HPLC Background Chem 250 F 2008
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Outline:
General and descriptive aspects of chromatographic retention and separation:
phenomenological k’, efficiency, selectivity.
Quantitative description of zone migration in partition chromatography: migration
velocity, partition coefficient and theoretical k’.
Theoretical description of efficiency: zone broadening and the Van Deemter
equation.
Solving the general elution problem: Gradient vs. isocratic elutions.
HPLC Instrumentation: pump, injector, column, detector, data collection.
Chromatographic modes: HPLC (reverse and normal phase or ‘flash’), gel
permeation (GPC), gel filtration, ion exchange.
Electrophoresis.
HPLC Background Chem 250 F 2008
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Chromatography: separation of chemicals in a mobile phase by differential flow
rates through stationary phase.
tM
tR
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
HPLC Background Chem 250 F 2008
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
Page 3 of 24
HPLC Background Chem 250 F 2008
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
Page 4 of 24
HPLC Background Chem 250 F 2008
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
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HPLC Background Chem 250 F 2008
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HPLC Background Chem 250 F 2008
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HPLC Background Chem 250 F 2008
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HPLC Background Chem 250 F 2008
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What changes in a
separation if alpha
changes?
How does that
look?
HPLC Background Chem 250 F 2008
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HPLC Background Chem 250 F 2008
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What changes in a
separation if N
changes?
How does that
look?
HPLC Background Chem 250 F 2008
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HPLC Background Chem 250 F 2008
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Theoretical Plates and Plate Count:
2
xi := i
100
1000
S ( x , μ , σ ) :=
1
2 ⋅π ⋅σ
⎛ 1000 ⎞ = 100
⎜
⎟
⎝ 100 ⎠
2
⎛ 1000 ⎞ = 400
⎜
⎟
⎝ 50 ⎠
50
1000
2
⎛ 1000 ⎞ = 1600
⎜
⎟
⎝ 25 ⎠
1000
2
⋅exp⎢
2
N:
⎡ − 1 ⋅( x − μ ) 2 ⎤
⎥
2
⎣ 2 ⋅σ
⎦
2
25
= 10
N
= 0.625
2
12.5
= 2.5
⎛μ⎞
⎜ ⎟
⎝σ⎠
1000
= 0.156
2
2
⎛ 1000 ⎞ = 6400
⎜
⎟
⎝ 12.5 ⎠
Illustration of different plate counts
0.03
6400 plates
0.025
signal
0.02
1600 plates
0.015
400 plates
0.01
0.005
0
100 plates
0
200
400
600
800
1000
1200
1400
retention time or column length
1600
1800
2000
HPLC Background Chem 250 F 2008
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Plate count depends on retention time:
Why does the plate count go up for the more retained peaks?
Because more retained peaks are expected to get broader by longitudinal
diffusion – but these do not.
In other words:
To produce a narrow peak at longer tR requires a higher performance column.
2
N:
2
⎛ 400 ⎞ = 1024
⎜
⎟
⎝ 12.5 ⎠
⎛ 1200 ⎞ = 9216
⎜
⎟
⎝ 12.5 ⎠
2
⎛ 1600 ⎞ = 16384
⎜
⎟
⎝ 12.5 ⎠
⎛ 800 ⎞ = 4096
⎜
⎟
⎝ 12.5 ⎠
N
⎛μ⎞
⎜ ⎟
⎝σ⎠
2
2
Illustration of different plate counts
0.03
0.025
signal
0.02
0.015
0.01
0.005
0
0
200
400
600
800
1000
1200
1400
retention time or column length
1600
1800
2000
HPLC Background Chem 250 F 2008
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The van Deemter Equation:
H = plate height
A = ‘multipath term’
B = longitudinal diffusion term
C = resistance to mass transfer
Plate height as a function of linear flow rate:
H
A+
B
u
+ C ⋅u
12
10
8
H ( 1 , 2 , 0.5 , u)
6
4
2
0
2
4
6
8
10
12
u
Rationalize why is there an optimum flow rate:
Very slow flow allows too much time for diffusional broadening.
