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HPLC and UHPLC Separations at Mid to High pH: Part 1

Introduction O Reviewed findings published by Kirkland,

Claessens and co-workers in the 1990s regarding the factors that can affect the stability, performance, and lifetime of silicabased RPLC columns at mid and high pH O Many experts have recommended that

reversed-phase method development begin using low pH mobile bases

Introduction O Many basic compounds O carry a full or partial positive charge at low pH, O will often be poorly retained, O have poor peak shape, or O may even be excluded (elute before void) on

most modern type B silica based packings O It is an advantage to be able to use mobile

phases with pHs at or above 7.0 for basic compounds

Advantages of Working at Mid or High pH O Some analytes are insoluble or insufficiently

soluble at low pH O Some analytes are unstable at low pH O Some analytes are not adequately retained or cannot be separated from closely eluting compounds at low pH O Improvement in response and signal-tonoise ratio is needed for analysis of basic analytes by LC-MS vs low pH

Advantages of Working at Mid or High pH O Analytes are not adequately retained or

cannot be separated from closely eluting compounds at low pH O Can observe improvement in LC-MS

response and signal-to-noise ratio for basic analytes at mid- or high pH vs. low pH

Column Stability O Degradation of silica-based columns at

intermediate or high pH O largely a function of dissolution of the silica

support O Rather than a loss of bonded phase due to hydrolysis (cause of column degradation at low pH)

Column Stability O Rate of silica degradation due to dissolution is

affected by: O O O O

Choice of mobile phase organic modifier Type of silica support Type and bonding density of bonded phase Type and concentration of buffer salts in mobile phase (NH4+, Na+, K+; PO4-3, CO3-2, acetate, formate, etc.) O Column temperature O Silica support solubility in the pH 6-8 range is

greatly increased in the presence of phosphate and carbonate buffers

Column Stability O Silica-based columns are rapidly degraded

when carbonate and phosphate buffers are used at pH 10 O Silica dissolution for untreated silica at pH

9-10 was reduced in high concentrations of organic modifier

Column Stability O Precolumns of unmodified silica greatly

extend the lifetime of columns used at high pH O Bonded-phase precolumns were much less


Column Stability O Longer chain bonded phases are more

stable than shorter chain phases O Certain silica-based, bonded-phase columns

can be used routinely for long periods to at least pH 9-10

HPLC and UHPLC Part 1