HPLC and UHPLC Part 2

1
HPLC and UHPLC Separations at Mid to High pH
Part 2
2
Introduction

• LabNote continues the discussion of the use of
  higher pH for method development and analysis
• There are many advantages to working at higher
  pH, but also some items to be aware of

3
Advantages

• Orthogonal selectivity (different elution order)
  can be obtained for many analytes
• Basic analytes are retained longer and have
  better loading capacity, linearity, and peak shape

4
Advantages

• Signal and signal-to-noise ratios are usually
  improved for basic analytes
• Chromatographic separations at higher pH allow
  compounds that are unstable at lower pH to be
  analyzed without on-column degradation

5
What to Watch Out For

• Analytes that are unstable at higher pH could
  degrade on column
• With LC-UV detection the UV spectra and lmax 
  values of ionizable analytes often change
  dramatically
• Can make peak tracking more challenging

6
What to Watch Out For

• Column lifetimes for all silica-based columns
  will be shorter using buffers
• On-column reaction or decomposition can occur for
  metal-sensitive analytes

7
Method Development

• Parameters that affect selectivity (relative
  retention) include
• Column bonded phase
• Organic modifier choice
• Mobile phase pH
• organic modifier or gradient steepness
• Column temperature
• Buffer identity and concentration
• Newest addition to the ACE column family the
  ACE SuperC18 phase

8
ACE SuperC18 Features and Benefits

• Unique bonding chemistry provides increased
  resistance to degradation
• Can be used at pHs ranging from 1.511 with all
  analytes types

9
ACE SuperC18 Features and Benefits

• Designed for use with LC-MS compatible mobile
  phase buffers
• Does not show hysteresis effects (memory effects)
  when changing between low and high pH mobile
  phases

10
ACE SuperC18 Features and Benefits

• The ACE SuperC18 column produces highly
  symmetrical peaks for bases, acids, and neutral
  analytes
• Baselines were observed to be better when NH4OH
  or NH4OAc were premixed with the organic modifier
  in the B solvent

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