Gas Chromatography

Gas Chromatography

an analytical separations technique useful for separating volatile organic compounds

consists of : – Flowing mobile phase (inert gas - Ar, Ne, N) – Injection port ( rubber septum - syringe injects

sample)• kept at a higher temperature than the boiling point

Principles

Separation due to differences in partitioning behavior

selective retardation

Key Information organic compounds separated due to

differences in their participating behavior between the mobile gas phase and the stationary phase in the column

in contrast to other types of chromatography, the mobile phase does not interact with molecules of the analyte; its only function is to transport the analyte through the column

Gas Chromatography

– Separation column containing stationary phase • since partitioning behavior independent of

temperature - kept in thermostat - controlled oven

– Detector

Schematic of a gas Chromatograph

The Beginning

concept of GC announced in 1941 by Martin and Synge (also did liquid partition chromatography)

10+ years later GC used experimentally 1955, first commercial apparatus for GC

appeared on the market

Today

estimate : 200, 000 gas chromatographs are currently used through out the world.

30+ instrument manufactures 130 different models cost 1,500 to 40,000 dollars improvements: computers- automatic control

open tubular columns-separate a multitude of analytes in relatively short times

Uses of Gas Chromatography

Determination of volatile compounds (gases & liquids)

Determination of partition coefficients and absorption isotherms

Isolating pure components from complex mixtures

Instrumentation

Instrumentation

flowing mobile phase injection port separation column detector

GC detectors

another powerpoint

Liquid Chromatography much slower diffusion in liquid as compared to gas

Liquid liquid extraction repeated extraction is basis for LC

Retardation of solutes in liquid onto a solid phase

Elution chromatographyElution chromatography

Increasing polarity of pure solvents

hexane ether acetone methanol water acetic acid

Solvents mixed %hexane and %

methanol miscible can be mixed

continuously (solvent programming)

Types of Liquid ChromatographyTypes of Liquid Chromatography

Liquid-solid: adsorption on solid which is generally polar (silica gel, alumina, magnesium silicates) or reverse phase (cellulose, poly amides)

Ion exchange: specific interactions with ionic species (change relative strengths of acid or base)

Types of Liquid ChromatographyTypes of Liquid Chromatography

Liquid-liquid: partition between 2 bulk phases (one immobilized) is highly selective

Liquid exclusion: molecular sieve separates molecules on basis of ability to diffuse into immobile support

Retardation based on size of molecule as it diffuses into porous solid

High Performance Liquid Chromatography

Once called High Pressure Liquid Chromatography

What is HPLC? The most widely used analytical separations technique Utilizes a liquid mobile phase to separate components

of mixture uses high pressure to push solvent through the column Popularity:

– sensitivity

– ready adaptability to accurate quantitative determination

– suitability for separating nonvolatile species or thermally fragile ones

HPLC is….

Popularity:– widespread applicability to substances that are of prime

interest to industry, to many fields of science, and to the public

Ideally suited for separation and identification of amino acids, proteins, nucleic acids, hydrocarbons, carbohydrates, pharmaceuticals, pesticides, pigments, antibiotics, steroids, and a variety of other inorganic substances

History lesson Early LC carried out in glass columns

– diameters: 1-5 cm

– lengths: 50-500 cm Size of solid stationary phase

– diameters: 150-200 m Flow rates still low! Separation times long! Eureka! Decrease particle size of packing causes increase in

column efficiency!– diameters 3-10 m

This technology required sophisticated instruments– new method called HPLC

Advantages to HPLC Higher resolution and speed of analysis HPLC columns can be reused without repacking or

regeneration Greater reproducibility due to close control of the

parameters affecting the efficiency of separation Easy automation of instrument operation and data

analysis Adaptability to large-scale, preparative procedures

Advantages to HPLC

Advantages of HPLC are result of 2 major advances:– stationary supports with very small particle sizes and

large surface areas

– appliance of high pressure to solvent flow

Schematic of liquid chromatograph

LC column

LC injector

Types of HPLC

Liquid-solid (adsorption) chromatography Liquid-liquid (partition) chromatography Ion-exchange chromatography Size exclusion chromatography

