HPLC and UHPLC Part 1
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Transcript of HPLC and UHPLC Part 1
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
http://www.mac-mod.com