1

11

Chapter 5Chapter 5

ORGANIC ANALYSISORGANIC ANALYSIS

22

Organic vs. InorganicOrganic vs. Inorganic�� The proper selection of analytical The proper selection of analytical

techniques that will allow the forensic techniques that will allow the forensic

scientist to identify or compare matter can scientist to identify or compare matter can

best be understood by categorizing all best be understood by categorizing all

substances into one of two broad groups: substances into one of two broad groups:

organics and organics and inorganicsinorganics..

�� In general, organic substances contain the In general, organic substances contain the

element carbon, commonly in combination element carbon, commonly in combination

with one or more other elements. with one or more other elements.

�� Inorganic materials encompass all other Inorganic materials encompass all other

known chemical substances.known chemical substances.

33

Qualitative vs. QuantitativeQualitative vs. Quantitative�� Another consideration in selecting an Another consideration in selecting an

analytical technique is the need for either a analytical technique is the need for either a

qualitative or a quantitative determination. qualitative or a quantitative determination.

�� The former relates just to the identity of the The former relates just to the identity of the

material, whereas the latter requires the material, whereas the latter requires the

determination of the percent composition of determination of the percent composition of

the components of a mixture.the components of a mixture.

�� Most of the evidence received by crime Most of the evidence received by crime

laboratories requires the identification of laboratories requires the identification of

organic compounds.organic compounds.

44

Chromatography vs. SpectrometryChromatography vs. Spectrometry

�� Chromatography, Chromatography, spectrophotometryspectrophotometry, and , and mass spectrometry are all readily used by mass spectrometry are all readily used by a forensic scientist to identify or compare a forensic scientist to identify or compare organic materials. organic materials.

�� Chromatography is a means of separating Chromatography is a means of separating and tentatively identifying the components and tentatively identifying the components of a mixture. of a mixture.

�� SpectrophotometrySpectrophotometry is the study of the is the study of the absorption of light by chemical absorption of light by chemical substances. substances.

55

What Is Chromatography?What Is Chromatography?

�� A family of laboratory techniques for A family of laboratory techniques for separating mixtures into their component separating mixtures into their component compoundscompounds

�� Uses some version of a technique in which Uses some version of a technique in which two phases, one mobile, one stationary, two phases, one mobile, one stationary, flow past one anotherflow past one another

�� The mixture separates as it interacts with The mixture separates as it interacts with the two phasesthe two phases

66

Basic PrinciplesBasic Principles

�� Different compounds will stick to a solid surface with Different compounds will stick to a solid surface with different degrees of strength or vary in the efficiency different degrees of strength or vary in the efficiency with which they dissolve in a liquidwith which they dissolve in a liquid

�� When a mixture of compounds flows over a surface, the When a mixture of compounds flows over a surface, the molecules will stick to the surfacemolecules will stick to the surface

�� If a molecule does not stick to the surface too strongly, If a molecule does not stick to the surface too strongly, the molecules stick & the molecules stick & unstickunstick many times as it is swept many times as it is swept along the surfacealong the surface

�� Over time, the molecules will become physically Over time, the molecules will become physically separated from each otherseparated from each other

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

2

77 88

Basic PrinciplesBasic Principles

�� A mobile phase sweeps the sample over a A mobile phase sweeps the sample over a

stationary phase stationary phase

–– like the wind sweeps the swarm over the like the wind sweeps the swarm over the

flower bedflower bed

99 1010

Chromatography-Types

Planar

• paper chromatography

• thin layer chromatography (TLC)

• Gel electrophoresis

Column chromatography

• Adsorption (SEC, IE Affinity)

• gas-liquid chromatography (GC)

• high-pressure liquid chromatography (HPLC -

high-performance liquid chromatography)

• Capillary electrophoresis (CE)

1111

Types of Chromatographic AttractionTypes of Chromatographic Attraction

�� Adsorption ChromatographyAdsorption Chromatography

–– depends on physical forces such as dipole attraction depends on physical forces such as dipole attraction

to cause the molecules to to cause the molecules to ““stickstick”” to the stationary to the stationary

phasephase

�� column, TLC, HPLCcolumn, TLC, HPLC

�� Partition ChromatographyPartition Chromatography

–– depends on the relative solubility of the mixturedepends on the relative solubility of the mixture’’s s

molecules in the stationary phase coatingmolecules in the stationary phase coating

–– polarity may also have some effectpolarity may also have some effect

�� gas chromatographygas chromatography

1212

Chromatography-Types

3

1313

Types of Chromatographic Types of Chromatographic

AttractionAttraction�� IonIon--exchangeexchange

–– depends on the relative strength with which depends on the relative strength with which

ions interact with an ionic resinions interact with an ionic resin

–– less strongly held ions are displaced by more less strongly held ions are displaced by more

strongly attaching ionsstrongly attaching ions

–– one kind of ion is exchanged for anotherone kind of ion is exchanged for another

