No thanks! I would like to know more about CHROMacademy

 Over 3000 E-Learning topics / 5000 Articles & Applications
Ion Pair Alternatives


The use of pH to suppress ionization, and therefore gain retention, for ionizable analytes in reversed phase HPLC is applicable to weakly acidic or basic compounds only.  If a separation of mixtures of weak acids and bases or analytes that are amphoteric is required, then ion suppression is of limited use.  When both acidic and basic functional groups are present, as the ionization of one functional group is suppressed, that of the other may be promoted, and a compromise is often very difficult to reach which results in non-robust methodology (Figure 1).

Similarly, if stronger acids or bases are to be analyzed, then the mobile phase pH at which the functional group is suppressed may lie outside the working range of traditional silica columns, precluding the use of ion suppression HPLC.

The analysis of mixtures of weak acids and bases or amphoteric analytes is possible using ion pair chromatography.  In this mode, a reagent is added to the mobile phase that contains both an ionic functional group and a hydrophobic portion, such as a hydrocarbon chain.  The hydrophobic section of the ion pairing reagent interacts with the stationary phase whilst the charged functional group undergoes an ion exchange or ion pairing interaction with the analyte (Figure 2).

The most common ion-pairing reagents are sulfonic acid derivatives such as hexane-, heptane-, octane-sulfonic acids, quaternary ammonium salts such as tetramethyl- or tetrabutylammonium hydroxide, and volatile reagents such as trifluoroacetic acid and triethylamine.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 1: Effect of pH on analyte ionization state.

Content on this page requires a newer version of Adobe Flash Player.

Get Adobe Flash player

Figure 2: Ion pair reagents and mechanisms.


  • Allows separation of analytes that cannot be separated by any other mode (let’s be fair this is the ultimate goal of any method)
  • Samples with a wide range of polar, non-polar, and ionizable compounds that would not otherwise be characterized under the same conditions can be analyzed
  • Ability to analyze ionized compounds which lack a chromophore for UV detection (some ion-pair conjugates absorb UV where the analyte itself would not)
  • Method development familiarity and abundance of literature to reference and extrapolate


  • Additional level of complexity in method development
  • Column contamination - ion pair reagents are difficult to remove from the column and it may be best to dedicate a column to a specific method
  • Increased problems with column blockages particularly with small sized particles due to salting out of ion pair reagents
  • Difficult coupling of methods with MS - ion pairing reagents can ionize creating high MS backgrounds, and strong ion pairing with an analyte can prevent ionization of the analyte.  This can be remedied by the use of LC-MS compatible ion pair reagents, such as, heptafluorobutanoic acid (HFBA) which is volatile
  • More complex to implement with gradient elution
  • Slow column equilibration (10 column volumes would be sufficient for reversed phase columns, whereas, with ion pair reagents 50+ column volumes may be required)
  • Many ion pair reagents have substantial UV absorbance which precludes use with low UV detection
  • Additional cost of ion pair reagents


There are alternatives that might be worth considering before developing an ion pair method. 

If the analyte in question is basic and requires high pH for ion suppression, consider using a high pH rated column.  Many manufacturers have columns which are rated to pH 11 or 12.  These pH stable stationary phases allow for improved retention and peak shape of basic analytes by running at basic mobile phase pH controlled with buffers instead of utilizing ion pair reagents.

Hydrophilic interaction liquid chromatography (HILIC) can be used to improve the retention of polar analytes under mass spectrometric compatible conditions.  HILIC is based on a mixed mode retention mechanism and can be considered as a type of normal phase chromatography, employing a polar stationary phase (for example, silica or a polar bonded phase) and an aqueous-organic mobile phase in which the aqueous content is the strong solvent.  Typically initial eluent composition will be 98:2 organic:water.  HILIC can be used in certain situations where reversed phase chromatography fails or is not efficient:

  • Samples with limited solubility in water or highly aqueous mobile phases
  • Samples that contain very polar analytes which are not retained adequately in revered phase
  • Hydrophilic water-soluble analytes, which are intractable to reversed phase and/or ion exchange chromatography

HILIC presents the added advantage of using acetonitrile, which has low UV absorbance (for better detection sensitivity) and low viscosity (for high chromatographic efficiency).  The highly organic mobile phase also makes HILIC amenable to hyphenation with mass spectrometric detection (MS).
Mixed mode chromatography is a method that uses more than one separation mode; mainly reversed phase combined with ion exchange interactions, which allows the retention and separation of both polar and nonpolar analytes in a single analysis.  The biggest benefit of this approach is that selectivity can be optimized by adjusting mobile phase ionic strength, pH, or organic solvent. As a result, the selectivity can be finely tuned for the separation of compounds with widely different physicochemical properties. For example, drug molecules and their counter ions may be separated in a single analysis.  Mixed mode chromatography requires no ion pair reagents in the mobile phase for separating highly hydrophilic charged analytes, which simplifies the mobile phase and is compatible with mass spectrometry (MS).1

