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Chromatographic separations. Chapter 26 The “stuff” you do before you analyze a “complex” sample. It is all about “Reducing Interferences”. Mobile and Stationary phase Retention - Migration Bands or zones Equilibrium!. Column vs. planar Liquid vs. gas vs. SF High vs. low resolution

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chromatographic separations

Chromatographic separations

Chapter 26

The “stuff” you do before you analyze a “complex” sample

chromatography basics
Mobile and Stationary phase

Retention - Migration

Bands or zones


Column vs. planar

Liquid vs. gas vs. SF

High vs. low resolution



Ion exchange

Size exclusion

Chromatography basics

A. Column Chromatograpgy, B. Planar Chromatograpgy

column chromatography
Column Chromatography

Dilution &

Peak broadening!


chromatography peak resolution
Chromatography: Peak Resolution

Poor resolution

More separation

Less band spread

chromatography distribution constant recommended by iupac old term partition coefficient
Chromatography: Distribution Constant(recommended by IUPAC)(old term: partition coefficient)

A mobile ↔ A stationary



CS = nS/VS, CM = nM/VM

K ~ constant  linear chromatography

>>>K >>> Retention in the stationary phase  Retention times

How to manipulate K?

chromatography retention times
Chromatography Retention Times

tM = retention time of mobile phase (dead time)

tR = retention time of analyte (solute)

tS = time spent in stationary phase (adjusted retention time)

L = length of the column

chromatography velocities linear rate of solute migration
Chromatography: VelocitiesLinear rate of solute migration!

Velocity = distance/time  length of column/ retention times

Velocity of solute:

Velocity of mobile phase:

Relative retention time:

RRT = tR/tRs

tRs = retention time of internal standard

chromatography selectivity factor can you separate from your neighbor
Chromatography Selectivity Factor: can you separate from your neighbor

B retained more than A  a >1

Distribution Constant

Retention factor

Retention time

chromatography column efficiency theoretical plates plate and rate theories
Chromatography Column Efficiency - Theoretical PlatesPlate andRate Theories

L = length of column packing

s standard deviation s2/L variance per unit length.

chromatography relation between column distance and retention times18
Chromatography Relation between column distance and retention times


 2

Tangent at

Inflection point

summary of plate theory
Summary of Plate Theory
  • Successfully accounts for the peak shapes and rate of movement
  • Does not account for the “mechanism” causing peak broadening
  • No indication of other parameters’ effects
  • No indication for adjusting experimental parameters
rate theory
Rate Theory
  • Zone broadening is related to Mass Transfer processes
zone broadening flow rate of mobile phase
Zone BroadeningFlow 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
Zone BroadeningKinetic Processes

Van - Deemter


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 processes25
Zone BroadeningKinetic Processes

Van - Deemter Equation

zone broadening multiple pathways
Zone BroadeningMultiple Pathways
  • Eddy Diffusion: band broadening process results from different path lengths passed by solutes.
    • Directly proportional to the diameters of packing
    • Offset by ordinary diffusion
    • Lower mobile-phase velocity, smaller eddy diffusion

Stagnant pools of mobile phase

retained in stationary phase.

quantitative analysis
Quantitative Analysis
  • Peak areas
  • Peak height
  • Calibration and standards
  • Internal Standard method
  • Relate to column chromatography
  • Retention times
  • Velocities of mobile and component
  • Height equivalent of theoretical plates
  • Peak or zone broadening
  • Resolution