ADV06-15_1-412

•

2007.01

Pharmaceutical

New

pHidelity pH-Stable HPLC Columns

(continued from page 3)

• Stable pH 12 – superior chromatography for basic compounds.

• Patented barrier technology protects silica particles.

• True C18 selectivity, for simpler and more reproducible analyses.

Figure 1

Improve SSRI retention, peak shape, and resolution using

an extended pH range pHidelity column.

Practically, the useable pH range for conventional

silica-based HPLC columns is pH 2.5 to 7.5.

Columns are used outside of this range only when

there is an extreme need for a separation, and the

inevitable price – a very short column lifetime –

must be accepted. pHidelity™ pH-stable HPLC

columns can be used far above the typical pH

range for silica-based stationary phases, with

mobile phases up to pH 12, giving more control

over analyte retention and resolution.

To illustrate the advantages of using high pH

mobile phases for assaying basic analytes, we first

analyzed selective serotonin reuptake inhibitors

(SSRIs). SSRIs are basic compounds with high

pKa values. Ideally, a high pH mobile phase would

be used for this analysis. A high pH mobile phase

will keep the analytes in their neutral forms and

allow better retention and resolution on an alkyl

C18 column. However, if using a mobile phase pH

appropriately above compound pKa values

(approximately 1.5-2 pH units) on a column with

a limited alkaline range, the caustic mobile phase

would rapidly degrade the silica particles – signifi-

cantly shortening column lifetime. Therefore,

with a conventional C18 column, an acidic or neu-

tral mobile phase pH must be used. As result,

when the compounds are assayed at a mobile

phase pH below their pKas, they are in their ion-

ized forms and their retention, peak shape, and

resolution is limited on a conventional C18 col-

umn (Figure 1A). An extended range pHidelity

C18 column allows the use of high pH mobile

phases, above the analytes pKa, without deleteri-

ous effects to the column. Under these conditions,

basic analytes are neutral, more hydrophobic and

better retained (Figure 1B). By using a pHidelity™

column mobile phase pH can be optimized,

improving retention, peak shape, and resolution

on a C18 column.

Another advantage to extending the pH range of

silica based columns, is improved analysis of multi-

component test mixes with high pH mobile phas-

es. When faced with a mixture of basic analytes,

choosing the appropriate mobile phase pH can be

problematic. An example of this is the mixture of

bases which vary in pKa value, as shown in Figure

2. If a conventional C18 column was employed to

assay this ionic mixture, a pH approximately 1.5 -

2 units below the lowest pKa would need to be used

(a pH above the highest pKa would be above the

operating range of conventional silica columns).

This would result in protonation of the basic ana-

lytes, making them more hydrophilic, and less

B) pHidelity columns allow use of mobile phases

above the SSRI’s pKa – for improved chromatography

A) Conventional C18 columns require mobile

phases below the SSRI’s pKa, limiting chromatography

LC_PH0362

LC_PH0361

Sample:

Inj.:

10µL

Conc.:

100µg/mL each component

Sample diluent: acetonitrile

Column:

C18

Dimensions:

150 x 4.6 mm

Particle size:

5µm

Pore size:

100Å

Conditions:

Mobile phase:

20mM potassium phosphate, monobasic

(pH 3)

:acetonitrile, 60:40

Flow:

1.0mL/min.

Temp.:

ambient

Det.:

UV @ 230 nm

Column:

pHidelity™ C18 (cat.# 9579365)

Dimensions:

150 x 4.6 mm

Particle size:

3µm

Pore size:

200Å

Conditions:

Mobile phase:

10mM ammonium bicarbonate

(pH 11)

:acetonitrile:tetrahydrofuran, 45:45:10

Flow:

1.5mL/min.

Temp.:

ambient

Det.:

UV @ 230 nm

1. uracil (marker)

2. fluvoxamine maleate

3. fluoxetine

4. sertraline HCl