Chemistry Reference
In-Depth Information
beginning of every gradient to accommodate transfer to systems with differing vol-
umes [20]. When UHPLC systems were first introduced in 2004, only high-pressure
mixing systems were available. Recently (2010), low-pressure mixing systems were
introduced that combine all the attributes of working with small particles at high
pressures with quaternary solvent mixing for method development [21].
3.4.5 S
AmPle
m
AnAgement
Conventional injection valves, either automated or manual, are not designed and
hardened to work at extreme pressure; and to protect the column from experienc-
ing extreme pressure fluctuations, the injection process must be relatively pulse-
free. The swept volume of the sample manager also must be minimized to reduce
potential band spreading. For UHPLC, a fast injection cycle time is needed to fully
capitalize on the speed of the analysis, which in turn requires a high sample capac-
ity. Low volume injections with minimal carryover are also required to realize the
increased sensitivity benefits. Temperature control and compatibility with a wide
range of sample formats (e.g., vials, microtiter plates) are also desirable features in
any sample management device used for method development.
3.4.6 d
etectIon
Detection plays an important role in method development systems, and the most
desirable configurations include a variety of complementary detectors to respond
to the widest range of analyte attributes. Depending on analyte properties, the most
commonly employed detectors in method development systems include UV (PDA),
evaporative light scattering (ELSD), corona charged aerosol (CAD), and mass spec-
trometry (MS-either single or triple quadrupole). Multiple detectors in a system can
be configured in series or parallel; often, the choice of which configuration to use
depends on whether or not the detector is destructive. Destructive detectors (e.g.,
ELSD, MS, CAD) must be placed last in the flow path and require splitting of the
flow stream.
Photodiode array (PDA) detection is commonly used during method development
to determine peak identity and purity/homogeneity. PDAs extend the utility of UV
detection by providing spectra of eluting peaks that can be used to aid in peak identi-
fication, and to monitor for co-elutions (peak homogeneity or purity), helpful during
method development. They can also serve as a multiwavelength UV/VIS detector. The
spectra collected at the chromatographic peak apex can be used to create a library
that can in turn be used to compare subsequent spectra for identification purposes,
and spectra collected across the peak at each data point can be compared to evaluate
peak homogeneity or purity. The added spectral resolution of modern PDA detectors,
coupled with chromatography data system (CDS) software algorithms, can quickly
compare fine differences in the spectra not clearly visible to the eye. Some compari-
sons are done by a simple direct point-to-point comparison of spectra, while in others,
complex vector analysis in multidimensional space is performed to look at spec-
tral fine structure. In order for PDA spectral comparisons to work, the compounds
must have some UV absorbance, and there must be some degree of spectral and
