1) Ion pair chromatography is a type of column chromatography that uses ion pairing agents to neutralize charged analytes and allow their separation on a reversed-phase column. 2) By adding counter ions with the opposite charge to the mobile phase, ion pairs form between the counter ions and analytes, neutralizing their charge and increasing their hydrophobicity. 3) The use of ion-pairing reagents as mobile phase additives allows the separation of ionic and highly polar substances that cannot otherwise be separated by reversed-phase chromatography.

1. ION PAIR CHROMATOGRAPHY
1st semister – seminar
M-Pharm (Pharmaceutics Dept)
Under the guidance of
Dr. (Mr.) M.T.MOHITE
(Dept of Pharmaceutics)

2. ION-PAIR CHROMATOGRAPHY
Definition :- A type of column chromatography
in which ions in solution can be 'paired' or
neutralised and separated as an ion pair on a
reversed-phase column. Ion-pairing agents
are usually ionic compounds that contain a
hydrocarbon chain

3. ION-PAIR CHROMATOGRAPHY
1) In reversed-phase chromatography, ionic compounds generally
form hydrates around charges and are not retained very much by
hydrophobic stationary phases.
2) By adding pair ions with the opposite charge to the mobile
phase, the pair ions form ion pairs with the ionic compounds,
which neutralizes the charge, increases hydrophobicity, and
increases retention.
3) If pair ions with a hydrophobic functional group
(heptafluorobutyric acid in the example shown below) are added
to the mobile phase, the hydrophobic functional groups are
retained by the stationary phase, as though a dynamic ion
exchange groups had formed on the surface of the stationary
phase.

4. Illustration of Silica Support with C18 Bonded Phase
With the addition of an ION PAIR REAGENT
Na+ Cl- + Analyte
Na+
O
O S
O
 The use of ion-pairing reagents as mobile phase
additives allows the separation of ionic and highly polar
substance
In addition to the aqueous buffer and an organic solvent
that is typical for reversed-phase, the mobile phase
contains a counter ion that combines with the analyte
ions to form an ion pair. This forms a neutral species that
can be separated in the normal way by the reversed
phase packing

5. QUATERNARY AMINE (Q-SERIES) ION PAIR
REAGENT INTERACTING WITH C-18 SUPPORT.

6. NORMAL PHASE LIQUID LIQUID ION PAIR
CHROMATOGRAPHY
 Stationary phase contains the counter ion and is present in an
aqueous coating on the solid support (usually silica particles)
 The mobile phase is an immiscible non-polar organic solvent
often with small amounts of additional organic modifier
 Reversed-phase chromatography employs a polar (aqueous)
mobile phase. As a result, hydrophobic molecules in the polar
mobile phase tend to adsorb to the hydrophobic stationary phase,
and hydrophilic molecules in the mobile phase will pass through
the column and are eluted first.
 Hydrophobic molecules can be eluted from the column by
decreasing the polarity of the mobile phase using an organic
(non-polar) solvent, which reduces hydrophobic interactions. The
more hydrophobic the molecule, the more strongly it will bind to
the stationary phase, and the higher the concentration of organic
solvent that will be required to elute the molecule

7. Basic Ion Pair Reagents
 The sulfonates are sodium salts that act as an anionic counterion for the
separation and resolution of positively charged analytes.
1) S5 (1-pentylsodiumsulfonate)
2) S6 (1-hexylsodiumsulfonate)
 Basic samples can be separated by addition of a straight-chain alkyl sulphonic
acid to the mobile phase. The pH of the mobile phase is adjusted to 3-4 and
diluted with the HPLC solvents (acetonitrile/water or methanol/water) to 5mM
ACIDIC ION PAIR REAGENTS
 Bulk powder — fine, purified crystals, for use as a buffer in large-scale
mobile phase preparation.
1) 1-Pentanesulfonate, Sodium Salt
2) 1-Hexanesulfonate, Sodium Salt
 Acidic samples can be separated with straight-chain alkyl
quaternary ammonium salts or alkyl amines. The pH of the mobile
phase needs to be ajdusted to 7.5 and diluted with the HPLC
solvents (acetonitrile/water or methanol/water) to 5mM. -

8. APPLICATIONS OF ION PAIR
CHROMAROGRAPHY

9. ADVANTAGES OF ION PAIR CHROMATOGRAPHY
OVER ION EXCHANGE CHROMATOGRAPHY
• Simple preparation of buffers
• Wide choice of carbon chain lengths for
improved retention and separation
• Significantly reduced separation time
• Simultaneous separation of both ionized and
nonionized solutes
• Highly reproducible results
• Improved peak shape

10. ADVANTAGES OF ION PAIR CHROMATOGRAPHY
 Ion Pair Chromatography is a method for improving the separation of
charged analytes.
 In the resolution of organic ions with conventional HPLC methods,use of
ion pair reagents can enhance peak shape and retention time when
common remedies such as modifying eluent ratios or changing stationary
phase fail.
 The columns used in ion pair chromatography possess high chromato
graphic efficiency
 After gradient elution the re- establishment of original conditions is rapid
in ion pair chromatography as compared with rather extended times as in
ion exchange chromatography
 Better chromatography of large ions (vs. ion exchange).
 Can separate neutral and charged ions at the same time.
 Analytes with very high or very low pKa values which are resistant to

11. REFERENCES
1) How Theory and Practice Meet in Ion Pair Chromatography, Jan
Ståhlberg,Academy of Chromatography.
2) Methods Development Using Ion-Pair Chromatographywith Suppressed
Conductivity Detection, Dionex International Subsidiaries
3) Weiss, J. Ion Chromatography, Second Edition; VCH Verlagsgesellschaft
mbH, Weinheim (Ger-many) and VCH Publisher, Inc., New York, 1995; pp.
239–289.
4) Small, H.; Ion Chromatography; Plenum Press, New York and London,
1989; pp. 106–118
5) Perry, J. A.; Glunz, L. G.; Szczerba, T. J.; Hocson, V. S.;Reagents For Ion
Pair Reversed-Phase HPLC;American Laboratory 1984, 16(10), 114—119.
6) Eksborg, S.; Lagerstrom, P.; Modin, R.; Schill, G.; Ion Pair
Chromatography of Organic Compounds J. Chrom. 1973, 83, 99.
7) Eksborg, S.; Schill, G.; Ion Pair Partition Chromatography of Organic
Ammonium Compounds Anal. Chem. 1973, 45, 209