Biomedical Engineering Reference
In-Depth Information
external field force is applied perpendicular to a laminar liquid flow of sample in a
carrier, forcing all analytes towards the ''accumulation wall'' of the flow channel and
so causing different species to be placed in different stream lines. Small molecules
and particles (less than *1 lm) are separated due to their different diffusivities: the
larger ones are less mobile and remain longer near to the accumulation wall, where
the flow velocity is smallest; hence, they are eluted later—''normal mode''. Particles
larger than *1 lm are, for geometric reasons, permanently exposed to the stream
lines of higher velocity and therefore eluted more quickly—''steric mode''. Useful
fields are gravity, temperature, cross flow, and electrical charge (among others). The
range of the (molecular) size of the analytes covered by FFF usually exceeds that
which can be determined by classical laboratory analytical methods such as size-
exclusion chromatography in a single run. Reports on investigated substances are
widespread and cover applications such as the separation and characterization of
proteins and enzymes, of viruses and mammalian cells, isolation of plasmid DNA, or
the molecular weight and particle size distribution of polymers. Langwost et al. [
68
]
have provided a comprehensive survey of various applications in biomonitoring.
Although not online, Hawe et al. [
69
] investigated the aggregation of IgG with an
asymmetrical-flow FFF.
3.3 Components Dissolved in the Medium
The biocatalysts consume substrates dissolved in the medium and produce prod-
ucts, many of which are excreted in soluble form into the medium. The masses or
concentrations of these components are important state variables and need to be
known. For many solutes online methods are available, as discussed in the fol-
lowing; however, the methods for estimating recombinant proteins are currently
laboratory techniques that are not fully automatically coupled to the processes
[
70
]; important exceptions are—at least potentially—fluorescence-tagged proteins
[
51
,
63
,
71
-
74
].
3.3.1 Flow Injection Analysis
Flow injection analysis (FIA) is an extremely versatile tool to (a) automate
(bio)chemical analyses, (b) combine various chemical or physical reaction steps
into a sequence, and (c) dilute samples appropriately. Any kind of appropriate
detector can be implemented at the end of the line. The automation may produce
systematic errors if calibration is done improperly and/or recalibration is omitted.
Frequent recalibration may be necessary if peristaltic pumps are used. FIA systems
are usually constructed as mini- or even microfluidic assemblies, and all the dif-
ficult problems associated with this technology apply for FIA as well, specifically
the formation and entrapment of gas bubbles in the system.
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