Biomedical Engineering Reference
In-Depth Information
During last decade, liquid chromatography (LC) column technology has
evolved exponentially, resulting in massive selection of commercially available
LC columns. In addition to traditional reverse and normal stationary phases such
as silica, phenyl, C8, or C18, a new generation of LC columns with various
modifications has emerged. These new columns attempt to improve the retention
of analytes, extend the lifetime of column and generally increase the throughput
of the LC analysis. A few representative columns belong to this category are
polar-embedded C8 or C18 columns, PFP (Pentaflourophenylpropyl) columns,
HILIC (Hydrophilic Interaction Liquid Chromatography) and monolithic col-
umns. Among these new columns, the properties of the PFP column are very
unique. To certain extent, the PFP columns mimic traditional reversed phase phe-
nyl columns in which molecules containing p - p interaction (such as aromatic ring
or resonance structure) but provide much greater retention due to the presence of
five strong electron-drawing atoms (i.e., pentafluorine). Because of the strong
retention, PFP column typically requires much higher organic content which
boosts ionization efficiency significantly. Another interesting phenomenon of PFP
column is that PFP column, like CN or NH
2
column, changes to normal phase
mechanism under very high organic conditions, thus provides additional and
unique selectivity.
In addition to the increased variety of different stationary phases, the size of
sorbent particles used for liquid chromatography has also evolved from the tradi-
tional 5 mm and 3 mm particles to smaller particle sizes such as 2.6 mm and 1.7 m m
for both true Ultrahigh Performance Liquid chromatography (UHPLC) and stan-
dard HPLC that can mimic UHPLC performance. Porous silica rod or monolithic
columns are also available to achieve higher throughput and/or better resolution;
however, the popularity of monolithic columns is relatively limited due to increased
solvent consumption, and their use has been larger superseded by the rapid growth
in UHPLC technology. Typical UHPLC analyses can achieve very high sample
throughput and excellent chromatographic peak resolution through the use of very
fine particle sizes (1.7 mm), high pressure solvent pumps (up to 15,000 psi.), and
limited void volumes throughout the system. UHPLC is very useful for multiple
analyte assays or the separation of isomers which have same selective reaction mon-
itoring (SRM) transition.
Most drug candidates are basic compounds with pKa values of around 9.5. In
order to keep basic substances uncharged in and increase the retention of the ana-
lytes on the column, it is common practice to use a mobile phase pH that is up to two
units higher than the pKa value of the analyte. Traditional silica based columns are
only suitable for acidic and neutral condition and have limited life time at high pH.
To meet the challenge and demand, several newer and modified LC column such as
Gemini
®
(Phenomenex), Xbridge
®
(Waters) and Kromasil
®
(Agilent) have merged
and can be used for mobile phases up to pH 12. While it is a well-known fact that
traditional basic modifiers such as Triethylamine (TEA) suffer ion suppression due
to ion pair, recently, it has been found that ammonium bicarbonate-ammonium
hydroxide (NH
4
HCO
3
-NH
4
OH) solution is an effective and great substitute buffer
at high pH range [
10
] .
