2 The Chromatogram A chromatogram is a graph showing the detector response as a function of elution time. The retention time, t R, for each component is the time needed after injection of the mixture until that component reaches the detector.
4 The Chromatogram (cont.) Retention volume, V R, is the volume of mobile phase required to elute a particular solute from the column: V R = t R F where F is the mobile phase flow rate The dead time, t m, is the time of travel of unretained mobile phase through the column.
5 The Chromatogram (cont.) The adjusted retention time, t R, for a solute is the additional time required for solute to travel the length of the column beyond the time required by unretained solvent: t R = t R t m In GC, t m is usually taken as the time needed for CH 4 to travel through the column.
6 The Chromatogram (cont.) For any two components 1 and, the relative retention, α, is the ratio of their adjusted retention times: where, so α > 1. ' 1 ' R R t t = α 1 R R t t = 1 R R t t = ' 1 ' R R t t = ' 1 ' R R t t >
7 The Capacity Factor For each peak in the chromatogram, the capacity factor, k, is defined as: k' = t R t m t m k'= time solute spends in stationary phase time solute spends in mobile phase
8 The Capacity Factor (cont.) time solute spends in stationary phase k' = = time solute spends in mobile phase moles of solute in stationary phase moles of solute in mobile phase k ' = C V C s m V s m k ' = K C C V V s m s m = K t = R t m t m = t t ' R m
9 Relative Retention Alternative Expressions α = t t ' R ' R1 = k k ' ' 1 = K K 1
10 Efficiency of Separation
11 Resolution Solute moving through a column spreads into a Gaussian shape with standard deviation σ. Common measures of breadth are: The width w ½ measured at half-height The width w at the baseline between tangents drawn to the steepest parts of the peak (inflection points).
13 Resolution (cont.) It can be shown that: and w ½ =.35σ w = 4σ
14 Resolution (cont.) In chromatography, the resolution of two peaks from each other is defined as R s = Δt w R av = ΔV w av R = 0.589Δt w 1 av R where Δt R or ΔV R is the separation between peaks and w av is the average width of the two peaks.
16 Resolution So, separation of mixtures depends on: width of solute peaks (want narrow) efficiency spacing between peaks (want large spacing) selectivity
17 Example What is the resolution of two Gaussian peaks of identical width (3.7 s) and height eluting at 67.3 s and 74.9 s, respectively? ANS: Resolution =.3
18 Diffusion A band of solute broadens as it moves through a column. Ideally, an infinitely narrow band applied to the inlet of the column emerges with a Gaussian shape at the outlet.
20 Diffusion (cont.) One main cause of band spreading is diffusion. The diffusion coefficient measures the rate at which a substance moves randomly from a region of high concentration to a region of lower concentration.
21 Diffusion (cont.) The number of moles crossing each square meter per second, called the flux, is proportional to the concentration gradient: flux mol m s J = D dc dx
23 Broadening of Chromatographic Band by Diffusion If solute begins to move through a column in an infinitely sharp layer with m moles per unit crosssectional area of the column and spreads by diffusion alone, then the Gaussian profile of the band is described by c = m e x ( 4Dt ) 4 π Dt
24 The standard deviation of the band is σ = Dt
26 The Theory of Chromatography: Column Efficiency Plate theory - older; developed by Martin & Synge Rate theory - currently in use
27 Plate Theory - Martin & Synge View column as divided into a number (N) of adjacent imaginary segments called theoretical plates within each theoretical plate complete equilibration of analytes between stationary and mobile phase occurs
29 Plate Theory - Martin & Synge Significance? Greater separation occurs with: greater number of theoretical plates (N) as plate height (H or HETP) becomes smaller L = N H or H = L / N where L is the length of column, N is the number of plates, and H is the plate height
30 First Important Prediction of Plate Theory Band spreading - the width of bands increases as their retention time (volume) increases: Plate height is the constant of proportionality between the variance of the band and the distance it has traveled: σ = Dt = D u x x D = x = u x Hx
31 Second Significant Prediction of Plate Theory The smaller HETP, the narrower the eluted peak
32 Plate Theory - Practical Considerations Not unusual for a chromatography column to have millions of theoretical plates Columns often behave as if they have different numbers of plates for different solutes present in same mixture
33 Number of plates on column: N V = 16 w w b base width of the peak R b This equation is a measure of the efficiency of a column.
