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Chapter 5 and 6

Organic and

Inorganic

Chemical Analysis

Ch 5/6: Organic and Inorganic

Analysis

• Elements and compounds.

• Solids, liquids, and gases.

• Phase Changes

• Organic vs. inorganic compounds.

• Qualitative vs. quantitative analysis.

• Chromatography.

• Retention time.

• Rf value.

• Electrophoresis.

• Spectrophotometry and spectrometry.

Phase Changes: (physical state changes)

– Melting: change from the solid directly into the liquid state

– Freezing: change from the liquid directly into the solid state

– Vaporization: change from the liquid directly into the gaseous state

– Condensation: change from the gas directly into the liquid state

– Sublimation: change from the solid directly into the gaseous state

– Deposition: change from the gas directly into the solid state

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Phase Diagrams:

Matter

Uniform Composition?

Heterogeneous

Can be separated

by physical methods

Pure Substance

Can it be broken down further ?

Compound Element

Yes No

No No Yes Yes

Homogeneous

(solution)

Mixture

Selecting an Analytical Technique

• Organic: a substance composed of

carbon and hydrogen, and, often, smaller

amounts of oxygen, nitrogen, chlorine,

phosphorus, or other elements

• Inorganic: a chemical compound not

based on carbon

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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 (e.g., ± 1.0% or ± 0.001%)

• Detection levels

• Destructive or non-destructive

• Availability of instrumentation

• Admissibility (e.g., are all lead pipes composed the same or are there sufficient variations among “known” Pb pipes of the world to link two samples)

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

• The mixture separates as it interacts with the two phases

• Chromatographic systems have a stationary phase (can be solid

or liquid) and a mobile phase (usually liquid or gas).

• The mixture to be separated is placed at the beginning of the

chromatographic system (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).

Chromatography Revisited

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• When the molecules reach the far end of

the surface, they are detected or

measured one at a time as they emerge

• Chromatography is non-destructive

(does not alter the molecular structure

of the compounds)

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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 7.0

Time (min)

0

20

40

60

80

1000

20

40

60

80

1000

20

40

60

80

100

Rela

tive

Abun

danc

e

0

20

40

60

80

1000

20

40

60

80

100 NL:7.91E6

m/z= 43.50-44.50 F: MS level4

NL:1.18E7

m/z= 57.50-58.50 F: MS level4

NL:5.92E5

m/z= 298.50-299.50 F: MS level4

NL:2.17E6

m/z= 81.50-82.50 F: MS level4

NL:2.40E5

m/z= 314.50-315.50 F: MS level4

Street Drugs in Real Time

Types of Chromatographic Attraction

• Adsorption Chromatography

depends on physical forces to cause the molecules to “stick”

to the stationary phase

ex. Paper, TLC, HPLC

• Partition Chromatography

depends on the relative solubility of the mixture’s molecules

ex. gas chromatography

• Size-exclusion

the relative sizes of the molecules determine how fast the

molecules move through the stationary phase

large molecules flow right through, small molecules spend

time trapped in the pores of the stationary phase

ex. gel filtration chromatography

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 through

stationary phase

• Components appear as separate spots spread

out on the paper after drying

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Thin Layer Chromatography (TLC)

• Stationary Phase

a thin layer of adsorbent coating on a sheet of plastic

or glass (usually Al2O3 (alumina) or SiO2 (silica))

• 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)

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

• Good indicator of whether an unknown and a known compound are similar, if not identical

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

• Rf = D1 / D2

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Gas Chromatography • Separates mixtures on basis of distribution between a

stationary liquid and a moving gas phase

• Stationary phase

a solid or very syrupy liquid lines a tube (column)

• silicone polymers (like Silly Putty) commonly used

• Mobile phase

an inert gas (carrier gas)

• Usually nitrogen or helium

GC Columns

A packed column A capillary column

Schematic of a GC

• A mixture is injected into the GC where the mixture is vaporized.

•The gas mixture travels through a GC column, where the compounds

become separated and enters a detector.

•Detectors use various methods to generate electrical signal which is

then recorded on a strip-chart recorder as a function of time.

•Each peak corresponds to a component

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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 present

allows quantitation of sample

Retention time: time between when the sample is injected

and when it exits the column reaching the detector

- Tm is the time taken for the mobile phase to pass

through the column

Retention Time

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Pyrolysis Gas Chromatography - Used when a sample

does not readily dissolve in a solvent (paint chips,

fibers)

High Performance Liquid Chromatography (HPLC)

• Moving phase is liquid pumped through a column

filled with fine solid particles which are chemically

treated

• Advantage: takes place at room temperature

• Used for organic explosives that are heat sensitive

as well as heat sensitive drugs such as LSD

• 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 Scale Underlying Ideas - Atomic and Molecular Weights

Creates charged particles (ions) from gas phase molecules.

