HPLC and UHPLC Separations at Mid to High pH: Part 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

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

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 oncolumn degradation

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

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

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

ACE SuperC18 Features and Benefits

O Unique bonding chemistry provides

increased resistance to degradation

O Can be used at pHs ranging from 1.511

with all analytes types

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

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

http://www.mac-mod.com