lecture 9 chromatographic separations the “stuff” you do before you analyze a “complex”...
TRANSCRIPT
![Page 1: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/1.jpg)
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
![Page 2: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/2.jpg)
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.
![Page 3: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/3.jpg)
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
![Page 4: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/4.jpg)
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
![Page 5: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/5.jpg)
GENERAL DESIGN OF OPTICAL INSTRUMENTS
![Page 6: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/6.jpg)
It is all about “Reducing Interferences”It is all about “Reducing Interferences”It is all about “Reducing Interferences”It is all about “Reducing Interferences”
![Page 7: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/7.jpg)
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
![Page 8: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/8.jpg)
ChromatographyChromatographyChromatographyChromatographyA. Column Chromatography, B. Planar Chromatography
![Page 9: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/9.jpg)
Column ChromatographyColumn ChromatographyColumn ChromatographyColumn Chromatography
Chromatogram
Dilution &Peak broadening!
![Page 10: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/10.jpg)
CHROMATOGRAPHYCHROMATOGRAPHY: : Peak separationsPeak separationsCHROMATOGRAPHYCHROMATOGRAPHY: : Peak separationsPeak separations
![Page 11: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/11.jpg)
CHROMATOGRAPHYCHROMATOGRAPHY: : Peak ResolutionPeak ResolutionCHROMATOGRAPHYCHROMATOGRAPHY: : Peak ResolutionPeak Resolution
Poor resolution
More separation
Less band spread
![Page 12: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/12.jpg)
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
![Page 13: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/13.jpg)
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
![Page 14: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/14.jpg)
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:
![Page 15: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/15.jpg)
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
![Page 16: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/16.jpg)
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
![Page 17: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/17.jpg)
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
![Page 18: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/18.jpg)
When k'A is <1.0, separation is poorWhen k'A is >30, separation is slowWhen k'A is 2-10, separation is optimum
![Page 19: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/19.jpg)
Relative retention time:RRT = tR/tRs
tRs = retention time of internal standard
![Page 20: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/20.jpg)
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
![Page 21: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/21.jpg)
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
![Page 22: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/22.jpg)
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
![Page 23: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/23.jpg)
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
![Page 24: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/24.jpg)
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
![Page 25: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/25.jpg)
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
![Page 26: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/26.jpg)
Rate TheoryRate Theory
Zone broadening is related to Mass Transfer Zone broadening is related to Mass Transfer processesprocesses
![Page 27: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/27.jpg)
Column EfficiencyColumn EfficiencyKinetic variablesKinetic variables
![Page 28: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/28.jpg)
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?
![Page 29: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/29.jpg)
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.
![Page 30: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/30.jpg)
Zone BroadeningZone BroadeningKinetic ProcessesKinetic Processes
)(/ MS CCBAH
Van - Deemter Equation
![Page 31: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/31.jpg)
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.
![Page 32: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/32.jpg)
CHROMATOGRAPHYCHROMATOGRAPHY ResolutionResolution
CHROMATOGRAPHYCHROMATOGRAPHY ResolutionResolution
BA
ARBRs
BAs
BAs
WW
ttR
WW
ZR
WW
ZR
])()[(2
2
2/2/
![Page 33: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/33.jpg)
ChromatographicChromatographic Separations with a twist Separations with a twist ChromatographicChromatographic Separations with a twist Separations with a twist
![Page 34: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/34.jpg)
Chromatographic DefinitionsChromatographic Definitions
![Page 35: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/35.jpg)
Chromatographic RelationshipsChromatographic Relationships
![Page 36: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/36.jpg)
QUANTITATIVE ANALYSISQUANTITATIVE ANALYSIS
Peak areasPeak areas Peak heightPeak height Calibration and standardsCalibration and standards Internal Standard methodInternal Standard method
![Page 37: LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample](https://reader033.vdocuments.net/reader033/viewer/2022050908/56649e4c5503460f94b4203f/html5/thumbnails/37.jpg)
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