hplc and uhplc part 2
DESCRIPTION
This presentation discusses the use of higher pH for method development & analysis. There are several advantages to using this method that can produce improved results. This can allow compounds to be separated which can not be done at a lower pH.TRANSCRIPT
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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