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A Guide to HPLC and LC-MS Buffer Selection

HPLC ColumnsJohn DolanA Guide to HPLC and LC-MS Buffer SelectionYour decision has lasting Inert Base-Deactivated HPLC ColumnsFor Performance, Selectivityand Guaranteed ReproducibilityACEperformanceguaranteeIf ACE does not outperform your existing column (of equivalent phase,particle size and dimensions), send in your comparative data within 60days and keep the ACE column FREE OF CHARGE. HPLC of ContentsPageWhy Control pH?2 Practical Implications4 Controlling pH4 What about LC-MS ?6 Just Interested in Low pH?7Or High pH?7 How Much?7 Solubility Issues10 Dilution Effects11 Buffer Preparation12 Precautions13 Summary15 References16 Using Buffers with HPLC and Buffers with HPLC and LC-MSWhen samples contain ionizable compounds, the mobile phase pH can be one ofthe most important variables in the control of retention in a reversed-phase HPLC (RP-HPLC) separation. However, if it is not controlled properly, pH can be a source of many problems.

silica-based columns. pKa (25°C) compound 0.3 trifluoroacetic acid2 2.15 phosphoric acid (pK1) 3.13 citric acid (pK1) 3.75 formic acid 4.76 acetic acid 4.76 citric acid (pK2) 4.86 propionic acid 6.35 carbonic acid (pK1) 6.40 citric acid (pK3) 7.20 phosphoric acid (pK2) 8.06 tris 9.23 boric acid 9.25 ammonia 9.78 glycine (pK2) 10.33 carbonic ...

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