�� ion exchange chromatographyion exchange chromatography

1414

Types of Chromatographic Types of Chromatographic

AttractionAttraction�� SizeSize--exclusionexclusion

–– the relative sizes of the molecules determine the relative sizes of the molecules determine

how fast the molecules move through the how fast the molecules move through the

stationary phasestationary phase

–– large molecules flow right throughlarge molecules flow right through

–– small molecules spend time trapped in the small molecules spend time trapped in the

pores of the stationary phasepores of the stationary phase

�� gel filtration chromatographygel filtration chromatography

1515

Chromatography-Types

1616

Paper ChromatographyPaper Chromatography

�� Stationary phase Stationary phase

–– a sheet or strip of papera sheet or strip of paper

�� mobile phasemobile phase

–– a liquid solventa liquid solvent

�� Sample mixture spotted onto the paperSample mixture spotted onto the paper

�� Capillary action moves mobile phase Capillary action moves mobile phase

through stationary phasethrough stationary phase

1717

Paper ChromatographyPaper Chromatography

�� Components appear Components appear

as separate spots as separate spots

spread out on the spread out on the

paper after dryingpaper after drying

�� Can be used for ink Can be used for ink

analysisanalysis

1818

Paper ChromatographyPaper Chromatography

�� 2D Chromatography2D Chromatography

–– accomplished by running another accomplished by running another

chromatography with the paper turned 90chromatography with the paper turned 90oo

–– Can complete separation of overlapping Can complete separation of overlapping

compoundscompounds

4

1919

TLCTLC

�� TLC uses a solid stationary phase usually coated onto a glass TLC uses a solid stationary phase usually coated onto a glass plate and a mobile liquid phase to separate the components plate and a mobile liquid phase to separate the components of the mixture. of the mixture.

�� The liquid will slowly rise up the plate by capillary action The liquid will slowly rise up the plate by capillary action causing the sample to become distributed between the causing the sample to become distributed between the stationary phase and the moving liquid phase.stationary phase and the moving liquid phase.

�� Because most compounds are colorless, the materials must be Because most compounds are colorless, the materials must be visualized by placing the plates under ultraviolet light or visualized by placing the plates under ultraviolet light or spraying the plate with a chemical reagent.spraying the plate with a chemical reagent.

�� The distance a spot travels up a thinThe distance a spot travels up a thin--layer plate can be layer plate can be assigned a numerical value known as the assigned a numerical value known as the RRff value.value.

2020

Thin Layer Chromatography Thin Layer Chromatography

�� Stationary PhaseStationary Phase

–– a thin layer of adsorbent coating on a sheet of a thin layer of adsorbent coating on a sheet of

plastic or glassplastic or glass

�� usually Alusually Al22OO33 (alumina) or SiO(alumina) or SiO22 (silica)(silica)

�� Mobile PhaseMobile Phase

–– a liquid solventa liquid solvent

�� Sample mixture spotted onto the Sample mixture spotted onto the

adsorbentadsorbent

2121

ThinThin--Layer Chromatography (TLC)Layer Chromatography (TLC)

2222

TLCTLC

�� Some components Some components

bind to the adsorbent bind to the adsorbent

strongly; some strongly; some

weaklyweakly

�� Components appear Components appear

as separate spots as separate spots

after developmentafter development

2323

Retention Factor (Retention Factor (RRff))

�� quantitative indication of how far a quantitative indication of how far a compound travels in a particular solventcompound travels in a particular solvent

�� good indicator of whether an unknown & a good indicator of whether an unknown & a known compound are similar, if not known compound are similar, if not identicalidentical

–– If the If the RRff value for the unknown compound is value for the unknown compound is close to or the same as that for the known close to or the same as that for the known compound, the two compounds are most compound, the two compounds are most likely similar or identicallikely similar or identical

2424

Retention Factor (Retention Factor (RRff))

�� RRff = distance the = distance the

solute (Dsolute (D11) moves ) moves

divided by the divided by the

distance traveled by distance traveled by

the solvent front (Dthe solvent front (D22) )

�� RRff = D= D11 / D/ D22

�� RRff <1.0<1.0

5

2525

Column Chromatography-

General Process

2626

Column Chromatography-

Output Chromatogram

2727

General Parts of Column

• Column

• copper tubing

• stainless steel tubing

• glass tubing

• Support

• finely divided solids (packed)

