Essential Parts of a Chromatograph

Chromatography is a physical process by which mixtures are separated into their component compounds.
The apparatus used to perform this separation is called a chromatograph. The physical data obtained from the apparatus is called a chromatogram.

A stationary phase
Solids (paper, silica gel, alumina, etc.) Viscous liquids (waxes)

A mobile phase
Liquids (water, methanol, ethanol, etc.) Gases (helium, argon, dinitrogen (N2), etc.)

Scientific Basis of Chromatography

Chromatographic separation of the components of a mixture is based on the difference in affinity of each component for the stationary phase of the chromatograph.

Differences in Affinity for the Stationary Phase

Differences in affinity of different molecules for the stationary phase are due to differences in-----size of the molecules, ---shape of the molecules, ---functional groups on the molecules.

Types of Chromatography
Paper Chromatography Thin-layer chromatography (TLC) High-performance liquid chromatography (HPLC) Gas Chromatography (GC)

Paper Chromatography: A Demonstration

Stationary Phase: a solid (cellulose) Mobile Phase: a liquid (methanol) Demonstration: separation of the components of ballpoint pen inks and tentative identification of the pen that produced the ink spot held as evidence.

Chromatographic Identification of Compounds

Chromatography separates mixtures into components. Chromatography does not identify these components. Identification of the components of a mixture can be accomplished by running standards (compounds of known identity) or controls (substances of known origin) in parallel with the unknown mixture. Components of the mixture are then identified by comparison.

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

Mechanism of Separation of Components of a Mixture by Chromatography

http://chemsite.lsrhs.net/a_Intro/chromatography.html

Simulation

Mechanism of Separation of Components of a Mixture by Column Chromatography

Mechanism of Separation of Components of a Mixture by Column Chromatography

Mechanism of Separation of Components of a Mixture by Column Chromatography

Mechanism of Separation of Components of a Mixture by Column Chromatography

Mechanism of Separation of Components of a Mixture by Column Chromatography

Mechanism of Separation of Components of a Mixture by Gas Chromatography

Thin Layer Chromatography

Thin Layer Chromatography = TLC Stationary Phase: solid (alumina or silica gel). Mobile Phase: a solvent (any liquid that does not dissolve the stationary phase).

Thin Layer Chromatography

The TLC plate is first spotted with a solution containing the substance to be analyzed. The chromatogram is developed with a solvent. Rf values for the component spots are compared to Rf values of spots produced by controls to guess at identity of the components.

Liquid Chromatography
Liquid Chromatography = LC Stationary Phase = insoluble solids Mobile Phase = a solvent(any liquid that does not dissolve the stationary phase) Types depend on the nature of the stationary phase:
Packed column capillary column High performance (HPLC)

Gas Chromatography
Gas Chromatography = GC Stationary Phase: solids or viscous liquids (waxes) Mobile Phase: inert gases (He is often used as the carrier gas.) Types depend on the nature of the stationary phase:
packed column capillary column

The Basic Gas Chromatograph

This figure represents the internal construction of conventional gas chromatographs. A liquid chromatograph is constructed by substituting a liquid reservoir and pump for the carrier gas cylinder. Retention time is measured for each component (the time from the point of injection to the point at which its maximum occurs on the chromatogram.

Gas Chromatograms of an Evidence Sample and a Control Sample

The top chromatogram is of an evidence sample. The bottom chromatogram is of a standard mixture. Comparison of the evidence sample to the control mixture shows that the evidence sample contains pentobarbital and secobarbital and a contaminant that is not one of the barbiturates.

Injection

Time since injection

Pyrograms
The method is used when the substance to be identified is non-volatile and insoluble in common solvents. Gas chromatography is done on the thermal decomposition products of the compound (pyrolysis products) rather than the compound itself. The substance itself in said to have been pyrolyzed or to have undergone pyrolysis.

Pyrogram of Auto Paint

Automobile paint is not volatile and is of limited solubility in almost all useful LC solvents. However, automobile paint may be pyrolyzed. The pyrogram of an automobile paint acts as a sort of a fingerprint, to allow identification of the type and source of the paint.

Gel Electrophoresis
Gel Electrophoresis - Separation of ionic substances based upon motion of charged particles (ions) in an electric field. Molecules that form ions:

Gel Electrophoresis Mass Spectrometry Spectrophotometry

Acids: -C-C=O O-H Bases: -C-N-H + H+ H

-C-C=O + H+ O1H+ -C-N-H H

Separation of Ions by Gel Electrophoresis

Control Sample #1 Evidence Sample Control Sample #2

Separation of Ions by Gel Electrophoresis

Separation of Ions by Gel Electrophoresis

Separation of Ions by Gel Electrophoresis

Mass Spectrometry (MS)

Mass spectrometry is an identification method. It is used with Gas Chromatography (GC-MS). Steps of GC-MS separation and identification:
Separate components of the mixture (GC) Fragment single components into ions Separate ionic fragments by mass/charge ratio Display fragment mass/charge ratios

Mass Spectrometry (MS)

Every different compound has its own fragmentation pattern. Compare fragmentation pattern of unknown compound to those of known compounds to find the identity of the unknown compound (This is usually done by having the instrument search a computer database of known compounds!). Method of identification of the unknown compound will stand up in court!

Mass Spectrometry (MS)

Every different compound has its own fragmentation pattern. Compare fragmentation pattern of unknown compound to those of known compounds to find the identity of the unknown compound (This is usually done by having the instrument search a computer database of known compounds!). Method of identification of the unknown compound will stand up in court!

