used to measure and record

the absorption spectrum of a chemical substance

UV Spectrophotometry

• Measures absorbance of UV and visible light as a function of wavelength or frequency

• Allows tentative identification

Ex. White powder with UV spectrum comparable to that of heroin results in a tentative identification

UV Spectrum

Infrared Spectrophotometry

• Different materials always have distinctively different infrared spectra

• Each IR spectrum is therefore equivalent to a “fingerprint” of that substance and no other

• Extensive catalogue of IR spectra of organic compounds allows for identification of organic substances

IR Spectrum

Atomic Emission Spectroscopy (AES)• Used to detect the types of elements present in a sample• Can use measurement of the emissions from excited atoms to

determine concentration. • Frequency of light given off

The Hydrogen Discharge Tube

• H2 molecules are excited by an electric discharge

• As the atoms return to lower energy states, light is emitted

Flame Tests Atomic Emission

Atomic Absorption Spectroscopy (AAS)

• Sample heated and atoms absorb radiation• Atoms become excited• The amount of radiation absorbed is recorded

• Can be used to quantitate amounts based on a calibration curve• Beer’s Law (Absorption is proportional to concentration)

Example:• Determination of the wavelength of light absorbed

by a sample of grape soda

Absorption of Grape Soda

Example:• Determination of the amount of dilution of a sample of

grape soda

Neutron Activation Analysis (NAA)

• Used for determining the concentrations of elements

Process:• Neutrons interact with a target nucleus to form a nucleus in an

excited state.

• The excited nucleus will decay immediately into a more stable configuration through emission of gamma rays

• This new configuration may yields a radioactive nucleus which continues to undergo decay but at a much slower rate (depending on the unique half-life of the sample). This decay is also measured.

• Allows quantitation in parts per billion but requires a nuclear reactor

Neutron Activation Analysis• Rate depends on half-life

– Prompt gamma ray formation• measurement taken during irradiation

– Delayed gamma ray formation• measurements taken after irradiation• more common

• About 70% of elements have properties suitable for measurement by NAA

Gamma-ray Spectra

Continuation of medium & long-lived elements