The “general” elution problem:
a single set of conditions is often unsatisfactory for
good separation of all components in a complex
mixture in an acceptable time (wide range of k values)
a) all components separated but time is long and peak
widths are broad at long times
b) a faster separation and good peak shapes for 5,6
but 1,2 and 3,4 are not resolved
c) intermediate case where 1,2 still not resolved
common solution: change separation conditions
dynamically – e.g. temperature gradient, solvent gradient
Partition Chromatography:
Liquid stationary phase bonded either by adsorption
(non-covalent) or covalent bonding.
Ex. of covalent bonded phase
€
R = C-8, C-18 hydrocarbon (often)
Bonded phases have longer lifetimes and are more
amenable to gradient elution. Generally, lower
sample loading capacities, however.
The analogy to temperature programming in GC is
the variation in solvent composition to dynamically
alter partitioning during elution,
Fixed solvent composition:
isocratic elution
Variation of solvent composition: gradient elution
Normal-phase HPLC
polar stationary phase, non-polar mobile phase
least polar solute elutes first, most polar solute last
Reverse-phase HPLC
non-polar stationary phase (e.g., hydrocarbon)
polar mobile phase (e.g., H2O, CH3OH, CH3CN)
most polar solute elutes first, least polar last
Gradient elution: change of solvent composition to change k values
during a given separation
normal phase:
polar solutes more strongly retained in stationary phase and
therefore they come out later. To reduce the time for elution of
slow eluting species, we want to decrease the k values. To do this,
the solvent gradient must increase polar composition of mobile
phase with time.
reverse phase:
To decrease k values for slow eluting solutes (non-polar), the
solvent gradient must decrease polar composition of mobile
phase with time.
Solvent
fluoroalkane
cyclohexane
n-hexane
carbon tetrachloride
diisopropyl ether
toluene
diethyl ether
dichloroethylene
Polarity Index, P
<-2
0.04
0.1
1.6
2.4
2.4
2.8
3.1
Solvent
tetrahydrofuran
chloroform
ethanol
dioxane
methanol
acetonitrile
nitromethane
water
P
4.0
4.1
4.3
4.8
5.1
5.8
6.0
10.2
A variety of means for detection of species that elute
from the column are available, some of which we
have studied in this course:
Liquid Chromatography –
variations:
liquid-liquid (partition)
adsorption (liquid-solid)
ion-exchange
size-exclusion
affinity
chiral
Gas chromatography (GC)
and liquid chromatography (LC):
The parameters describing chromatograms for both
GC and LC are calculated in the same way. The
concepts developed thus far for extractions and
column chromatography apply to both. The species
for which GC and LC are useful, as well as
technological aspects, differ. (some overlap)
GC – elements of a gas chromatograph system
Carrier gases (mobile phase) – usually He, sometimes
Ar, N2, H2
flow rates – packed columns 1-25 mL/min
open tubular
25-150 mL/min
Columns
length – 2-50 m
elution times – typically 2-30 min.
Temperature – since partitioning is between the gas
and a liquid, efficiencies and elution times are highly
dependent upon temperature. It is usually controlled
to within a few tenths of a degree.
Effect of temperature:
a) isothermal at 45 oC
b) isothermal at 145 oC
c) programmed 30-180 oC
short column + temperature programming can lead to
fast GC (with compromise in resolution)