Chromatography

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)

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.

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.

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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. 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

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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Van Deemter Equation: H = A + B/u + Csu + Cmu

Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

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.

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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.

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.

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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 = t’r/tm K = βk’ where β is phase ratio

Measures of chromatographic performance Selectivity factor (α α) is a measure of the ability of a column to resolve two solutes. α = KB/KA = k’B/k’A where A is the first peak and B is the second peak. α is always greater than one.

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Measures of chromatographic performance Resolution (Rs) is the separation of two peaks. Rs = [(tr)B – (tr)A]/W (assuming peaks are of equal width) = [N1/2/4] [(α α-1)/α α] [k’B/(1+k’B)] α/(α α-1)]2 [(1+k’B)/k’B]2 N = 16Rs2 [α

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.

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.

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Source: Skoog, Holler, and Nieman, Principles of Instrumental Analysis, 5th edition, Saunders College Publishing.

Examples of methods to quantitatively measure peak area.

Source: Rubinson and Rubinson, Contemporary Instrumental Analysis, Prentice Hall Publishing.

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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. Rs = [(tr)B – (tr)A]/W = [N1/2/4] [(α α-1)/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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