ChromatographyChapter 4

Best Broken into four categories

Theoretical Background

Gas Chromatography

HPLC

Quantitation, Calibration,

Standardisation and Validation

TheoryReview of PartitioningYou need to be aware of the following concepts in order to have any idea about this chapter!

Partitioning between two liquids (aqueous/organic)

Why does the analyte partition?o Dynamic Process – Constant exchange at the interfaceo Partition Coefficientso Hydrophobic and Polar Functional Groupso Ions and Solvationo Influence through pH – changes ionisation state of

molecule

DefinitionsChromatographyphysical method of separation in which the components to be separated are distributed between two phases, one of which is stationary (stationary phase) while the other (the mobile phase) moves in a definite direction.

Stationary Phaseone of the two phases forming a chromatographic system. It may be a solid, a gel or a liquid. If a liquid, it may be distributed on a solid. The liquid may also be chemically bonded to the solid (Bonded Phase) or immobilized onto it (Immobilized Phase).

Mobile Phasefluid which percolates through or along the stationary bed, in a definite direction. It may be a liquid (Liquid Chromatography) or a gas (Gas Chromatography) or a supercritical fluid (Supercritical-Fluid Chromatography). In gas chromatography the expression Carrier Gas may be used for the mobile phase. In elution chromatography the expression Eluent is also used for the mobile phase.

Chromatographic Process

Solid – Liquid Interface

ChromatogramsCompound elution as a function of time

Each component is characterised by its retention time at peak maximum tr

In constant mobile phase tr can be converted into retention volume Vr

Vr = Fvtr where Fv is flow rate

Chromatogram

tr Retention timet0 Hold-up time (void time)t’r Adjusted retention timew Peak width

Types of TheoryPlate TheoryUseful chromatographic characteristicsNeglects influence of diffusion and flow paths

Rate TheoryAccounts influence of diffusion and flow pathsPredicts effects on column performance factors

Plate TheoryPartition Coefficient

Assumption:Independent on concentration, affected by temperature

Large K means more time spent on the columnTherefore:Increased elution time = Larger K

Plate TheoryRetention factor (k’)

Measure of impact on stationary phase on analyte (how retained in column it is)

Related directly to K

Thermodynamic property

Plate TheorySeparation Factor α

How to increase α?Longer Column – Better SeparationSide effects of Longer Column

Peak broadens

Increase in time for separation and quantity of mobile phase

Plate TheoryPlate Number N

Number of theoretical platesSeparation power assessed by plate numberNotetr and w MUST be measured in same units

HETP

Height equivalent to a theoretical plate

L Length of column

Plate TheoryResolution Rs

Same separation, different resolution

Rate TheoryBand BroadeningAffects peak widthGoverned through Kinetic Processes

DiffusionEddy DiffusionMolecular Diffusion

Mass TransferTime taken for partition between stationary

and mobile phases

Rate TheoryEddy Diffusion

Effected by particle size and flow rateTherefore peak broadening

Rate TheoryMolecular Diffusion

Effected by diffusion coefficient and flow rate

Rate TheoryMass Transfer

Rate of partitioning

Faster Partitioning, decreased band broadening

Rate TheoryVan Deemter EquationA Eddy Diffusion ConstantB Molecular Diffusion Constant effected by flow rateC Mass Transfer Constant effected by flow rateu Flow RateH Height of theoretical plates

Rate TheoryVan Deemter Plot

Determine optimum flow rate

What Do We WantMaximum Resolution in Minimum TimeThey counter-act eachother – oh dear How does N, k’ and α impact these?

Key Points What are k’, α, Rs, N (H)? How do you calculate them? Different factors contributing to band broadening

and column efficiency The van Deemter equation – what do the different

terms represent? The effect of altering different parameters on

separation ability

Best Broken into four categories

Theoretical Background

Gas Chromatography

HPLC

Quantitation, Calibration,

Standardisation and Validation

Gas Chromatography Only works for volatile chemical species

o Gas-Solid – ADSORPTION chromatography analysis of permanent gases (e.g O2 or N2O)

o Gas-Liquid – PARTITION chromatography analysis of organic species

Carrier Gas Nitrogen, Hydrogen or Helium

Must be of high purityHydrogen preferred but generated in situ as needed

Injectors Injected directly into heated port using micro-syringe

Split Injection (left)Only 0.1-1% of sample enters column, remainder waste

Splitless injection (right)All sample to columnGood for trace analysis

ColumnsPacked (top)Liquid coated silica particles in glass tubeBest for large scaleSlow and Inefficient

Capillary / Open Tubular (bottom)Wall coated thick liquid on inside of silica tube WCOTSupport coated support particles coated with stationary phase SCOTBest for speed and efficiencyOnly small particles

Stationary PhasesImmobilized ‘Liquid’ Stationary Phases Low volatility High decomposition temperature Chemically Inert Chemically attached to support Appropriate k’ and α for good resolution

Stationary Phases Usually bonded or cross-linked

Like attracts LikeNon-Polar stationary phase for non-polar analytesPolar stationary phase for stationary analytes

Elution Control using Temp

Minimum TemperatureRequired for analytes to get into vapour phase

Higher TemperatureFaster the analytes run

DetectorsExamplesThermal ConductivityFlame IonisationElectron captureFlame photometricNitrogen-PhosphorusPhotoionizationHall DetectorMass SpectrometerFourier Transform Infrared

Thermal ConductivityTC Detector (TCD)SimpleBulk property detector (responds to components and mobile phase)Universal (Sensitive to near all compounds)Non-DestructiveConcentration based signalNot very sensitive

Good for detecting permanent gasses (O2 or N2O etc)

Thermal ConductivityWhat is it?Measures change in thermal conductivity due to analyte gases eluting from columnHow?Pass elute over heated wireTemperature of wire changes as thermal conductivity of the effluent changesSignal is based on change in temperatureCarrier Gas needs VERY LARGE thermal conductvityHydrogen Highest of all – analyte will reduce thermal

conductivityHelium Analyte detected as a negative (overall thermal conductivity increase)

Flame IonisationSimpleSelectiveDestructiveSignal dependent on Mass-Flow

High temperature flame ionises the componentsIons are collected and records a current

Flame IonisationResponseApproximately proportional to number of carbon atoms in the compoundExample ethane would be twice response of methane (per mole)

Complex compounds, use table

Electron Capture Detector

Contains Ionised GasCreates conductive system

Decrease in conductivity relates to compounds with high electron affinity

E.G halogenated compounds, aromatics, alcohols

Sample Prep - GCPre-concentratingVery dilute analytes to get a high enough concentration to measure

If its non-volatile – use HPLC

DerivatisationYou can react analyte with compounds to make them volatile – HPLC simpler

GC UsesAnalysis of Permanent Gases Volatile mixtures

o Petrol, Perfumes Purity and content of volatile small molecules

o Pesticides, Drug compounds Production processes

o Alcohol in fermentation, conversions of petrochemicals Anything Volatile