TABLE OF CONTENTS - BGB Analytik AG

pH and Mobile Phase
The choice of mobile phase for a reversed-phase system starts with selecting the organic
modifier. Selectivity differences and sample retention will vary significantly among mobile
phases containing acetonitrile, methanol, and tetrahydrofuran (THF). Sample solubility is likely
to differ in such solvents and dictate use of a specific solvent or solvents. UV detection at
certain wavelengths is not possible with certain modifiers (e.g., methanol at 200 nm).
Both pH and ionic strength of the aqueous portion of mobile phases are important parameters
in developing rugged methods that are not sensitive to small variations in conditions. With
ionic compounds, retention of typical species shows significant changes with pH. It is very
important to control pH in such reversed-phase systems to stabilize retention and band
spacing. A pH set between 2 and 4 generally provides the most stable conditions for retention
vs. small changes in pH and this pH is recommended for starting method development for
most samples, including basic compounds and typical weak acids.
Column Choice Relative to Application Objective
www.agilent.com/chem/supplies
Column Selection
Application Objective Column Diameter (mm)
Very high sensitivity, LC/MS, peptides and proteins 0.1, 0.075
Very high sensitivity, limited sample, LC/MS,
peptides and proteins
0.3, 0.5
High sensitivity, limited sample, LC/MS 1.0
Save solvent; special low-volume instrumentation
is available
2.1
Special detectors, e.g., mass spec 2.1
High sensitivity, limited sample 2.1
Save solvent; standard HPLC equipment available, LC/MS 3.0
Standard separations 4.6
Small-scale (mg) preparative separations 9.4
Large-scale preparative separations (100 mg-gram) 21.2
Large-scale preparative separations (up to 100 mg-gram)
Consult the Column Hardware section for guard column configurations
30, 50
LC and LC/MS
565