B/u term
Excessive flow rate can exceed the rate of partitioning into and out of the
stationary phase.
C*u term
HPLC Background Chem 250 F 2008
H
B
A + + C ⋅u
u
A
2λ ⋅dp
B
2γ ⋅DM
Page 16 of 24
multipath term
longitudinal diffusion
C = complex function of particle
size, coating, diffusion coefficient,
favored in general by large
diffusivity and small particle size
resistance to mass
transfer
HPLC Background Chem 250 F 2008
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Retention and efficiency
together.
The General Elution Problem and Gradient Elution
A mixture of molecules with strongly differing k’ values is hard to separate
because:
If the solvent is weak enough to separate 1 and 2:
a.
Separation takes too long
b.
Peaks 5 and 6 broaden into baseline
If the solvent is strong enough to elute 5 and 6 in a reasonable time:
Peaks 1 and 2 and 3 and 4 co-elute
HPLC Background Chem 250 F 2008
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isocratic (LC) and isothermal (GC) separations are vulnerable to the general
elution problem
the solution to the general elution problem:
o solution gradient elution
(liquid chromatography)
o temperature gradient elution
(gas chromatography)
the gradient methods
o begin with weakly eluting conditions and this helps to separate the
weakly retained species
o finish with strongly eluting conditions to expedite the elution of
strongly retained species and to limit diffusional broadening
in LC gradient elution is a bit more difficult because
o the columns require a fairly significant equilibration time between runs
o the detector baseline can drift significantly if the different solvents have
different UV absorbance at the detection wavelength – if this is a problem,
then one can use
o a different wavelength
or
o a different set of solvents
HPLC Background Chem 250 F 2008
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Example: GC of a complex mixture.
Isothermal:
45°C
Isothermal:
145°C
T-programmed:
30° - 180° C
HPLC Background Chem 250 F 2008
High Performance Liquid Chromatography
Skoog Holler and Nieman Principles of Instrumental Analysis 5th ed.
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HPLC Background Chem 250 F 2008
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Types of LC, all can be performed as HPLC in ‘high performance’
instrumentation if necessary.
Normal Phase:
stationary phase: hydrophilic (SiO2 particles)
mobile phase:
hydrophobic mobile phase (hexane, methanol).
weak solvent:
hexane
strong solvent:
methanol
retained:
polar compounds
eluted first:
non-polar compounds
Reverse Phase:
stationary phase: hydrobic (SiO2 particles coated with hexadecane
monolayer)
mobile phase:
hydrophilic mobile phase (water, methanol)
weak solvent:
water
strong solvent:
methanol
retained:
non-polar compounds
eluted first:
polar compounds
Exclusion Gels
‘GPC’
Gel permeation chromatography: porous polymer gel particles – small
molecules penetrate and are retained, large molecules elute more quickly.
PAGE polyacrlamide gel electrophoresis:
Aqueous gel: -CH2-CONH- used for electrophoresis, large molecules
experience more drag and are retained.
HPLC Background Chem 250 F 2008
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HPLC modes continued Ion Exchange:
Stationary phase is an ionic polymer – e.g. polystyrenesulfonic acid:
SO3-
*
*
n
The solid or gel phase polymer transiently binds cations from solution.
Cations that have a higher affinity for the resin are retained longer and
vice-versa.
HPLC Background Chem 250 F 2008
HPLC System overview:
Injection valve
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HPLC Background Chem 250 F 2008
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Example of Reverse-phase HPLC stationary phase:
Ideal qualities of HPLC stationary phase:
Very fast partitioning between mobile and stationary phase.
1. Ultimate thin film: single molecular layer.
2. ‘Perfect’ – defect free film with no sites for strong adsorption.
Mechanically strong to withstand high pressure w/o collapse.
Chemically inert.
Partition coefficients of solute analytes are all different in single solvent –
i.e. the SP provides a measure of selectivity without being limited to just a
few chosen analytes.