– ground firebrick

– alumina, specially treated

• walls of column for capillary columns2828

Parts of Column

Stationary Phase

• stationary phase evenly dispersed on

surface of support

– column chromatography

• non-volatile, viscous liquids dispersed evenly on

surface of support

2929

Parts of Column

Stationary Phase

• stationary phase evenly dispersed on

surface of support

– column chromatography

• non-volatile, viscous liquids dispersed evenly on

surface of support

3030

Parts of Column

Mobile Phase

• sample mixture carried through

stationary phase by mobile phase

• non-reactive gas in glc (gas-liquid

chromatography, gc)

• non-reactive liquid in llc (liquid-liquid

chromatography, lc)

6

3131

Introduction Gas ChromatographyIntroduction Gas Chromatography

�� GCs are the most widely used analytical GCs are the most widely used analytical instruments in the worldinstruments in the world

�� Premiere technique for separation and Premiere technique for separation and analysis of volatile compoundsanalysis of volatile compounds

–– Organic and inorganicOrganic and inorganic

–– Weights up to 1,000 g/molWeights up to 1,000 g/mol

�� Often coupled to MS for both qualitative Often coupled to MS for both qualitative and quantitative analysisand quantitative analysis

3232

Gas ChromatographyGas Chromatography

�� Stationary phaseStationary phase

–– a solid or very syrupy liquid lines a tube a solid or very syrupy liquid lines a tube

(column)(column)

�� silicone polymers (like Silly Putty) commonly usedsilicone polymers (like Silly Putty) commonly used

�� Mobile phaseMobile phase

–– an inert gasan inert gas

�� nitrogennitrogen

�� heliumhelium

3333

Advantages of GCAdvantages of GC

�� Fast Fast –– minutesminutes

�� High Resolution. Record of N~1 x 10High Resolution. Record of N~1 x 1066

�� Sensitive detectors (Sensitive detectors (ppmppm and ppb range)and ppb range)

�� Highly accurate quantification (RSD of Highly accurate quantification (RSD of 1.5%)1.5%)

�� AutomatedAutomated

�� NonNon--destructivedestructive–– Coupled to other techniques, especially MSCoupled to other techniques, especially MS

�� ReliableReliable

�� Low cost ($25 K)Low cost ($25 K)

�� Small sample sizes (Small sample sizes (microlitersmicroliters)) 3434

GC InstrumentationGC Instrumentation

Carrier gas: Nitrogen

Helium Inlet Pressure:10-50 psi

Flow

25-150 mL/min packed

1-25 mL/min open 2-50 m column

0-400oC operating range, accurate to 1oC Many detectors

FID, TCD, ECD, MS

3535 3636

GC ColumnsGC Columns

A packed column A capillary column

7

3737

Retention TimeRetention Time

3838

Retention TimeRetention Time

�� The time between when the sample is The time between when the sample is

injected & when it exits the column injected & when it exits the column

reaching the detectorreaching the detector

�� Tm is the time taken for the mobile phase Tm is the time taken for the mobile phase

to pass through the columnto pass through the column

3939

Analysis Using the GCAnalysis Using the GC

�� Retention time can be used as an Retention time can be used as an identifying characteristic of a substanceidentifying characteristic of a substance

–– retention times may not be uniqueretention times may not be unique

–– GC is not an absolute method of identificationGC is not an absolute method of identification

�� An extremely sensitive techniqueAn extremely sensitive technique

–– area under a peak is proportional to the area under a peak is proportional to the quantity of substance presentquantity of substance present

–– allows quantization of sampleallows quantization of sample

4040

ApplicationsApplications--Solid Phase Solid Phase

MicroextractionMicroextraction (SPME)(SPME)

4141

ApplicationsApplications--Solid Phase Solid Phase

MicroextractionMicroextraction (SPME)(SPME)

4242

Identification of AccelerantsIdentification of Accelerants

unevaporated gasoline

90% evaporated gasoline

unevaporated kerosene

90% evaporated kerosene

8

4343

Disadvantages of GCDisadvantages of GC

�� Limited to volatile samplesLimited to volatile samples

–– <380<380ooCC

–– Need Need PPvapvap ~ 60 ~ 60 TorrTorr at 380at 380oo CC

�� Not suitable for thermally labile samplesNot suitable for thermally labile samples

�� Some samples and those les volatile require Some samples and those les volatile require

extensive sample prep (extraction, extensive sample prep (extraction,

derivatizationderivatization))

�� Requires secondary technique (MS) to confirm Requires secondary technique (MS) to confirm

peak identitypeak identity

4444

GC and MassGC and Mass�� A direct connection between the GC column A direct connection between the GC column

and the mass spectrometer allows each and the mass spectrometer allows each

component to flow into the mass spectrometer component to flow into the mass spectrometer

as it emerges from the GC. as it emerges from the GC.

