ACHIRAL CHROMATOGRAPHIC METHOD
DEVELOPMENT STRATEGIES WITH
CARBON DIOXIDE MOBILE PHASES
Paula Hong, Isabelle Durieux, Michael Jones and Patricia McConville
Waters Corporation, Milford, MA 01757
INTRODUCTION
1
AU
0.20
5
2
BEH
0.10
3
4
0.00
0.15
1
5
AU
0.05
3
BEH 2-EP
2
0.10
In convergence chromatography, retention of non
polar compounds can be challenging. Given these
considerations, the method development strategy
should evaluate those factors which have the
greatest impact on retention. These include
modifier, gradient, and physical parameters
(temperature and pressure).
4
2 5
1, 2
3,4
4
3
5
0.10
1, 2
1.00
0.00
1
0.50
0.00
AU
AU
AU
HSS C18 SB
1
0.10
CSH FP
4
0.50
1.00
AU
4
2.00
3.00
4.00
5.00
6.00
RESULTS AND DISCUSSION
Method Development Strategies
for Polar Compounds
0.50
4
1
5
1-caffeine
2-thymidine
3-amitriptyline
4-noritriptyline
5-prednisolone
2
0.10
1, 2
0.50
0.00
0.10
0.20
0.40
AU
1
4
2
1:1
Methanol
Acetonitrile
2
0.05
0.00
0.20
5
3
0.10
1.00
2
0.2% NH4OH in
Methanol
4
2.00
1-propiophenone
2-valerphenone
3-diethyl phthalate
4-dioctyl phthalate
50 °C
USP Res= 0.85
0.40
3
4
0.40
40 °C
USP Res= 1.24
0.20
0.60
3.00
4.00
0.00
5.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
Minutes
Figure 5. Effect of temperature. A neutral standard
mix was screened on a non-polar (HSS C18 SB) stationary phases with acetonitrile as a modifier.
Earlier elution for phthalates (3,4) was observed with
lower temperatures. In addition, selectivity differences
for phenones (1,2) resulted in increased resolution
with lower temperature.
CONCLUSIONS
127.05
195.05
30 °C
USP Res= 1.36
0.40
Combined - SQ1: MS Scan 1: 50.00-750.00 ES+, Continuum, CV
1.101 Combined - SQ1: MSScan 1: 50.00-750.00 ES+, Continuum
2.0x107
1.5x107
3x107
Intensity
Intensity
1.20
0.20
Figure 2. Effect of mobile phase additives on peak
shape of basic compounds. A basic/neutral mix was
screened on a bare silica (BEH) stationary phase with
different additives. Improved peak shape for strong
bases was observed with mobile phases containing
basic additives (0.2% NH4OH and 15mM ammonium
formate with 2% HCOOH in methanol). A buffered/
acidic mobile phase provided lowest peak widths and
greatest peak height for the basic analytes.
2x107
1.0x107
265.21
1x107
5.0x106
87.07
227.28283.61
200.00
483.85
400.00
649.62
600.00
m/z
98.87
100.00
167.10181.16 243.21 281.21322.44
200.00
4
3
86.99
0.0
2.0x109
Intensity
1.10
0.60
Minutes
0
1.00
0.00
1
5x107
0.90
0.00
0.00
300.00
TIC ESI+
m/z
1
1.5x109
5
1.0x109
2
5.0x108
0.20
UV @ 254nm
0.15
AU
1
AU
5
0.10
15mM Ammoni2 3 4 um Formate with
2% HCOOH in
Methanol
AU
AU
1
2.5x109
0.80
0.20
0.00
3.0x109
0.60
0.70
Minutes
0.60
AU
AU
5
0.50
Figure 4. Column screening of non-polar compounds.
A neutral standard mix was screened on polar (BEH 2EP and CSH FP) and non-polar (HSS C18 SB) stationary phases with 1:1 methanol/acetonitrile modifier
blend. Co-elution and poor retention were observed
for pheones (1,2). The greatest retention was observed on a hydrophobic (HSS C18 SB) stationary
phase.
4x107
Method development strategies often begin
with a
screening protocol. This
process
typically evaluates a set of stationary phases
selected to provide differences in selectivity.
