Page 129 - Multidimensional Chromatography

Coupled-Column Liquid Chromatography 121

suggested and reversed-phase HPLC is frequently the technique of choice for these
types of analyses (30). However, considering that these compounds may be present
at low levels in natural and drinking water ( 1 g/l), a preconcentration step before
one-dimensional HPLC analysis is usually required (31–33). Furthermore, the pro-
teins present in samples containing cyanobacteria are particularly troublesome since
they tend to denature on reversed-phase packing and render the column useless (dis-
torted peak shape, multiple peaks, etc.) (34). In the proposed LC–LC system, a pre-
column consisting of a Zorbax CN cartridge was used for simultaneous enrichment
and clean-up of the microcystins in water. The sample (100 ml) was passed through
the cartridge on the enrichment side at a flow rate of 3 ml/min, and the microcystins
were strongly retained in a narrow band on the top of the cartridge, due to their high
hydrophobicity. At the same time, the analytical column was equilibrated with the
starting mobile phase consisting of 10 mM phosphate buffer (pH, 2.5), containing
25% (vol/vol) acetonitrile. Desorption was performed by coupling the cartridge on-
line with the analytical column and starting the gradient. Gradient elution was per-
formed in the opposite direction to the sample preconcentration as follows: 0% of B
at 0 min, 20% of B at 38 min, 60% of B at 42 min, and then isocratic under the same
conditions until a period of 50 min. In this system, the mobile phase B was acetoni-
trile and the elution was carried out at a flow rate of 1 ml/min. Microcystins are pep-
tides of differing hydrophobicity that can be readily chromatographed by using
reversed-phase (RP)-HPLC. An example of the determination of microcystins (-LR,
-RR and -YR) from a water sample by using the developed procedures is reported in
Figure 5.3. Considering the two examples reported above regarding sample enrich-
ment, the most important parameter is the sensitivity of the method (or minimal
detectable concentration of analytes), determined by the sensitivity of the detector
used, the adsorption capacity of the precolumn, the sample volume, the desorption
and the chromatographic procedures. Two distinct processes are involved, i.e. (i) a
frontal chromatography during the enrichment step, and (ii) a displacement chro-
matography during the desorption step.
A fundamental parameter characterizing the usefulness of a given precolumn for
enrichment purposes is the breakthrough volume, V B . This volume can be deter-
mined by monitoring continuously or discretely the detector signal at the outlet of
the precolumn (35–37). The breakthrough volume can be defined by the following
expression (37):
(5.21)
V B V R 2.3 V
where V is the standard deviation depending on the axial dispersion of analyte
along the bed of particles in the precolumn. If the capacity factor, k S of the analyte
eluted with a mobile phase that corresponds to the sample solvent, wash solvent or
elution solvent can be predicted and if V 0 , the dead volume of the precolumn, is
determined, then V R can be calculated by using the following expression:

V R V 0 (1 k S ) (5.22)
while if the number of theoretical plates, N, of the precolumn is known, V can be