�� The separation of a mixtureThe separation of a mixture’’s components is s components is

first accomplished by the GC.first accomplished by the GC.

�� Then, fragmentation of each component by Then, fragmentation of each component by

highhigh--energy electrons in the mass energy electrons in the mass

spectrometer, will produce a distinct pattern, spectrometer, will produce a distinct pattern,

somewhat like a somewhat like a ““fingerprint,fingerprint,”” of the substance of the substance

being examined.being examined.

4545

GC/MSGC/MS

�� As individual compounds elute from the As individual compounds elute from the

GC column, they enter the MS detectorGC column, they enter the MS detector

�� Fragmented by electron bombardmentFragmented by electron bombardment

–– fragments are charged ions with a certain fragments are charged ions with a certain

massmass

–– mass to charge ratio (M/Z)mass to charge ratio (M/Z)

�� Z is usually +1Z is usually +1

�� M/Z represents that molecular weight of the M/Z represents that molecular weight of the

fragmentfragment

4646

The Mass SpectrometerThe Mass Spectrometer

�� A detectorA detector

�� Allows the identification of a chemical Allows the identification of a chemical compound compound

�� In the MS, a compound is bombarded with In the MS, a compound is bombarded with a stream of electronsa stream of electrons

–– compound breaks into fragmentscompound breaks into fragments

–– each compound gives a unique set of each compound gives a unique set of fragmentsfragments�� ““fingerprintfingerprint””

4747

Mass Spectrometer (MS)Mass Spectrometer (MS)

4848

Mass SpectrometerMass Spectrometer

9

4949 5050

Mass SpectrumMass Spectrum

5151 5252

5353 5454

High Pressure Liquid High Pressure Liquid

Chromatography (HPLC)Chromatography (HPLC)�� Stationary PhaseStationary Phase

–– fine solid particlesfine solid particles

�� Mobile PhaseMobile Phase

–– a liquid solventa liquid solvent

�� The solvent is pumped through the The solvent is pumped through the columncolumn

�� The sample components are retarded by The sample components are retarded by different amounts by interaction with the different amounts by interaction with the column packingcolumn packing

10

5555

HPLCHPLC�� HPLC separates compounds using a HPLC separates compounds using a stationary phase, a column filled with fine stationary phase, a column filled with fine solid particles, and a mobile liquid phase.solid particles, and a mobile liquid phase.

�� As the liquid carries the sample through the As the liquid carries the sample through the column, different components are retarded to column, different components are retarded to different degrees, depending on their different degrees, depending on their interaction with the stationary phase. interaction with the stationary phase.

�� The major advantage of HPLC is that the The major advantage of HPLC is that the entire process takes place at room entire process takes place at room temperature.temperature.

�� Substance, such as organic explosives, which Substance, such as organic explosives, which are heat sensitive are more readily separated are heat sensitive are more readily separated by HPLC.by HPLC.

5656

5757

Stationary Phase Structure

5858

Schematic of HPLCSchematic of HPLC

5959

HPLC Separation Process

6060

TYPICAL HPLC Instrument

11

6161 6262

Advantages of HPLCAdvantages of HPLC

�� Separation occurs at room temperatureSeparation occurs at room temperature

�� Does not decompose heat sensitive Does not decompose heat sensitive

materialsmaterials

–– explosivesexplosives

–– heat sensitive drugs like LSDheat sensitive drugs like LSD

6363

Analysis of Components of SakeAnalysis of Components of Sake

�� Sake is composed of several chemical Sake is composed of several chemical

componentscomponents

–– sugarsugar

–– organic acidsorganic acids

–– amino acidsamino acids

�� Each component relates to the taste of the Each component relates to the taste of the

sakesake

6464

Sugar & organic acids can be analyzed

by HPLC

6565 6666

Advantages and Disadvantages of Advantages and Disadvantages of

HPLCHPLC

�� AdvantagesAdvantages–– SpeedSpeed

–– High ResolutionHigh Resolution

–– Sensitivity (Sensitivity (ngng--fgfg))

–– Reproducibility (1%)Reproducibility (1%)

–– AutomatedAutomated

�� DisadvantagesDisadvantages–– ComplexityComplexity

–– Irreversibly bound species not detectedIrreversibly bound species not detected

–– Need secondary detector, not applicable to all Need secondary detector, not applicable to all analytesanalytes

–– Co eluting species difficult to analyzeCo eluting species difficult to analyze

12

6767 6868

Size Exclusion Chromatography

Molecules smaller than the exclusion limit

of the gel become trapped and elute last

Larger molecular weight not trapped

and flow through more quickly.

6969 7070

ElectrophoresisElectrophoresis

�� A technique analogous to TLC is A technique analogous to TLC is electrophoresis. electrophoresis.

