Principles of Chromatography

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Chapter 21

• Principles of Chromatography

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• Chromatography is the most powerful tool for
  separating measuring the components of a
  complex mixture.
• Quantitative qualitative analysis

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21-1 What is Chromatography?

• 1) Solvent Extraction
• transfer of a solute from phase 1? phase 2
• S (in phase1) ? S (in phase 2)
• partition coefficient

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21-1 What is Chromatography?

• 2) Chromatography same as extraction
• a) One phase held in place
• ? stationary phase.
• solid material (packing material)
• Another phase fluid phase
• ? mobile phase.
• sample gas (GC)
• liquid (LC)

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21-1 What is Chromatography?

• b) A solute equilibrates between a mobile and a
  stationary phase.
• The more it interacts with the stationary phase,
  the slower it is moved along a column.
• Xm ? Xs
• Ks Xs / Xm
• Solutes with a large Ks value will be retained
  more strongly by the stationary phase.

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21-1 What is Chromatography?
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Color Plate 22
COLOR PLATE 22 Thin-Layer Chromatography (a)
Solvent ascends past mixture of dyes near bottom
of flat plate coated with solid adsorbent. (b)
Separation achieved after solvent has ascended
most of the way up the plate. 
P.459
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21-1 What is Chromatography?

• c) The science art of separation
• d) Originator adsorption chromatography by
  M.Tswett in 1903
• e) Eluent, eluate, elution.

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21-1 What is Chromatography?

• elution always (100) dilution

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21-1 What is Chromatography?

• 3) Types of Chromatography
• Is divided into categories on the basis of the
  mechanism of interaction of the solute v.s. the
  stationary phase.

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21-1 What is Chromatography?
polar s.p.
for GC LC for GC
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21-1 What is Chromatography?
resin-SO3- gel
filtration resin-N(CH3)3
by size
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21-1 What is Chromatography?
the most selective one
pH, and ionic strength
Ask Yourself 20-A p.461
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21-2 How do we describe a chromatogram

• 1) Chromatogram
• A graph showing the detectors response as a
  function of elution time bands shapes,
  position, resolution.

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21-2 How do we describe a chromatogram

• 2) For individual band
• Retention time (tr) the time needed after
  injection for an individual solute to reach
  detector.
• An ideal chromatographic peak? Gaussian shape.
  w½ 2.35s, w 4s

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21-2 How do we describe a chromatogram
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21-2 How do we describe a chromatogram
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21-2 How do we describe a chromatogram

• For pairs of bands
• Efficiency two factors contribute to how well
  components are separated
• the widths of the peaks
• the wider the peak, the poorer
  separation.
• the spacing in time
• the further apart, the better separation.

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21-2 How do we describe a chromatogram

• Theoretical plates (N) (from distillation)
• the more plates on a column, the more
  equilibration steps, and the better the
  separation.
• Number of plates on column
• N 5.55(tr/w½)2
• Plate height H L/N
• The smaller plate height
• ? narrower peaks ? better separation

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21-2 How do we describe a chromatogram

• c) Resolution (Rs)

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21-2 How do we describe a chromatogram
d) Qualitative Quantitative analysis

• Qualitative
• Co-chromatography
• Detector
• Mass spectrometer
• IR, UV-VIS spectrophotometer

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Qualitative and Quantitative Analysis

• Figure 21-5 illustrates the point that computers
  and humans may not choose the same baseline for
  measuring area.

P.464
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5-4 Internal Standards (p119)

• An internal standard is known amount of a
  compound, different from analyte, that is added
  to an unknown.
• To use an internal standard, we prepare a known
  mixture of standard and analyte and measure the
  relative response of the detector to the two
  species. In Figure 5-6, the area under each peak
  is proportional to the concentration of each
  compound injected into the column.

P.119
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• X and S are the concentrations of analyte and
  standard after they have been mixed together.

P.119
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• Example Using an Internal Standard
• In a chromatography experiment, a solution
  containing 0.083 7 M X and 0.066 6 M S gave peak
  areas of Ax423 and AS347.
• To analyze the unknown, 10.0 mL of 0.146 M S were
  added to 10.0 mL of unknown, and the mixture was
  diluted to 25.0 mL in a volumetric flask.
• This mixture gave the chromatogram in Figure 5-6,
  with peak areas Ax533 and AS582. Find the
  concentration of X in the unknown.

