Chromatographic Chromatographic separationsseparations

The “stuff” you do The “stuff” you do before you analyze a before you analyze a “complex” sample“complex” sample

It is all about “Reducing It is all about “Reducing Interferences”Interferences”

It is all about “Reducing It is all about “Reducing Interferences”Interferences”

Chromatography Chromatography basicsbasics

Chromatography Chromatography basicsbasics

MobileMobile and and StationaryStationary phase phase

Retention - Retention - MigrationMigration

Bands or zonesBands or zones Equilibrium!Equilibrium!

Column vs. planarColumn vs. planar Liquid vs. gas vs. Liquid vs. gas vs.

SFSF High vs. low High vs. low

resolutionresolution PartitionPartition AdsorptionAdsorption Ion exchangeIon exchange Size exclusionSize exclusion

ChromatographyChromatographyChromatographyChromatographyA. Column Chromatograpgy, B. Planar Chromatograpgy

Column Column ChromatographyChromatography

Column Column ChromatographyChromatography

Chromatogram

Dilution &Peak broadening!

Chromatography: Chromatography: Peak Peak separationsseparations

Chromatography: Chromatography: Peak Peak separationsseparations

Chromatography: Chromatography: Peak Peak ResolutionResolution

Chromatography: Chromatography: Peak Peak ResolutionResolution

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

Chromatography Chromatography Retention TimesRetention Times

Chromatography Chromatography Retention TimesRetention 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:

Chromatography Chromatography Velocity/Retention time and KcVelocity/Retention time and Kc

Chromatography Chromatography Velocity/Retention time and KcVelocity/Retention time and Kc

SSMM

MM

VcVc

Vcv

v

v

solute of moles total

phase mobile in solute of moles

phase mobile in timeof fraction

Chromatography Chromatography Velocity RelationshipsVelocity Relationships

Chromatography Chromatography 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

Relative retention time:RRT = tR/tRs

tRs = retention time of internal standard

Chromatography Chromatography Selectivity Factor: can you separate from Selectivity Factor: can you separate from

your neighboryour neighbor

Chromatography Chromatography Selectivity Factor: can you separate from Selectivity Factor: can you separate from

your neighboryour 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

Chromatography Chromatography Column Efficiency - Theoretical Column Efficiency - Theoretical

PlatesPlatesPlate andPlate and Rate TheoriesRate Theories

Chromatography Chromatography Column Efficiency - Theoretical Column Efficiency - Theoretical

PlatesPlatesPlate 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

Chromatography Chromatography Relation between column Relation between column

distance and retention timesdistance and retention times

Chromatography Chromatography Relation between column Relation between column

distance and retention timesdistance and retention times

R

R

R

tL

tL

t

L

/

in timedeviation standard

timeretention

distancein deviation standard

(distance)length column

Chromatography Chromatography Relation between column Relation between column

distance and retention timesdistance and retention times

Chromatography Chromatography Relation between column Relation between column

distance and retention timesdistance and retention times

2

22

16

4

4

R

R

R

R

t

LW

LH

t

LW

W

t

L

tL

~96% 2Tangent at

Inflection point

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 Successfully accounts for the peak shapes and rate of movementshapes and rate of movement

Does not account for the Does not account for the “mechanism” causing peak “mechanism” causing peak broadeningbroadening

No indication of other parameters’ No indication of other parameters’ effectseffects

No indication for adjusting No indication for adjusting experimental parametersexperimental parameters

Rate TheoryRate Theory

Zone broadening is related to Zone broadening is related to Mass Transfer processesMass Transfer processes

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 diffusion

3. Lower mobile-phase velocity, smaller eddy diffusionStagnant pools of mobile phase

retained 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 Chromatographic DefinitionsDefinitions

Chromatographic Chromatographic RelationshipsRelationships

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 Height equivalent of theoretical

platesplates Peak or zone broadeningPeak or zone broadening ResolutionResolution