–– Here, materials are forced to move Here, materials are forced to move across a gelacross a gel--coated plate under the coated plate under the influence of an electrical potential. influence of an electrical potential.

�� In this manner, substances such as In this manner, substances such as proteins and DNA can be separated proteins and DNA can be separated and characterized.and characterized.

7171

ElectrophoresisElectrophoresis

7272

�� Wavelength: Wavelength: the distance between crests the distance between crests of adjacent wavesof adjacent waves

�� Frequency: Frequency: the number of waves that the number of waves that pass a given point per secondpass a given point per second

�� Electromagnetic spectrum: Electromagnetic spectrum: the entire the entire range of radiation energy from the most range of radiation energy from the most energetic cosmic rays to the least energetic energetic cosmic rays to the least energetic radio wavesradio waves

�� XX--ray: ray: a high energy, short wavelength a high energy, short wavelength form of electromagnetic radiationform of electromagnetic radiation

Spectroscopy: Basic Terms

13

7373

�� Laser: Laser: light amplification by the simulated light amplification by the simulated

emission of radiation. Light that has all its emission of radiation. Light that has all its

waves pulsating in unisonwaves pulsating in unison

�� Photon: Photon: a small pocket of electromagnetic a small pocket of electromagnetic

radiation energy. Each photon contains a radiation energy. Each photon contains a

unit of energy equal to the product of unit of energy equal to the product of

PlanckPlanck’’s constant and the frequency of s constant and the frequency of

radiation: E= radiation: E= hfhf

�� MonochromatorMonochromator: : a device for isolating a device for isolating

individual wavelengths or frequencies of individual wavelengths or frequencies of

lightlight

�� Monochromatic light: Monochromatic light: light having a light having a

single wavelength or frequencysingle wavelength or frequency 7474

�� Ultraviolet: Ultraviolet: Invisible long frequencies of Invisible long frequencies of

light beyond violet in the visible spectrumlight beyond violet in the visible spectrum

�� Infrared:Infrared: invisible short frequencies of invisible short frequencies of

light before red in the visible spectrumlight before red in the visible spectrum

�� Ion: Ion: an atom or molecule bearing a an atom or molecule bearing a

positive or negative chargepositive or negative charge

7575

Electromagnetic SpectrumElectromagnetic Spectrum

7676

Theory of LightTheory of Light

�� Two models describe the behavior of light.Two models describe the behavior of light.

–– Light is described as a continuous wave.Light is described as a continuous wave.

–– Light is depicted as a stream of discrete energy Light is depicted as a stream of discrete energy particles.particles.

�� When white light passes though a prism, it is dispersed When white light passes though a prism, it is dispersed into a continuous spectrum of colors.into a continuous spectrum of colors.

�� Visible light ranges in color from red to violet in the Visible light ranges in color from red to violet in the electromagnetic spectrum.electromagnetic spectrum.

�� Waves are described in terms such as:Waves are described in terms such as:

–– Wavelength, the distance between two successive Wavelength, the distance between two successive crests (or one trough to the next trough).crests (or one trough to the next trough).

–– Frequency, the number of crests (or troughs) passing Frequency, the number of crests (or troughs) passing any one given point per unit of time.any one given point per unit of time.

7777

Wave Nature of LightWave Nature of Light

�� WavelengthWavelength–– distance between crestsdistance between crests

�� FrequencyFrequency–– number of crest that pass a given point in one secondnumber of crest that pass a given point in one second

7878

Wave Nature of LightWave Nature of Light

�� EnergyEnergy

–– frequency & energy are proportionalfrequency & energy are proportional

14

7979

Theory of LightTheory of Light�� Frequency and wavelength are inversely Frequency and wavelength are inversely

proportional to one another. proportional to one another.

�� The electromagnetic spectrum is the entire The electromagnetic spectrum is the entire range of radiation energy from the most range of radiation energy from the most energetic cosmic rays to the least energetic energetic cosmic rays to the least energetic radio waves.radio waves.

–– Visible light is only a small part of the Visible light is only a small part of the electromagnetic spectrum.electromagnetic spectrum.

�� As electromagnetic radiation moves through As electromagnetic radiation moves through space, its behavior can be described as that of space, its behavior can be described as that of a continuous wave; however, once radiation is a continuous wave; however, once radiation is absorbed by a substance, it is best described as absorbed by a substance, it is best described as discrete particles of light known as photons.discrete particles of light known as photons.

8080

The Electromagnetic SpectrumThe Electromagnetic Spectrum

8181 8282

AbsorptionAbsorption�� Just as a substance can absorb visible light to Just as a substance can absorb visible light to produce color, many of the invisible produce color, many of the invisible radiations of the electromagnetic spectrum radiations of the electromagnetic spectrum are likewise absorbed.are likewise absorbed.

