A Global Approach to HPLC Column Selection Using Reversed Phase and HILIC Modes: What to Try When C18 Doesn't Work

Mar 01, 2010
Volume 28, Issue 3, pg 234–244

Aqueous–organic mobile phases (for example, water–acetonitrile) have become preferred for high performance liquid chromatography (HPLC) analysis due to their wide availability, high quality, low toxicity, and compatibility with popular detection methods. Selecting the optimum column to work with aqueous mobile phases is an important part of HPLC method development. Given the vast number of choices and the lack of chemical detail about many column products, many analysts do not take time to explore column options in any systematic way. Most prefer to simply use familiar columns having traditional C18 or closely related phases. Published column selection guides and other resources are available; however, they tend to be biased toward specific products or involve mathematical treatments that can be too complex and time-consuming for the busy analyst.

Recommendations for column selection have appeared in the literature and virtually every LC textbook. Early column selection schemes were uncomplicated, because there were relatively few column choices. However, as the number of columns and separations has grown, it has become difficult to organize the information for use in developing new methods. Historically, HPLC applications have highlighted success stories for individual columns. If one has different compounds, success depends upon the similarity of the analyte to the compounds in the published application. If one could not acquire the same column or preferred to evaluate another brand, information about the relative behavior of different column phases toward the analyte would be needed, but is seldom available. Classification schemes are sometimes helpful but often are focused on the characteristics of the stationary phase (for example, polar, nonpolar, deactivated, and so forth) rather than the behavior of target analytes. For example, while nonpolar solutes are separated readily on reversed-phase columns, water-soluble analytes can benefit from consideration of a separation mode such as hydrophilic interaction liquid chromatography (HILIC), which also is referred to as aqueous normal phase (ANP). This article will employ the name HILIC/ANP for this mode, which is growing in popularity because it is complementary to the reversed-phase mode.

A global approach to predicting retention patterns has been developed around three highly orthogonal phases. C18, silica, and pentafluorophenylpropyl (PFP or F5) have very different surface polarity and are compatible with popular aqueous-organic mobile phases. C18 and silica columns were obvious selections as the boundary conditions for global screening, while PFP was selected as a good column example that exhibits strong retention in both reversed-phase and HILIC/ANP modes. Test compounds were selected to illustrate a wide range of chemical properties, including neutral, acidic, basic, and zwitterionic functional groups. When this column screening method is applied to any target mixture, results should indicate whether reversed-phase, HILIC/ANP, or both modes can be useful.

By systematically varying organic-water content, pH, and ionic strength, it becomes possible to draw useful conclusions about retention patterns on very different phases for a wide range of compound polarities. This more global approach allows one to determine where a particular stationary phase–analyte combination is likely to be successful, or, conversely, where a particular combination is unlikely to produce useful chromatographic results.


Reagents and Chemicals: Test probes were obtained from Sigma-Aldrich (St. Louis, Missouri) and were reagent grade or better. Acetonitrile for mobile phase use was HPLC grade (Acros Chemicals, Geel, Belgium). Water was produced by a local purification system (Culligan Water Systems). Mobile phase additives (ammonium formate and trifluoroacetic acid) were reagent grade and obtained from Sigma-Aldrich. Standards were dissolved in 50% buffer–acetonitrile at a concentration of approximately 20 mg/mL. This 50:50 blend is not always ideal for evaluating retention and peak shape under both reversed-phase (often >50% water) and HILIC/ANP (often >50% acetonitrile) mobile phases; however, one can usually avoid problems by injecting small sample volumes of 5 μL or less into standard-bore columns. During method optimization, samples should be dissolved when possible in solvents that are slightly weaker than the mobile phase for the mode selected.

Equipment: All HPLC experiments used an Agilent 1100 system (Agilent Technologies, Santa Clara, California), consisting of an on-line degasser, binary pump, autosampler, temperature-controlled column compartment, and diode- array detector with semimicro flow cell. Columns were maintained at 25 °C for all experiments. Injection volume was 5 μL. Instrument control was provided by ChemStation software (Agilent Technologies), version B.04.01.

Table I: Global screening columns
Columns: All columns were supplied by Supelco Division of Sigma-Aldrich, Inc. (Bellefonte, Pennsylvania). The stationary phases, dimensions, and operating conditions are summarized in Table I. To maintain consistent separation conditions, column flow was adjusted to produce constant, optimum linear velocity for the particle size and column internal diameter.

Procedure: Each column was equilibrated with the appropriate mobile phase for a minimum of 5 min. For the short columns used in this study, this represented a minimum of 10 column volumes. Suggestions for starting conditions during method development are discussed in the Recommendations section. The diode array collected spectra and monitored several wavelengths to allow detection and confirmation of identity for all analytes. For convenience, chromatograms are displayed at 230 nm, but this does not necessarily represent the optimum setting for each compound.

Because it can be difficult to select true void volume markers for columns and analytes under global conditions, void times were estimated from column dimensions (t M = 0.25 × e × π × d c 2 × L/F c), where e is the column porosity, d c is column diameter, L is length, and F c is flow rate). The value of e was set at 0.64 for this study, which represented a midrange value for the column types being studied. Thus, calculated retention factors k can be different from actual measured values for some columns. However, since only relative retention factors are being compared, these differences do not affect any study conclusions. Only retention factors between 1 and 10 are reported for comparison, as values outside this range are not chromatographically useful.

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