Pharmaceutical Articles

Optimize Selectivity & Efficiency in UHPLC Separations

With More Stationary Phase Choices on 1.9µm Pinnacle™ DB HPLC Columns

By Rick Lake, Pharmaceutical Innovations Chemist

Largest variety of stationary phases for UHPLC.
Faster analyses, uncompromised chromatography.
100% Restek manufactured—from base silica to final packed column.

Since the late 1960s continual advancements have been made in HPLC column technology, and over time the trend has been toward smaller particle sizes. This trend has led us to where we are today—Ultra-High Performance Liquid Chromatography (UHPLC). UHPLC is a milestone in the evolution of LC in that columns packed with <2µm particles, used with instrumentation capable of handling the resulting high back pressures, make possible extremely fast and efficient separations. UHPLC is a very powerful tool for today’s practicing chromatographer, as it can significantly increase the efficiency of a chromatographic separation. In addition, the wider range of usable flow rates makes high speed separations possible. However, in light of this new technology, it is important that we do not forget the importance of selectivity. In this article, we will review the significance of selectivity in obtaining acceptable resolution and demonstrate how having choices in stationary phase allows you to maximize the benefits of UHPLC.

Although small particles have made faster separations possible, selectivity has the greatest effect on resolution. Selectivity, in turn, is governed predominantly by analyte interactions with both the stationary and mobile phases. UHPLC, through the use of small particle columns, does maximize efficiency (e.g. theoretical plates), but the stationary phase is still the most important consideration when attempting to resolve mixtures of compounds. Ideally, a stationary phase that produces optimum selectivity or allows for resolution of compounds in a timely manner should be selected.

Previously, some advantages of selectivity in specific separations have been noted. For example, the use of a unique Biphenyl stationary phase has shown excellent selectivity for aromatic or fused ring compounds. When using the Biphenyl stationary phase and combining it with the heightened efficiencies of the 1.9µm Pinnacle™ DB column, we can produce highly selective and fast separations of steroids (Figure 1). A Pinnacle™ DB 1.9µm Biphenyl column can separate a test mix of seven hormones in under 2 minutes, a feat not possible through C18 selectivity.

Another example of unique selectivity available on a 1.9µm particle size column is the PFP Propyl (pentafluorphenyl propyl) stationary phase for halogenated drug compounds. This phase is very selective and retentive for organohalogens or other compounds containing basic or electronegative functionalities. To demonstrate heightened selectivity for halogenated drug compounds, we assayed a test mix of eight benzodiazepines and two metabolites, a mix commonly assayed on a C18 colum, in just over 4 minutes with complete resolution (Figure 2). To get the same level of selectivity from a C18 column, a shallower gradient would be needed, prolonging the analysis time. Since the selectivity of the Pinnacle™ DB 1.9µm PFP Propyl column elutes the benzodiazepines in quick succession, a simple gradient still allows for the earlier elution of the more polar metabolites, while maintaining a fast overall run time.

Restek is committed to giving the practicing chromatographer choices, and has therefore sought to deliver the widest selection of stationary phases available with <2µm particle sizes. The goal of chromatography is always to resolve compounds of interest in the fastest time possible. By combining the benefits of UHPLC with Restek’s complement of unique stationary phase choices, faster separations become a reality.

Figure 1 Restek’s 1.9µm Pinnacle™ DB Biphenyl columns are highly selective for steroids, making an extremely fast and selective analysis.

estriol
17β-estradiol
17α-estradiol
ethynyl estradiol
testosterone
estrone
norethindrone

Conditions:

Mobile phase:

A: Water
B: Acetonitrile

Time (min)		%B
0		30
1		30
3		70

Flow:

0.8mL/min.

Temp.:

30°C

Det.:

UV @ 220nm

Sample:

Inj.: 1µL; Conc.: 100µg/mL each component; Sample diluent: water:methanol (50:50)

Column:

1.9µm Pinnacle™ DB Biphenyl; Cat. # 9409252; Dimensions: 50mm x 2.1mm;

Particle size:

1.9µm; Pore size: 140Å

Instrument:

Jasco X-LC

LC_PH0453

Figure 2 Fast, selective analysis of benzodiazepines is made possible by combining the speed of UHPLC with the enhanced selectivity of the 1.9µm Pinnacle™ DB PFP Propyl column.

Peak List:

Conc (µg/ml)