Unit 7 Chromatography

UNIVERSITY OF TECHNOLOGY, TECHNOLOGY, JAMAICA FACULTY OF HEALTH AND APPLIED SCIENCES

Laboratory Instrumentation Instrumentation Unit 7: Chromatography

7.1

What is chromatography?

A broad range of physical methods used to separate and/or analyse complex mixtures. The components in a mixture are distributed between two phases: a stationary phase bed and a mobile phase which percolates through the stationary bed. The method relies on differences in partitioning behaviour between the flowing mobile phase and the stationary phase to separate the components in a mixture.

A column (or other support for TLC) holds the stationary phase and the mobile phase carries the sample through it. Sample components that partition strongly into the stationary phase spend a greater amount of time in the column and are separated from components that stay predominantly in the mobile phase and pass through the column faster.

Applications Wide range of applications include: Qualitative analysis

• Used as criteria of purity for organic compounds – contaminants if present are revealed by the appearance of additional peaks •

Used to evaluate the effectiveness of purification procedures

•

Retention times or volumes are employed for qualitative identification

Quantitative analysis

•

peak areas or heights provide quantitative information

•

Industrial- PAH, aromatics, pesticides

•

Pharmaceutical/therapeutic drug testing

•

Trace analysis in biological and environmental samples

• Quantification of aromatic, molecules present at trace concentrations in biological and environmental samples • Extended to a wide variety of organic and inorganic compounds via chemical labelling and derivatization procedures

Factors that affect analyte separation and analysis of mixtures: •

Compound volatility

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Contamination of mobile phase

•

Gas flow rate

•

Oven temperature programme

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Stationary phase:

Stationary phase

Trade name

Maximum temperature

Common applications

Polydimethyl siloxane

HP 1/ OV 1

350

Non polar phase; hydrocarbons; polynuclear  aromatics; drugs; steroids;

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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PCBs Stationary phase

Trade name

Maximum temperature

Common applications

Poly (phenylmethyldimethyl) siloxane (10% phenyl)

HP 3 / OV 3

350

Fatty acid methyl esters; alkaloids; drugs; halogenated compounds

Poly (phenylmethyl) siloxane (50% phenyl)

HP17/ OV17

250

Drugs; steroids; pesticides; glycols

Poly (trifluoropropyldimethyl) siloxane

OV 210

200

Chlorinated aromatics; nitroaromatics; alkylsubstituted benzenes

Polyethylene glycol

Carbowax 20 M

250

Free acids; alcohols; ethers; essential oils; glycols

7.3

Classification of Column Chromatographic Methods

General Classification

Specific Method

Gas Chromatography (GC)

1. Gas-liquid (GLC)

2. Gas-solid Liquid Chromatography (LC)

Stationary Phase Liquid adsorbed or  bonded to a solid surface -

Solid

1. Liquid-liquid or  partition

Liquid adsorbed or  bonded to a solid surface

2. Liquid-solid or  adsorption

-

Solid

-

Ion exchange resin

3. Ion exchange 4. Size Exclusion 5. Affinity

Liquid in interstices of a polymeric solid Group specific liquid bonded to a solid surface

Supercritical Fluid Chromatography (SFC)

7.3

Organic species bonded to a solid surface

Types of Chromatography Methods

(source: http://www.rpi.edu/dept/chem-eng/Biotech-Environ/CHROMO/be_types.htm )

• o

Adsorption Chromatography one of the oldest types of  chromatography. utilizes a mobile or gaseous phase that is adsorbed onto the surface of a stationary solid phase. o

The equilibration between the mobile and stationary phases accounts for the separation of different solutes. o

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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• o

o

Based on a thin film formed on the surface of a solid support by a liquid stationary phase. Solute equilibrates between the mobile phase and the stationary liquid.

•

Ion Exchange Chromatography

o

A resin (solid stationary phase) is used to covalently attach anions or cations onto it.

o

• o

o

o

o

• o

o

o

7.4

Partition Chromatography

Solute ions of the opposite charge in the mobile phase are attracted to the resin by electrostatic forces.

Molecular Exclusion Chromatography Also known as gel permeation or gel filtration. This type lacks an attractive interaction between the stationary phase and the solute. The liquid or gaseous phase passes through a porous gel which separates molecules by size. The pores are small and exclude larger  solute molecules, causing the larger  molecules to pass through the column at a faster rate than the smaller ones. Affinity Chromatography The most selective type of  chromatography. Utilizes the specific interaction between one kind of solute molecule and a second molecule that is immobilized on a stationary phase. The specific solute molecule is bound to the stationary phase and later  extracted by changing ion strength or  pH.

Basis of Chromatography

The Mobile and Stationary Phases • The mobile phase is comprised of a solvent into which the sample is injected. The solvent and sample flow through the column together; thus the mobile phase is often referred to as the "carrier fluid." • The stationary phase is the material in the column for which the components to be separated have varying affinities. Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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• The materials which comprise the mobile and stationary phases depend on the general type of chromatographic process being performed.

The Column

• Most modern applications of chromatography employ a column. This is where the separation takes place. • Usually a glass or metal tube of sufficient strength to withstand the pressures applied across it. • Contains the stationary phase. The mobile phase runs through the column and is adsorbed onto the stationary phase. Flow Meter  Detector 

C O L U M N

Oven

Feed Injection Pump Solvent Tank

Basic Layout of a Chromatograph

Gas Chromatography •

The mobile phase is generally an inert gas.

