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How to choose an HPLC Column

Tony Edge (Thermo Scientific) shares some great general hints and tips for HPLC column selection.

Read the literature
It is very rare that a separation has never been performed before and so checking the literature, refereed journals, trade magazines and even dare I say it the manufacturer's webs sites is an excellent use of resources

Run a generic method
So you have read the literature and still have not found a separation. At this point there is an option to go to the method development drawer and bring out a trusted column. There is a possibility that the column will work based on the fact that the molecules which are being separated are probably similar to previous molecules. It may require a small tweak of the gradient but it is an approach that is employed by many laboratories.

Developing a method from scratch
To develop a method from scratch it is important to understand the physiochemical properties of the molecule and also how the column works.

Understanding the chemistry
A key parameter, that is often quoted for a particular molecule is log P, or the log partition coefficient. Log P describes how hydrophobic a compound is, with positive numbers indicating that the molecule is hydrophobic, whereas negative numbers indicate that the compound is hydrophilic. The definition of log P applies only to the neutral forms of a molecule, however many compounds have acidic (proton donator) or basic (proton acceptor) functionality and so log D, log distribution, is a better measure of the hydrophobicity.

Equation 1
Equation 1

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As well as understanding the hydrophobicity of a molecule, there are also a variety of
different modes in which a molecule can interact with the HPLC stationary phase, including:




  • Dispersive – these interactions will exist between all organic molecules to a
    certain extent and is the major retention mechanism for alkyl phases.
    In general the degree of retention on a C18 phase will be dependent on the
    hydrophobicity, log P, of the molecule.

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  • Charge transfer – sometimes referred to as π-π or dipole-dipole interactions,
    this mode of interaction is primarily seen with aromatic compounds or with
    compounds that are unsaturated. These modes of interactions are enhanced
    with methanol and are clearly dominant with aromatic phases (Phenyl, PFP, etc.).

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  • Hydrogen bonding and dipole-dipole interactions – the analyte acts as a proton
    donor with proton accepting groups on the stationary phase surface.

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  • Ion exchange – the analyte can lose or gain one or more protons resulting
    in a charged species which can interact with the oppositely charge species
    on the surface of the stationary phase.

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An understanding of the shape, polarity, and the hydrophobicity of the molecule will help substantially in determining which mode of chromatography to employ, whether that is HILIC, normal phase, reversed phase, supercritical fluid etc.

Choosing the column chemistry from first principles
Even if the mode of chromatography has been determined to be hydrophobic, there are still many columns to select from. The differences in retention and selectivity relate to the silica substrate and also the bonding moiety. The different types of silica surface sites are listed in Table 1 and the amount of each surface site in a column will be determined by the manufacturing process. It is important to stress at this point that the separation will depend on the column-compound combination, and so a column that is good for one separation may not be so good for another. Thus it would be wrong to say that a column is bad, merely that it does not work for a particular selection of compounds.

Table 1: List of different forms of silica present at the surface ordered
in terms of their activity towards basic compounds »

Surface Site Activity level
Internal metal activated Most
Free or isolated  
Associated / vicinal  
Siloxane Least
silica surface silanol groups



In terms of fingerprinting how a column works, there has been a substantial amount of academic work done in this area.1-3 The most notable is by Tanaka1 who came up with a series of test probes that characterized the following modes of interaction (Table 2- below).

« Figure 1: Types of silica surface silanol groups.

Test # Parameters Property of stationary phase Factors in preparation of
the stationary phase
1 k (Pentylbenzene) in 80% MeOH Retention capacity (A) Amount of alkyl chains Silica surface
Silica coverage
k (Pentylbenzene) /
k (Butylbenzene)
in 80% MeOH
Hydrophobicity (B) Hydrophobic capacity
(CH2-group selectivity)
Surface coverage
k (Triphenylene) /
k (o-Terphenyl) in 80% MeOH
Steric selectivity (C) Steric (shape) selectivity Silane functionality
Surface coverage
2 k (Caffeine) / k (Phenol) in 30% MeOH Silanol capacity (D) Silanol capacity (content and type of silanols) Residual silanols
Surface coverage
3 k (Benzylamine) / k (Phenol)
30% MeOH /70% Phosphate buffer pH 7.6
Ion exchange capacity (E) Ion exchange capacity at
pH 7
Residual silanols
Active sites at pH 7
4 k (Benzylamine) / k (Phenol)
30% MeOH / 70% Phosphate buffer pH 2.7
Ion exchange capacity (F) Ion exchange capacity at
pH 3
Active sites at pH 3
Level of metal impurities

Table 2: A list of the tests used to characterize a column.

The US Pharmacopeia has taken a similar approach to the classification of HPLC reversed phase columns, and has placed this information on their web site 4. This tabulated information is an ideal way to compare and contrast a variety of different columns, but more importantly they allow a mechanism for determining what interactions exist between a stationary phase and a compound.

Effect of physical parameters
There are also a range of physical parameters that need to be considered, including column length, column diameter, pore diameter, and particle size.
The column length will increase the separation performance but it should be noted that there is a diminishing return on the resolution, Rs, as the length is increased; there will also be an increase in the pressure drop across the column in proportion to the length.

Eq 2 Equation 2

As the column diameter is altered, so the flow rate and also the injection volume should be scaled to ensure that over pressurization of the column does not occur or that the column is not overloaded. The scaling relationship is based on the ratio of the square of the column diameters.

Eq 3 Equation 3

The pore size will determine the surface area which in turn will give an indication of the retentiveness of the stationary phase, with smaller pores giving a larger surface area and as a result having greater retentivity. If the pore size is reduced too much then the molecule will not be able to fit into the pore and this will effectively reduce the surface area.


  1. N. Tanaka, "Chromatographic Characterization of Silica C18 Packing Materials. Correlation between a Preparation Method and Retention Behavior of Stationary Phase," vol. 27, no. 12, pp. 721-728, 1989.
  2. M. Euerby and P. Petersson, "Chromatographic classification and comparison of commercially available reversed-phase liquid chromatographic columns using principal component analysis," J. Chromatogr. A., vol. 994, pp. 13-36, 2003.
  3. L. R. Snyder, J. W. Dolan and P. W. Carr, "The Hydrophobic-subtraction Model of Reversed-phase Column Selectivity," J. Chromatogr. A., vol. 1060, pp. 77-116, 2004.
  4. USP. [Online]. Available:

Tony Edge is currently the R&D Principal for the chromatography consumables division within Thermo Scientific. 
Tony has over 15 publications in refereed journals including a book chapter on turbulent flow chromatography.  In 2008 he was fortunate enough to be awarded the Desty memorial lecture for his contributions to innovating separation science, and in the same year also won a clinical excellence award from AstraZeneca.  Tony's current interests are centered around improving the extraction process and high temperature chromatography.


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