Liquid chromatography with C18-bonded packings and aqueous-organic eluents is a mature, ubiquitous, and incredibly
efficient method for the separation and analysis of botanicals and natural products.
Recent improvements in pump technology (1,200bar pressure) and silica chemistry (very
stable, bridged-ethylene hybrids) have allowed the use of very small particles (<2µm)
to achieve high efficiency separations of complex samples very rapidly. Despite the
proven efficacy of UPLC, no overarching theory has emerged to describe the basic retention
mechanism(s) operative in these complex chromatographic systems. The eluent usually
consists of water with varying concentrations of an organic component, small concentrations
of a polar acid, as well as buffers to control pH. The stationary phase often consists
of a silica base with hydrophilic silanol and hydrophobic siloxane groups on the surface
of the solid. The octadecane coating is chemically-bonded to the silica so one end
is tethered and acts like a solid while the other end is free to act as a liquid.
It is an unsettled issue whether the bonded octadecane on the silica exists in a collapsed
state that supports adsorption or an extended (solvated) state that facilitates absorption
of analytes. The eluent and stationary phases are complex enough by themselves. However,
in a chromatographic column the stationary phase and eluent interact in dynamic equilibrium.
In particular, almost all of the eluent components will be taken up in varying degrees
by the stationary phase. It is this complex, in situ, dynamic equilibrium condition that is so difficult to model. The technique known
as mass spectrometric tracer pulse chromatography1 was used to measure the uptake of eluent components by typical UPLC packing material.
The ultimate goal is the determination of the effect of such eluent uptake on the
retention of analytes. Unfortunately, it is impossible to directly measure the amount of anything absorbed by a liquid (C18) or adsorbed by a solid (silica or C18) from liquid eluents. However, the excess amount of eluent absorbed in or adsorbed
on the stationary phase can be determined. An example of an excess isotherm of acetonitrile
taken up by a C18-bonded BEH column as a function of eluent composition is given in Figure 1. The maximum absolute amount of acetonitrile taken up by the stationary phase can, however, be estimated
from the isotherm data. It was found that for a UPLC column (2.1×150mm), the following
amounts of eluent were either absorbed or adsorbed by the bonded stationary phase.
The volume of bonded C18 material in the same column (˜40µL) can be estimated from the bonding density (3.1µmol/m2), the surface area (185m2/g) and the amount of packing in the column (˜300mg). Thus, the isotherm measurements
indicate that the volume of ACN that acts as a part of the stationary phase (˜40µL)
is essentially equal to the volume of octadecane chemically bonded to the packing
material. The exact effect of this uptake of the retention of analytes is very difficult
to isolate from the effects of the multiple other factors influencing the chromatographic
performance of UPLC systems.
Figure 1. Excess Isotherms of Acetonitrile on or in C18-bonded BEH (1.7µm) particles at 35 and 55 °C.
|
Temperature
(0C)
|
Volume (µL) taken up by
the stationary phase
|
Volume Fraction of Acetonitrile
|
|
|
Acetonitrile
|
Water
|
|
|
35
|
43
|
7
|
0.86
|
|
55
|
37
|
8
|
0.82
|
Acknowledgements: This research is supported in part by Science Based Authentication of Dietary Supplements
and Botanical Dietary Supplement Research funded by the Food and Drug Administration
grant numbers 5U01FD002071–10 and 1U01FD003871–02, and the United States Department
of Agriculture, Agricultural Research Service, Specific Cooperative Agreement No.
58–6408–2-0009.
References: [1] Wang M, Mallette J, et al. (2008) Anal Chem, 80: 6708–6714.
