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 installment, 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 toward 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 N ∝ L/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 downward 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).
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).