Teori Kolom Kromatografi 2

Outline
Separation Techniques
Theory of Column Chromatography
Definitions
Elution Chromatography
Partition Chromatography Theory
Vaq
KD
Countercurrent Fractionation
Partition coefficient
Capacity
Selectivity
M. Guilhaus UNSW 1999 - All rights reserved.
Types of Chromatographic Equilibria
Vorg
Band Broadening and Plate Theory

Resolution
www.chem.unsw.edu.au/UGNotes/Guilhaus/
CHEM2801 Analytical & Physical Chemistry for Food Science - Separations -2
Definitions
Chromatography (from Greek chromatos
meaning colour) - named by Tswett who
separated plant pigments in a column.
Based on dynamic phase equilibrium (counter

current fractionation).
Solute carried through a stationary medium as

a constantly flowing mobile phase passes
through.
Solute is held up by interaction with stationary

phase.
Mobility depends on strength of interaction with

stationary phase.
Michael Tswett
(1872-1919)
Russian Botanist
Elution Chromatography
1
2
4
5
mixture (cyan and magenta dyes)

introduced at top of column ...
Mobile phase supplied at constant rate at

top of column ...
mixture moves down column (forced by

mobile phase) and begins to separate into
bands ...
bands continue to separate ...

finally separated fractions are collected at
end of column
Chromatographic Equilibria
Adsorption: solute
adsorbed on surface of
stationary phase
Partition: solute dissolved
in liquid phase bonded to
surface of solid support
Ion Exchange: mobile

anions retained by cations
bonded to stationary phase
(anion-exchange resin)
Converse also available

(cation-exchange resin)
Molecular Exclusion:
large molecules are
excluded and are therfore
not retained.
Affinity: based on
molecular recognition
between covalently bound
molecule having specific
donor-receptor attraction.
Other molecules are not
retained.
Partition Chromatography -Theory

Separations based on phase equilibria.
The partition coefficient Kd determines how much

solute is in the mobile and stationary phases at any
one time.
This is just like countercurrent fractionation in

solvent extraction.
mobile phase
stationary phase

Retention Time & Velocity
CHROMATOGRAM
v=
L
tR
u=
L
tM
For a column of length L:
A is not retained
B is retained by the
column
solute velocity
mobile phase
velocity
10

Retention Time & Kp
Solute velocity is related to fraction of
time spent in mobile phase
Let CM be conc solute. in mobile
phase, VM vol of mobile phase
CS and VS are the corresponding

parameters for the stationary phase.
Note: VM and VS usually known from

column preparation method
v = u (fraction of time in mobile phase)

moles in mobile phase
v =u
total moles
v =u
The mobility of the solute is

fundamentally linked to the
partition coefficient
CM VM
1
1
=u
=u
V
C V
CM VM + CS VS
1+ S S
1+ Kp S
VM
CM VM
11

Capacity Factor k for Retained Solute
k measures the mobility of a
retained solute in the column.
k' R =
1
1+ Kp
VS
VM
KVS
VM
Readily obtained from

chromatogram.
v =u
1
L
L
=
t R t M 1 + k' R
k' R =
tR - tM
tM
12

Selectivity Factor a for 2 Retained Solutes
k' B K B t RB - t M
a=
=
=
k' A K A t RA - t M
For 2 solutes (A & B) is the ratio of the

capacity factor of the more strongly
retained solute to that for the less
strongly retained solute.
Measures how well separated A and B
will be.
Measured directly from chromatogram.
Used to determine resolving power.
13

Band Broadening
One important purpose of
chromatography is to separate
mixtures.
Solutes migrate in bands as

they pass through a column.
The band always broadens.
Broadening limits the ability
separation (resolution)
because it causes overlap of
peaks.
Why do bands broaden?
14

Band Broadening from Longditudinal Diffusion
Diffusion of solute occurs along the column.
Broadening increases with time.
Inversely proportional to velocity of mobile

phase
COLUMN
stationary phase
mobile phase
peak width B/u

15

Band Broadening from Slow Equilibration
Equilibration not instantaneous.
Broadening increases with speed of mobile

phase (u).
COLUMN
stationary phase
mobile phase
peak width C u
16

Band Broadening from Random Paths
Statistical distribution of path lengths.
Broadening independent of speed of mobile

phase (u).
COLUMN
stationary phase
mobile phase
peak width A
17

Van Deempter Equation
The combined effect of
the three sources of
broadening reveals an
optimum mobile phase
velocity.
The vertical axis

increases with
increasing broadening.
The vertical axis is

usually labeled height
of the equivalent
theoretical plate (H or
HETP)
y = H = A + B/u + Cu
y = Cu
y=A
y = B/u
u
18

Plate Height
Plate theory of chromatography is attributed to
martin and Synge (Nobel Prize in 1952).
They used a fractionation column model to

explain chromatographic behavior.
More plates in a fractionating column allows

compounds with smaller differences in boiling
points to be separated.
Thus a column is imagined to be composed of

N plates, each of height H.
The smaller the plate height the greater the

number of plates in a length of
chromatographic column and the better the
ability to separate compounds
19

Number of Plates
The number of
theoretical plates
is estimated from
the width of the
peak
t
N = 16 R
W
20

Resolution
A quantitative
measure of column
ability to separate
solutes with similar
mobilities
R=
2Dt R
WA + WB
21

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Outline

M. Guilhaus UNSW 1999 - All rights reserved.

Types of Chromatographic Equilibria

Band Broadening and Plate Theory

Based on dynamic phase equilibrium (counter

Solute carried through a stationary medium as

Solute is held up by interaction with stationary

Mobility depends on strength of interaction with

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

M. Guilhaus UNSW 1999 - All rights reserved.

mixture (cyan and magenta dyes)

M. Guilhaus UNSW 1999 - All rights reserved.

Mobile phase supplied at constant rate at

mixture moves down column (forced by

bands continue to separate ...

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

M. Guilhaus UNSW 1999 - All rights reserved.

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

M. Guilhaus UNSW 1999 - All rights reserved.

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

Ion Exchange: mobile

Converse also available

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

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

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

M. Guilhaus UNSW 1999 - All rights reserved.

Partition Chromatography -Theory

The partition coefficient Kd determines how much

This is just like countercurrent fractionation in

M. Guilhaus UNSW 1999 - All rights reserved.

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

Partition Chromatography -Theory

M. Guilhaus UNSW 1999 - All rights reserved.

For a column of length L:

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

Partition Chromatography -Theory

CS and VS are the corresponding

Note: VM and VS usually known from

v = u (fraction of time in mobile phase)

The mobility of the solute is

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

Partition Chromatography -Theory

M. Guilhaus UNSW 1999 - All rights reserved.

Readily obtained from

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

Partition Chromatography -Theory

For 2 solutes (A & B) is the ratio of the

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

Partition Chromatography -Theory

Solutes migrate in bands as

Partition Chromatography -Theory

Broadening increases with time.

Inversely proportional to velocity of mobile

M. Guilhaus UNSW 1999 - All rights reserved.

peak width B/u

Partition Chromatography -Theory

Equilibration not instantaneous.

Broadening increases with speed of mobile

Partition Chromatography -Theory

Statistical distribution of path lengths.

Broadening independent of speed of mobile

Partition Chromatography -Theory

The vertical axis