12A Overview of Analytical Separations

In Chapter 7 we examined several methods for separating an analyte from potential

interferents. For example, in a liquidliquid extraction the analyte and interferent are
initially present in a single liquid phase. A second, immiscible liquid phase is introduced,
and the two phases are thoroughly mixed by shaking. During this process the analyte
and interferents partition themselves between the two phases to different extents,
affecting their separation. Despite the power of these separation techniques, there are
some significant limitations.
12A.1 The Problem with Simple Separations
Suppose we have a sample containing an analyte in a matrix that is incompatible with
our analytical method. To determine the analytes concentration we first separate it from
the matrix using, for example, a liquidliquid extraction. If there are additional analytes,
we may need to use additional extractions to isolate them from the analytes matrix. For
a complex mixture of analytes this quickly becomes a tedious process. Furthermore, the
extent to which we can effect a separation depends on the distribution ratio of each
species in the sample. To separate an analyte from its matrix, its distribution ratio must
be significantly greater than that for all other components in the matrix. When the
analytes distribution ratio is similar to that of another species, then a separation
becomes impossible. For example, lets assume that an analyte, A, and a matrix
interferent, I, have distribution ratios of 5 and 0.5, respectively. In an attempt to separate
the analyte from its matrix, a simple liquidliquid extraction is carried out using equal
volumes of sample and a suitable extraction solvent. Following the treatment outlined in
Chapter 7, it is easy to show that a single extraction removes approximately 83% of the
analyte and 33% of the interferent. Although it is possible to remove 99% of A with three
extractions, 70% of the interferent. Although it is possible to remove 99% of A with three
extractions, 70% of I is also removed. In fact, there is no practical combination of number
of extractions or volume ratio of sample and extracting phases that produce an
acceptable separation of the analyte and interferent by a simple liquidliquid extraction.
12A.2 A Better Way to Separate Mixtures
The problem with a simple extraction is that the separation only occurs in one direction.
In a liquidliquid extraction, for example, we extract a solute from its initial phase into
the extracting phase. Consider, again, the separation of an analyte and a matrix
interferent with distribution ratios of 5 and 0.5, respectively. A single liquidliquid
extraction transfers 83% of the analyte and 33% of the interferent to the extracting
phase (Figure 12.1). If the concentrations of A and I in the sample were identical, then
their concentration ratio in the extracting phase after one extraction is Thus, a single
extraction improves the separation of the solutes by a factor of 2.5. As shown in Figure
12.1, a second extraction actually leads to a poorer separation. After combining the two
portions of the extracting phase, the concentration ratio decreases to
[ ...A] [I]
0 97
0 55
18
[.
.
.
A]
[I]
0 83
0 33
25

Thus, a single extraction improves the separation of the solutes by a factor of 2.5. As
shown in Figure 12.1, a second extraction actually leads to a poorer separation.
After combining the two portions of the extracting phase, the concentration ratio
decreases to
[.
.
.
A]
[I]
0 97
0 55
18

We can improve the separation by first extracting the solutes into the extracting phase,
and then extracting them back into a fresh portion of the initial phase (Figure 12.2).
Because solute A has the larger distribution ratio, it is extracted to a greater extent
during the first extraction and to a lesser extent during the second extraction. In this
case the final concentration ratio of in the extracting phase is significantly greater. The
process of extracting the solutes back and forth between fresh portions of the two
phases, which is called a countercurrent extraction, was developed by Craig in the
1940s.1* The same phenomenon forms the basis of modern chromatography.
Chromatographic separations are accomplished by continuously passing one sample-free
phase, called a mobile phase, over a second sample-free phase that remains fixed, or
stationary. The sample is injected, or placed, into the mobile phase. As it moves with the
mobile phase, the samples components partition themselves between the mobile and
stationary phases. Those components whose distribution ratio favors the stationary
phase require a longer time to pass through the system. Given sufficient time, and
sufficient stationary and mobile phase, solutes with similar distribution ratios can be
separated. The history of modern chromatography can be traced to the turn of the
century when the Russian botanist Mikhail Tswett (18721919) used a column packed
with a stationary phase of calcium carbonate to separate colored pigments from plant
extracts. The sample was placed at the top of the column and carried through the
stationary phase using a mobile phase of petroleum ether. As the sample moved through
the column, the pigments in the plant extract separated into individual colored bands.
Once the pigments were adequately separated, the calcium carbonate was removed from
the column, sectioned, and the pigments recovered by extraction. Tswett named the
technique chromatography, combining the Greek words for color and to write.
There was little interest in Tswetts technique until 1931 when chromatography was
reintroduced as an analytical technique for biochemical separations. Pioneering work by
Martin and Synge in 1941 2 established the importance of liquidliquid partition
chromatography and led to the development of a theory for chromatographic
separations; they were awarded the 1952 Nobel Prize in chemistry for this work. Since
then, chromatography in its many forms has become the most important and widely
used separation technique. Other separation methods such as electrophoresis, effect a
separation without the use of a stationary phase.