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Introduction to Chromatography Theory

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Plate theory - older; developed by Martin & Synge. Rate theory - currently in use today ... into a number (N) of adjacent imaginary segments called theoretical plates ... – PowerPoint PPT presentation

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Title: Introduction to Chromatography Theory


1
Introduction to Chromatography Theory
  • Bioanalytical Chemistry
  • Spring 2004

2
The Theory of Chromatography
  • Plate theory - older developed by Martin Synge
  • Rate theory - currently in use today

3
Plate Theory - Martin Synge 1954 Nobel Laureates
  • 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

4
Plate Theory - Martin Synge 1954 Nobel Laureates
  • 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 / Nwhere L is length of
    column, N is number of plates, and H is height of
    plates

5
N can be Estimated Experimentally from a
Chromatogram
  • N 5.55 tr2 / w1/22 16 tr2 / w2wheretr is
    retention timew1/2 is full width at maximumw
    is width measured at baseline

6
Choice of Column Dimensions
  • Nmax 0.4 L/dpwhereN - maximum column
    efficiencyL - column lengthdp - particle size
  • So, the smaller the particle size the higher the
    efficiency!

7
Efficiency Relative to Analysis Time
today90 mm L 3 um
today150 mm L 5 um
N
1970s300 mm L 10 um
Analysis Time, min
10
100
8
First Important Prediction of Plate Theory
  • Bandspreading - the width of bands increases as
    their retention time (volume) increases

9
Problem
  • A band exhibiting a width of 4 mL and a retention
    volume of 49 mL is eluted from a column. What
    width is expected for a band with a retention
    volume of 127 mL eluting from the same analyte
    mixture on the same column?
  • ANS 10.4 mL

10
Second significant prediction of plate theory
  • The smaller HETP, the narrower the eluted peak

11
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

12
Rate Theory
  • Based on a random walk mechanism for the
    migration of molecules through a column
  • takes into account
  • 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

13
Van Deemter Equation
  • H A n1/3 B/n C nwhereH is HETP
    (remember want a minimum!)n is mobile phase
    velocityA, B, and C are constants

14
Van Deemter Equation
  • H A n1/3 B/n C n
  • first term - rate of mobile phase movement
    through column (often just a constant)
  • second term - longitudinal solute diffusion
    solute concentration always lower at edges of
    column so solute diffuses longitudinally
  • third term - equilibration is not instantaneous

15
Resolution
  • Ideal chromatogram exhibits a distinct separate
    peak for each solute
  • reality chromatographic peaks often overlap
  • we call the degree of separation of two peaks
  • resolution peak separation
    average peak width

16
Resolution
  • Resolution D tr / wavg
  • lets take a closer look at the significance of
    the problem

17
Resolution
  • So, separation of mixtures depends on
  • width of solute peaks (want narrow)efficiency
  • spacing between peaks (want large
    spacing)selectivity

18
Example
  • What is the resolution of two Gaussian peaks of
    identical width (3.27 s) and height eluting at
    67.3 s and 74.9 s, respectively?
  • ANS Resolution 2.32
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