Utilizing Xbridge? HPLC Columns for Method Development at pH Extremes - XBridge" C18 columns provide outstanding phosphate buffer stability at pH 2, 7 and 12. - Chromatography Online
Utilizing Xbridge? HPLC Columns for Method Development at pH Extremes
XBridge" C18 columns provide outstanding phosphate buffer stability at pH 2, 7 and 12.


The Application Notebook


Introduction


Table 1: Gradient profile for selectivity study.
For the best peak shape, retention and loading of basic analytes in reversed-phase HPLC, the pH of the mobile phase should be at least 2 units higher than the pK a of the molecule. For many bases, this means the pH of the mobile phase should be 10 or higher. Volatile buffers and additives such as ammonium bicarbonate and ammonium hydroxide are commonly used in high pH mobile phases, but their upper buffering capacity is pH 11. Phosphate buffer, a favourite of chromatographers because of its three pK a's of 2.15, 7.2 and 12.3 and transparency at low UV wavelengths, is rather aggressive at pH 12. Standard silica-based column packings are unusable at pH 12 with phosphate buffer, and many columns designed to work at high pH suffer from reduced lifetimes. With the introduction of the new XBridge™ family of HPLC columns, phosphate buffer at all three buffering ranges can now be routinely used.

Chromatographic Conditions Stability Testing

Column: XBridge™ C18 5Ám 4.6 x 150 mm

Mobile Phase A: 200 mM K3PO4 (pH 12):MeCN (80:20)

Mobile Phase B: Water

Mobile Phase C: Acetonitrile

Isocratic Mobile Phase Composition: 10% A; 37% B; 53% C

Flow-rate: 1.0 mL/min

Injection Volume: 10ÁL of 440Ág/mL (total concentration)

Detection: UV @ 254 nm

Selectivity Study

Column: XBridge™ C18 3.5Ám 4.6 x 100 mm

Mobile Phase A1: 30 mM Potassium phosphate, pH 2

Mobile Phase A2: 30 mM Potassium phosphate, pH 7

Mobile Phase A2: 30 mM Potassium phosphate, pH 12

Mobile Phase B: Acetonitrile

Flow-rate: 1.4 mL/min

Gradient: See table 1.

Column Temperature: 30░C

Injection Volume: 20ÁL of 5Ág/mL (each) standard

Detection: UV @ 210 nm (pH 2, 7); 220 nm (pH 12)

Instrument (both studies): Waters Alliance« 2695 with 2996 PDA


ADVERTISEMENT

blog comments powered by Disqus
LCGC E-mail Newsletters
Global E-newsletters subscribe here:




 

LCGC COLUMNISTS 2014

Sample Prep Perspectives | Ronald E. Majors: Ron Majors, established authority on new column technologies, keeps readers up-to-date with new sample preparation trends in all branches of chromatography and reviews developments.
LATEST: UV Detector Problems


Perspectives in Modern HPLC | Michael W. Dong: Michael W. Dong is a senior scientist in Small Molecule Drug Discovery at Genentech in South San Francisco, California. He is responsible for new technologies, automation, and supporting late-stage research projects in small molecule analytical chemistry and QC of small molecule pharmaceutical sciences. LATEST: Superficially Porous Particles: Perspectives, Practices, and Trends


MS — The Practical Art | Kate Yu: Kate Yu brings her expertise in the field of mass spectrometry and hyphenated techniques to the pages of LCGC. In this column she examines the mass spectrometric side of coupled liquid and gas-phase systems. Troubleshooting-style articles provide readers with invaluable advice for getting the most from their mass spectrometers. LATEST: Radical Mass Spectrometry as a New Frontier for Bioanalysis


LC Troubleshooting | John Dolan: LC Troubleshooting sets about making HPLC methods easier to master. By covering the basics of liquid chromatography separations and instrumentation, John Dolan is able to highlight common problems and provide remedies for them. LATEST: Problems with Large-Molecule Separations


More LCGC Chromatography-Related Columnists>>

LCGC North America Editorial Advisory Board>>

LCGC Europe Editorial Advisory Board>>

LCGC Editorial Team Contacts>>


Source: The Application Notebook,
Click here