Ion-pair chromatography is an alternative to ion exchange chromatography, utilizing a mobile phase that contains ion-pair reagents to neutralize and separate ions. This method allows for the effective separation of both cationic and anionic components in a sample by employing counter ions and adjusting pH levels. Key applications include the separation of charged analytes such as catecholamines, artificial sweeteners, and water-soluble vitamins.

1. ION-PAIR
CHROMATOGRAPHY
PRESENTED BY,
JIKHILA MACHADO
2ND SEM M.PHARM

2. ION-PAIR CHROMATOGRAPHY
 It represents an alternative to ion exchange
chromatography.
 The column and mobile phase used are similar-addition
of an ion pair reagent to mobile phase in case of ion pair
chromatography.
 It is a type of column chromatography in which ions in
solution can be paired or neutralized and seperated as an
ion pair on a reversed phase column.
 Ion-pairing agents are usually ionic compounds that
contain a hydrocarbon chain.

3.  In ion-pair chromatography, the solute ion is distributed
between the mobile phase and the stationary phase together
with an ion of opposite charge(counter ion).
Sample+counter ion (sample counter ion) pair
 This method is often used in reverse phase chromatography
as a convenient method to control the retention of solutes.
 A sample containing both cationic and anionic components
has one type masked by counter ion and the other supressed
by a suitable pH level.

4. ION PAIR REAGENT
 In ion-pair chromatography an ion-pair reagent(organic
counter ion) is added at a low concentration to the
mobile phase usually at 0.005 M.
 The ion-pair reagent itself is ionized.
BASIC ION- PAIR REAGENT
 Sulfonates –sodium salts that act as anionic counter ion.
-S5(1-pentylsodiumsulfonate)
-S6(1-hexylsodiumsulfonate)
 Basic samples seperated by addition of straight chain
alkyl sulphonic acid to M.P
 pH of M.P adjusted to 3-4 and diluted with HPLC
solvents to 5mM.

5. ACIDIC ION-PAIR REAGENTS
 Bulk powders
-1-pentanesulfonate sodium salt
-1-Hexanesulfonate sodium salt
 Acidic samples seperated with straight chain alkyl
quaternary ammonium salts or alkyl amines.
 pH of M.P adjusted to 7.5 and diluted with HPLC
solvents.
 Counter ion quaternary amines suitable for strong and
weak acids.
 Alkyl and aryl sulphonates used for strong and weak
bases.

6. MECHANISM OF ION-PAIR
CHROMATOGRAPHY
 Two fundamental models:-
 The solute molecule forms an ion-pair with the counter
ion in the mobile phase.
-this uncharged ion pair then partition into the
lipophilic stationary phase.
 The othermechanism postulate that the counter ion
partition into stationary phase or loaded onto the
stationary phase
- this produce two possibilities for tha material to be
chromatographed,
a) It can be attracted to the hydrocarbon portion in the
usual reverse phase manner.
b) It can be in an ion exchange mode.

7. FACTORS INFLUENCING RETENTION
 The extent to which the ionized sample and counter ion
form an ion-pair complex and its binding strength affect
retention.
 The relative size of lipophilic group on counter ion affect
degree of retention.
- longer the alkyl chain – denser the ion population in the
S.P – yields greater retention of given ions.
 Increasing the concentration of counter ion increases
retention
 Control of pH

8. EXPERIMENTAL CONDITION
 Stationary phase(bonded phase column ) –monolayer C-18 or
C-8.
 Starts with 0.001 M concentration of shorter chain counter ion
or if it contains decyl or longer chain alkyl groups -0.005 M
solution.
 Most common solvent combinations are,
water/methanol and water/acetonitrile
 buffer components should be selected with poor ion-pair
properties but good solubilities.
 Ion pair partition can separate non-ionic and ionic compounds
in the same sample.
first, optimizes the separation of non-ionic solutes then selects
and add counter ion to M.P where ion solutes become
retained.

9. EXAMPLE:
Separation of water soluble vitamins.
 At pH 3-4 thiamine is strongly ionic, pyridoxine and
niacinamide are less and riboflavin is non-ionic.
 Two step procedure,
 In the first step, methanol/water ratio adjusted to obtain
good retenion of non-ionic compound riboflavin.
 In the second stage, organic counter ion I chosen and
added to eluent to separate three ionic compounds.
o The optimum separation is achieved with 50/50 mixture
of C-5/C-7 alkyl sulfonic acids.

10.  Mixture of counter ion added to mobile phase
produces a retention proportional to the
concentration of each counter ion.

11. ADVANTAGES
 Is the method for improving the separation of charged
analytes.
 Use of ion-pair reagents can enhance peak shape and
retention time.
 The column used in ion-pair chromatography posses
high chromatographic efficiency.
 After gradient elution the re-establishment of original
conditions is rapid in ion-pair chromatography.
 Better chromatography of larger ions.
 Can separate neutral an charged ions at the same time.

12. APLICATIONS:
 Separation of catecholamines
 Separation of artificial sweetners
 Separation of water soluble vitamins.

13. REFERENCES
 Text book of instrumental methods of analysis,7th