While this approach can be utilized in
convergence chromatography, there are also
other considerations. For example, in liquid
CO 2
chromatography,
hydrogen-bonding
interactions can affect both peak shape and
retention of polar analytes. To address these
undesired effects, additives in the mobile
phase can improve peak shape.1,2 Given these
factors, any method development strategy
should evaluate additives in the mobile phases,
preferably those compatible with MS for multidetection method development.
0.30
0.00
0.10
CSH FP
3 4
Methanol
3
3
0.00
AU
Figure 1. Column screening of polar compounds. A
basic/neutral mix was screened on polar (BEH 2-EP
and CSH FP) and non-polar (HSS C18 SB) stationary
phases with methanol as the modifier. The basic compounds exhibited poor peak shape on all stationary
phases. The greatest peak height for bases (3,4) was
observed on the polar stationary phase, BEH 2-EP.
Alternative selectivity was observed on the HSS C18
SB stationary phase.
AU
Columns: (3.0 x 100 mm)
ACQUITY UPC2 BEH Column, 130Å, 1.7 µm,
ACQUITY UPC2 HSS C18 SB Column, 100Å, 1.8
µm,
ACQUITY UPC2 BEH 2-Ethylpyridine Column,
130Å, 1.7 µm
ACQUITY UPC2 CSH Fluoro-Phenyl Column,
130Å, 1.7 µm
Mobile Phase A: CO2
Mobile Phase B:
B1:Methanol; B2: 1:1 Methanol/
Acetonitrile; B3: 15mM Ammonium Formate
with 2% HCOOH in Methanol; B4: 0.2%
NH4OH in Methanol
Gradient:
Non polar compounds: 2-40 % modifier in 5
minutes
Polar compounds: 5- 40% modifier in 5
minutes
Column Temperature: 50 °C
Injection volume: 1 µL
Flow rate: 2 mL/min
Wavelength: 254 nm or 220nm: Compensated
350-450
ABPR: 2000 psi (3600-3900 starting system
pressure)
Sample Diluent: 9:1 Heptane/2-Propanol
HSS C18 SB
1, 2
1.00
System: ACQUITY UltraPerformance
Convergence Chromatography™ (UPC2®)
with PDA and SQD
BEH 2-EP
0.00
Minutes
Conditions
3
0.00
2
3
1.00
METHODS
1-propiophenone
2-valerphenone
3-diethyl
phthalate
4-dioctyl phthalate
BEH
1.00
0.00
AU
Convergence
chromatography
is
a
chromatographic technique that uses
super/sub critical carbon dioxide to
provide alternative selectivity to reversed
-phase chromatography. This normal
phase technique is based on the
numerous modes of interaction between
the stationary phase and the solute.
These interactions can be manipulated by
a
number
of
variables
column
chemistries,
organic
modifiers
and
physical
factors.
Combining
and
optimizing the impact of these factors can
be a complex and time-consuming
method development process. In order to
streamline
this
process,
we
are
developing a systematic approach to
achiral
convergence
chromatography
method development.
Method Development Strategies
for Non-polar compounds
1-caffeine
2-thymidine
3-amitriptyline
4-noritriptyline
5-prednisolone
0.10
0.05
 For
both
polar
and
non-polar
compounds, a streamlined approach
allows for rapid method development in
convergence chromatography.
 This
systematic
approach
should
evaluate stationary phases, additives
for peak shape, and physical factors
(temperature
and
pressure)
for
selectivity and retention.
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
Minutes
Figure 3. Comparison of UV and Mass Spectrum chromatograms. A basic/neutral mix was analyzed on a
UPC2 BEH column with 15mM ammonium formate,2%
HCOOH in methanol. The MS compatible mobile phase
allows for confirmation by mass spectrometry.
References
1.
Ventura, M.; Murphy, B.; Goetzinger, W.; Journal of Chromatography A
2012, 1220 (0), 147-155.
2.
Grand-Guillaume Perrenoud, A.; Boccard, J.; Veuthey, J.-L.; Guillarme, D.,
Journal of Chromatography A 2012, 1262 (0), 205-213.
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