June 2013 » Chromatography Techniques CT17
and regulates pressure, temperature and mobile phase gradient composi-
tion. This is key to maintaining fluid density and stabilizing analyte reten-
tion on sub-two micron particle columns. These parameters may be used
in conjunction with a broad polarity spectrum of co-solvents and available
column chemistries to fine-tune resolution and selectivity for successful sep-
arations. Furthermore, low system dispersion enables an increase in speed,
sensitivity and resolution inherent in small-particle columns.
Flurbiprofen is an anti-inflammatory used in pain management. It
has one chiral site, as indicated in the structures in
Figure 1. Using an UPC2 System with photodiode
array (PDA) detection, a simple mobile phase (CO2:
methanol 75:25) at a back pressure set point of 130
bar, and a column temperature of 40 C, an excellent
separation of flurbiprofen enantiomers is obtained.
As shown in Figure 1, smaller particle size not only
speeds the analysis but also reduces solvent con-
Clenbuterol is a potent bronchodilator used to
treat people with chronic breathing disorders, such
as asthma. Typical daily dosage is 2 to 40 mg/day, so
sensitive methods of analysis are needed to determine its enantiomers at low concentration. Using
a UPC2 system, a matrix of 16 gradient separations
using combinations of four different columns and
four co-solvents was run in less than three hours.
Within a few more minutes, the best combination
was used to obtain the optimized isocratic separation shown in Figure 2.
Retention time and peak area reproducibility
were excellent (% RSDs [n=6] of 0.012 and 0.016
for retention time, 0.34 and 0.33 for area, of peaks
1 and 2, respectively). If more specificity and sensitivity is desired for bioanalysis, the volatile mobile
phase is compatible with MS detection.
Figure 2: UPC2 analysis of clenbuterol enantiomers was done at 40 C using CO2:methanol 85:15 at
2 mL/min and a back pressure of 1500 psi.
Figure 3: A mixture of two chiral sulfoxides analyzed with an UPC2–PDA system coupled to a proprietary 3100 MS detector.
Unlike NPLC, UPC2 separations use MS-compat-ible co-solvents, such as alcohols, thereby enabling
the application of highly specific MS detection to
chiral analysis. By tuning in to a selected mass characteristic of a known chiral analyte, MS detection
can locate, within a mixture of compounds, its pair
of enantiomers, though it cannot distinguish which
of the pair each peak represents. Such specificity
may confirm if the enantiomers co-elute with a
related compound or process impurity. Further, the
high sensitivity of MS detection may detect very
low levels of one enantiomer in the presence of the
other. This capability can be used to ensure that
enantiomeric excess falls within acceptable levels.
When sample mixtures are complicated by the
presence of several enantiomeric pairs that must
be determined, there are two options for increasing
analytical throughput. With a significant capital
investment, a sample containing several chiral compounds may be screened simultaneously on multiple systems, each of which uses a column/mobile
phase combination specific for one of the analytes.
A more sophisticated and less costly approach uses
a single UPC2–MS combination to screen a sample
for multiple analytes in a single analysis, identifying
the enantiomeric pairs by examining the extracted
ion chromatograms. This idea is illustrated in principle by the model experiment in Figure 3.