Partition Chromatography

Most widely used Bonded-phase Chromatography Silica Stationary Phase: OH OH OH OH O O O Si Si Si Si Siloxanes: O CH3

Si O Si R R= C8, C18

O CH3

Partition Chromatography II

Reverse Phase Chromatography– Nonpolar Stationary Phase

– Polar Mobile Phase

Normal Phase Chromatography– Polar Stationary Phase

– Nonpolar Mobile Phase

Column Selection Mobile-Phase Selection

Partition Chromatography III

Research Applications– Parathion in Insecticides: O

– CH3CH2O P O NO2

CH3CH2O

– Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa

Adsorption Chromatography

Classic Solvent Selection Non-polar Isomeric Mixtures Advantages/ Disadvantages Applications

What is Ion Chromatography?

Modern methods of separating and determining ions based on ion-exchange resins

Mid 1970s Anion or cation mixtures readily resolved on HPLC

column Applied to a variety of organic & biochemical systems

including drugs, their metabolites, serums, food preservatives, vitamin mixtures, sugars, pharmaceutical preparations

The Mobile Phases are...

Aqueous solutions

– containing methanol, water-miscible organic solvents

– also contain ionic species, in the form of a buffer

– solvent strength & selectivity are determined by kind and concentration of added ingredients

– ions in this phase compete with analyte ions for the active site in the packing

Properties of the Mobile Phase

Must– dissolve the sample

– have a strong solvent strength leads to reasonable retention times

– interact with solutes in such a way as to lead to selectivity

Ion-Exchange Packings

Types of packings– pellicular bead packing

• large (30-40 µm) nonporous, spherical, glass, polymer bead

• coated with synthetic ion-exchange resin

• sample capacity of these particles is less

– coating porous microparticles of silica with a thin film of the exchanger

• faster diffusion leads to enhanced efficiency

Ion-Exchange Equilibria

Exchange equilibria between ions in solution and ions on the surface of an insoluble, high molecular-weight solid

Cation exchange resins– sulfonic acid group, carboxylic acid group

Anion exchange resins– quaternary amine group, primary amine group

CM CelluloseCation Exchanger

DEAE CelluloseAnion Exchanger

Eluent Suppressor Technique

Made possible the conductometric detection of eluted ions.

Introduction of a eluent suppressor column immediately following the ion-exchange column.

Suppressor column– packed with a second ion-exchange resin

Cation analysis Anion analysis

Size Exclusion Chromatography(SEC) Gel permeation(GPC), gel filtration(GFC)

chromatography Technique applicable to separation of high-molecular

weight species Rapid determination of the molecular weight or

molecular-weight distribution of larger polymers or natural products

Solute and solvent molecules can diffuse into pores -- trapped and removed from the flow of the mobile phase

Specific pore sizes.average residence time in the pores depends on the effective size of the analyte molecules– larger molecules

– smaller molecules

– intermediate size molecules

SEC(continued)

SEC Column Packing

Small (~10 µm) silica or polymer particles containing a network of uniform pores

Two types (diameters of 5 ~ 10 µm)– Polymer beads

– silica-based particles

Advantages of Size Exclusion Chromatography Short & well-defined separation times Narrow bands--> good sensitivity Freedom from sample loss, solutes do not interact

with the stationary phase Absence of column deactivation brought about by

interaction of solute with the packing

Disadvantages

Only limited number of bands can be accommodated because the time scale of the chromatogram is short

Inapplicability to samples of similar size, such as isomers. – At least 10% difference in molecular weight is required

for reasonable resolution

Instrumentation

Instruments required:– Mobile phase reservoir

– Pump

– Injector

– Column

– Detector

– Data system

Schematic of liquid chromatograph

Mobile phase reservoir

Glass/stainless steel reservoir Removal of dissolved gases by degassers

– vacuum pumping system

– heating/stirring of solvents

– sparging

– vacuum filtration

Elution methods

Isocratic elution– single solvent of constant composition

Gradient elution– 2 or more solvents of differing polarity used

Pumping System I

Provide a continuous constant flow of the solvent through the injector

Requirements– pressure outputs up to 6000 psi

– pulse-free output

– flow rates ranging from .1-10 mL/min

– flow control and flow reproducibility of .5% or better

– corrosion-resistant components

Pumping System II

Two types:– constant-pressure

– constant-flow Reciprocating pumps

– motor-driven piston

– disadvantage: pulsed flow creates noise

– advantages: small internal volume (35-400 L), high output pressures (up to 10,000 psi), ready adaptability to gradient elution, constant flow rates

