HPLC and UHPLC Separations at Mid to

High pH: Part 1

IntroductionO Reviewed findings published by

Kirkland, Claessens and co-workers in the 1990s regarding the factors that can affect the stability, performance, and lifetime of silica-based RPLC columns at mid and high pH

O Many experts have recommended that reversed-phase method development begin using low pH mobile bases

IntroductionO 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-to-noise 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 StabilityO Degradation of silica-based columns at

intermediate or high pHO largely a function of dissolution of the

silica supportO Rather than a loss of bonded phase due

to hydrolysis (cause of column degradation at low pH)

Column StabilityO Rate of silica degradation due to dissolution

is affected by:O Choice of mobile phase organic modifierO Type of silica supportO Type and bonding density of bonded phaseO 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 StabilityO 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 StabilityO Precolumns of unmodified silica

greatly extend the lifetime of columns used at high pH

O Bonded-phase precolumns were much less effective

Column StabilityO 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

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