Using Selectivity to Enhance Separation of Analgesics
Selectivity is the most powerful tool to optimize separations in HPLC. This parameter is changed by using different bonded
phases, including C18, polar embedded, phenyl bonded phases and perflorophenyl, or by changing the mobile phase. In this work,
4.6 × 50 mm Poroshell 120 columns are used to quickly evaluate method development choices for the analysis of non-steroidal
anti-inflammatory drugs (NSAIDS). The short column length and high efficiency provide short analysis times and rapid equilibration,
leading to fast investigations of selectivity.
Instrument: Agilent 1260 Infinity Binary LC System
Columns: Noted below
Flow rate: 2 mL/min
Mobile Phase: A: 20 mM NH4HCO2pH 3.0 B: Acetonitrile
Temperature: 40 °C
Detection: UV, 254 nm
Time % Organic
The Agilent 1260 Infinity Binary LC System was configured as follows:
- G1312B Binary Pump SL, capable of delivering up to 600 bar
- G1316C Thermostatted Column Compartment (TCC)
- G1376D High Performance Autosampler SL Plus
- G4212A Diode Array Detector equipped with a G4212-60008 10 mm path length, 1 µL volume flow cell
The following columns were used in this study.
- Agilent Poroshell 120 PFP, 4.6 × 50 mm, 2.7 µm (p/n 699975-408)
- Agilent Poroshell 120 EC-C18, 4.6 × 50 mm, 2.7 µm (p/n 699975-902)
- Agilent Poroshell 120 Bonus-RP, 4.6 × 50 mm, 2.7 µm (p/n 699968-901)
- Agilent Poroshell 120 Phenyl-Hexyl, 4.6 × 50 mm, 2.7 µm (p/n 699975-912)
A generic gradient separation was used to evaluate these columns consisting of ammonium formate (20 mM NH4HCO3pH 3.0) using either methanol or acetonitrile.
Table I: Retention time, Log P, and pKa data for selected analgesics
The analgesic materials all possess a wide variety of functional groups including fluorine (sulindac and diflunisal) and chlorine
(diclofenac). The structures of the compounds examined are shown in Figure 1 and Table I. All samples were prepared at 10
mg/mL in acetonitrile and were diluted in water to a final concentration of 0.1 mg/mL.
Figure 1: Structures of selected analgesics.
Column Choice to Enhance Selectivity
The columns were chosen to improve selectivity in the separation. They included a highly end capped C18 column recommended
as a first choice in method development (Poroshell120 EC- C18).
Figure 2: Separation of analgesics using Agilent Poroshell 120 columns using methanol.
Poroshell 120 Bonus-RP can be used for many of the same separations as a C18 column while avoiding some of the disadvantages
of C18, such as poor wettability in high aqueous mobile phases. In addition, it is much more retentive for those molecules
that can interact by hydrophobic interactions and also by H-bonding with the amide group. Compared to alkyl only phases, Bonus-RP
has enhanced retention and selectivity for phenols, organic acids, and other polar solutes due to strong H-bonding between
polar group (H-bond acceptor) and H-bond donors, like phenols and acids. Bonus-RP gives retention slightly less than a C18
allows, for easy column comparison without the need to change mobile phase conditions. The Bonus-RP phase gives different
selectivity than C18 for polar compounds. It is also compatible with 100% water.
Poroshell 120 Phenyl-Hexyl columns deliver unique selectivity for compounds with aromatic groups, providing superior resolution
for these samples. Poroshell 120 Phenyl-Hexyl can also provide optimum separations of moderately polar compounds where typical
alkyl phases (C18 and C8) do not provide adequate resolution. Acetonitrile tends to decrease the π–π interactions between
aromatic and polarizable analytes and the phenyl-hexyl stationary phases, but methanol enhances those same interactions, giving
both increased retention and changes in selectivity. This does not mean that acetonitrile should not be used with a phenyl
bonded phase or that it might not provide an acceptable separation, but methanol is more likely to deliver the different selectivity
that is desired from a phenyl phase.
Poroshell 120 PFP columns possess a pentafluorophenyl ligand. This can provide an orthogonal separation mechanism to traditional
reverse phase columns. By specifically targeting many polar retention mechanisms, PFP phases can separate analytes based on
small differences in structure, substitution, and steric access to polar moieties. The resulting selectivity for positional
isomers, halogenated compounds, and polar analytes is particularly useful in the analysis of complex mixtures, and small molecule