• Electron Ionization (EI)- Uses electron impact

• Chemical Ionization (CI)- First ionizes a molecular gas

which in turn ionizes the molecule of interest. A

“gentler” method of ionization.

• Fast Atom Bombardment (FABS)- Mainly for nonvolatile

compounds - very harsh.

The MS analyzes ions to provide information about the

molecular weight of the compound and its chemical

structure.

Mass Spectrometry (MS or mass spec) Basic Ideas

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Mass Spectrometer (MS) • As the gas leaves the GC, it enters the MS

• 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 on a graph

• Each substance breaks down into its own characteristic pattern

Mass Spectrometer Atomic Spectra

mass number (amu)

Int.

Mass

Spectrum

Cl

mass number (amu)

Int.

Mass

Spectrum

C

mass number (amu)

Int.

Mass

Spectrum

P 35

37

35Cl: 75% abundant 37Cl: 24% abundant

31

12Cl: 98.9% abundant 13Cl: 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

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

Intensity

Mass

Mass Spectrometer

Unknown 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

Intensity

Mass

Heroin other peaks at 327 and 369

43

94146

204

215

268

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

Intensity

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

Intensity

Mass

42

82

122 150

182

272

303

Cocaine

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

Intensity

Mass

Caffeine

42

55

67

82

109

194

MS of Unknown

MS

Library

Cocaine

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Mass Spectrometer

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

Intensity

Mass

Caffeine

42

55

67

82

109

194

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

Intensity

Mass

Caffeine

42

55

67

82

109

194

MS of Unknown

MS Library Caffeine

Mass Spectrometer

N

NN

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

Intensity

Mass

GC-Mass Spectrometry

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Electrophoresis

• Separates materials

based on their

migration rates on a

stationary solid

phase

• Passes an electrical

current through

medium and allows

for classification of

proteins

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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

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• 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 energy equal to the product of Plank’s constant and the frequency of radiation.

E = hν

E = energy of photon

ν = frequency of radiation

h = Plank’s constant

The Spectrophotometer

• Instrument used to measure and record

the absorption spectrum of a chemical

substance

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UV Spectrophotometry

• 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-VIS Spectrophotometer

UV-VIS Spectrum

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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 Spectrophotometer

IR Spectrum

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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.

The Hydrogen Discharge Tube

• H2 molecules are

split into excited H

atoms by an electric

discharge

• As the atoms return

to lower energy

states, light is

emitted

Flame Tests

Atomic Emission

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Atomic Absorption Spectroscopy (AAS)

• Sample heated to vaporize atoms

• Vaporized atoms exposed to radiation emitted from a discharge tube

• Atoms absorb radiation & become excited

• The amount of radiation absorbed is recorded

• The amount absorbed is determined based on a calibration curve using Beer’s Law (Absorption is proportional to concentration)

Beer’s Law

Absorption is proportional to concentration

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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

Atomic Absorption (AAS)

Typical Problem –

• A child becomes quite ill and is taken to the

hospital.

• It is found that the child is suffering from lead

poisoning.

• A forensic laboratory is contacted and asked if it

can determine the source of the lead which the

child has ingested.

• Paint samples from a number of objects with which

the child has had repeated contact are collected.

Paint on the child's crib, paint from his toys, and

paint from the child's swing, to name a few, are sent

to the laboratory.

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Neutron Activation Analysis (NAA)

• Neutrons interact with a target nucleus to form a compound

nucleus in an excited state.

• The compound nucleus will decay into a more stable

configuration through emission of one or more gamma rays.

• This new configuration may yields a radioactive nucleus

which also decays by emission of delayed gamma rays, but

at a much slower rate according to the unique half-life of the

radioactive nucleus.

• Quantitation in parts per billion but requires a nuclear

reactor

Neutron Activation Analysis

• Rate depends on half-life of isotope

– 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

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Gamma-ray Spectra

Continuation of medium- & long-lived elements

Comparison of Techniques

Technique Qual.* or Quant.

Sample Size

Detection levels

Destructive Instr. Avail.

Mass Spec. Qual. 0.1 mL to 10-8 mL

* Yes Easy

Infrared Qual. 0.001 g * No Easy

UV-visible Qual. 0.001 g No Easy

AES Quant. 10-4 g/L Yes Moderate

AAS Quant. 10-4 g/L Yes Easy

NAA Quant. 1 x 10-9 g Possibly Difficult

* Primary use is in qual. analysis, although it can be used quantitatively in some cases.