An%20Introduction%20to%20Chromatographic%20Separations

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An Introduction to Chromatographic Separations

• Lecture 36

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• Dividing both nominator and denominator by kB
• R (N1/2/4)(1 kA/kB)/(1kB)/kB
• However, a kB/kA
• R (N1/2/4)(1 1/a) (kB)/(1kB)
•  

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• Therefore, resolution can be viewed as a
  composite contribution of three terms
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• a.       Efficiency term where R is proportional
  to N1/2
• How to increase efficiency?
• 1. Increase column length as N L/H

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• 2. Decrease H
•  a. In liquid chromatography
• Resistance to mass transfer terms (K2 ds2 V/Ds
  and K3 dp2 V/DM) are most important in liquid
  chromatography and thus should be particularly
  minimized. This can be done by
• Decreasing particle size
• Decreasing the thickness of stationary phase
• Working at low flow rates
• Increase DM by using mobile phases of low
  viscosities.

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•  a. In gas chromatography
• Longitudinal diffusion term (k1DM/V) is the most
  important one in gas chromatography. Reducing
  this term involves
• Working at higher flow rates
• Decreasing DM by using carrier gases of higher
  viscosities

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• b.      Retention term where R is proportional to
  k/(1k) which suggests that the retention
  parameter should be optimized. A value for k in
  the range from 5-10 is preferred as smaller
  values (low retention) results in bad resolution
  while a very high k value means very long
  retention with exceedingly small improvements in
  resolution

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These data can be better viewed as a plot where
as k was increased, almost a plateau was
realized.

•  

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• The overlap of two Gaussian peaks of equal area
  and amplitude, at various values of resolution
  (R) is presented below
•  
• (1) R 0.50                                   
• Overlap of two peaks 16
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• (2) R 1.00                         
• Overlap of two peaks 2.3 
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• (3) R 1.50
• Overlap of two peaks 0.1
• c.       Resolution is dependent on a selectivity
  term (a 1)/a). As the selectivity is
  increased, resolution increases as well. When a
  1, resolution is zero.

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• When a is close to unity, optimising k' and
  increasing N is not sufficient to give good
  separation in a reasonable time. In these cases,
  a is increased by one of the following
  procedures
• 1.      Changing mobile phase composition
• 2.      Changing column temperature
• 3.      Changing composition of stationary phase
• 4.      Using special chemical effects (such as
  incorporating a species which complexes with one
  of the solutes into the stationary phase or use
  of surfactants)

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Effect of Other Parameters on Resolution

• a. The resolution of a column is proportional to
  the square root of its length since N L/H.
•  LA/LB RA2/RB2
• b. Retention time as related to resolution can be
  obtained by the following treatment
• N L/H
• tM NH/u

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• tR,B tM (1kB)
• tR,B (NH/u) (1kB)
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• N 16R2(1 kB)/kB2a /(a - 1)2
• Substitution gives
• tR,B (16R2H/u)(1 kB)3 /kB2a /(a - 1)2
• Therefore, one can also write
• tR,A/tR,B RA2/RB2

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• To obtain a high resolution, the three terms must
  be maximized. An increase in N, the number of
  theoretical plates can simply be done by
  lengthening the column. This leads to two
  opposing effects where resolution is increased
  but at the same time this causes an increase in
  retention time and thus increased band
  broadening. In addition, a longer column may not
  always be available. An alternative is to
  increase the number of plates, the height
  equivalent to a theoretical plate by adjusting
  elution variables (mobile phase composition), and
  other factors affecting selectivity.

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Chromatographic Relationships
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• It is often found that by controlling the
  capacity factor, k', separations can be greatly
  improved. This can be achieved by changing the
  temperature (in Gas Chromatography) or the
  composition of the mobile phase (in Liquid
  Chromatography).

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The General Elution Problem

• Look at the chromatogram below in which six
  components are to be separated by an elution
  process
•  

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Qualitative Analysis

• Usually, the retention time of a solute is the
  qualitative indicator of a specific analyte. The
  retention time of an analyte is thus compared to
  that of a standard. If both have the same
  retention time, this may be a good indication
  that the identity of the analyte is most probably
  that of the standard. However, there can be
  important uncertainties since some different
  compounds have similar retention. In such cases,
  it is not wise to use the retention time as a
  guaranteed marker of the identity of compound,
  except in cases where the sample composition is
  known.

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