Ion pair chromatography is a reversed-phase liquid chromatography technique used to separate ionized and partly ionized organic compounds. It utilizes reversed-phase columns and mobile phases with an added ion pair reagent that interacts with the analytes to change their retention times. The technique has advantages over ion exchange chromatography like higher efficiency columns and ability to use water-rich mobile phases. It has wide applications in analysis of biomolecules, food, drugs and more.

1. ION - PAIR , REVERSED - PHASE
LIQUID CHROMATOGRAPHY
Name – URVI SHARMA
Class – M.Sc. MICROBIOLOGY (3rd sem )
Reg . No – 19MSRMB039
Subject – Microbiological Techniques & Research
Methodology

2. INTRODUCTION
• Ion pair chromatography (IPC) is an effective reversed-phase liquid
chromatographic (RPLC) technique for separation of
organic ions and partly ionized organic analytes.
• The purpose of adding an ion pair reagent to the mobile phase is
usually to change the retention time of ionic analytes.
• The technique utilizes the same types of stationary phases
and mobile phases as RPLC; the main characteristic for IPC is that
an ion pair reagent is added to the mobile phase.
• The ion pair reagent is usually an alkylsulfonate, an alkylsulfate or
an alkylammonium salt.
• The high efficiency of RPC columns compared with columns used in
ion exchange or ion chromatography also makes IPC a valuable
alternative to these techniques.
• IPC has been applied in almost all areas of analytical chemistry
where chromatography is used.

3. Cont…
• In IPC, water-rich mobile phases can be employed with a
variety of buffers and ionic and non-ionic additives and the
technique is therefore suitable for separation of important
classes of biomolecules and specifically amino acids,
peptides, proteins and nucleic acids.
• In the food industry, IPC is used for the analysis of water-
soluble vitamins, caffeine, theobromine, amines, etc.
• Since many drugs are basic or acidic, the driving force for
the development of IPC came from the pharmaceutical
industry where today it is used on a routine basis.
• Ion-pairing chromatography (IPC) can be used for both
positively and negatively charged analytes.
 Negatively charged reagent can be used to retain
positively charged ionic bases.
 Positively charged reagent can be used to retain
negatively charged ionic acids.

4. lon Pair Reagent
• Added to mobile phase at low concentration (usually
0.05M) and is itself ionized.
• lon pair reagent are molecules containing a hydrophobic
region and an ionizable functioality.
• •One ion of the reagent is retained by the stationary phase
providing the otherwise neutral stationary phase with its
charge.
• •This charged stationary phase can then retain and separate
organic solute ions of the opposite charge by forming a
reversible ion pair complex with the ionized sample.
 Example –
RCOO- + R4N+ ---→ [R4N+-OOCR] ion pair

5. MECHANISM
 There are two fundamental models are as follow –
1) The solute molecule forms an ion pair with counter ion in
the mobile phase. This uncharged ion pair partitions into the
lipophilic stationary phase.
2) The counterion partitions into the stationary phase or is
loaded onto the bonded reverse phase packing, with its ionic
group oriented at the surface generating two possibilities:
a) Analyte can be attracted to the hydrocarbon portion in the
usual reverse phase monomer, or
b) Analyte can interact in an ion exchange mode.

6. Cont…
• True mechanism involves both the postulates and is further
complicated by adsorption and micelle formation allowing the
unique separation.

7. ADVANTAGES
• IPC separation involves two additional variables
that can be used for control of selectivity (IPC-
reagent type and concentration) .
• Simple preparation of buffers.
• Wide choice of carbon chain lengths for
improved retention and separation.
• Significantly reduced separation time (vs. ion
exchange).
• Simultaneous separation of both ionized and non-
ionized molecules.
• High reproducibility of results.
• Improved peak shapes.
• Better separation of large ions (vs. ion exchange).

8. DISADVANTAGES
• Greater complexity of operation and more
challenging interpretation of results.
(e.g., more variables to choose from or to
control).
• Slow column equilibration after changing the
mobile phase.
Specially slow when IPC reagent is more
hydrophobic.
Possible that not all of the IPC reagent will be
washed from the column.

9. APPLICATION
1) Biological analysis
 Ion pair reverse phase chromatography: a versatile platform
for the analysis of RNA.
• Ability to separate large RNA molecules with higher
resolution.
• In addition to efficient isolation and analysis of RNA species,
IP, RP, HPLC provides valuable structural information.
2) Food Analysis
 Determination of water-soluble vitamins in infant milk by
High-Performance Liquid Chromatography.
• Separation of molecules with different chemical properties
such as stability, polarity, and acidity.
• Can control pH and concentration of mobile phase.
• Ability to separate multiple vitamins in a single run.
• Better resolution in comparison to ion exchange
chromatography.

10. APPLICATION
3) Pharmaceutical Analysis
 Separation and determination of impurities in paracetamol,
codeine and pitophenone in the presence of fenpiverinium
in combined suppository dosage form.
• Can determine impurities in dosage form.
• Sensitive- ability to measure low concentrations of analytes.
• Great accuracy- highly reproducible results.
• Manipulation in pH of buffer leads to better resolved peaks.
• Improved peak shape

11. CONCLUSION
• It is also known as ION INTERACTION or
DYNAMIC ION EXCHANGE
CHROMATOGRAPHY.
• Based on the greater complexity of Ion-pair
Chromatography compared to Reverse Phase
Chromatography it is recommended that IPC
should be considered only after RPC techniques
are not effective.
• Ion-pairing liquid chromatography is a
promising means of increasing analyte
retention.

12. REFERENCES
 J.Dolan. LCGC Europe, Volume 21, Issue 5, pp 258-263.
 L. Snyder, J. Kirkland, and J. Dolan. (2010). Introduction to
Modern Liquid Chromatography 3rd Edition. Wiley & sons,
Inc., Publications. (pages. 331-347).
 Vailaya, C. Horvath, J Chromatogr. A 829 (1998) 1–27.
Dickman, M.J.Chromatography Today, 2011, 22-26.
Erich, S.; Anzmann, T.; Fischer, L. J. Food chemistry, 2012,
135, 2393-2396.
Vojta, J.; Hanzlíka, P.; Jedličkaa, A.; Coufal, P. J.
Pharmaceutical and Biomedical Analysis, 2014, 102, 85-62.
https://www.researchgate.net/publication/215520397_Ion-
pair_reversed-phase_high-
performance_liquid_chromatography_of_ondansetron_hydroc
hloride_using_sodium_heptanesulphonate_as_a_counterion
https://www.sciencedirect.com/topics/chemistry/ion-pair-
chromatography