system. It is important to note that while altering the tempera-
ture of a separation can lower back pressure, it also lowers
retention and can change selectivity. pH can also be used to
control the selectivity and retention of ionizable compounds.
Acid-base equilibrium can be employed to directly affect the
retention characteristics of acidic andbasic compounds,mainly
in reversedphase chromatography (RPC).ThepH limit ofmost
silica columns is between2 and8.
StationaryPhases
Stationary phase, or the specific chemical ligand bonded to the
silica support, plays a primary role in resolving compounds.
Through selectivity, the major contributor to resolution, a sta-
tionary phase can control the retention characteristics of the
solutes. Identifying the appropriate stationary phase can greatly
easemethoddevelopment and create less need formobilephase
additives.Thedecision tree inFigure2 (nextpage) canhelpana-
lysts select appropriate stationary phases, based upon analyte
solubility andpolarity. Liquid chromatography employs specific
modes of separationwhich are denoted by the polarity distinc-
tion between the stationary and mobile phases; the most
common are reversedphase, normal phase andHILIC.
Reversed phase chromatography (RPC) consists of a nonpolar
stationary phase and a polar mobile phase. RPC is the most
commonly usedmode andworks well for the analysis of water-
soluble hydrophobic compounds. The most common types of
columns used inRPC are alkyls (most often a C18, also known
asoctadecylorODS).End-capping isoftenemployed in reversed
phase columns. End-capping refers to the dense bonding or
modificationof the silica surface to further limit silanol activity.
This acts to provide better peak symmetry, especially for basic
compounds. Alternate ligands and bonding chemistries can be
applied to RPC columns to incorporate phenyl, cyano, amino,
and other polar groups into the stationary phase, providing
alternate selectivity to aC18.
Normal phase chromatography (NPC), named because it was
the first typeof liquidchromatography,not forbeingmorecom-
mon, employs a polar stationary phase and a nonpolar mobile
phase. NPC is suited for the analysis of fat soluble compounds
andcanalsoprovidemore selectivity forpositional isomers than
is commonly observed in RPC. Bare silica columns are most
commonly used for NPC. Other phases for NPC include cyano
and amino.
Hydrophilic Interaction Chromatography (HILIC) employs a
polar stationary phase and a less polar mobile phase. HILIC
differentiates itself fromRPCandNPCas ituses traditionalNPC
stationary phases and RPC mobile phases. HILIC is recom-
mended for the analysis of very polar compounds, oftenhaving
negative log P values, and for analysis by LC/MS. Bare silica,
cyano and amino columns are also commonly used in HILIC
mode. Some stationary phases, like IBD, PFPpropyl and cyano,
incorporate both nonpolar and polar functionality and can be
used inmultiple ormixed-mode separationmechanisms.
Restek stationary phases and recommended uses are presented
in Figure 3 (page 149). For additional help selecting a column,
contact Restek at
or call your local Restek
representative.
800-356-1688or 814-353-1300
147
HPLCCOLUMNS
Column Selection
Figure1
Relative retentioncapabilitiesof commercially
availablecolumns forhydrophobiccompounds.
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