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

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

Key Information

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 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 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 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

Outline
What is HPLC?
– An Overview

Types of HPLC
– – – – Partition Chromatography Adsorption Chromatography Ion Chromatography Size-Exclusion 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 µ m

Flow rates still low! Separation times long! Eureka! Decrease particle size of packing causes increase in column efficiency!
– diameters 3-10 µ 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

Liquid chromatography
Instrumentation
– Mobile Phase Reservoir – Pumping Systems – Sample Injection Systems – Liquid-Chromatographic Columns – Detectors

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 O Si R CH3 R= C8, C18

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 CH3CH2O – Cocaine in Fruit Flies: A Study of Neurotransmission by Prof. Jay Hirsh, UVa

NO2

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 Cellulose Cation Exchanger

DEAE Cellulose Anion 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 molecularweight 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

SEC(continued)
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 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 µ 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 µ L Sampling loops
– interchangeable loops (5-500 µ 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 λ = 254nm λ = 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 field Separation based on charge or mobility

Capillary electrophoresis higher voltages can be used as the heat can be dissipated

Capillary electrophoresis