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.

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

2. Introduction
O 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

3. Advantages
O 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

4. Advantages
O 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

5. What to Watch Out For
O Analytes that are unstable at higher pH
could degrade on column
O With LC-UV detection the UV spectra and
values of ionizable analytes often
change dramatically
max
O Can make peak tracking more challenging

6. What to Watch Out For
O Column lifetimes for all silica-based columns
will be shorter using buffers
O On-column reaction or decomposition can
occur for metal-sensitive analytes

7. Method Development
O Parameters that affect selectivity (relative
retention) include:
1.
2.
3.
4.
5.
6.
Column bonded phase
Organic modifier choice
Mobile phase pH
% organic modifier or gradient steepness
Column temperature
Buffer identity and concentration
O Newest addition to the ACE column family:
the ACE® SuperC18 phase

8. ACE SuperC18 Features and
Benefits
O Unique bonding chemistry provides
increased resistance to degradation
O Can be used at pHs ranging from 1.5—11
with all analytes types

9. ACE SuperC18 Features and
Benefits
O 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

10. ACE SuperC18 Features and
Benefits
O 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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