Analytical Session
David H Thomas, Thermo Fisher Scientific, Chelmsford, MA, USA
Co-author(s): Paul Ullucci and Ian Acworth, Thermo Fisher Scientific, Chelmsford, MA, USA
ABSTRACT: Carbohydrates are a structurally diverse group
of compounds that can be categorized as monosaccharides, disaccharides,
oligosaccharides, glycoproteins, etc. Carbohydrates are difficult to
analyze because they have similar physical and chemical characteristics
and do not have a suitable chromophore for UV detection. Several
different HPLC methods using various detector strategies (pulsed
amperometric electrochemical, fluorescence following derivatization,
charged aerosol detection, and mass spectrometry) were developed to help
study carbohydrates, and examples for each approach are presented.
Although fluorescent tags improve chromatographic resolution and
detector sensitivity, they can lead to increased assay variability.
Various HPLC modes can be used for carbohydrate separations, with ion
exchange, hydrophilic interaction liquid chromatography (HILIC), and
reversed phase (RP) on porous graphite column (PGC) being the most
common. HPLC provides for simple chromatographic methods; direct
detection using PAD or mass detectors such as ELSD, MS, and CAD are
employed. CAD is an ideal detector when combined with HILIC or RP/PGC
for measuring different carbohydrates. It is a mass-sensitive detector
that can measure any non-volatile, and many semi-volatile compounds,
typically with low ng sensitivity. Unlike ELSD, it shows high
sensitivity, wide dynamic range, high precision, and more consistent
inter-analyte response independent of chemical structure. For the
analysis of glycans liberated from glycoproteins, the utility of the
LC-MS-CAD platform is presented; CAD is used for quantitative analyses,
while MS provides structural verification. The advantage of this
approach over methods using fluorescent tags is discussed.
Dave
Thomas received a Ph.D. degree in analytical chemistry from the
University of Nebraska-Lincoln in 1994 for his work developing
high-performance immunoaffinity chromatography. In post-doctoral
appointments at Midwest Research Institute-California Operations and
Sandia National Laboratories, he worked to implement HPIAC and HPLC
approaches on miniaturized electrochromatographic separation and
analysis platforms. Later, he spent several years developing a variety
of IC, HPLC, and LC/MS applications at Dionex Corporation and Thermo
Fisher Scientific, where he also served as manager of the HPLC and LCMS
applications laboratory. After a few years in vaccine analytical
development at Wyeth and Pfizer, Dave returned to Thermo Fisher
Scientific, where he continues to develop applications for HPLC with
charged aerosol and electrochemical detection.