34 Sometimes the number of plates is measured at the bandwidth at half-height w 1/ : N = 5.54 V w R 1
35 Estimating the Plate Number for Asymmetric Peaks The Dorsey-Foley equation: N = 41.7 t R w0.1 A B
37 N can be Estimated Experimentally from a Chromatogram Knowing the number of theoretical plates and the length of the column, we can determine the HETP, height equivalent to a theoretical plate: H = HETP = L N = L 16 w V b R = L 16 w t b R
38 Effective Number of Theoretical Plates Introduced to characterize open tubular columns uses adjusted retention volume V R in lieu of total retention volume V R : b ' b ' eff = = w t w V N R R
39 Effective Number of Theoretical Plates (cont.) The N eff value is useful for comparing a packed and an open tubular column when both are used for the same separation. N and N eff are related by the expression N eff = N k' k' + 1
40 Rate Theory Based on a random walk mechanism for the migration of molecules through a column Takes into account: mechanism of band broadening effect of rate of elution on band shape availability of different paths for different solute molecules to follow diffusion of solute along length
41 Van Deemter Equation for Plate Height B H = A + + u x Cu x Multiple paths Longitudinal diffusion Equilibration time
42 In packed columns, all three terms contribute to band broadening In open tubular columns, A is zero In capillary electrophoresis, both A and C go to zero
43 H = A + min B C H = A + min BC u opt = B C
44 Longitudinal Diffusion Gives rise to B/u x term Solute continuously diffuses away from the concentrated center of its zone The greater the flow rate, the less time is spent in the column and the less longitudinal diffusion occurs
45 Longitudinal Diffusion (cont.) The variance resulting from diffusion is σ = D m t = D u m x L Plate height due to longitudinal diffusion: H D = σ L = D u x m B u x
46 Longitudinal Diffusion (cont.) In packed columns, the tortuosity coefficient γ is used to account for irregular diffusion patterns and is usually less than unity (γ ~ 0.6), because molecular diffusivity is smaller in packed columns than in open tubes (γ = 1): H D = γd u x m
48 Longitudinal Diffusion (cont.) Because longitudinal diffusion in a gas is much faster than in a liquid, the optimum flow rate in gas chromatography is higher than in liquid chromatography
49 Nonequilibrium (Resistance to Mass Transfer Term) This term comes from the finite time required for the solute to equilibrate between the mobile and stationary phases Some solute is stuck in the stationary phase, but the remainder in the mobile phase moves forward resulting in spreading of the zone The slower the flow rate, the more complete equilibration is and the less band broadening occurs
51 Resistance to Mass Transfer (cont.) Plate height due to finite equilibration time: H = Cu = + mass transfer x ( C ) s Cm ux where C s describes the rate of mass transfer through the stationary phase and C m describes the rate of mass transfer through the mobile phase. Specific equations for C s and C m depend on the type of chromatography.
52 Resistance to Mass Transfer (cont.) For gas chromatography in an open tubular column, the terms are: Mass transfer in stationary phase: Mass transfer in mobile phase: ( ) s f s D d k k C 1 ' 3 ' + = ( ) m m D r k k k C 1 ' 4 ' 11 ' =
53 Resistance to Mass Transfer (cont.) k the capacity factor d f the thickness of stationary phase film D s the diffusion coefficient of solute in the stationary phase r the column radius D m the diffusion coefficient of solute in the mobile phase
54 Resistance to Mass Transfer (cont.) Efficiency is increased by: Decreasing stationary phase thickness Reducing column radius Increasing temperature
56 Multiple Flow Paths (Eddy Diffusion) The term A accounts for the multitude of pathways that could be followed through the column by one molecule: A = λd p λ dimensionless constant characteristic of packing d p the particle diameter
58 Multiple Flow Paths (Eddy Diffusion) (cont.) The A term can be minimized by: Eliminating column packing (open tubular columns) Reducing the particle size of the packing Packing the column more uniformly (spherical particles of similar size)
61 Alternative Plate Height Equation: The Knox Equation Used to compare column efficiencies Makes use of so-called reduced parameters (dimensionless quantities): Reduced plate height: h = H/d p Reduced velocity: v = ud p /D m 1 b 3 h = aν + + ν cν
62 The Knox Equation (cont.) For well-packed columns of varying particle size and differing conditions, the coefficients a, b and c will be roughly constant: e.g. a =1, b =, and c = 0.05 for porous particles.
63 Modification of the van Deemter Equation: the Giddings Equation Giddings realized that the eddy diffusion and resistance to mass transfer in the mobile phase must be treated dependently: 5 1 B H = + + Csu + Cmu + 1 i + 1 = 1 u A C u 1 H e
64 The Giddings Equation H e extracolumn band broadening C 1 = c b d D m p c b a proportionality constant; c b 1
65 The Giddings Equation (cont.) The five possible mechanisms of band broadening are: Through channels between particles Through particles Resulting from uneven flow channels Between inhomogeneous regions Throughout the entire column length
67 Factors Affecting Resolution A more usable expression for resolution is R s α 1 1 k' = N 4 α k' + 1 efficiency selectivity retention
68 Required Plate Number If k and α are known, the required number of plates can be calculated: The R s value is set at the 6σ level or 1.5 ' ' req = k k R N s α α ' ' req = k k R N s α α
69 Required Column Length The N req parameter can be used to determine the length of column necessary for a separation. We know that N = L/H; thus ' ' req = k k H R L s α α
70 Minimum Analysis Time The minimum analysis time t min is t min = 16R s H u α α 1 ( ' k ) + 1 ( ' k ) 3
71 The Major Objective in Chromatography The goal in chromatography is the highest possible resolution in the shortest possible time. Optimization techniques aim at choosing conditions that lead to a desired degree of resolution with a minimum expenditure of time
72 Optimization Techniques Minimizing plate height: Reducing d p Reducing column diameter Changing column temperature Reducing the thickness of the liquid film Optimizing the flow rate of the mobile phase
73 Optimization Techniques (cont.) Resolution also improves with L, but it expensive in terms of time of analysis Variation in the selectivity factor: Changing the composition of the mobile phase (in HPLC) Changing the column temperature Changing the stationary phase Using special chemical effects
75 Effect of Capacity Factor on Resolution and Elution Time Increases in k enhance resolution but at the expense of elution time The optimum k values are from 1 to 5 The easiest way to improve R s is by optimizing k In GC, k can be improved by temperature programming In LC, k is improved by gradient elution
78 Advantages of Open Tubular Columns Compared with packed columns, open tubular columns can provide: Higher resolution Shorter analysis time Increased sensitivity Lower sample capacity
79 Advantages of Open Tubular Columns (cont.) Compared with packed columns, open tubular columns allow Increased linear flow rate or a longer column or both Decreased plate height, which means higher resolution
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