pharmaceutical
Applications
note
RestekCorporation • (800)356-1688 • (814)353-1300 •
#59118A
pharmaceutical
Allure
™
PFP Propyl and Ultra PFP HPLC Columns Provide Improved
Analyses of Basic Compounds
High performance liquid chromatography (HPLC) methods
must be optimized to provide the greatest selectivity and
sensitivity, and the best peak shape. Unfortunately, many
analysts consider switching the stationary phase—the heart of
the HPLC system—only as a last resort. Too often analysts
coax the stationary phase to perform a non-native separation
by using modifiers and ion pairing agents, which leads to
reduced sensitivity and equilibration problems (the C18 phase
is the most often misused). Selection of the proper stationary
phase for your separation can improve LC sensitivity, analyte
retention, and peak shape without the use of modifiers or ion
pairing agents. For example, Restek’s Allure
™
PFP Propyl and
Ultra PFP HPLC columns easily perform separations of many
basic analytes.
Research shows the Allure
™
PFP Propyl stationary phase not
only provides the greatest retention and capacity factor (k')
(Figure 1) for basic analytes such as beta blockers and tricyclic
antidepressants, but also the best peak shape. “…The results
indicate that both the fluorine groups and the propyl chain are
important on the phenyl ring to obtain the best peak shape
and retention of the basic solutes when ammonium
formate:acetonitrile (10:90) is used as the mobile phase.”
1
As
peak asymmetry is improved, sensitivity is increased.
Basic analytes are difficult to retain on C18 phases if the
analytes have pKa’s greater than 8. They can be retained on
C8 or C18 columns by using modifiers, but at the expense of
sensitivity.
2
Sensitivity can be reduced further on an LC/mass
spectrometer (MS) ESI interfaces when the buffer concentra-
tions exceed 50mM. TheAllure
™
PFP Propyl and Ultra PFP
columns eliminate the need for modifiers, and analytes such as
cocaine (COC) and its metabolite, ecgonine methyl ester
(EME), can be separated and retained using 90% acetonitrile
in under 4.5 minutes (Figure 2). As the concentration of the
organic solvent in the mobile phase increases, the desolvation
process becomes more effective and the LC/MS ESI signal
increases.
3
Because of this interaction, using the Allure
™
PFP
Propyl column with a high organic concentration increases the
response of COC and other basic solutes by as much as twelve
times over that from a C18 column.
4
Proper retention also is needed to separate basic analytes from
naturally occurring substances in the blood, urine, or body
tissues. If the analytes elute too closely to the void volume,
ionization suppression can occur.
5
To be cost effective,
however, the analytes should be separated in less than 6
minutes.The high selectivity of the Allure
™
PFP Propyl
column more than adequately separates EME from COC and
the column void volume, respectively. (Figure 2).
6
The Ultra PFP column is similar to theAllure PFP Propyl. The
PFP column features a 100 angstrom pore size and is available
in 3 and 5 micron particle sizes. Like the PFP Propyl, the PFP
has high retention for basic and multi-halogenated analytes.
Table I displays the retention of many basic beta-blockers on
the PFP phase versus many other phases using 90% acetoni-
trile as the mobile phase. The HPLC/MS ESI analysis of beta-
blockers using the Ultra PFP column shows good intensity
(Figure 6). The PFP phase is also able to retain multi-
halogenated analytes such as the thyroid hormone,
levothyroxine, and purines and pyrimidines (Figures 4 & 5).
The PFP phase has the greatest retention, which will improve
the separation of one analyte from another.
The attraction mechanism of pi-acid HPLC phases, such as
PFPP and PFP, allow stronger retention of bases than alkyl
phases like C18. Alkyl phases like C18 cannot adequately
retain strongly basic analytes unless they have significant
non-polar functional groups to allow hydrophobic retention.
100
50
k'
Figure1
Allure
™
PFPPropyl demonstrates increased
retention of basic analytes
5mMNH
4
OAc, pH 4.5:ACN; 30x2.1mm; 0.4mL/min.
Acebutolol
Data Courtesy of Shane Needham, Pfizer Inc
0
50
100
%ACN
30
15
k'
Amitriptyline
PLC-0001
0
50
100
%ACN
Column
PFP Propyl
C4
CN
OH
Column
PFP Propyl
C4
CN
OH
