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2007 vol. 3

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 chro-

matographer, 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.

In past articles we have discussed the physical

advantages that are driving interest in small particles,

mainly the influence of particle size on usable flow

rates and peak efficiency. 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 res-

olution 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 sta-

tionary 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 resolu-

tion (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 sta-

tionary 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 sep-

arations become a reality.

Figure 1

Restek’s 1.9 μm Pinnacle™ DB Biphenyl columns are highly

selective for steroids, making an extremely fast and selective analysis.

Peak List:

1. estriol

2. 17

-estradiol

3. 17

-estradiol

4. ethynyl estradiol

5. testosterone

6. estrone

7. norethindrone

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

Conditions:

Mobile phase:

A: Water

B: Acetonitrile

Time (min) %B

Flow:

0.8mL/min.

Temp.:

30°C

Det.:

UV @ 220nm