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LECTURE 9 LECTURE 9 CHROMATOGRAPHIC SEPARATIONSCHROMATOGRAPHIC SEPARATIONS
The “stuff” you do before you The “stuff” you do before you analyze a “complex” sampleanalyze a “complex” sample
Problem SetProblem Set
26-3, 26-12, 26-16, 26-18, 26-2526-3, 26-12, 26-16, 26-18, 26-25
due Thurs. May 12.due Thurs. May 12.
What is CHROMATOGRAPHY?
Many determinations involve Many determinations involve separationseparation followed by analysis followed by analysis chromatographychromatography electrophoresiselectrophoresis
CHROMATOGRAPHYCHROMATOGRAPHY::
sample transported by sample transported by mobile phasemobile phase
electrostatic or van der Waals'electrostatic or van der Waals'
some components in sample interact more strongly some components in sample interact more strongly with with stationary phase stationary phase and are more strongly and are more strongly retainedretained
sample separated into zones or bandssample separated into zones or bands
CHROMATOGRAPHY
The The MOBILE PHASE MOBILE PHASE – (the solvent moving through the column) – (the solvent moving through the column) is either a liquid or a gas.is either a liquid or a gas.
The The STATIONARY PHASE STATIONARY PHASE – (the one that stays in place inside a – (the one that stays in place inside a column) – is a viscous liquid chemically bonded to the inside of a column) – is a viscous liquid chemically bonded to the inside of a capillary tube unto the surface of solid particles.capillary tube unto the surface of solid particles.
Fluid entering the column is the Fluid entering the column is the ELUENTELUENT
Fluid emerging from the end of the column is Fluid emerging from the end of the column is ELUATEELUATE
GENERAL DESIGN OF OPTICAL INSTRUMENTS
It is all about “Reducing Interferences”It is all about “Reducing Interferences”It is all about “Reducing Interferences”It is all about “Reducing Interferences”
CHROMATOGRAPHY BASICSCHROMATOGRAPHY BASICSCHROMATOGRAPHY BASICSCHROMATOGRAPHY BASICS
MobileMobile and and StationaryStationary phasephase
Retention - MigrationRetention - Migration Bands or zonesBands or zones Equilibrium!Equilibrium!
Column vs. planarColumn vs. planar Liquid vs. gas vs. SFLiquid vs. gas vs. SF High vs. low resolutionHigh vs. low resolution PartitionPartition AdsorptionAdsorption Ion exchangeIon exchange Size exclusionSize exclusion
ChromatographyChromatographyChromatographyChromatographyA. Column Chromatography, B. Planar Chromatography
Column ChromatographyColumn ChromatographyColumn ChromatographyColumn Chromatography
Chromatogram
Dilution &Peak broadening!
CHROMATOGRAPHYCHROMATOGRAPHY: : Peak separationsPeak separationsCHROMATOGRAPHYCHROMATOGRAPHY: : Peak separationsPeak separations
CHROMATOGRAPHYCHROMATOGRAPHY: : Peak ResolutionPeak ResolutionCHROMATOGRAPHYCHROMATOGRAPHY: : Peak ResolutionPeak Resolution
Poor resolution
More separation
Less band spread
CHROMATOGRAPHY: CHROMATOGRAPHY:
Distribution ConstantDistribution Constant (recommended by IUPAC)(recommended by IUPAC)(old term: partition coefficient) (old term: partition coefficient)
CHROMATOGRAPHY: CHROMATOGRAPHY:
Distribution ConstantDistribution Constant (recommended by IUPAC)(recommended by IUPAC)(old term: partition coefficient) (old term: partition coefficient)
M
Sc c
cK stationary
mobile
A mobile ↔ A stationary
K ~ constant linear chromatography
>>>K >>> Retention in the stationary phase Retention times
How to manipulate K?
CS = nS/VS, CM = nM/VM
CHROMATOGRAPHYCHROMATOGRAPHY Retention Times Retention Times CHROMATOGRAPHYCHROMATOGRAPHY Retention Times Retention Times
tM = retention time of mobile phase (dead time)tR = retention time of analyte (solute)tS = time spent in stationary phase (adjusted retention time)L = length of the column
Chromatography: Chromatography: VelocitiesVelocitiesLinear rate of solute migration!Linear rate of solute migration!
Chromatography: Chromatography: VelocitiesVelocitiesLinear rate of solute migration!Linear rate of solute migration!
M
R
t
L
t
Lv
Velocity = distance/time length of column/ retention times
Velocity of solute:
Velocity of mobile phase:
ChromatographyChromatography Velocity/Retention time and KcVelocity/Retention time and Kc
ChromatographyChromatography Velocity/Retention time and KcVelocity/Retention time and Kc
SSMM
MM
VcVc
Vcμv
solute of moles total
phase mobilein solute of molesμv
phase mobilein timeoffraction μv
ChromatographyChromatography Velocity RelationshipsVelocity Relationships
ChromatographyChromatography Velocity RelationshipsVelocity Relationships
MS
M
S
MMSS
SSMM
MM
VVKv
c
cK
VcVcv
VcVc
Vcv
/1
1
Constanton Distributi
/1
1
Chromatography Chromatography Retention Factor : are we there yet?Retention Factor : are we there yet?
Chromatography Chromatography Retention Factor : are we there yet?Retention Factor : are we there yet?
