68
[40]. Historically, in the event that analytes of interest are not sufficiently volatile,
derivatizations such as alkylation have been necessary to increase volatility for
effective analysis by GC [40]. While effective, these derivatization techniques can
be laborious and complicate data [41]. For these reasons, GC is not as widely used
in metabolomics workflows as LC.
4.3.3 Capillary Electrophoresis
Another form of separation which has been gaining popularity in the field of metabolomics is capillary electrophoresis (CE). CE is not a form of chromatography
because it lacks a stationary phase, a defining component of all chromatography
[42]. Instead, CE separation is achieved by applying high voltage to a capillary,
inducing an electrophoretic migration of ions. The electrophoretic mobility of the
analytes is dependent upon the ions charge-to-size ratio [42], wherein separation of
ions with differing electrophoretic mobilities is achieved. One strength of CE is its
high resolution, which is directly correlated to the potential applied to the column
as well as narrow peak widths provided in part by the inherent electroosmotic flow,
rather than laminar flow as in traditional chromatography [43, 44]. This resolution
coupled to mass spectrometry is conducive to both targeted and untargeted metabolomics. In the past, the integration of these two technologies was a limiting factor
[45]. In recent years however, advancements have been made which allow for easy
coupling of CE to mass spectrometry [46]. Current limitations of CE include a lack
of robustness, especially related to clogging [47].
4.3.4 Ion Mobility
Another separation technology which has been demonstrated to be effective for the
analysis of metabolites is ion mobility (IM) [48]. By applying a high voltage gradient opposing a gas flow, charged analytes are driven by the voltage gradient in one
direction, and by the gas flow the opposite direction. These competing forces allow
gas-phase separation of ions based on differing size-to-charge [49]. Because IM
operates in the gas phase, it is frequently coupled with mass spectrometry, often
being integrated within the mass analyzers of an instrument [50]. Ion mobility provides a degree of separation which can be comparable to that of LC-MS or GC-MS,
on a much shorter timescale. Where chromatographic methods separate metabolites
in a matter of minutes to hours, [51, 52] IM operates on the order of milliseconds
[53]. IM is usually measured in drift time, and can be used to calculate an ion’s collision cross section with proper calibration [54]. When coupled to mass spectrometry, IM provides a high degree of separation, having been shown to separate
isobaric species, as well as offering this orthogonal drift time information for each
analyte. Moreover, IM can be utilized in conjunction with chromatography
E. S. Rivera et al.
[40]. Historically, in the event that analytes of interest are not sufficiently volatile,
derivatizations such as alkylation have been necessary to increase volatility for
effective analysis by GC [40]. While effective, these derivatization techniques can
be laborious and complicate data [41]. For these reasons, GC is not as widely used
in metabolomics workflows as LC.
4.3.3 Capillary Electrophoresis
Another form of separation which has been gaining popularity in the field of metabolomics is capillary electrophoresis (CE). CE is not a form of chromatography
because it lacks a stationary phase, a defining component of all chromatography
[42]. Instead, CE separation is achieved by applying high voltage to a capillary,
inducing an electrophoretic migration of ions. The electrophoretic mobility of the
analytes is dependent upon the ions charge-to-size ratio [42], wherein separation of
ions with differing electrophoretic mobilities is achieved. One strength of CE is its
high resolution, which is directly correlated to the potential applied to the column
as well as narrow peak widths provided in part by the inherent electroosmotic flow,
rather than laminar flow as in traditional chromatography [43, 44]. This resolution
coupled to mass spectrometry is conducive to both targeted and untargeted metabolomics. In the past, the integration of these two technologies was a limiting factor
[45]. In recent years however, advancements have been made which allow for easy
coupling of CE to mass spectrometry [46]. Current limitations of CE include a lack
of robustness, especially related to clogging [47].
4.3.4 Ion Mobility
Another separation technology which has been demonstrated to be effective for the
analysis of metabolites is ion mobility (IM) [48]. By applying a high voltage gradient opposing a gas flow, charged analytes are driven by the voltage gradient in one
direction, and by the gas flow the opposite direction. These competing forces allow
gas-phase separation of ions based on differing size-to-charge [49]. Because IM
operates in the gas phase, it is frequently coupled with mass spectrometry, often
being integrated within the mass analyzers of an instrument [50]. Ion mobility provides a degree of separation which can be comparable to that of LC-MS or GC-MS,
on a much shorter timescale. Where chromatographic methods separate metabolites
in a matter of minutes to hours, [51, 52] IM operates on the order of milliseconds
[53]. IM is usually measured in drift time, and can be used to calculate an ion’s collision cross section with proper calibration [54]. When coupled to mass spectrometry, IM provides a high degree of separation, having been shown to separate
isobaric species, as well as offering this orthogonal drift time information for each
analyte. Moreover, IM can be utilized in conjunction with chromatography
E. S. Rivera et al.