Columns that contain a polar embedded group, sometimes called "Aq" columns, allow the use of 100% aqueous mobile phases under reversed phase conditions. These conditions will sometimes provide enough retention of polar basic molecules which would be retained poorly at 5% organic, the lower limit for most reversed phase columns. Basic compounds may still not exhibit enough retention, even in 100% aqueous mobile phases or at any pH within the column's pH-stable range. Sometimes these columns under 100% aqueous conditions (with buffer or pH adjustment) will give sufficient retention for polar compounds without the need for ion pairing.2

Porous graphitic carbon (PGC) phases are composed of flat sheets of hexagonally arranged carbon atoms.  There are no surface silanol groups and these phases offer total pH stability (0-14).  They can be applied to the separation of a wide variety of molecules, and are particularly useful for the separation of highly polar species which show poor retention under reversed phase conditions.  PGC phases can be used under reversed and normal phase modes as well as for LC-MS applications. Contrary to traditional reversed phase stationary phases, PGC exhibits increased retention of polar analytes (with retention increasing as polarity increases).  This effect has been termed the polar retention effect on graphite (PREG), and it is this retention mechanism which makes PGC particularly applicable to the separation of highly polar compounds, such as, carbohydrates and compounds which contain several hydroxyl, carboxyl, and amino groups.3,4

Trifluoroacetic (TFA) acid can be used as an alternative to traditional sulfonate ion pair reagents.  TFA is already widely used in many different supplication areas, including for ion pairing with biological molecules.  0.1% TFA is added to both mobile phase A and B allowing for isocratic and gradient methods to be developed as normal.  Gradient equilibration times are fast and TFA can be utilized for low wavelength applications.  It should be noted that TFA has been successfully and extensively utilized with LC-MS applications; however, TFA can cause ion suppression, but if a little bit of sensitivity can be sacrificed then this is a viable option.

If an ion pairing method is inevitable follow a few precautions:

  • Allow sufficient time for the mobile phase and column to equilibrate
  • Avoid changes in temperature - this could disrupt the analyte/stationary phase interactions
  • Avoid changes in mobile phase organic - this could disrupt the analyte/stationary phase interactions
  • Peak shape can be improved by addition of the ion pair reagent o the sample diluent
  • Have the ion pair in both mobile phase A and B if implementing a gradient to ensure consistent concentrations


  1. Taylor, T. LCGC North America, 2014, 32, 226
  2. Dolan, J. W. LCGC North America 2008, 26, 170-174
  4. Knox, J.; Ross, P. Adv. Chromatogr. 1997, 37, 73-119


You may also like...

Quick Guides

The Basics of HILIC »

Why Is pH Important for HPLC Buffers »

Buffer Choice for HPLC Separations »

How to Choose an HPLC Column »

Webcasts & Tutorials

HILIC Method Development - Making HILIC Work for You »

Mixed Mode Chromatography - The Answer to Everything? »

Developing Better HPLC(MS) Methods »

Critical Evaluation of HPLC Methods »


Column Chemistry »

Ion Pair Chromatography »


loading data
loading data
loading data
loading data
loading data

Dr. Dawn Watson

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

group  subsCHROMacademy can deliver to corporate clients on a multi-user subscription basis.
Served up from secure servers to the corporate intranet or individual desktops.

  • Microsite - your own learning site powered by CHROMacademy
  • Your Landing Pages -with your logo and branding
  • Customized Assessments - Based on content agreed upon Certificate of Completion
  • Certification Programs - Offer your learners a goal to strive towards
  • Our Learning Management System is S.C.O.R.M. compliant and will connect to your system
  • Engagement Package - E-newsletter stimulation program derived from your content and ours
  • Full archive of Essential Guide webcasts & tutorials
  • 1000’s of eLearning topics - HPLC / GC / Sample Prep / Mass Spec
  • Ask the Expert - our experts will answer your chromatography questions within 24 hrs.
  • Assessments - test your knowledge
  • Application notes & LCGC articles
  • Troubleshooting and virtual lab tools

Request a quote


 Home | About UsContact Us | SubscribeTerms and Conditions | Advertise | Privacy Policy 

  • loading data

loading data

loading data


loading data

loading data