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INTRODUCTION TO CHROMATOGRAPHIC SEPARATION

Lecture 9

PROBLEM SET

26-4, -12, -16, -25 27-3, -20, -25 28-6, -15 Due Thursday, May 17

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!!

Methods(of(Elimination(Interferences(in(a(Chemical(Analysis!

Method( Basis(of(Method(1.!!!!Masking! Immobilization!of!an!interferent!as!a!nonreactive!

complex!2.!!!!Mechanical!Phase!Separation! !! a.!!Precipitaion!/!Filtration! Difference!in!solubility!of!compounds!formed!! b.!!Distillation! Difference!in!volatility!of!compounds!! c.!!Extraction! Difference!in!solubility!in!two!immiscible!liquids!! d.!!Ion!Exchange! Difference!in!stability!of!reactants!in!with!ion!

exchange!resins!3.!!Chromatography! Difference!in!a!rate!of!movement!of!a!solute!

through!a!stationary!phase!4.!!Electrophoresis! Difference!in!migration!rate!in!an!electric!field!

gradient!!!

IT IS ALL ABOUT “REDUCING INTERFERENCES”

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Many determinations involve separation followed by analysis v  chromatography v  electrophoresis CHROMATOGRAPHY:

sample transported by mobile phase electrostatic or van der Waals' some components in sample interact more

strongly with stationary phase andare more strongly retainedsample separated into zones or bands

CHROMATOGRAPHY

The MOBILE PHASE – (the solvent moving through the column) is either a liquid or a gas. The STATIONARY PHASE – (the one that stays in place inside a column) – is a viscous liquid chemically bonded to the inside of a capillary tube unto the surface of solid particles. Fluid entering the column is the ELUENT Fluid emerging from the end of the column is ELUATE

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CHROMATOGRAPHY

COLUMN CHROMATOGRAPHY

Dilution & Peak broadening!

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CHROMATOGRAPHY: PEAK SEPARATIONS

PEAK RESOLUTION

Poor resolution

More separation

Less band spread

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DISTRIBUTION CONSTANT (recommended by IUPAC) (old term: partition coefficient)

M

Sc c

cK = stationary mobile

A mobile ↔ A stationary

K ~ constant è linear chromatography >>>K >>> Retention in the stationary phase à Retention times How to manipulate K?

CS = nS/VS, CM = nM/VM

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

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CHROMATOGRAPHY: VELOCITIES LINEAR RATE OF SOLUTE MIGRATION!

Velocity = distance/time à length of column/ retention times Velocity of solute: Velocity of mobile phase: M

R

tLtLv

=

=

µ

VELOCITY/RETENTION TIME AND KC

SSMM

MM

VcVcVcv

v

v

+×=

×=

×=

µ

µ

µ

solute of moles totalphase mobile in solute of molesphase mobile in timeof fraction

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VELOCITY RELATIONSHIPS

MS

M

S

MMSS

SSMM

MM

VVKv

ccK

VcVcv

VcVcVcv

/11

Constanton Distributi

/11

+×=

=

+×=

+×=

µ

µ

µ

RETENTION FACTOR :

M

MRA

AMR

A

MSAA

MS

tttk

ktL

tL

kv

VVKkVVK

v

−=

+×=

+×=

=

+×=

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11

Factor) (Retention /

/11

µ

µ

Adjusted retention time

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Relative retention time: RRT = tR/tRs

tRs = retention time of internal standard

SELECTIVITY FACTOR:

MAR

MBR

M

MBRB

M

MARA

A

B

A

B

tttt

tttkand

tttk

kkKK

−−

=

−=

−=

=

=

)()(

)()(

α

α

α

B retained more than A à α >1

Distribution Constant

Retention factor

Retention time

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COLUMN EFFICIENCY - THEORETICAL PLATES Plate and Rate Theories

LH

HLN

NH

2

plates ofnumber height plate

σ=

=

=

=

σ à standard deviation σ2/Là variance per unit length.

L = length of column packing

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RELATION BETWEEN COLUMN DISTANCE AND RETENTION TIMES

R

R

R

tL

tL

t

L

/

in timedeviation standard timeretention

distancein deviation standard (distance)length column

στ

τστ

σ

=

=

=

=

=

=

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RELATION BETWEEN COLUMN DISTANCE AND RETENTION TIMES

2

22

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4

4

R

R

R

R

tLW

LH

tLW

WtLtL

==

=

=

=

=

σ

σ

τ

τσ

τσ

~96% ± 2τ Tangent at

Inflection point

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DETERMINING THE NUMBER OF THEORETICAL PLATES

2

2/1

2

54.5

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pates ofnumber

⎟⎟⎠

⎞⎜⎜⎝

⎛=

⎟⎠

⎞⎜⎝

⎛=

=

WtN

WtN

N

R

R

W1/2

SUMMARY OF PLATE THEORY

§  Successfully accounts for the peak shapes and rate of movement

§  Does not account for the “mechanism” causing peak broadening

§  No indication of other parameters’ effects §  No indication for adjusting experimental parameters

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RATE THEORY

§  Zone broadening is related to Mass Transfer processes

COLUMN EFFICIENCY: Kinetic variables

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ZONE BROADENING : Flow Rate of Mobile Phase

Liquid chromatography Gas chromatography Note the differences in flowrate and plates height scales

Why GC normally has high H, but also high overall efficiency?

ZONE BROADENING: Kinetic Processes

µµ )(/ 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.

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ZONE BROADENING: Kinetic Processes

µµ )(/ MS CCBAH +++=

Van Deemter Equation

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ZONE BROADENING: Multiple 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 diffusion

Stagnant pools of mobile phase retained in stationary phase.

ZONE BROADENING: Longitudinal Diffusion

§  The higher the µ, the smaller the H

§  Much smaller in LC than in GC

Column

Diffusion

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ZONE BROADENING: Mass Transfer between Phases

§  Slow equilibrium of solute between mobile and stationary phases

§  Time is required for solute molecules to diffuse from the

interior of these phase to there interface where transfer occur

ZONE BROADENING: Longitude vs.Mass Transfer

§  Longitude o  Parallel to the flow o  Inversely proportional to the flow of the

mobile phase §  Mass Transfer

o  Diffusion tends to be right angles to the flow o  The faster the mobile phase moves, the larger

the band broadening

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CHROMATOGRAPHY: Resolution

BA

ARBRs

BAs

BAs

WWttR

WWZR

WWZR

+

−=

+

Δ=

+

Δ=

])()[(2

22/2/

CHROMATOGRAPHIC SEPARATIONS

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CHROMATOGRAPHIC DEFINITIONS

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CHROMATOGRAPHIC RELATIONSHIPS

QUANTITATIVE ANALYSIS

o  Peak areas o  Peak height o  Calibration and standards o  Internal Standard method

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SUMMARY

v  Relate to column chromatography v  Retention times v  Velocities of mobile and component v  Height equivalent of theoretical plates v  Peak or zone broadening v  Resolution