�� SpectrophotometrySpectrophotometry, an important analytical , an important analytical tool, measures the quantity of radiation that tool, measures the quantity of radiation that a particular material absorbs as a function of a particular material absorbs as a function of wavelength and frequency.wavelength and frequency.

�� The quantity of light absorbed at any The quantity of light absorbed at any frequency is directly proportional to the frequency is directly proportional to the concentration of the absorbing species. This concentration of the absorbing species. This is known as Beeris known as Beer’’s Law.s Law.

8383

The Hydrogen AtomThe Hydrogen Atom

�� The electron structure of an atom is quantizedThe electron structure of an atom is quantized–– electrons can only exist in discrete energy levelselectrons can only exist in discrete energy levels

8484

Excitation/AbsorptionExcitation/Absorption

�� When a When a ““packetpacket”” of energy equal to the energy of energy equal to the energy

difference between two energy levels is absorbeddifference between two energy levels is absorbed

–– electron is promotedelectron is promoted

15

8585

Excitation/Absorption Excitation/Absorption

Excitation causes wavelengths to be absorbed

& removed

8686

8787

DeexcitationDeexcitation

�� As the electrons falls back to the ground stateAs the electrons falls back to the ground state

–– an energy an energy ““packetpacket”” is emittedis emitted

8888

DeexcitationDeexcitation

Deexcitation causes wavelengths to be emitted

8989

Absorbance of Light

9090

Beer’s Law

Describes the linear relationship between absorbance of light and concentration

cbaA **=

a is the absorptivity in L/g cm

When concentration is molar (mol/L) then the equation is:

bcA ε=

Where epsilon is the molar absorptivity in L/mol cmMolar absorptivity describes the probability of absorbing a photonIt is wavelength dependent

16

9191

BeerBeer’’s Laws Law

Absorption is proportional to concentration

9292

The SpectrophotometerThe Spectrophotometer�� The spectrophotometer is the instrument The spectrophotometer is the instrument used to measure and record the absorption used to measure and record the absorption spectrum of a chemical substance.spectrum of a chemical substance.

�� The components of a spectrophotometer are:The components of a spectrophotometer are:–– A radiation sourceA radiation source

–– A A monochromatormonochromator or frequency selectoror frequency selector

–– A sample holderA sample holder

–– A detector to convert electromagnetic radiation A detector to convert electromagnetic radiation into an electrical signalinto an electrical signal

–– A recorder to produce a record of the signalA recorder to produce a record of the signal

�� Absorption spectra can be done in the visible, Absorption spectra can be done in the visible, ultraviolet (UV) or infrared (IR) regions. ultraviolet (UV) or infrared (IR) regions.

9393

Visible SpectroscopyVisible Spectroscopy

9494

Photodiode Array Spectrometer

No monochromatorall wavelengths at once

Simple FastLimited Resolution

9595

ExampleExample

�� Determination of the wavelength of light Determination of the wavelength of light

absorbed by a sample of grape sodaabsorbed by a sample of grape soda

�� Determination of the amount of dilution of Determination of the amount of dilution of

a sample of grape sodaa sample of grape soda

9696

Absorption of Grape SodaAbsorption of Grape Soda

17

9797

Dilution of Grape SodaDilution of Grape Soda

9898

Typical Organic UV/Vis SpectraTypical Organic UV/Vis Spectra

Note how the same

molecule displays

markedly different

spectral features

depending upon the

environment in which

it finds itself.

9999

Types of LuminescenceTypes of Luminescence

• Fluorescence

– Happens quickly after initial photon absorption (µs to ps lifetime).

• Phosphorescence

– Happens slowly after initial photon absorption (min to ms lifetime).

• Chemiluminescence

– Excitation arises from a chemical reaction, instead of

photoabsorption.

All of these techniques can be used for analytical procedures. Best applied for trace analyses, having detection limits 100

to 1000 times lower than corresponding absorption techniques.

100100

Radiative DecayRadiative Decay

101101

Filter Fluorometer DesignFilter Fluorometer Design

A basic fluorometer compares fluorescence intensity while monitoring incident

light intensity. The most simple instruments use filters to select specific bands,

thus dedicating the instrument to a specific task.

Excitation

SourceFilter

Filter

Attenuator

Sample

Detector

Amplifier

Detector

This unit is the AU-10 Field Fluorometer from

Turner Designs. Different filters allow it to

perform different experiments in the field. For

example, its detection limit for chlorophyll in

water is 30 parts per trillion; for crude oil in water

it is 10 parts per billion.

