Basic HTML Version
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 and basic compounds, mainly
in reversed phase chromatography (RPC). The pH limit of most
silica columns is between 2 and 8.
Stationary Phases
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
ease method development and create less need for mobile phase
additives. The decision tree in Figure 2 (next page) can help ana-
lysts select appropriate stationary phases, based upon analyte
solubility and polarity. Liquid chromatography employs specific
modes of separation which are denoted by the polarity distinc-
tion between the stationary and mobile phases; the most
common are reversed phase, normal phase and HILIC.
Reversed phase chromatography (RPC) consists of a nonpolar
stationary phase and a polar mobile phase. RPC is the most
commonly used mode and works well for the analysis of water-
soluble hydrophobic compounds. The most common types of
columns used in RPC are alkyls (most often a C18, also known
as octadecyl or ODS). End-capping is often employed in reversed
phase columns. End-capping refers to the dense bonding or
modification of 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 a C18.
Normal phase chromatography (NPC), named because it was
the first type of liquid chromatography, not for being more com-
mon, employs a polar stationary phase and a nonpolar mobile
phase. NPC is suited for the analysis of fat soluble compounds
and can also provide more selectivity for positional 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 from RPC and NPC as it uses traditional NPC
stationary phases and RPC mobile phases. HILIC is recom-
mended for the analysis of very polar compounds, often having
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, PFP propyl and cyano,
incorporate both nonpolar and polar functionality and can be
used in multiple or mixed-mode separation mechanisms.
Restek stationary phases and recommended uses are presented
in Figure 3 (page 149). For additional help selecting a column,
contact Restek at
support@restek.com
or call your local Restek
representative.
800-356-1688 or 814-353-1300
www.restek.com
147
HPLC COLUMNS
Column Select ion
Figure 1
Relative retention capabilities of commercially
available columns for hydrophobic compounds.
Website : www.chromtech.net.au E-mail : info@chromtech.net.au TelNo : 03 9762 2034 . . . in AUSTRALIA
Mar 2011