Biomedical Engineering Reference
In-Depth Information
is utilized. LLE for bioanalysis is typically conducted in tubes. The transfer can be
achieved by either pipetting or freeze-pour. For freeze-pour approach, the tubes are
merged in acetone or methanol-dry ice bath for approximately 1 min, the aqueous
layer which is normally at the bottom layer is frozen, thus the top organic layer can
be pour out easily. Compared to manual pipetting transfer, the freeze-pour is much
easier and quicker which is quite commonly used in bioanalytical laboratory. LLE
can also be conducted in 96-well plate format in which the extraction was achieved
by mixing using automation robots via pipette tips for 30-40 times. This extraction
approach is labor free and efficient. However, it is less forceful than by mixing using
shaker or vortexer. Thus, the recovery is normally lower than tube extraction.
In recent years, supported-liquid extraction (SLE) has merged and become
increasingly popular [
9
]. Commercial SLE plate consists of 96 individual wells
each packed with proprietary and modified form of diatomaceous earth which has a
high capacity for retaining aqueous samples. When the plasma sample is loaded
onto the extraction well, the analytes absorb over the surface of the support in a very
thin layer. Unlike manual LLE in which the emulsions could potentially cause prob-
lem, in SLE extraction, emulsion is completely eliminated for the sample and water
immiscible extraction solvent are never in direct contact. It also removes all manual
steps such as capping, mixing, centrifuging, and decapping et al., thus greatly reduc-
ing contamination. Because all procedural steps can be fully automated, SLE is as
efficient as using PPE plate but provides much cleaner extracts. The only disadvan-
tage for SLE is the limited sample aliquot volume (<400 mL) in order to maintain in
96-well plate format.
2.3
Of fl ine and Online Solid Phase Extraction (SPE)
Another popular and selective extraction technique widely used in bioanalysis is
solid phase extraction (SPE). SPE is a separation process utilizing the affinity of the
analytes to a solid stationary phase. By manipulating the polarity and pH of the
mobile phase, the analytes of interest or undesired impurities pass through station-
ary phase sequentially according to their physical and chemical properties. For a
SPE procedure, a wash step refers to the elution of the unwanted impurities which
are discarded and the elution step refers to the elution of the analytes of interest
which are collected. While the fundamental remains the same in decades, the con-
tinuing invention and introduction of new commercial stationary phases and acces-
sory devices have boosted the application of SPE in bioanalysis and many other
fi elds.
SPE initially comes in the form of a packed syringe-shaped cartridge for manual
extraction, and then followed by 96-well plate each of which can be mounted on its
specific type of extraction manifold. The first generation of SPE stationary phases was
made of silica backbone bonded to hydrocarbon chain of variable length such as C18,
C8, and phenyl. The second generation of SPE stationary phases was still primarily
made of silica backbone but with more modification for these functional groups.