102102

Spectra of 9,10Spectra of 9,10--diphenylanthracenediphenylanthracene

These spectra were taken with

cyclohexane as the solvent. This

molecule has an extremely high

quantum yield: 0.90. 90% of

absorbed incident photons show

up as fluorescent photons.

Hamal and Hirayama, J. Phys. Chem. 87, 83 (1983).

18

103103

Fluorescence ApplicationsFluorescence Applications

Since fluorescence depends linearly on concentration, it can be used to measure

concentration just like absorption experiments.

Because the fluorescence can be increased by increasing the incident power,

fluorometry can be used to detect very low concentrations – much better than

absorption experiments.

Applied to a species which fluoresces or chemically attach something that does

fluoresce. Fluorescent tags are commonly used in biochemistry.

Choose and excitation and emission wavelength, if known. Otherwise, measure

their spectra and make appropriate selections.

Create a calibration curve for concentration range of interest.

Make measurements and employ statistics the same as with other experiments.

104104

ChemiluminescenceChemiluminescence

Here are two containers of tris(2,2’-bipyridyl)ruthenium (II). Sodium hydroxide

is being added to the one of the left and codeine is being added to the one on the

right. This produces orange light (at 610 nm). The reaction is used to detect and

monitor the presence of certain amines, alkaloids, and oxalates.

Image from Dr. S.W. Lewis of Deakin University.

105105

Chemiluminescence Detector DesignChemiluminescence Detector Design

Chemiluminescence instrumentation is considerably simpler than fluorescence; it

needs no excitation source – the chemical reaction provides the input energy –

and it needs no wavelength selection device. Essentially one only needs a light

detector and a sample holder/introduction system.

This unit is the PMT-FL from FIAlab

Instruments. It has a detection limit of 2 parts

per trillion for fluorescein.

An example application uses a compound called

Luciferase Photinus pyralis. The bioluminescent

reaction of this enzyme with luciferin, ATP, and

O2 results in the emission of light. Luciferase

can be used to detect trace amounts of ATP.

106106

IRIR

�� Probes different molecular vibrations Probes different molecular vibrations

–– absorption occurs when the frequency of the absorption occurs when the frequency of the

IR wave matches a IR wave matches a vibrationalvibrational frequency of frequency of

the molecule the molecule

�� Most molecules have numerous vibrations Most molecules have numerous vibrations

–– bond stretching vibrations bond stretching vibrations

�� detect different kinds of Adetect different kinds of A--B bonds B bonds

–– bond bending involves several atoms at oncebond bending involves several atoms at once

107107

IR RadiationIR Radiation

�� Exposing molecules to the correct Exposing molecules to the correct

frequencies of infrafrequencies of infra--red light will result in red light will result in

some of that light being absorbedsome of that light being absorbed

–– gives that molecule more energygives that molecule more energy

–– results in more energetic motion of the atoms results in more energetic motion of the atoms

in the moleculein the molecule

108108

19

109109

Types of VibrationsTypes of Vibrations

• molecular dipole moment must change during a vibration to be IR active.

• this oscillating dipole interacts with the oscillating E-M field of the photon,

leading to absorption.

+ – + +

–

Stretching Vibrations Bending Vibrations

symmetric anti-symmetric rocking scissoring twisting wagging

In-Plane Out-of-Plane

Changes in bond length Changes in bond angle

110110

Types of VibrationsTypes of Vibrations

111111

Types of VibrationsTypes of Vibrations

112112

IR SpectrophotometerIR Spectrophotometer

113113

Typical SpectraTypical Spectra

• spectra are usually presented as %transmittance against wavenumber. Because

we are plotting transmittance, the spectrum dips down when there is a strong

absorption. This is the opposite when plotting absorbance.

114114

Qualitative AnalysisQualitative Analysis

IR Spectroscopy is widely employed as an identification technique.

• many narrow peaks

• subtle shifts are measurable with good resolution

– analyze wavenumber shifts in specific group

CH stretches 3000 - 2800 cm-1

OH stretches 3600 - 3200 cm-1

CO stretch 1690 - 1760 cm-1

– fingerprint region 1200 - 700 cm-1

– computer search libraries >100,000 spectra

20

115115

Sample PrepSample Prep

116116

Raman SpectroscopyRaman Spectroscopy

Another spectroscopic technique which probes the rotational and vibrational

structure of molecules.

C.V. Raman discovered in 1928; received Nobel Prize in 1931.

Can probe gases, liquids, and solids.

Must use a laser source for excitation.

Resurgence in recent years due to the development of new detectors with

improved sensitivity and diode lasers that are stable and cheap.