Mass Spectrometry (MS)

Mass Spectrometry (MS)

Mass Spectrometry (MS)

Mass Spectrometry (MS)

Mass Spectrometry (MS)

Atomic and Molecular Mass: the Molecular Mass of CH3NH2

Atomic Masses:
H C N O 1 amu (atomic mass unit) 12 amu 14 amu 16 amu

Fragmentation of CH3NH2
Molecule CH3NH2 Fragment CH3NH+ CH2NH2+ CH3+ NH2+ H+ 15
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Molecular Mass: the sum of all atoms atomic masses M =1(12) + 3(1) + 1(14) + 2(1) = 31 amu/molecule
for CH3NH2.

Mass(amu) 30 30 15 16 1 30
30 40

1
0

16
20 Mass Spectrum

GC-MS: A Separation and Identification Method

Internal Construction of a Gas Chromatograph-Mass Spectrometer

Inject Fragment and Ionize Separate Fragment Ions

Time since injection. Separation!

Mass spectrum of each Component of the mixture. Identification!

Separate Components Of the Mixture

Display Spectrum Detect Fragment Ions

Mass Spectra of Heroin and Cocaine

Spectrophotometry
Spectrophotometry - Analysis of samples by measuring the absorption of light by that sample. Spectrophotometer a device for measuring the percentage of light absorbed by a sample as a function of the wavelength or frequency of the light within some specific region of the spectrum. Spectrophotometry is a method of identifying and measuring the quantity of a pure chemical compound.

General construction of a Spectrophometer

Sample absorbs some light Displays amount of light absorbed versus wavelength of the light

Light: Wave/particle duality

Light as a wave form: electromagnetic radiation, or crossed, fluctuating, electric and magnetic fields. Light as a particle: a classical particle having no mass and annihilated in or produced by one interaction with matter (a zero rest-mass particle).

Detects amount of light absorbed Selects the color of light to be used Separates white light into a spectrum of light

Light: Wave Properties

Properties: = wavelength of light f = frequency of oscillation of light c = speed of light = 3.00 x 108 m/s = energy of light Relationships: c = f and = hf or = hc/ h = 6.626 x 10-34 Js (Plancks constant)

Wave Properties of Light: Wavelength and Frequency

Absorption of Light by Matter: Beers Law

Beers Law: the extent to which light is absorbed by matter is proportional to the concentration of the absorbing unit of the matter. A = kC where A = absorbance k = proportionality constant C = concentration

UV/Visible Spectrum of Heroin

The wavelength of The maximum is Characteristic of heroin

Concentration is Proportional to Absorbance at this maximum

Regions of the Electromagnetic Spectrum and Absorption Processes

Region Radio Microwave Infrared Visible Ultraviolet X-Ray Gamma Rays Molecular Process nuclear spin rotation vibration electronic electronic, bond dissociation bond dissociation, ionization ionization

Infrared Spectra of the Xylenes

Ortho-xylene

Meta-xylene

Para-xylene

Inorganic Analysis versus Organic Analysis

Organic Analysis analysis of molecular compounds that contain covalently bonded nonmetallic atoms. Chapter 6 Criminalistics: An Introduction to Forensic Science Inorganic Analysis analysis of elemental substances containing atoms and ionic compounds containing both metal ions and nonmetal ions.

Atomic Spectroscopy
Emission Spectroscopy
Flame emission Arc emission X-ray emission (fluorescence)

Atomic Spectroscopy Neutron Activation Analysis X-ray Diffraction

Absorption Spectroscopy
Flame absorption Arc (plasma) absorption

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Flame Emission by Elements

Arc Emission by Elements

Li Lithium

Na Sodium

Cu Copper

Ne Neon

Ar Argon

Hg Mercury

Arc and Flame Emission Spectra

White Light

Schematic of an Emission Spectrograph

Na H

Ca Hg
400 500 600 700

Arc Emission

Emission Spectrum

Wavelength, nm

Emission Spectrometers vs. Absorption Spectrometers

Schematic of a Flame Absorption Spectrophotometer

Vaporized sample in the flame. Sample absorbs red light. No sample in the flame. No light absorbed.

Emission

Sample is in the flame.

No sample in the flame.

Absorption

Sample dissolved in a solvent

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The Energy of Emitted or Absorbed Light

The light absorbed or emitted by atoms must have an energy equal to the difference between the final electronic state and the initial electronic state.
1H

Neutron Activation Analysis

1. The sample to be analyzed is bombarded with neutrons and energy-rich isotopes form. 23Na + n0 24Na* 2. 3. The energy-rich isotopes slowly decay to stable isotopes by emission of gamma ray photons. 24Na* 24Na + -ray photon 4. 5. The gamma ray photon spectrum of the irradiated sample is measured.

1s1

2s1

1H*

2s1
higher

1s1
lower Light out

lower higher Light in

Electron jumps to higher state Absorption of Light

Electron falls to lower state Emission of Light

Neutron Activation Analysis: Gamma Ray Spectrometer

Neutron Activation Analysis: Gamma Ray Spectrum

Neutron Activation Analysis

6. The wavelengths of the gamma ray photons emitted by the sample indicate which elements are present in the sample. Each isotope of every element has its own gamma ray emission. 7. The intensity (peak height) of the emission line for each element indicates how much of that element is present in the sample.

X-Ray Diffraction
X-ray diffraction is a method for identifying pure crystalline solids (elements, compounds or minerals). X-rays are diffracted by the regular pattern of atoms or ions in crystalline solids. Diffraction patterns are specific to the particular atom or ion packing arrangement of the crystalline solid.

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X-ray Crystallography

X-Ray Powder Camera and Powder Pattern

Reflections occur off parallel planes of atoms. Note the large number of different planes that exist!

X-ray Diffraction Patterns for Potassium Nitrate and Chlorate

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