• The stationary phase is generally an adsorbent or liquid distributed over the surface of a porous, inert support.

Liquid Chromatography • The mobile phase is a liquid of low viscosity which flows through the stationary phase bed. This bed may be comprised of an immiscible liquid coated onto a porous support, a thin film of liquid phase bonded to the surface of a sorbent, or a sorbent of controlled pore size.

7.5

Theory of GC and HPLC

Gas Chromatography (GC)

• Gas chromatography involves a sample being vapourised and injected onto the head of  a chromatographic column. The sample is transported through the column by the flow of  inert, gaseous mobile phase. • In GLC, the column itself contains a liquid stationary phase which is adsorbed onto the surface of an inert solid.

Schematic of a gas chromatograph

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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Carrier Gas

• The carrier gas must be chemical inert. Commonly used gases include N, He, Ar and CO2. The choice of carrier gas often depends on type of detector used. •

Carrier gas system also contains a molecular sieve to remove water and impurities.

Sample Injection Port

• For optimum column efficiency, the sample should not be too large and should be introduced onto the column as a “plug” of vapour – slow injection of large samples leads to band broadening and loss of resolution.

vs. • Most common injection method is where a microsyringe is used to inject sample through a rubber septum into a flash vapouriser port at head of column. Temperature of port usually ~50° > BP of least volatile sample. •

Sample size for packed columns 0.1 – 20 µ L. Capillary columns ~10 -3 µ L.

• Injector contains a heated chamber – sample vapourises to form a mixture of carrier gas, vapourised solvent and vapourised solutes:

Columns Packed Bed Columns

•

Comprised of a finely divided, inert, solid support material coated with liquid stationary phase (GLC). This stationary phase completely fills the column.

•

1.5 – 10 m long; internal diameter of 2 – 4 mm.

Capillary or Open Tubular Columns

•

The liquid stationary phase is a thin film or layer on the column wall. There is a passageway through the centre of the column.

•

Internal diameter < 1 mm.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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Column temperature

•

For precise work, column temperature must be controlled to within 0.1 °.

• The optimum column temperature depends on the boiling point of the sample. As a rule of thumb, a temperature slightly above the average boiling point of the sample results in an elution time of 2 – 30 minutes. Minimal temperatures give good resolution, but increase elution times. • If a sample has a wide boiling range, then temperature programming can be useful. The column temperature is increased (either continuously or in steps) as separation proceeds.

Detectors

•

Different detectors will give different types of selectivity: o

A non-selective detector responds to all compounds except the carrier gas.

A selective detector responds to a range of compounds with a common physical or chemical property. o

o

A specific detector responds to a single chemical compound.

• Detectors can also be grouped into concentration dependant detectors and mass flow  dependant detectors: The signal from a concentration dependant detector is related to the concentration of solute in the detector, and does not usually destroy the sample. o

Mass flow dependant detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector. o

Detectors must be able to respond quickly to low solute concentrations (a few ppt) as they are eluted from the column. Other properties include: •

Linear response

•

Stability

• Uniform response to wide variety of species or predictable responses to one or more classes of chemicals.

No one detector possesses all the desirable properties but the three most popular are: •

Flame ionisation detectors

•

Thermal conductivity detectors

•

Electron capture detectors

GC may also be coupled with other methods such as mass spectrometry (GC-MS) and infrared spectrometry (GC-IR).

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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High-Performance Liquid Chromatography (HPLC)

In early liquid chromatography, separation times were long – several hours. Column packings (solid coated with thin liquid film) were 50 – 500 cm in length, with internal diameter of 10 – 50 mm and particle sizes >150 – 200 µ m. For increased column efficiency, decrease particle size (decrease plate height or increase plate count). Since the 1960,s HPLC was developed.

HPLC – most popular of the analytical separation techniques. •

High sensitivity

•

Good adaptability

•

Ease of automation

•

Can be used for non-volatile compounds

Different methods are used depending on the nature of solutes to be separated:

• High molecular mass compounds (>10,000 g mol -1) utilise size-exclusion chromatography. •

Low molecular mass ionics utilise ion-exchange or reverse-phase chromatography.

•

Non-polar species utilise adsorption chromatography.

Instrumentation

•

Pumping pressures of several hundred atmospheres are employed.

•

Column particle sizes ≤ 10 µ m.

•

The instruments are elaborate and expensive.

Mobile-Phase Reservoirs and Solvent Treatment Systems •

One or more mobile-phase reservoirs used.

•

The treatment systems remove bubbles and particulates.

• Sparging is a process in which dissolved gases are swept out of a solvent by bubbles of  an inert, insoluble gas. •

Isocratic (simple) or gradient elution is used.

Information provided here was compiled from various sources by Dr. D. Gordon-Smith and Dr. K. Bartley-Hynes (University of Technology, Jamaica)

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Pumping Systems A pump should have the following characteristics: •

Pressures up to 6000 psi

•

Pulse-free output

•

Flow rates 0.1 – 10 mL/min

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Good flow reproducibilities

•

Resistant to corrosion by a variety of solvents

Two types of pumps used: mechanical or pneumatic pumps. NOTE: High pressures are not an explosion hazard because liquids are not very compressible. If there is a rupture in a component, leading to leakage then there is a fire hazard.

Sample Injection Systems • Syringe injection through a septum is used. However, it is are not very reproducible and used for pressures