M
MRA
AMR
A
MSAA
MS
t
ttk
kt
L
t
L
kv
VVKk
VVKv
1
1
1
1
Factor) (Retention /
/1
1
Adjusted retention time
When k'A is <1.0, separation is poorWhen k'A is >30, separation is slowWhen k'A is 2-10, separation is optimum
Relative retention time:RRT = tR/tRs
tRs = retention time of internal standard
Chromatography Chromatography Selectivity Factor: can you separate from your neighborSelectivity Factor: can you separate from your neighbor
Chromatography Chromatography Selectivity Factor: can you separate from your neighborSelectivity Factor: can you separate from your neighbor
MAR
MBR
M
MBRB
M
MARA
A
B
A
B
tt
tt
t
ttkand
t
ttk
k
k
K
K
)(
)(
)()(
B retained more than A >1
Distribution Constant
Retention factor
Retention time
LARGER =BETTER SEPARATION
CHROMATOGRAPHY CHROMATOGRAPHY Column Efficiency - Theoretical PlatesColumn Efficiency - Theoretical Plates
Plate andPlate and Rate TheoriesRate Theories
CHROMATOGRAPHY CHROMATOGRAPHY Column Efficiency - Theoretical PlatesColumn Efficiency - Theoretical Plates
Plate andPlate and Rate TheoriesRate Theories
LH
H
LN
N
H
2
plates ofnumber
height plate
standard deviation 2/L variance per unit length.
L = length of column packing
CHROMATOGRAPHYCHROMATOGRAPHY Relation between column distance and retention Relation between column distance and retention
timestimes
CHROMATOGRAPHYCHROMATOGRAPHY Relation between column distance and retention Relation between column distance and retention
timestimes
R
R
R
tL
tL
t
L
/
in timedeviation standard
timeretention
distancein deviation standard
(distance)length column
CHROMATOGRAPHYCHROMATOGRAPHY Relation between column distance and retention Relation between column distance and retention
timestimes
CHROMATOGRAPHYCHROMATOGRAPHY Relation between column distance and retention Relation between column distance and retention
timestimes
2
22
16
4
4
R
R
R
R
t
LW
LH
t
LW
W
t
L
tL
~96% 2
Tangent atInflection point
Efficient column has small plate height - less zone broadening
Chromatography Chromatography Determining the Number Determining the Number
of Theoretical Platesof Theoretical Plates
Chromatography Chromatography Determining the Number Determining the Number
of Theoretical Platesof Theoretical Plates
2
2/1
2
54.5
16
pates ofnumber
W
tN
W
tN
N
R
R
W1/2
Summary of Plate TheorySummary of Plate Theory
Successfully accounts for the peak shapes and Successfully accounts for the peak shapes and rate of movementrate of movement
Does not account for the “mechanism” causing Does not account for the “mechanism” causing peak broadeningpeak broadening
No indication of other parameters’ effectsNo indication of other parameters’ effects No indication for adjusting experimental No indication for adjusting experimental
parametersparameters
Rate TheoryRate Theory
Zone broadening is related to Mass Transfer Zone broadening is related to Mass Transfer processesprocesses
Column EfficiencyColumn EfficiencyKinetic variablesKinetic variables
Zone BroadeningZone BroadeningFlow Rate of Mobile PhaseFlow Rate of Mobile Phase
Liquid chromatography Gas chromatography
Note the differences in flowrate and plates height scales
Why GC normalluy has high H, but also high overall efficiency?
Zone BroadeningZone BroadeningKinetic ProcessesKinetic Processes
)(/ MS CCBAH
Van - Deemter Equation
λ and γ are constants that depend on quality of the packing.
B is coefficient of longitudinal diffusion.
Cs and Cm are coefficients of mass transfer in stationary and mobile phase, respectively.
Zone BroadeningZone BroadeningKinetic ProcessesKinetic Processes
)(/ MS CCBAH
Van - Deemter Equation
Zone BroadeningZone BroadeningMultiple PathwaysMultiple Pathways
Eddy Diffusion: band broadening process results from different path lengths passed by solutes.
1. Directly proportional to the diameters of packing
2. Offset by ordinary diffusion3. Lower mobile-phase velocity,
smaller eddy diffusion
Stagnant pools of mobile phaseretained in stationary phase.
CHROMATOGRAPHYCHROMATOGRAPHY ResolutionResolution
CHROMATOGRAPHYCHROMATOGRAPHY ResolutionResolution
BA
ARBRs
BAs
BAs
WW
ttR
WW
ZR
WW
ZR
])()[(2
2
2/2/
ChromatographicChromatographic Separations with a twist Separations with a twist ChromatographicChromatographic Separations with a twist Separations with a twist
Chromatographic DefinitionsChromatographic Definitions
Chromatographic RelationshipsChromatographic Relationships
QUANTITATIVE ANALYSISQUANTITATIVE ANALYSIS
Peak areasPeak areas Peak heightPeak height Calibration and standardsCalibration and standards Internal Standard methodInternal Standard method
SUMMARYSUMMARY
Relate to column chromatographyRelate to column chromatography Retention timesRetention times Velocities of mobile and componentVelocities of mobile and component Height equivalent of theoretical platesHeight equivalent of theoretical plates Peak or zone broadeningPeak or zone broadening ResolutionResolution