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Beyond C18

Increase Retention of Hydrophilic Compounds Using Biphenyl Columns

By Amanda Rigdon, Pharmaceutical Innovations Chemist and Rick Lake, Pharmaceutical Market Development Manager

Searching for a better way to retain hydrophilic aromatic drug compounds? Biphenyl phases, such as the Pinnacle™ DB Biphenyl column, provide greater retention than alkyl phases. Use a Biphenyl column to separate difficult-to-retain polar aromatics from unretained matrix contaminants.

Many drug classes include compounds with aromatic ring structures, some of which also contain a sulfone or sulfoxide group. Both sulfur groups have dipole moments, adding a hydrophilic character to compounds containing these functional groups. The analysis of hydrophilic compounds on a traditional alkyl column (e.g., C18) can be problematic, since alkyl columns depend on hydrophobic (dispersive) interactions for retention. Since the sulfone and sulfoxide groups contain ? bonds, the Biphenyl column’s affinity toward compounds containing these bonds makes it a logical choice when increased retention of compounds containing these groups is desired.

To explore the selectivity of the biphenyl phase towards sulfur-containing aromatic compounds, phenyl sulfone, a simple probe, was analyzed on alkyl (C18), phenyl, phenyl hexyl, and Biphenyl columns to determine the relative retention of each phase, as measured by capacity factor (k'). In order to ensure separation of analytes from unretained contaminants, a minimum k' value of 2 is recommended for most analyses, however in cases where there is little to no matrix interference, a k' of 1 may be acceptable. The data in Figure 1 show that phenyl sulfone is retained to a much greater degree on the Pinnacle™ DB Biphenyl column, than on the other phases tested (k' = 2.08). This is due to the unique retention mechanism of the biphenyl stationary phase, which can interact with both the hydrophobic aromatic ring and the hydrophilic sulfone group through ?-? interactions. Although the phenyl stationary phase also allows for the use of ?-? interactions, the biphenyl phase has a larger electron cloud and is significantly more retentive.

To further test the retention of the Biphenyl column, a second set of probes, consisting of compounds in the NSAID family, was analyzed. Tenoxicam, which contains a sulfone group, and sulfinpyrazone, which contains a sulfoxide group, were analyzed along with a void marker (uracil). Although these compounds are more complex than the probe used in the first experiment, the same pattern of retention was observed (Figure 2). The Pinnacle™ DB Biphenyl column exhibited the greatest retention for tenoxicam. With k' values of 0.33 on the C18 and 0.49 on the phenyl columns, tenoxicam shows almost no retention on these stationary phases. The phenyl hexyl phase performed slightly better with a k' value of 1.52 for tenoxicam. However, when tenoxicam was analyzed on the Biphenyl column under the same conditions, the k' value increased to 2.22, a value much more likely to provide adequate resolution from matrix components. Sulfinpyrazone, a less polar compound, also followed the same pattern of retention (Table I).

The improved retention for hydrophilic aromatics shown here is due to the unique ?-? interaction retention mechanism of the Biphenyl phase. This mechanism is particularly useful for analysis of sulfone- and sulfoxide-containing drug compounds, which are not easily retained on alkyl or phenyl phases. The Biphenyl phase provides greater retention than alkyl and phenyl phases and is ideal for separating difficult-to-retain polar aromatics from unretained matrix contaminants.

Figure 1 The Biphenyl phase is more retentive for phenyl sulfone than other alkyl and phenyl phases.

Figure 2 Only the Biphenyl phase retains both test probes to k? > 2, the level recommended to ensure separation from unretained matrix contaminants.

1. Uracil (void marker)
2. Tenoxicam
3. Sulfinpyrazone
NSAIDs on Pinnacle<sup>®</sup> DB Biphenyl
Column Pinnacle® DB Biphenyl (cat.# 9409565)
Dimensions: 150 mm x 4.6 mm ID
Particle Size: 5 µm
Pore Size: 140 Å
Temp.: 30 °C
Diluent: 0.1% formic acid in water:methanol (40:60)
Conc.: 100 µg/mL each component (see peak list)
Inj. Vol.: 10 µL
Mobile Phase
A: 0.1% formic acid in water
B: methanol
Time (min)%B
0 60
2.0 60
8.0 90
20.0 90
20.1 60
Flow: 1.0 mL/min.
Detector UV/Vis @ 254 nm
Instrument Shimadzu Prominence
Notes Column B: phenyl
Column C: phenyl hexyl
Column D: C18
Detector: Shimadzu PDA (SPD-M20A)
Note: a minimum k' value of 2 is generally recommended to fully separate target analytes from matrix contaminants.

Table I Biphenyl columns show improved retention of sulfone- and sulfoxide-containing aromatic drugs.

K' Value
Biphenyl Phenyl
Phenyl C18
Tenoxicam 2.23 1.39 0.637 0.235
Sulfinpyrazone 4.18 3.90 1.88 1.89

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