Introduction to Chromatographic Separations

1
Introduction to Chromatographic Separations

• Due to lack of analytical specificity,
  separations are often necessary
• Chromatography is about separations
• need minimum of two phases
• stationary phase
• mobile phase
• analyte and matrix must have differing affinities
  for one or more phases

2
Example of Chromatographic Experiment
The compounds A and B which are attracted to
the stationary phase form bands, or zones, along
the length of the stationary phase
In this example, compound B is more attracted
to the stationary phase than is compound A
3
Intro to Band Broadening, I

• As analytes migrate farther through the
  stationary phase, band broadening occurs

4
Intro to Band Broadening, II

• Band broadening decreases chromatographic
  resolution
• ? we need to optimize the chromatographic method

5
Chromatographic 'Figures of Merit'

• Table 26-5 in text, 'Calculation of Derived
  Quantities'
• a series of formulas that describe, in various
  ways, aspects of chromatographic performance
• we will not examine the derivation of these here,
  but you may find it helpful to follow the
  derivation in the text as an aid to understanding
  Table 26-5
• many of the derived quantities in Table 26-5 are
  themselves functions of derived quantities
• the question becomes, what can we measure? (and
  how does that relate to the derived quantities)

6
What we can measureExperimental Quantities (see
Table 26-4)

• migration time of unretained species
• (dead time) tM
• retention time (species A and B) (tR)A, (tR)B
• adjusted retention time (species A) (tR)A- tM
• peak width (species A) WA
• length of column packing L
• flow rate of mobile phase F
• volume of stationary phase VS
• concentration of analyte in mobile
  and stationary
  phases cM, cS
• actually, cM, cS aren't practical to measure

Chromatographic figures of merit

• resolution
• capacity factor
• selectivity

7
Resolution

• From experimental data
• As a derived quantity (rearranged from last
  equation in Table 26-5)
• u is the linear velocity of the mobile phase,
  also related to the Height Equivalent of a
  Theoretical Plate (HETP) or H
• ? is the selectivity factor
• k' is the capacity or retention factor

The Capacity or Retention Factor

• Experimentally
• gives relative value for attraction of analyte to
  the stationary phase

The Selectivity Factor

• Experimentally
• Derived
• Info on the preferential attraction of A for the
  stationary phase relative to B (1? ? ? ?)

8
Plates, u and the van Deempter Equation

• plate conceptually separations unit
• the more plates, N, the better the separation
• the shorter the plate, the more that can be
  stuffed onto fixed length, L, column
• experimentally
• empirically (the van Deempter equation)

9
van Deempter Plots
Liquid Chromatography
Gas Chromatography
10
Laundry List of Experimental Parameters of Concern

• number of plates, HETP ? L
• stationary phase ? column type, head pressure
• mobile phase ? F, head pressure, mixture,
  gradient
• temperature (programmable) ? T
• quality of column packing ? column type

11
The 'General Elution Problem'