P.119
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• SOLUTION

Because X was diluted from 10.0 to 25.0 mL when
the mixture with S was prepared, the original
concentration of X in the unknown was
(25.0/10.0)(0.057 21 M)0.143 M.
P.120
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21-2 How do we describe a chromatogram

• e) Scaling up (rule at p.464)
• Analytical chromatography long thin column.
  For a small scale separate, identify, or
  measure.
• Preparative chromatography short, fat column.
  For large scale purify

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21-3 Why do bands spread ?

• 1) Why broadening?
• a) diffusion
• b) slow equilibration of solute between the m.p
  and s.p.
• c) irregular flow paths.

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21-3 Why do bands spread ?

• Longitudinal diffusion
• the faster the flow
• ? the less a band spends in column.
• ?the less time for diffusion.
• ? broadening

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21.3 Why do bands spread ?

• solute requires time to equilibrate between
  phases.
• (s.p.?m.p.)? with temp.?
• broadening ? u
• Cant equilibrate rapidly enough.

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21-3 Why do bands spread ?
Figure 21-8 Solute requires a finite time to
equilibrate between the mobile and stationary
phases.
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21-3 Why do bands spread ?

• 4) A Separation Has an Optimum Flow Rate
• The rate of mass transfer between phases
  increases with temperature.

Figure 21-9 Optimum resolution (minimum plate
height) occurs at an intermediate flow rate.
Curves show measured plate height in
gas chromatography of n-C17H36 at 175C, using
N2, He, or H2 mobile phase.
P.466
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21-3 Why do bands spread ?

1. Multiple paths

Figure 21-10 Band spreading from multiple flow
paths. The smaller the stationary-phase
particles, the less serious is this problem. This
process is absent in an open tubular column.
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21-3 Why do bands spread ?

• 6) Plate height equation

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Plate height equation
21-3 Why do bands spread ?
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21-3 Why do bands spread ?

• 7) open tubular columns
• Packed column (A, B, C ? 0 in van Deemters eqn.)
• Open tubular column (A 0 in van Deemters eqn.)
  
• ? resolution? (? H? column length?)
• ? sample capacity? (? less s.p.)

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21-3 Why do bands spread ?

• 8) Funny shapes

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21-4 Mass Spectrometry
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21- 4 Mass Spectrometry

• Mass spectrometry measures the masses and
• abundances of ions in the gas phase.
• A Mass Spectrometer
• Figure 21-13 shows a transmission quadrupole mass
  spectrometer, which is the most common mass
  separator in use today.
• The mass separator consists of four parallel
  metal rods to which a constant voltage and a
  radio-frequency oscillating voltage are applied.

P.470
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Figure 21-13
Figure 21-13 Transmission quadrupole mass
spectrometer.
P.470
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21-4 Mass Spectrometry

• Ionization
• 1) Electron ionization
• 2) Chemical ionization

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1) Electron ionization
M e- ? M e- e- 70 eV
-55 eV 0.1eV
Molecular ion break into fragments. Base peak
most intense peak.
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2) Chemical ionization
CH4 e- ? CH4 2e- CH4 CH4 ? CH5
CH3 CH5 M ? CH4 MH CH4 ? CH3 H CH3
CH4 ? C2H5 H2
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• Total ion Chromatograms
• 21-16a is a reconstructed total ion chromatogram
  showing all ions from seven opium alkaloids found
  in street heroin.
• Selected ion Chromatograms
• Simplify analysis
• improve S/N

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21-5 Information in a Mass Spectrum

• Nominal Mass C4H9Br is 136

P.473
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21-5 Information in a mass spectrum
Rxn CH3(CH2)2CH2OH Br- ? CH3(CH2)2CH2Br
1Butanol
1Bromobutane
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21-5 Information in a mass spectrum
Fragmentation Patterns
C4H979Br 50.0 C4H981Br
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21-5 Information in a mass spectrum
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21-5 Information in a mass spectrum

• Isotope Patterns
• CnHxOyNz
• 12C/13C
• Intensity n x 1.1
• Ex C6H6
• (M1)/M 6 x 1.1
• Nitrogen Rule
• A compound odd nominal mass / odd number of
  N atoms even nominal mass/ even number of N
  atoms