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Chromatography
Chromatography
Mechanisms of Separation:
•Partitioning
•Adsorption
•Exclusion
•Ion Exchange
•(Affinity)
Classification of Chromatography
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Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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.
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
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Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
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.
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Theoretical plates
N = 16 (tr/W)2 = 5.54 (tr/W1/2)2
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
4
Van Deemter Equation:
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
5
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.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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Measures of chromatographic performance
tr = retention time of analyte
α = KB/KA = k’B/k’A
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Measures of chromatographic performance
Resolution (Rs) is the separation of two peaks.
α-1)/α
= [N1/2/4] [(α α] [k’B/(1+k’B)]
α/(α
N = 16Rs2 [α α-1)]2 [(1+k’B)/k’B]2
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
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Improving Resolution
Improve efficiency (narrower peaks)
Or
Improve separation (peaks farther apart)
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
9
Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.
Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.
10
Summary of Chromatography Equations
Partition Coefficient (K) is related to the stationary phase (β β ) and mobile phase (via
retention time) K = Cs/Cm K = β k’
Capacity Factor (k’) reflects retention of solute on column
k’ = (tr-tm)/tm = t’r/tm
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.
L = NH N = 16 (tr/W)2 = 5.54 (tr/W1/2)2
Plate height (H) is related to mobile phase linear velocity (υ υ) 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.
H = A + B/υ υ + Csυ + Cmυ
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.
α-1)/α
Rs = [(tr)B – (tr)A]/W = [N1/2/4] [(α α] [k’B/(1+k’B)]
Selectivity is the measure of how well one peak is retained in preference to another.
α = KB/KA = k’B/k’A
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