Chromatography

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• Chromatography
• Physical separation method based on the
  differential
• migration of analytes in a mobile phase as they
  move
• along a stationary phase.
• Mechanisms of Separation
• Partitioning
• Adsorption
• Exclusion
• Ion Exchange
• (Affinity)

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Classification of Chromatography Column
Chromatography the stationary phase is held in
a narrow tube through which the mobile phase is
forced under pressure or by gravity. Planar
Chromatography the stationary phase is
supported on a flat plate or the interstices of a
paper and the mobile phase moves through the
stationary phase by capillary action or by
gravity.
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Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
4
Chromatographic Separations Based on the
distribution (partitioning) of the
solutes between the mobile and stationary phases,
described by partition coefficient, K K
Cs/Cm where Cs is the solute concentration in the
stationary phase and Cm is its concentration in
the mobile phase.
Distribution isotherms (Cs vs. Cm) and the
expected peak shape resulting from these
isotherms. Ideally, Cs is proportional to Cm.
Source Rubinson and Rubinson, Contemporary
Instrumental Analysis, Prentice Hall Publishing.
5
Source Rubinson and Rubinson, Contemporary
Instrumental Analysis, Prentice Hall Publishing.
6
Chromatographic Efficiency Theoretical plates,
borrowed from distillation theory. At
each plate, it is assumed that an equilibrium of
the solute between the mobile and stationary
phases takes place. Solute movement is viewed as
a series of stepwise transfers from one plate
to the next. Height equivalent to a theoretical
plate (H or HETP) and number Of theoretical
plates (N) are measures of column efficiency L
NH where L is the column length. As H
decreases, efficiency increases since there will
be more equilibrations in along the column.
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Theoretical plates N 16 (tr/W)2 5.54
(tr/W1/2)2
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Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
9
Van Deemter Equation H A B/u Csu Cmu
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
10
Example of A term of van Deemter Equation,
depicting peak broadening due to multiple flow
paths. Note that in capillary columns, this term
reduces to zero.
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
11

• Other peak-broadening factors
• Longitudinal diffusion (B term)
• Due to diffusion away from (concentrated) center
  of flow.
• Only significant in GC, not LC.
• Directly proportional to diffusion of analyte in
  mobile phase.
• Inversely proportional to linear velocity of
  mobile phase.
• Mass Transfer (C terms)
• Related to diffusion of analyte between phases.
• Inversely proportional to diffusion.

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Van Deemter plot, showing contributions of A, B,
and C terms to column efficiency, H.
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
13
Measures of chromatographic performance tr
retention time of analyte tm retention time of
unretained peak k capacity factor, widely
used to characterize performance k KVs/Vm
or k (tr-tm)/tm tr/tm K bk where b is
phase ratio
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Measures of chromatographic performance Selectivi
ty factor (a) is a measure of the ability of a
column to resolve two solutes. a KB/KA
kB/kA where A is the first peak and B is the
second peak. a is always greater than one.
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Measures of chromatographic performance Resolutio
n (Rs) is the separation of two peaks. Rs
(tr)B (tr)A/W (assuming peaks are of equal
width) N1/2/4 (a-1)/a
kB/(1kB) N 16Rs2 a/(a-1)2
(1kB)/kB2
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Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
17
Improving Resolution Improve efficiency
(narrower peaks) Or Improve separation (peaks
farther apart)
Source Rubinson and Rubinson, Contemporary
Instrumental Analysis, Prentice Hall Publishing.
18

• Chromatography Elution Problem
• Efficiency decreases for later eluting solutes.
• Improve by increasing the mobile phase strength
  during
• The course of the chromatographic run (i.e.,
  programming)
• GC Temperature Programming
• LC Gradient Elution
• SFC Pressure (density) Programming, some
  gradient elution

Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
19
Source Skoog, Holler, and Nieman, Principles of
Instrumental Analysis, 5th edition, Saunders
College Publishing.
20
Examples of methods to quantitatively measure
peak area.
Source Rubinson and Rubinson, Contemporary
Instrumental Analysis, Prentice Hall Publishing.
21
N 16 (tr/W)2 5.54 (tr/W1/2)2
Summary of Chromatography Equations Partition
Coefficient (K) is related to the stationary
phase (b) and mobile phase (via retention
time) Capacity Factor (k) reflects retention of
solute on column Column efficiency is defined by
height equivalent to a theoretical plate (plate
height, H) or plate number (N). More plates or
smaller plate height means greater
efficency. plate number can be determined from
retention time and peak width. Plate height (H)
is related to mobile phase linear velocity (u)
and diffusion. Diffusion explains why capillary
columns are more efficient than packed columns,
GC is more efficient than LC, and capillary
(open-tubular) columns are not used in LC
often. Resolution (Rs) describes how well
separated two peaks are and can be measured
from retention time and peak width. It is
related to the square root of efficiency (e.g.,
if you double the column length, resolution only
improves by the square root of two), selectivity,
and retention. Selectivity is the measure of how
well one peak is retained in preference to
another.
K Cs/Cm
K bk
k (tr-tm)/tm tr/tm
L NH
H A B/u Csu Cmu
Rs (tr)B (tr)A/W
N1/2/4 (a-1)/a kB/(1kB)
a KB/KA kB/kA