Biomedical Engineering Reference
In-Depth Information
9
Liquid Chromatography
High-performance liquid chromatography (HPLC) is a well-established separation
technique; it is able to solve numerous analytical problems and there is the possibil-
ity of acting on the mobile phases with appropriate additives to improve the quality
of the peak. Of course, any additive must be compatible with the MS detector: non-
volatile buffer or eluent additives cannot be used; strong acids such as trifluoroacetic
acid (TFA) may cause significant signal suppression in positive ionization. Different
stationary phases are used as an alternative to the classical C18: Phenyl, HILIC,
fl uorinated, etc.
9.1
UHPLC and Fast Chromatography
Enhancing productivity and reducing costs could be a driving force for faster sepa-
rations [
78
]. Several development have been done recently to improve the perfor-
mances but the most important can be considered ultra high performance liquid
chromatography (UHPLC) [
79
]. UHPLC systems are commercially available since
2004 and, regardless to major costs of instrumentation, are spreading in forensic
laboratories [
80
]. Due to the cost of UHPLC hardware, chromatographic research
has been addressed to improve HPLC performance in terms of faster separation and
efficiency. So, when UHPLC is not available, alternative devices and technologies
have been developed for
fast chromatography
: high-temperature liquid chromatography
(HTLC), use of monolithic supports, use of column with super fi cially porous packing
materials based on silica particles with nonporous cores [
81
] .
HTLC
operates chromatographic separation at elevated temperature (60-120 °C)
resulting in a significant reduction of mobile phase viscosity, leading to higher dif-
fusion coefficients for the compounds and improved mass transfer [
82
] . The authors
also highlighted the practical limitations due to the instrumentation; moreover, lim-
ited number of stable stationary phases compatible with elevated temperature makes
HTLC to be rarely used in routine analysis and only investigated in academic labo-
ratories [
81
] .
Monolithic supports
consist of a single rod of porous material with several unique
features in terms of permeability and efficiency. The low backpressure generated
and good mass transfer enable use of elevated flow rates (3-10 times larger) [
83
] .
First publication with the use of this support in LC was in 1989 by Hjerten et al.
[
84
], but to date monoliths are not widely used.
The use of column with
superficially porous packing materials
based on silica
particles with nonporous cores is the most recently reported strategy for improving
chromatographic performance. This technology, originally developed by Kirkland in
the 1990s to limit diffusion of macromolecules into the pores [
85
] , became commer-
cially available in 2007 [
86
]. In comparison with totally porous particles of similar
diameters, the both A and C term of the Van Deemter curve are reduced [
87,
88
] .
