ADV01-05_1-298

Over the past several years, HPLC column manufac-

turers have been creating new stationary phases in

attempts to address some of the separation prob-

lems encountered by analytical chemists.

Stationary phases in traditional reversed phase

columns are strictly non-polar alkyls, like C18. In

contrast, many newer specialty reversed phase

columns have stationary phases that are primarily

alkyls, but with some secondary polar functionality.

The polar functionality offers several advantages,

including: unique selectivity, enhanced retention of

polar compounds, and compatibility with complete-

ly aqueous mobile phases.

These specialty reversed phase columns can differ

either in the type of polar group they incorporate

or in how the polar group is incorporated into the

stationary phase. Restek offers two specialty

reversed phase columns that represent two different

approaches to introducing secondary polar groups

into a straight chain alkyl ligand (Figure 1). The

stationary phase in the Ultra Aqueous C18 column

has small polar groups attached to the silica sur-

face, between the C18 chains. In contrast, the Ultra

IBD stationary phase is a “polar embedded” type

• 4 •

www.restekcorp.com

800-356-1688

by Terrence S. Reid, HPLC Innovations Chemist

Specialty Reversed Phase HPLC

Columns for Polar Analytes

Ultra Aqueous C18 and Ultra IBD Columns

Solve Retention Dilemmas

✔

Both columns provide sharp peaks for basic analytes.

✔

Both columns compatible with 100% aqueous mobile phases.

✔

Complementary selectivity for acidic and basic analytes.

stationary phase, because its polar groups are

embedded within a straight alkyl chain.

One drawback to these specialty columns is that

their potential for mixed mode interactions makes

it more difficult to predict which column will per-

form best for a particular application. With this in

mind, we used a series of simple tests to directly

compare the performance characteristics of Ultra

Aqueous C18 and Ultra IBD columns. From the

results of these tests, we can offer some useful

guidelines for selecting a specialty reversed phase

column.

The first test measured the hydrophobic retention

of each column, using a sample mixture of com-

pletely nonpolar analytes and a mobile phase con-

taining a high proportion of organic solvent. For

pure alkyl stationary phases, hydrophobic retention

usually is directly proportional to the percent car-

bon (%C) in the bonded phase silica, if the phases

are bonded on silica particles of comparable sur-

face area. Figure 2 shows that the hydrophobic

retention of Ultra Aqueous C18 columns is approxi-

mately twice that of Ultra IBD columns, based on

capacity factors for pyridine, despite the two bond-

ed phases' similar surface area and %C (Ultra

Aqueous C18: 100Å pores, 14%C; Ultra IBD: 100Å

pores, 12%C). The hydrophobic retention of Ultra

Aqueous C18 columns is equivalent to that of con-

ventional C18 columns with the same surface area

and %C. The considerably reduced hydrophobic

retention of Ultra IBD columns can be attributed to

the embedded polar group in the stationary phase

shielding the lower portion of the alkyl chain from

the nonpolar analytes.

We compared the columns’ base deactivation by

measuring the peak shape for a basic analyte, pyri-

dine (Figure 3). Both Ultra Aqueous C18 and Ultra

IBD columns show excellent base deactivation, with

pyridine peak symmetry values better than those for

highly base-deactivated C18 phases made through

conventional chemistry. Although they are similarly

base-deactivated, Ultra Aqueous C18 columns

exhibit much greater retention of pyridine than do

Ultra IBD columns.

Next, we compared Ultra Aqueous C18 and Ultra

IBD columns’ ability to separate small carboxylic

acids. It is difficult for conventional reversed phase

columns to retain these molecules. A very weak,

highly aqueous mobile phase is required. Many C18

phases are not compatible with highly aqueous

mobile phases, and show a gradual or sudden loss

of retention that is attributed to “chain folding” or

“phase collapse.” Both Ultra Aqueous C18 and Ultra

IBD columns are completely compatible with 100%

aqueous mobile phases, as shown in Figure 3A.

Neither column showed any loss of retention, even

after mobile phase flow was temporarily stopped.

(Absence of pressure maximizes the potential for

phase collapse, thus exposure to 100% aqueous

mobile phase under no flow is the most extreme

test of phase integrity.) This comparison did reveal

Conventional Alkyl

Ultra Aqueous C18

Ultra IBD

Figure 1

The stationary phase in an Ultra Aqueous

C18 column has small polar groups attached

to the silica surface; in the Ultra IBD station-

ary phase polar groups are embedded in the

alkyl chain.

Peak List:

Conc. (mg/mL)

1. uracil

0.02

2 benzene

3.00

3. naphthalene

0.50

4. biphenyl

0.06

LC_0233

Sample:

Inj.:

1.3µL

Solvent: methanol:water

(75:25, v/v)

Mobile Phase:

A: water

B: methanol

Isocratic: 80%B

Flow:

1.0mL/min

Temp.:

ambient

Det.:

UV @ 254nm

LC_0234

Column:

Ultra Aqueous C18

Ultra IBD

Catalog #:

9178565

9175565

Dimensions:

150 x 4.6mm

Particle Size:

5µm

Pore Size:

100Å

Figure 2

Hydrophobic retention for Ultra Aqueous C18 columns is approximately twice that for Ultra IBD

columns, despite similar surface areas and % carbon.

polar group