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HPLC and UHPLC Part 1

The presentation discusses a set of findings from the 1990's that can affect the stability of RPLC columns at a high

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HPLC and UHPLC Part 1

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

  2. Introduction • 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 • Many experts have recommended that reversed-phase method development begin using low pH mobile bases

  3. Introduction • Many basic compounds • carry a full or partial positive charge at low pH, • will often be poorly retained, • have poor peak shape, or • may even be excluded (elute before void) on most modern type B silica based packings • It is an advantage to be able to use mobile phases with pHs at or above 7.0 for basic compounds

  4. Advantages of Working at Mid or High pH • Some analytes are insoluble or insufficiently soluble at low pH • Some analytes are unstable at low pH • Some analytes are not adequately retained or cannot be separated from closely eluting compounds at low pH • Improvement in response and signal-to-noise ratio is needed for analysis of basic analytes by LC-MS vs low pH

  5. Advantages of Working at Mid or High pH • Analytes are not adequately retained or cannot be separated from closely eluting compounds at low pH • Can observe improvement in LC-MS response and signal-to-noise ratio for basic analytes at mid- or high pH vs. low pH

  6. Column Stability • Degradation of silica-based columns at intermediate or high pH • largely a function of dissolution of the silica support • Rather than a loss of bonded phase due to hydrolysis (cause of column degradation at low pH)

  7. Column Stability • Rate of silica degradation due to dissolution is affected by: • 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.) • Column temperature • Silica support solubility in the pH 6-8 range is greatly increased in the presence of phosphate and carbonate buffers

  8. Column Stability • Silica-based columns are rapidly degraded when carbonate and phosphate buffers are used at pH 10 • Silica dissolution for untreated silica at pH 9-10 was reduced in high concentrations of organic modifier

  9. Column Stability • Precolumns of unmodified silica greatly extend the lifetime of columns used at high pH • Bonded-phase precolumns were much less effective

  10. Column Stability • Longer chain bonded phases are more stable than shorter chain phases • Certain silica-based, bonded-phase columns can be used routinely for long periods to at least pH 9-10 http://www.mac-mod.com

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