The keys to understanding reversed-phase liquid chromatography (LC) are provided at the molecular mechanism level as determined
by high accuracy molecular simulation. The essential features of C
stationary-phase chains in contact with methanol–water and acetonitrile–water mixtures are discussed in the context of bonded-chain
geometry, spatial distribution of alkane and alcohol solutes, retention mechanism, and retention thermodynamics. This tutorial
is intended to be applicable to a wide audience ranging from occasional users of liquid chromatography to separation scientists.
The technique referred to as reversed-phase liquid chromatography (LC) is the workhorse of all LC techniques. Continued improvements in columns, instrumentation, and application methodologies
have led to reversed-phase LC being used in more laboratories and delivering results faster, with higher resolution. We confine
the scope of our discussion of reversed-phase LC to column materials that contain a bonded-phase layer of a hydrophobic material,
usually dimethyl octadecylsilane (C18), bound to a porous silica support. The C18 chains function as the retentive material, yet details of chain conformation, how the solutes of interest interact with the
chains, where the solvent lies in proximity to the chains, and the thermodynamics of this process have been lacking for many
years. It is ironic that with the high popularity of this technique, the details of the retention mechanism have been so difficult
to obtain. The purpose of this tutorial is to enlighten readers about the detailed inner workings of reversed-phase LC.
For many years researchers believed they could figure out the mechanism of reversed-phase LC by examining the pattern of retention
times produced by injecting different but chemically related solutes into columns with different stationary-phase chemistries
using a range of mobile-phase compositions. This is a fallacy because retention time experiments cannot give a microscopic
picture of the bonded-phase chains and inference of the structure of these chains is largely circumstantial. The retention
time experiment is thermodynamic in nature — it is determined by the distribution of the solute between the mobile-phase solvent
system and the stationary phase. This distribution is the subject of what is often referred to as "phase equilibria." No microscopic
mechanism can be gleaned from data of this type (that is, thermodynamic data). In essence, thermodynamics is just a bookkeeping
system, nothing more and nothing less.
Spectroscopic investigations have also been inconclusive and have not revealed details of the retention mechanism. This is
because spectroscopic techniques like nuclear magnetic resonance (NMR), infrared (IR), and fluorescence spectroscopy detect
bonded-phase chain conformations and solute associations for a large number of molecules simultaneously; these conformations
and interactions are complex, the signals are difficult to decompose into single molecule information, and widely diverse
signals lead to spectroscopic broadening and loss of resolution. Hence, reversed-phase LC retention mechanisms have not been
revealed by these techniques. Some spectroscopic techniques have given outstanding structural information, for example, determining
the distribution and types of silanol groups on silica and revealing the silica bonding details of derivatizing agents by
solid-state NMR spectroscopy (1).
We have learned a great deal about the reversed-phase LC system through the use of high-accuracy molecular simulations (2–4).
Simulation offers a unique methodology to understand disordered and microheterogeneous systems and this has been the primary
technique used for many years in the study of liquids (5,6). Simulation is necessary because of the complexity from the large
number of simultaneous interactions that take place (the so-called "n-body problem" of liquids). These types of problems cannot be understood or formulated mathematically unless drastic simplifications
are made. These simplifications render such theories of limited use. With the advent of high-speed computers and advances
in simulation methodology, this method of investigation has become more practical and has now evolved to the point where simulation
can reproduce the energetics of reversed-phase LC accurately. Since simulation gives mechanical details of reversed-phase
LC on an atomic scale (that is, chain conformation, solute and solvent locations) the mechanism of the reversed-phase LC process
can be revealed using simulation methodology. As we will show, the comparison of simulation with experiment is impressive
and provides an assurance that these computational results for model systems carry a sufficient amount of realism to learn
about real chromatographic systems.
In this tutorial we will guide readers through a number of our simulation findings that give the microscopic details needed
to understand reversed-phase LC. This is not only useful for chromatographers in understanding practical separations, but
it may prove useful toward designing new stationary phases for liquid-based separations other than reversed-phase LC. A detailed
description of the simulation methodology, the bonding procedure, and further details are given in two review articles (3,4).
We refer readers to these references and others given here for the detailed workings of this methodology.