Biomedical Engineering – From Theory to Applications

1. Introduction
Column Coupling
Electrophoresis in Biomedical Analysis
5
Peter Mikuš and Katarína Maráková
Faculty of Pharmacy, Comenius University,
Slovakia
Biomedical analysis is one of the most advanced areas solved in analytical chemistry due to
the requirements on the analyzed samples (analyte vs. matrix problems) as well as on the
overall analytical process regarding automatization and miniaturization of the analyses.
Separation methods for the biomedical analysis are requested to provide high resolution
power, high separation efficiency and high sensitivity. This is connected with such conditions
that analytes are present in the samples in very low (trace) amounts and/or are present in
multicomponent matrices (serum, plasma, urine, etc.). These complex matrices consist from
inorganic and organic constituents at (very) differing concentrations and these can overlap the
analyte(s) peak(s) due to migration and detection interferences. In addition, a column
overloading can occur in such cases. It can be pronounced especially for the microscale
separation methods such as the capillary electrophoresis (CE). Hence, it is obvious that there is
the need for the sample preparation: (i) preconcentration – lower limits of detection and
quantification; (ii) purification of the sample and isolation of analytes – elimination of sample
matrix; (iii) derivatization – improvement of physical and/or chemical properties of the
analytes, before the CE analysis in these situations to reach relevant analytical information.
Sample pretreatment can be performed either off-line (before injection of analyzed sample
into the analyzer) or on-line (after the injection). The conventional separation systems (single
column) use mostly external (off-line) sample pretreatment, even though this analytical
approach has many limitations. These are (i) a loss of the analytes, (ii) time consuming and
tedious procedure, (iii) problematic manipulation with minute amounts of the samples, (iv)
problematic for automatization, (v) decreased precision of the analyses, etc. On the other
hand, on-line sample pretreatment has many advantages as (i) elimination of random
and/or systematic errors caused by external sample handling, (ii) simplification of an
overall analytical process (less number of an external steps), (iii) reduction of the total
analysis time and (iv) possibility of the automatization and miniaturization of the analytical
process (routine precise microanalyses). A significant enhancement of sensitivity and
selectivity is one of the main benefits of the on-line sample pretreatment. An on-line
pretreatment is crucial when there are only micro amounts of the samples for the analysis
and/or when analytes/samples have lower stability.
The advanced single column electrophoretic techniques (transient isotachophoresis, fieldenhanced
sample stacking, dynamic pH junction, sweeping, in-capillary solid/liquid phase
extraction-CE, in-capillary dialysis-CE, etc.), representing the CE with the on-line (incolumn)
sample preparation, were described and successfully applied for trace analytes and