Comprehensive two-dimensional separations are a definite trend in chromatography today. Comprehensive two-dimensional gas
chromatography (GC×GC) emerged some 15 years ago (1). After a sluggish start — arguably attributable to a limited availability
of instrumentation and software — this is now a widely accepted and commonly used technique for the separation of complex
mixtures of volatile analytes. The roots of comprehensive two-dimensional liquid chromatography (LC×LC ) are older. Two-dimensional
thin-layer chromatography can be considered a form of LC×LC, dating the technique back at least some 50 years. The contemporary
version of LC×LC — based upon two columns and an elegant valve-switching concept — was first demonstrated by Erni and Frei
(2) and it, too, preceded GC×GC by a considerable period of time.
Figure 1
The common way to perform LC×LC involves a first-dimension separation that is relatively slow, with a typical analysis time
of 1 h or longer. Fractions of the effluent from this first-dimension column are collected in a loop of a switching valve.
When this valve is switched, the fraction is injected onto a second column, which provides a much faster separation, typically
with an analysis time of a minute or less. While one fraction is being analyzed on the second-dimension column, a new fraction
is being collected in another loop connected to the same valve (or by using a second valve). One popular configuration employs
a two-way 10-port switching valve, as illustrated in Figure 1. To construct an LC×LC system requires (at least) two columns
and two pumping systems. Detection typically takes place after the second-dimension column (and not after the first dimension),
so that "one-and-a-half" liquid chromatographs are needed to assemble one comprehensive two-dimensional one.
Figure 2
The result of a comprehensive two-dimensional separation is a series of many (for example, 100) fast second-dimension chromatograms.
These usually are combined into a data matrix, which then can be displayed as a contour plot (see Figure 2) or color plot
that displays peak intensity as a function of the retention times in the first and second dimensions (3). In truly comprehensive
separations, such a two-dimensional chromatogram is representative for the entire sample.
There are two criteria for calling a two-dimensional separation "comprehensive" (4). One is that every bit of the sample is
being analyzed in both dimensions, without anything going to waste. The second is that the resolution obtained in the first
dimension be maintained, despite cutting the chromatogram in a limited number of fractions. The first criterion is met by
using a valve-switching system like that shown in Figure 1 (5). The second criterion is more difficult to meet. It has long
been assumed (6) that about four "cuts" would need to be taken along every first-dimension peak. Recent results from Tanaka's
group (7) suggest that two cuts per peak can be optimal.
The questions that will be addressed in this communication involve the value of LC×LC in practice. Why would one want to employ
the technique, given that the equipment is more complicated (an additional LC pumping system is required) and that a valve-switching
system and dedicated software are needed? The answer is twofold, and it will be provided in the next two sections. One deals
with the peak capacity that can be obtained from comprehensive two-dimensional chromatographic separations. In the second
one, the concept of sample dimensionality will be discussed and we will see how structured, readily interpretable two-dimensional
chromatograms can be obtained.
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