117117

How It WorksHow It Works

Excited electronic state

Ground electronic state

0

123

0

123

Virtual electronic states

ExcitationRayleigh

Scattering

Raman

Scattering

Anti

-Sto

kes

Sto

kes

118118

The SpectrumThe Spectrum

A complete Raman spectrum

consists of:

• a Rayleigh scattered peak

(high intensity, same

wavelength as excitation)

• a series of Stokes-shifted

peaks (low intensity, longer

wavelength)

• a series of anti-Stokes shifted

peaks (still lower intensity,

shorter wavelength)

• spectrum independent of

excitation wavelength (488,

632.8, or 1064 nm)Spectrum of CCl4, using an Ar+ laser at 488 nm.

119119

Compare IR and RamanCompare IR and Raman

Spectra of PETN explosive. 120120

Some Raman AdvantagesSome Raman Advantages

Here are some reasons why someone would prefer to use Raman Spectroscopy.

• Non-destructive to samples (minimal sample prep)

• Higher temperature studies possible (don’t care about IR radiation)

• Easily examine low wavenumber region: 100 cm-1 readily achieved.

• Better microscopy; using visible light so can focus more tightly.

• Easy sample prep: water is an excellent solvent for Raman. Can probe sample

through transparent containers (glass or plastic bag).

21

121121

SourcesSources

Raman intensity is weak and the excitation source must be strong to generate

sufficient signal.

Source must be monochromatic so that spectrum is sufficiently uncomplicated.

Intense lamps work, but when monochromatized, have very little power.

Scattering efficiency increases as ν4: the bluer the light, the more the scattering.

The bluer the light, the greater the chance of producing fluorescence.

Lasers are used almost exclusively.

Ar+ Ion: 488.0 and 514.5 nm

Kr+ Ion: 530.9 and 647.1 nm

He:Ne: 632.8 nm

Diode Lasers: 782 and 830 nm

Nd: YAG: 1064 (532 when doubled) nmHere is a 500 mW Ar ion laser

122122

SourcesSources

Experiment used to require considerable excitation power

Ion lasers, 40 W cw

He:Ne, 10 W cw

YAG, 1 J/10 ns pulse (100 MW average pulse)

But detectors have improved so much, the source power requirements have been

decreased.

Diode laser, 25 mW

other lasers can be made correspondingly smaller.

123123

Multichannel Raman SpectroscopyMultichannel Raman Spectroscopy

124124

Chemical MappingChemical Mapping

Focus laser to small spot. Tune spectrometer to particular Raman transition peak.

Raster scan the sample under the laser beam, record intensity changes. Resultant

map correlates with substance. Acquire an entire spectrum at every point, then

choose the feature with which to key the image.

Motorized stage from Renishaw

for chemical mapping.

This is a drug tablet.

The yellow

corresponds to the

active ingredient.

Particles are in the

10’s of µm range.

125125

Chemical ImagingChemical Imaging

Mixture of cocaine and sugar. Bright spots are cocaine. 126126

Applications Applications -- Art RestorationArt Restoration

This12 century fresco on a church wall in

Italy needed to be restored. What paints to

use?

Raman analysis clearly identified the paints

and pigments that were originally present,

permitting a correct choice of cleaning

materials and subsequent repainting to

restore its original condition.

22

127127

Applications Applications -- Paint ChipsPaint Chips

Forensic analysis of paint chips in vehicle accidents. Often multiple layers. Can

analyze with IR by stripping successive layers. Image edge with microRaman.

Layers 1 and 3 turned out to be rutile phase TiO2 - a

white paint. Layer 2 was a Goethite, a red pigment

and corrosion inhibitor. Layer 4 was molybdate

orange, a common red paint in the 70’s in North

America and still used in the U.K. today. Layer 5

was a silicate based paint. Data arising from a case

investigated by LAPD.

128128

Applications Applications -- Gem ForgeryGem Forgery

In 1999 a new process was developed – called GE POL – whereby brown type IIa

diamonds could be treated to become indistinguishable from naturally clear

diamonds. Raman presented way to distinguish them.

Naturally clear diamondOriginally brown diamond

129129

Applications Applications -- Bullet Proof GlassBullet Proof Glass

Identify poly(carbonate) from poly(methylmethacrylate).

Both used for shatter-proof glass

130130

Applications Applications -- Sunscreen FormulationsSunscreen Formulations

Here are the spectra of 5 common

sunscreen ingredients. Raman is able to

determine from a spectrum on the arm

the nature of the sunscreen being used.

A: ODPABA (octyl N,N-dimethyl-p-

aminobenzoic acid)

B: OMC (octyl p-methoxycinnamate)

C: BZ3 (oxybenzone)

D: OCS (octyl salicylate)

E: DBM (dibenzoylmethane)

G.R. Luppnow et

al., J. Raman. Spec.

34, 743 (2003).