Speciation analysis by liquid chromatography–inductively coupled plasma-mass spectrometry (LC–ICP-MS) has been growing rapidly
in popularity and application over the past several years. Not only have people begun looking at different elements and species,
but there has been an increase in the variety of matrices that speciation analysis is being performed on: various types of
environmental, waste, and process waters; both solid and liquid foods; and biological fluids. With this increased interest,
more laboratories are offering speciation analysis as service. When deciding upon a speciation method, many factors must be
The chromatographic separation of components is based upon competition between the column, mobile phase components, species,
and the sample matrix. With the variety of matrices now being analyzed, there are a variety of chromatographic options available.
This column will discuss three column types and the relative advantages and disadvantages of each.
Ion-Exhange Versus Reversed-Phase Ion-Pairing Chromatography
Two common types of separation schemes used in chromatography are ion-exchange and reversed-phase ion-pairing chromatography.
These schemes differ primarily in the column stationary phase, which affects the way components compete for active sites on
Ion-exchange chromatography involves the interaction of ionic (that is, charged) components in the mobile phase with charged
stationary groups on the column packing material. Charged species in the sample compete with mobile phase components for sites
on the column. Species with a stronger attraction to column sites than mobile phase components will be retained on the column
longer and have longer elution times. Species with a weaker attraction to the column than the mobile phase components will
have shorter elution times. This is the mechanism by which separation is achieved and is represented in Figure 1.
Figure 1: Schematic of the ion-exchange separation mechanism.
Reversed-phase ion-pairing chromatography involves the use of reversed-phase columns that are characterized by nonpolar stationary
phases composed of carbon chains, typically 18 carbons (C18), although other columns exist with different length carbon chains
(for example, four and eight carbons). Because most inorganic species exist in charged states in solution, it appears that
reversed-phase columns would not be able to perform the separation.
This situation is rectified by incorporating an ion-pairing reagent in the mobile phase. Ion-pairing reagents consist of both
nonionic and ionic parts: The nonionic sections interact with the carbon chain on the column, and the ionic sections interact
with the charged species in solution by mechanisms similar to those in ion-exchange chromatography. In this way, a reversed-phase
column can be made to perform like an ion-exchange column. The mechanism of reversed-phase ion-pairing chromatography is shown
in Figure 2.
Figure 2: Diagram illustrating the reversed-phase ion-pairing mechanism.
Both ion-exchange and reversed-phase ion-pairing chromatography each have their advantages and disadvantages. One of the main
advantages of ion exchange is that there is only one interaction involved in the separation: the analytical species interacting
with the stationary phase. With reversed-phase ion pairing, two interactions are involved with the separation: the ion-pairing
reagent interacting with stationary phase of the column and the species interacting with the ion-pairing reagent. As a result,
ion-exchange chromatography may have more matrix tolerance.
The downside of ion-exchange chromatography is that these columns typically are much more expensive than reversed-phase columns.
Also, because reversed-phase columns have been in use for many years, they are reliable and established, meaning that results
will be reproducible from one column to the next. This may not always be true with ion-exchange columns because new columns
are constantly being developed with new ionic groups bound to the stationary phase. Because of this constant evolution, some
ion-exchange columns may not always be well established, which could lead to irreproducibility from column to column.
The decision to use either ion-exhange or reversed-phase ion-pairing chromatography depends upon the application: the elements
and species of interest, the sample matrix, and the levels of the different species that need to be measured.