Pumping System III

Displacement pumps– syringe-like chambers activated by screw-driven

mechanism powered by a stepper motor

– advantages: output is pulse free

– disadvantage: limited solvent capacity (~20 mL) and inconvenience when solvents need to be changed

Flow control and programming system– computer-controlled devices

– measure flow rate

– increase/decrease speed of pump motor

Sample Injection Systems

For injecting the solvent through the column Minimize possible flow disturbances Limiting factor in precision of liquid chromatographic measurement Volumes must be small .1-500 L Sampling loops

– interchangeable loops (5-500 L at pressures up to 7000 psi)

LC column

LC injector

Liquid Chromatographic Column

Smooth-bore stainless steel or heavy-walled glass tubing

Hundreds of packed columns differing in size and packing are available from manufacturers ($200-$500)

Add columns together to increase length

Liquid Chromatographic Columns II Column thermostats

– maintaining column temperatures constant to a few tenths degree centigrade

– column heaters control column temperatures (from ambient to 150oC)

– columns fitted with water jackets fed from a constant temperature bath

Detector

Mostly optical Equipped with a flow cell Focus light beam at the center for

maximum energy transmission Cell ensures that the separated

bands do not widen

Some Properties of Detector

Adequate sensitivity Stability and reproducibility Wide linear dynamic range Short response time Minimum volume for reducing zone broadening

More Properties of Detector

High reliability and ease of use Similarity in response toward all analytes Selective response toward one or more classes of

analytes Non-destructive

Types of Detector

Refractive index UV/Visible Fluorescence Conductivity Evaporative light scattering Electrochemical

Refractive Index I

Measure displacement of beam with respect to photosensitive surface of dectector

Refractive Index II

Advantages– universal respond to nearly all solutes

– reliable

– unaffected by flow rate

– low sensitive to dirt and air bubbles in the flow cell

Refractive Index III

Disadvantages– expensive

– highly temperature sensitive

– moderate sensitivity

– cannot be used with gradient elution

UV/Visible I

Mercury lamp = 254nm = 250, 313, 334 and 365nm with filters Photocell measures absorbance Modern UV detector has filter wheels for rapidly

switching filters; used for repetitive and quantitative analysis

UV/Visible II

UV/Visible III

Advantages– high sensitivity

– small sample volume required

– linearity over wide concentration ranges

– can be used with gradient elution

UV/Visible IV

Disadvantage– does not work with compounds that do not absorb light

at this wavelength region

Fluorescence I

For compounds having natural fluorescing capability

Fluorescence observed by photoelectric detector

Mercury or Xenon source with grating monochromator to isolate fluorescent radiation

Fluorescence II

Advantages– extremely high sensitivity

– high selectivity

Disadvantage– may not yield linear response over wide range of

concentrations

Conductivity

Measure conductivity of column effluent

Sample indicated by change in conductivity

Best in ion-exchange chromatography

Cell instability

Evaporative Light Scattering I

Nebulizer converts eluent into mist Evaporation of mobile phase leads to formation of

fine analyte particles Particles passed through laser beam; scattered

radiation detected at right angles by silicon photodiode

Similar response for all nonvolatile solutes Good sensitivity

Evaporative Light Scattering II

Electrochemical I

Based on reduction or oxidation of the eluting compound at a suitable electrode and measurement of resulting current

Electrochemical II

Advantages– high sensitivity

– ease of use

Disadvantages– mobile phase must be made conductive

– mobile phase must be purified from oxygen, metal contamination, halides

Data System

For better accuracy and precision Routine analysis

– pre-programmed computing integrator Data station/computer needed for higher control levels

– add automation options

– complex data becomes more feasible

– software safeguard prevents misuse of data system

Electrophoresis…charged species migrate in electric fieldSeparation based on charge or mobility

Capillary electrophoresishigher voltages can be used as the heat can be dissipated

Capillary electrophoresis