HPLC and UHPLC Separations at Mid to

High pH: Part 2

IntroductionO LabNote continues the discussion of

the use of higher pH for method development and analysis

O There are many advantages to working at higher pH, but also some items to be aware of

AdvantagesO Orthogonal selectivity (different

elution order) can be obtained for many analytes

O Basic analytes are retained longer and have better loading capacity, linearity, and peak shape

AdvantagesO Signal and signal-to-noise ratios are

usually improved for basic analytes

O Chromatographic separations at higher pH allow compounds that are unstable at lower pH to be analyzed without on-column degradation

What to Watch Out ForO Analytes that are unstable at higher

pH could degrade on column

O With LC-UV detection the UV spectra and lmax  values of ionizable analytes often change dramatically

O Can make peak tracking more challenging

What to Watch Out ForO Column lifetimes for all silica-based

columns will be shorter using buffers

O On-column reaction or decomposition can occur for metal-sensitive analytes

Method DevelopmentO Parameters that affect selectivity

(relative retention) include:1. Column bonded phase2. Organic modifier choice3. Mobile phase pH4. % organic modifier or gradient steepness5. Column temperature6. Buffer identity and concentration

O Newest addition to the ACE column family: the ACE® SuperC18 phase

ACE SuperC18 Features and BenefitsO Unique bonding chemistry provides

increased resistance to degradation

O Can be used at pHs ranging from 1.5—11 with all analytes types

ACE SuperC18 Features and BenefitsO Designed for use with LC-MS

compatible mobile phase buffers

O Does not show hysteresis effects (memory effects) when changing between low and high pH mobile phases

ACE SuperC18 Features and BenefitsO The ACE SuperC18 column produces

highly symmetrical peaks for bases, acids, and neutral analytes

O Baselines were observed to be better when NH4OH or NH4OAc were premixed with the organic modifier in the B solvent

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