HPLC Column Selection

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HPLC Column Selection

May 01, 2013
By LCGC Editors
LCGC Europe
Volume 26, Issue 5
An excerpt from LCGC's e-learning tutorial on HPLC column selection at
CHROMacademy.com [1]
As chromatographers we face a bewildering number of choices when it comes to
selecting the correct high performance liquid chromatography (HPLC) column for an
analysis. True, we may be able to find a suggested stationary phase in the literature,
but are the column dimensions applicable, will that column geometry work well with
our system and allow us to attain our required detection levels? This instalment of
"The Essentials" establishes a number of "rules of thumb" that can be applied when
selecting stationary-phase type and column dimensions to somewhat ease the burden
that comes with such a proliferation of choice.
Column length is typically predicated by the resolution required from a "traditional"
HPLC system and in this sense we mean systems which have not been designed or
adapted to minimize extra column volume or cannot achieve system back pressures
above 400 bar. High resolution is typically required when separating samples with
many components or when many chemically similar compounds are being separated.
Beware that HPLC is a diffusion-limited technique and that above analyte retention
factor (k) values of around 10, increasing retention will have little or no effect on
resolution because of decreases in efficiency caused by an increase in longitudinal
molecular diffusion. So you may need to adjust the eluotropic strength of the eluent to
reduce overall retention time.
Column internal diameter is typically chosen depending on analytical requirements and
system limitations. It dictates the speed of analysis and influences method sensitivity.
Use 4.6-mm i.d. columns when working with traditional HPLC systems described
above, or 3-mm i.d. columns if efficiency loss because of extracolumn volume is not
too great. Use 2.1-mm i.d. columns when you are using low-extracolumn-volume
systems to save solvent, increase sensitivity (such as when working with a limited
sample) or to reduce flow rate while still working at a reasonable eluent linear velocity
(when working with electrospray ionization mass spectrometry [ESIMS] systems, for
example).
The particle size of the stationary phase support affects the efficiency of a separation
and one typically needs high efficiency when trying to separate a few components in a
short time or many components in a reasonable time. Smaller particle sizes bring
higher efficiency; however, this is usually at the expense of an increase in system back
pressure, unless flow rate is adjusted downward. The most widely used particles are 5
m in diameter and provide very reasonable efficiencies when used with traditional
HPLC systems. Other particles with diameters such as 3.0 m or 3.5 m provide
higher resolution and shorter analysis times, but beware of increases in system back
pressure and also note that as particle size reduces so does the retaining frit porosity
so greater care must be taken with sample and eluent preparation. Sub-2.0-m
particles are typically used with ultrahigh-pressure liquid chromatography (UHPLC)
systems, which are capable of dealing with high system back pressures (1000 bar and
greater) and can generate very high efficiencies for high resolution or very fast
separations. We now also have a choice in particle morphology with the availability of
coreshell particles, which give rise to high efficiencies (approaching those of
sub-2-m particles) with much lower system back pressures. The lower back pressure
provides the advantage of allowing their use on traditional HPLC systems.
Pore size describes the average pore diameter of the pores on the surface of the silica
packing material. Pores of 612 nm are used with analytes below ~2000 Da and 30
nm pores are used for analytes above 4000 Da. For the
20004000 Da range you should experiment to see which pore size gives the best
retention and efficiency. Wider-pore columns tend to provide a higher surface area and
are therefore more "loadable" that is, more analyte mass can be applied before
peak shape deterioration (asymmetry) becomes a problem.
Type II (B) silicas are less acidic than Type I (A) and therefore give rise to less peak
tailing when analysing polar or ionizable analytes (especially basic molecules).
Select hydrophobic stationary phases (C18, C8) when differences in analyte
hydrophobicity are large and can be exploited to affect a separation. Typically, this is
when the carbon to heteroatom ratio within the analyte is large. Hydrophobic stationary
phase ligands do not work well in 100% aqueous eluents and are not well suited for
the retention of more polar analytes.
Modified alkyl phases (those with more polar functional groups embedded within the
ligand) are used when separating analytes with different (polar) functional groups and
where exploiting differences in the bulk hydrophobic properties of analytes does not
produce an effective separation. Typically, these phases can be used with 100%
aqueous eluent systems and are better at retaining polar analytes.
Phenyl-containing phases interact strongly with analytes containing p electron systems
(aromatic, unsaturated) and will retain these analytes in preference to those with no p
electrons.
Cyano and fluorinated phases interact strongly with basic, nitrogen-containing and
halogenated species and should be used when the analytes contain these species.
Amino, diol and silica phases are traditionally used for the separation of polar analytes
in both reversed-phase and normal-phase modes. Lately they have also found utility in
hydrophilic interaction liquid chromatography (HILIC) mode in which highly organic

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HPLC Column Selection

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eluent systems are used to retain polar analytes using polar stationary phases.
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