Various Gradient Elution
Methods in Supercritical Fluid
Chromatography
and
their Applications to the Separation of
Polystyrene Oligomers
Introduction
Chromatogram
(c) was obtained by the
negative temperature
programming
method, decreasing the
temperature as a function of
We have
demonstrated three types of
gradient elution
methods
time.
The polystyrene oligomer
was separated effectively
with
applicable
to Supercritical Fluid Chromatography
(SFC) as they
similar
spacing between peaks and
peaks of higher than 10-mers
are
pertain to
the separation of polystyrene
oligomers. The three
modes
clearly
observed. In addition, there is no
appreciable baseline drift.
of gradient
elution are as follows: 1)
The modifier
programming
Chromatogram
(d) was obtained by the
pressure programming
method
(LC-like), 2) The temperature gradient
method (GC-like),
method,
elevating the temperature as a
function of time.
This
and 3)
The pressure programming
method that is unique to
SFC. In
chromatogram
(d) is similar to chromatogram
(c) obtained by using
the
modifier programming method,
the content of organic
modifier in
the
negative temperature programming
method, and shows
clearly
the
mobile phase is varied as a
function of time. This is
similar to
separated
peaks. However, the monomer
peak is not resolved
from
binary
gradient elution in HPLC.
There are two basic
methods that
the
solvent peak.
both
give similar effects in the
temperature gradient method.
One is
the
positive temperature programming
method that elevates
tempera-
Conclusion
ture
versus time. This is
comparable to gas chromatography.
The
other is
negative temperature programming
method that lowers
As we have
demonstrated, SFC has three
different gradient
temperature
versus time. When sample
solutes are volatile,
the
methods.
The most effective method
can be selected easily to
obtain
positive
temperature programming method
can be applied
because
the
most efficient separations of
various compounds suitable for
use
the
solutes` vapor pressures
increase as the temperature is
elevated,
with
SFC.
and
when the temperature goes
high enough for the solutes
to be
vaporized in
the pressurized mobile
phase, the solutes elute in
the
Injector or
auto sampler
same
manner as in GC. On the
contrary, if the solutes are
non-
volatile,
they require the solvating
power of the supercritical
mobile
Oven
phase to be
eluted. Higher solvating
power is obtained by
increasing
Liquid CO2
delivery
pump
the
density of the supercritical
mobile phase. In the
isobaric mode,
Preheating
Column
the
density of the mobile phase
increases as the temperature of
the
coil
mobile
phase decreases, offering
the higher solvating power.
Thus,
Back
pressure
regulator
both
the negative and positive
programming methods can be
effective
CO2 cylinder
for
separation of moderately volatile
solutes. In the isothermal
mode,
the
higher the pressure the
higher the density of the
mobile phase
becomes,
increasing the mobile
phase's strength. This
pressure
Modifier
Pump
PDA
detector
programming
method is unique to SFC. We
have applied all three
of
with
high-pressure cell
these
methods to the separation of
polystyrene oligomers up to
10-mer
and have reported the
results of each.
Figure 1:
Flow Diagram of SFC
System
Experimental
n=5
3.0E+05
(a)
Isobaric
The
apparatus used in this
experiment was a JASCO SFC
system.
n=10
The
system consisted of a
PU-2080-CO2
carbon
dioxide pump, a
0.0
PU-2080
modifier pump, an AS-2059-SF
autosampler, a preheating
3.0E+05
n=5
(b) Solvent
program
coil, a GL
Science GC-4000 gas chromatograph
oven, a SFCpak SIL
n=10
column
(4.6mmI.D. x 250mmL. packed with
5µm silica gel), a
MD-
2010 PDA
detector with a high pressure
cell and a BP-2080
0.0
n=5
1.2E+06
automatic
back-pressure regulator. The
oven was used to control
the
(c)
Negative temp. program
column
temperature to perform the
temperature programming
n=10
methods,
and the back-pressure
regulator was used to
control the
0.0
n=5
pressure to
perform the pressure
programming method. Figure
1
3.0E+05
(d) Pressure
program
shows
the flow diagram of the
SFC system used for the
experiment.
n=10
0.0
4.0
8.0
12.0
Results
and Discussion
Time
(min)
Figure 2:
Chromatograms of Polystyrene
Oligomer
Figure 2
shows chromatograms of polystyrene
oligomers
obtained by
the isobaric method (a)
and different gradient
methods,
Isobaric
mode
Chromatogram
(a)
i.e.,
solvent programming (b),
negative temperature
programming
CO2:
3.0
mL/min
(c),
and the positive temperature
programming (d) methods.
Gradi-
Solvent
(EtOH):
0.4
mL/min
Pressure:
20.0
MPa
ent
conditions are listed in
Table 1.
Temperature:
60ºC
As can be
seen in Chromatogram (a) by the
isobaric method, the
distances
between peaks becomes longer
and the peak
height
Gradient elution
mode:
Solvent
program
Chromatogram
(b)
CO2:
3.0
mL/min
becomes
lower as the degree of
polymerization goes high,
and the
Solvent
(EtOH):
0.4 mL/min -> 0.7
mL/min (10min)
peaks of
higher than 10-mer peaks
are hardly seen.
Pressure:
20.0
MPa
Temperature:
60ºC
Chromatogram
(b) was obtained by the
modifier programming
method,
increasing the ethanol
content as a function of time in
the
Gradient elution
mode:
Negative temperature
program
Chromatogram
(c)
same
manner as in HPLC gradient
elution method. By this
method,
CO2:
3.0
mL/min
Solvent
(EtOH):
0.4
mL/min
the
overall separation time is
shortened; however, baseline
drift is
Pressure:
20.0
MPa
observed
and increases with increasing
modifier concentration. It
Temperature:
100ºC ->
-5ºC/min
should
also be noted that the
column must be re-equilibrated to
the
Pressure
program
Chromatogram
(d)
initial
modifier concentration before
the next injection can
be
CO2:
3.0
mL/min
performed.
This is analogous to the
requirement when using
binary
Solvent
(EtOH):
0.4
mL/min
HPLC
gradient methods. This
yields a longer cycle time
between
Pressure:
15.0 MPa
-> 25.0 MPa
(10min)
Temperature:
80ºC
injections.
Table 1:
Gradient Conditions
