Download presentation
Presentation is loading. Please wait.
Published byEarl Bates Modified over 9 years ago
1
LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample
2
Problem Set 26-3, 26-12, 26-16, 26-18, 26-25 due Thurs. May 12.
3
What is CHROMATOGRAPHY? Many determinations involve separation followed by analysis chromatography electrophoresis CHROMATOGRAPHY: sample transported by mobile phase electrostatic or van der Waals' some components in sample interact more strongly with stationary phase and are more strongly retained sample separated into zones or bands
4
CHROMATOGRAPHY The MOBILE PHASE – (the solvent moving through the column) is either a liquid or a gas. The STATIONARY PHASE – (the one that stays in place inside a column) – is a viscous liquid chemically bonded to the inside of a capillary tube unto the surface of solid particles. Fluid entering the column is the ELUENT Fluid emerging from the end of the column is ELUATE
5
GENERAL DESIGN OF OPTICAL INSTRUMENTS
6
It is all about “Reducing Interferences”
7
CHROMATOGRAPHY BASICS Mobile and Stationary phase Mobile and Stationary phase Retention - Migration Retention - Migration Bands or zones Bands or zones Equilibrium! Equilibrium! Column vs. planar Liquid vs. gas vs. SF High vs. low resolution Partition Adsorption Ion exchange Size exclusion
8
ChromatographyChromatography A. Column Chromatography, B. Planar Chromatography
9
Column Chromatography Chromatogram Dilution & Peak broadening!
10
CHROMATOGRAPHY : Peak separations
11
CHROMATOGRAPHY : Peak Resolution Poor resolution More separation Less band spread
12
CHROMATOGRAPHY: Distribution Constant (recommended by IUPAC) (old term: partition coefficient) stationary mobile A mobile ↔ A stationary K ~ constant linear chromatography >>>K >>> Retention in the stationary phase Retention times How to manipulate K? C S = n S /V S, C M = n M /V M
13
CHROMATOGRAPHY Retention Times t M = retention time of mobile phase (dead time) t R = retention time of analyte (solute) t S = time spent in stationary phase (adjusted retention time) L = length of the column
14
Chromatography: Velocities Linear rate of solute migration! Velocity = distance/time length of column/ retention times Velocity of solute: Velocity of mobile phase:
15
Chromatography Velocity/Retention time and Kc
16
Chromatography Velocity Relationships
17
Chromatography Retention Factor : are we there yet? Adjusted retention time
18
When k' A is <1.0, separation is poor When k' A is >30, separation is slow When k' A is 2-10, separation is optimum
19
Relative retention time: RRT = t R /t Rs t Rs = retention time of internal standard
20
Chromatography Selectivity Factor: can you separate from your neighbor B retained more than A >1 Distribution Constant Retention factor Retention time LARGER = BETTER SEPARATION
21
CHROMATOGRAPHY Column Efficiency - Theoretical Plates Plate and Rate Theories standard deviation 2 /L variance per unit length. L = length of column packing
22
CHROMATOGRAPHY Relation between column distance and retention times
23
~96% 2 Tangent at Inflection point Efficient column has small plate height - less zone broadening
24
Chromatography Determining the Number of Theoretical Plates W 1/2
25
Summary of Plate Theory Successfully accounts for the peak shapes and rate of movement Successfully accounts for the peak shapes and rate of movement Does not account for the “mechanism” causing peak broadening Does not account for the “mechanism” causing peak broadening No indication of other parameters’ effects No indication of other parameters’ effects No indication for adjusting experimental parameters No indication for adjusting experimental parameters
26
Rate Theory Zone broadening is related to Mass Transfer processes Zone broadening is related to Mass Transfer processes
27
Column Efficiency Kinetic variables
28
Zone Broadening Flow Rate of Mobile Phase Liquid chromatography Gas chromatography Note the differences in flowrate and plates height scales Why GC normalluy has high H, but also high overall efficiency?
29
Zone Broadening Kinetic Processes Van - Deemter Equation λ and γ are constants that depend on quality of the packing. B is coefficient of longitudinal diffusion. Cs and Cm are coefficients of mass transfer in stationary and mobile phase, respectively.
30
Zone Broadening Kinetic Processes Van - Deemter Equation
31
Zone Broadening Multiple Pathways Eddy Diffusion: band broadening process results from different path lengths passed by solutes. 1.Directly proportional to the diameters of packing 2.Offset by ordinary diffusion 3.Lower mobile-phase velocity, smaller eddy diffusion Stagnant pools of mobile phase retained in stationary phase.
32
Zone Broadening Longitudinal Diffusion The higher the , the smaller the H Much smaller in LC than in GC Column Diffusion
33
Zone Broadening Mass Transfer between Phases Slow equilibrium of solute between mobile and stationary phases Time is required for solute molecules to diffuse from the interior of these phase to there interface where transfer occur
34
Zone Broadening Longitude vs. Mass Transfer Longitude Parallel to the flow Inversely proportional to the flow of the mobile phase Mass Transfer Diffusion tends to be right angles to the flow The faster the mobile phase moves, the larger the band broadening
35
CHROMATOGRAPHY Resolution
36
Chromatographic Separations with a twist
37
Chromatographic Definitions
38
Chromatographic Relationships
39
QUANTITATIVE ANALYSIS Peak areas Peak areas Peak height Peak height Calibration and standards Calibration and standards Internal Standard method Internal Standard method
40
SUMMARY Relate to column chromatography Retention times Velocities of mobile and component Height equivalent of theoretical plates Peak or zone broadening Resolution
Similar presentations
© 2025 SlidePlayer.com. Inc.
All rights reserved.