chapter 4. theoretical background gas chromatography hplc quantitation, calibration, standardisation...
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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