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Pharmaceutical Articles

New pHidelity™ pH-Stable HPLC Columns

For Analyses at Extreme pH Conditions

By Becky Wittrig, Ph.D., HPLC Product Marketing Manager, Frank Dorman Ph.D., HPLC Innovations Manager, Rick Lake, Pharmaceutical Innovations Chemist, Vernon Bartlett, HPLC Innovations Scientist, Bruce Albright, HPLC Innovations Chemist, and Randy Romesberg, HPLC Innovations Chemist
  • Stable to pH 12 — superior chromatography for basic compounds.
  • Patented barrier technology protects silica particles.
  • True C18 selectivity, for simpler and more reproducible analyses.

We are pleased to introduce pHidelity™ pH-stable HPLC columns, designed for analyses that require, or benefit from, extreme pH conditions. pHidelity™ columns incorporate a proprietary barrier layer that protects the base silica particle, and a secondary layer that provides the functional stationary phase ligand. pHidelity™ columns can be used routinely up to pH 12 — a significant improvement over the typical pH 2.5 to 7.5 range for silica-based materials. pHidelity™ columns give you more control over analyte retention and resolution; mobile phase pH can be increased to enhance retention of basic analytes — without sacrificing column lifetime.

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 — significantly shortening column lifetime. Therefore, with a conventional C18 column, an acidic or neutral 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 ionized forms and their retention, peak shape, and resolution is limited on a conventional C18 column (Figure 1A). An extended range pHidelity C18 column allows the use of high pH mobile phases, above the analytes pKa, without deleterious 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 phases. 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 analytes, making them more hydrophilic, and less retained (Figure 2A). In contrast, using an extended range pHidelity™ C18 column and a mobile phase pH above the highest pKa of the analytes, the compounds will be uncharged and more hydrophobic, resulting in greater retention (Figure 2B). The analysis of basic compounds using a high pH mobile phase is an easy way to increase retention and to enhance resolution and peak shape. This makes a simpler task of method development, especially for complex test mixtures.

Non-silica-based chemistries have been developed in attempts to overcome the pH constraints of conventional silica-based HPLC columns, but silica-based phases offer a number of advantages, including high efficiencies, consistent lot-to-lot reproducibility, and predictable selectivities. Silica-based pHidelity™ pH-stable columns offer selectivity similar to conventional materials, but with dramatically increased column lifetime, even under the most harsh conditions. Figure 3 shows equivalent comparisons of a pHidelity™ C18 column and a conventional C18 column in an accelerated lifetime test under high pH conditions, at pH 10 and 60°C. This test demonstrates that pHidelity™ columns have a much greater lifetime when used in caustic environments than conventional C18 columns. Additionally, the pHidelity™ packing material is based on a true silica particle, ensuring a more C18-like selectivity than any competitive column based on non-silica or hybrid materials.

If your separation would benefit from extended pH conditions, we recommend you take advantage of pHidelity™ column for extreme-pH stability, C18-like selectivity, and long lifetimes. To discuss your separation, or for more information, please contact Restek’s HPLC technical service group, and we will be happy to discuss how you can improve your analysis, and make fewer column replacements, by using pHidelity™ column.


Figure 1  Improve SSRI retention, peak shape, and resolution using an extended pH range pHidelity column.

  1. uracil (marker)
  2. fluvoxamine maleate
  3. fluoxetine
  4. sertraline HCl

Sample:

Inj.:

10µL

Conc.:

100µg/mL each component

Sample diluent:

acetonitrile

A)  Conventional C18 columns require mobile phases below the SSRI’s pKa, limiting chromatography



LC_PH0362

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

B)  pHidelity columns allow use of mobile phases above the SSRI’s pKa — for improved chromatography



LC_PH0361

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


Figure 2  Using a high pH mobile phase is an easy way to improve peak retention, resolution, and symmetry in a test mix of compounds varying in pKa values.

  1. lidocaine
  2. verapamil
  3. diphenhydramine
  4. nortriptyline

Sample:

Inj.:

10µL

Conc.:

100µg/mL each component

Sample diluent:

acetonitrile

A)  Poor separation of bases under typical conditions



LC_PH0392

Column:

C18

Dimensions:

150 x 4.6 mm

Particle size:

3µm

Pore size:

200Å

Conditions:

Mobile phase:

20mM ammonium acetate (pH 4):acetonitrile 50:50

Flow:

0.75mL/min.

Temp.:

ambient

Det.:

UV @ 254 nm

B)  Complete separation of bases on a pHidelity™ column at pH 11



LC_PH0363

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, 35:55:10 (v/v/v)

Flow:

1.5mL/min.

Temp.:

ambient

Det.:

UV @ 254 nm


Figure 3  pHidelity™ C18 columns - exceptional performance under accelerated high pH stability testing conditions.