LECTURE 9 CHROMATOGRAPHIC SEPARATIONS The “stuff” you do before you analyze a “complex” sample

Problem Set 26-3, 26-12, 26-16, 26-18, due Thurs. May 12.

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

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

GENERAL DESIGN OF OPTICAL INSTRUMENTS

It is all about “Reducing Interferences”

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

ChromatographyChromatography A. Column Chromatography, B. Planar Chromatography

Column Chromatography Chromatogram Dilution & Peak broadening!

CHROMATOGRAPHY : Peak separations

CHROMATOGRAPHY : Peak Resolution Poor resolution More separation Less band spread

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

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

Chromatography: Velocities Linear rate of solute migration! Velocity = distance/time  length of column/ retention times Velocity of solute: Velocity of mobile phase:

Chromatography Velocity/Retention time and Kc

Chromatography Velocity Relationships

Chromatography Retention Factor : are we there yet? Adjusted retention time

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

Relative retention time: RRT = t R /t Rs t Rs = retention time of internal standard

Chromatography Selectivity Factor: can you separate from your neighbor B retained more than A   >1 Distribution Constant Retention factor Retention time LARGER  = BETTER SEPARATION

CHROMATOGRAPHY Column Efficiency - Theoretical Plates Plate and Rate Theories   standard deviation  2 /L  variance per unit length. L = length of column packing

CHROMATOGRAPHY Relation between column distance and retention times

~96%  2  Tangent at Inflection point Efficient column has small plate height - less zone broadening

Chromatography Determining the Number of Theoretical Plates W 1/2

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

Rate Theory Zone broadening is related to Mass Transfer processes Zone broadening is related to Mass Transfer processes

Column Efficiency Kinetic variables

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?

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.

Zone Broadening Kinetic Processes Van - Deemter Equation

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.

Zone Broadening Longitudinal Diffusion The higher the , the smaller the H Much smaller in LC than in GC Column Diffusion

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

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

CHROMATOGRAPHY Resolution

Chromatographic Separations with a twist

Chromatographic Definitions

Chromatographic Relationships

QUANTITATIVE ANALYSIS Peak areas Peak areas Peak height Peak height Calibration and standards Calibration and standards Internal Standard method Internal Standard method

SUMMARY  Relate to column chromatography  Retention times  Velocities of mobile and component  Height equivalent of theoretical plates  Peak or zone broadening  Resolution