Introduction
Tanaka plots are among the most widely recognized tools for characterizing and comparing reversed phase HPLC columns. They provide a standardized approach for evaluating stationary phase properties such as hydrophobicity, steric selectivity, and secondary interactions.
However, chromatographers occasionally attempt to use Tanaka plot data when evaluating columns for HILIC applications. This can lead to incorrect assumptions because the retention mechanisms that govern HILIC separations are fundamentally different from those measured by the Tanaka test protocol.
When developing HILIC methods on Cogent™ TYPE-C™ columns, method developers should evaluate columns using HILIC-relevant conditions rather than relying on reversed phase characterization metrics.
What Is a Tanaka Plot?
The Tanaka test protocol was developed as a standardized method for comparing reversed phase column characteristics.
The test utilizes a series of carefully selected probe compounds under prescribed reversed phase chromatographic conditions to measure characteristics such as:
- Hydrophobicity
- Shape selectivity
- Steric recognition
- Hydrogen-bonding interactions
- Residual silanol activity
The resulting data are often displayed graphically as a Tanaka plot, allowing direct comparison of reversed phase stationary phases.
Because these tests are performed under reversed phase conditions, the information generated is highly useful for evaluating reversed phase chromatography.
Why Tanaka Plots Work Well for Reversed Phase Columns
In reversed phase chromatography, retention is dominated primarily by:
- Hydrophobic interactions
- Stationary phase surface characteristics
- Solute shape recognition
- Secondary polar interactions
The Tanaka protocol was specifically designed to evaluate these properties.
As a result, Tanaka plots can be effective for:
- Comparing C18 columns
- Evaluating alternative reversed phase chemistries
- Assessing method-transfer suitability
- Understanding reversed phase selectivity differences
Why Tanaka Plots Do Not Translate to HILIC
HILIC separations operate in a chromatographic environment that differs greatly from reversed phase chromatography.
Important factors affecting HILIC retention often include:
- Analyte polarity
- Ionization state
- Mobile phase water content
- Buffer concentration
- Surface charge effects
- Adsorption interactions
- Organic solvent composition
These variables are not directly evaluated by the Tanaka test protocol.
Consequently, a column that appears similar to another column on a Tanaka plot may behave very differently under HILIC conditions.
Different Retention Mechanisms Require Different Evaluation Methods
The central limitation of Tanaka plots in HILIC is that they measure retention characteristics associated with reversed phase chromatography rather than HILIC retention processes.
Because the dominant retention forces differ, reversed phase characterization cannot reliably predict:
- HILIC retention strength
- HILIC selectivity
- Elution order
- Retention-time control
- Method robustness
For this reason, columns intended for HILIC applications should be evaluated using HILIC methods rather than reversed phase benchmarking tools.
Evaluating Columns for HILIC Applications
A more effective strategy is to evaluate columns under actual HILIC operating conditions.
Use HILIC Screening Gradients
Compare columns using:
- High-organic starting conditions
- Appropriate volatile additives
- Representative analytes
Monitor:
- Retention
- Selectivity
- Resolution
- Peak shape
Use Application-Relevant Probe Compounds
Rather than relying on reversed phase probe molecules, evaluate compounds representative of the intended application.
Examples include:
- Amino acids
- Organic acids
- Metabolites
- Polar pharmaceuticals
- Sugars
- Zwitterionic compounds
This approach produces information that is directly useful for method development.
Evaluate Operational Performance
For HILIC methods, practical metrics often provide the most useful information.
Examples include:
- Retention reproducibility
- Equilibration speed
- LC-MS compatibility
- Signal-to-noise performance
- Carryover
- Long-term method stability
These factors frequently have a greater impact on method success than traditional reversed phase characterization data.
Application-Driven Column Selection
When evaluating HILIC columns, the most useful comparisons are typically those based on actual applications.
Questions to consider include:
- Which column provides the best retention of target analytes?
- Which column produces the most reproducible retention times?
- Which column achieves the required resolution?
- Which column performs best in LC-MS workflows?
Application-specific testing generally provides far more actionable information than reversed phase characterization metrics.
HILIC Method Development Using TYPE-C™ Columns
Cogent™ TYPE-C™ columns are frequently selected for HILIC applications involving:
- Polar metabolites
- Organic acids
- Amines
- Pharmaceutical compounds
- Biological samples
- LC-MS workflows
For these applications, direct HILIC evaluations using relevant analytes and mobile phases provide the most meaningful information regarding column performance.
Key Takeaways
- Tanaka plots were developed exclusively for reversed phase chromatography.
- The tests measure reversed phase selectivity characteristics and not HILIC retention mechanisms.
- HILIC retention depends on factors that are largely outside the scope of the Tanaka protocol.
- Tanaka plot data should not be used to predict HILIC performance.
- HILIC columns should be evaluated using HILIC screening methods and application-relevant analytes.
- Practical metrics such as retention, selectivity, reproducibility, equilibration, and LC-MS performance are more useful than reversed phase benchmarking data for HILIC method development.
