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