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CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -2

© M

. Guilhaus U

NS

W 1999 - A

ll rights reserved.

1

Outline

Separation Techniques‘Theory of Column Chromatography’

• Definitions

• Elution Chromatography

• Types of Chromatographic Equilibria

• Partition Chromatography Theory

– Countercurrent Fractionation

– Partition coefficient

–Capacity

–Selectivity

–Band Broadening and Plate Theory

–Resolution

Vaq

p

KD

D

www.chem.unsw.edu.au/UGNotes/Guilhaus/

q

Vorg


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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Definitions

• Chromatography (from Greek ‘chromatos’meaning colour) - named by Tswett whoseparated plant pigments in a column.

• Based on dynamic phase equilibrium (countercurrent fractionation).

• Solute carried through a stationary medium asa constantly flowing mobile phase passesthrough.

• Solute is held up by interaction with stationaryphase.

• Mobility depends on strength of interaction withstationary phase.

Michael Tswett(1872-1919)Russian Botanist


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. Guilhaus U

NS

W 1999 - A

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1Elution Chromatography

Mobile phase supplied at constant rate attop of column ...

2 mixture (cyan and magenta dyes)introduced at top of column ...

3 mixture moves down column (forced bymobile phase) and begins to separate into

bands ...

4 bands continue to separate ...

5 finally separated fractions are collected atend of column


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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Chromatographic Equilibria

Adsorption: soluteadsorbed on surface ofstationary phase


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. Guilhaus U

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Partition: solute dissolvedin liquid phase bonded tosurface of solid support


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. Guilhaus U

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Ion Exchange: mobileanions retained by cationsbonded to stationary phase(anion-exchange resin)

Converse also available(cation-exchange resin)


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. Guilhaus U

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Molecular Exclusion:large molecules areexcluded and are therforenot retained.


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. Guilhaus U

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Affinity: based onmolecular recognitionbetween covalently boundmolecule having specificdonor-receptor attraction.Other molecules are notretained.


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. Guilhaus U

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Partition Chromatography -Theory

• Separations based on phase equilibria.

• The partition coefficient Kd determines how muchsolute is in the mobile and stationary phases at anyone time.

• This is just like countercurrent fractionation insolvent extraction.

mobile phase stationary phase


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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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For a column of length L:v

Lt

uL

t

R

M

=

=

solute velocity

mobile phasevelocity

Retention Time & Velocity

A is not retainedB is retained by thecolumn

CHROMATOGRAM


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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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Solute velocity is related to fraction oftime spent in mobile phase

Let CM be conc solute. in mobilephase, VM vol of mobile phase

CS and VS are the correspondingparameters for the stationary phase.

v u= ´ (fraction of time in mobile phase)

v u= ´moles in mobile phase

total moles

v uC V

C V C Vu C V

C V

uK

V

V

M M

M M S S S S

M Mp

S

M

= ´+

= ´+

= ´+

1

1

1

1

The mobility of the solute isfundamentally linked to thepartition coefficient

Retention Time & Kp

Note: VM and VS usually known fromcolumn preparation method


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. Guilhaus U

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k’ measures the mobility of aretained solute in the column.

Readily obtained fromchromatogram.

Capacity Factor k’ for Retained Solute

kKV

VRS

M' =

v uK

V

VpS

M

= ´+

1

1

Lt

Lt kR M R

= ´+

11 '

kt t

tRR M

M' =

-


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. Guilhaus U

NS

W 1999 - A

ll rights reserved.

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For 2 solutes (A & B) is the ratio of thecapacity factor of the more stronglyretained solute to that for the lessstrongly retained solute.

Measures how well separated A and Bwill be.

Measured directly from chromatogram.

Used to determine resolving power.

Selectivity Factor a for 2 Retained Solutes

a = = =-

-kk

KK

t t

t tB

A

B

A

R M

R M

B

A

''


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. Guilhaus U

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One important purpose ofchromatography is to separatemixtures.

Solutes migrate in bands asthey pass through a column.

The band always broadens.

Broadening limits the abilityseparation (resolution)because it causes overlap ofpeaks.

Why do bands broaden?

Band Broadening


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. Guilhaus U

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• Diffusion of solute occurs along the column.

• Broadening increases with time.

• Inversely proportional to velocity of mobilephase

Band Broadening from Longditudinal Diffusion

mobile phasestationary phase

COLUMN

peak width µ B/u


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. Guilhaus U

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• Equilibration not instantaneous.

• Broadening increases with speed of mobilephase (u).

Band Broadening from Slow Equilibration


COLUMN

peak width µ C ́ u


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. Guilhaus U

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• Statistical distribution of path lengths.

• Broadening independent of speed of mobilephase (u).

Band Broadening from Random Paths


COLUMN

peak width µ A


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. Guilhaus U

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• The combined effect ofthe three sources ofbroadening reveals anoptimum mobile phasevelocity.

• The vertical axisincreases withincreasing broadening.

• The vertical axis isusually labeled ‘heightof the equivalenttheoretical plate’ (H orHETP)

Van Deempter Equation

y = B/u

y = Ay = Cu

y = H = A + B/u + Cu

u

H


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. Guilhaus U

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• Plate theory of chromatography is attributed tomartin and Synge (Nobel Prize in 1952).

• They used a fractionation column model toexplain chromatographic behavior.

• More plates in a fractionating column allowscompounds with smaller differences in boilingpoints to be separated.

• Thus a column is imagined to be composed ofN plates, each of height H.

• The smaller the plate height the greater thenumber of plates in a length ofchromatographic column and the better theability to separate compounds

Plate Height

T

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