Seven Common Faux Pas in Modern HPLC

Jul 31, 2014

The seven faux pas discussed here are common practices in high performance liquid chromatography (HPLC) that may no longer make sense because of improved technologies or other changes in modern HPLC. Therefore, careful consideration should be given to alternative approaches.

The expression faux pas is French in origin and literally means "false step", or "misstep". Although in English the phrase typically refers to an embarrassing social blunder rather than to a technical error, I am borrowing this expression, and stretching its meaning a bit, as a way to highlight some common practices in high performance liquid chromatography (HPLC) — legacy practices that have been in use for many years — that may no longer make sense because of improved technologies or other reasons in modern HPLC. In this instalment, I highlight seven such questionable practices and show why they should not be followed without first considering some alternative approaches:

  • Using columns packed with 5-µm particles
  • Using 4.6-mm i.d. columns at 1 mL/min
  • Filtering HPLC mobile phases
  • Using buffered mobile phases
  • Preparing fresh reference standard solutions with every assay
  • Shaking HPLC sample vials
  • Using stainless steel ferrules for column connections

As each topic is discussed, I will offer rationales and references to support why the practice is being called into question — and in many cases, I will offer exceptions to the rule.

Using HPLC Columns Packed with 5-µm Particles

For many years, the "standard" HPLC analytical column has been a 250 mm × 4.6 mm column packed with 5-µm particles that could deliver ~20,000 plates (1,2). In a survey on column trends published in 2012, Ron Majors reported that the "standard" workhorse column was likely to be shifting towards a 150 mm × 4.6 mm column packed with 3-µm particles, which offers comparable efficiencies with a 40% or more reduction in analysis time (3). This trend is not surprising for experienced practitioners because plate count (N) is proportional to the ratio of column length (L) to particle size (d p), expressed mathematically as NL/d p. The 3-µm particle has proven to be as reliable as its 5-µm counterpart and offers higher performance (lower plate height [H]) while maintaining compatibility with conventional HPLC equipment (1,2). As ultrahigh-pressure liquid chromatography (UHPLC) and liquid chromatography–mass spectrometry (LC–MS) systems are becoming standard equipment in many laboratories, the "standard" column particle sizes are expected to trend downwards to sub-3-µm or sub-2-µm particles (4). Another emerging trend is the increased acceptance of superficially porous packings, which have improved particle morphology and substantially higher performance (~20–30%) compared with totally porous materials (5).

Exceptions: Although columns packed with 3-µm particles are generally superior to those packed with 5-µm materials, they do use finer inlet frits (0.5 µm versus 2 µm), which are more prone to clogging from "dirtier" samples. Another exception to this rule may come from specialized columns (for example, size-exclusion chromatography columns for protein analysis) where higher pressure and higher temperature from frictional heating may lead to increased on-column protein aggregation (6).