The separation and analysis of the chemical components of natural products is critically
dependent upon modern liquid chromatographic techniques and instrumentation. The use
of high efficiency LC with modern bridged-ethylene hybrid, small particle packings
combined with fragmentation patterns and accurate mass determination has produced
truly impressive results in the various fields of natural products research. However,
despite the obvious successes of reversed-phase liquid chromatography, the exact retention
mechanisms controlling resolution and retention are unclear [1].
There are currently three principle chromatographic methods for measuring the uptake
of multicomponent eluents by a given packing: i) concentration pulse (CP) chromatography,
ii) frontal analysis (FA) chromatography and iii) tracer pulse (TP) chromatography.
These techniques are complementary and the selection of methods is usually dictated
by the available detection system. The objectives of the present investigation were
i) to compare and validate concentration and tracer pulse experimental methods for
the determination of the excess volumes of eluent components taken up by RPLC packings
over a range of temperature, pressure, flow rate and eluent composition, ii) to determine
the effect of various experimental parameters on the accuracy of the measured excess
volumes, and iii) to measure the excess volumes of acetonitrile and methanol on modern
BEH UHPLC packings.
In our study, excess volume isotherms of acetonitrile and methanol sorbed on a C18 BEH UHPLC packing were determined over a range of pressure, temperature, flow rate
and eluent composition. The isotherm measurements were carried out by concentration
pulse and tracer pulse chromatografies. Isotherms were measured with both experimental
techniques at 30, 45 and 60°C. The excess isotherms increased with decreasing temperature
although the variations were relatively small. Direct comparison of the two experimental
techniques showed that the measured void volumes were identical within experimental
error. The measured excess volumes by both techniques were comparable with the concentration
pulse experiments producing slightly higher excess volume data with highly aqueous
eluents as shown in Fig.1. Both experimental techniques show some variations of the
retention volumes with sample volume, sample composition, flow rate and column inlet
pressure. The results confirmed the validity of both concentration and tracer pulse
chromatografies for the determination of column void volumes and the excess volume
of eluent taken up by UHPLC packings [2].
Fig.1 . Excess Isotherms for Methanol and Acetonitrile Experiments at 30°C and 0.2 mL/min.
(○) CP: Acetonitrile; (·) TP: Acetonitrile; (△) CP: Methanol; (▲) TP: Methanol
Acknowledgements
: This research is supported in part by Science Based Authentication of Dietary Supplements
funded by the Food and Drug Administration grant No. 1U01FD004246–01; the United States
Department of Agriculture, Agricultural Research Service, Specific Cooperative Agreement
No. 58–6408–2-0009, and the Global Research Network for Medicinal Plants (GRNMP),
King Saud University. References: [1] Wang M, Mallette J, et al. (2008) Anal Chem 80: 6708–6714 [2] Wang M, Avual B,
et al. (2011)J Chromatogr A doi:10.1016/j.chroma.2011.11.051.