Effective sample preparation method during the HPLC

Indian Institute of Chromatography and Mass Spectrometry
No.7, Chakrapani road, Guindy, Chennai 600 032, Tamil Nadu, India
Keep learning
Introduction to
Ion-Exchange Chromatography
MAR ‘2012
at a glance
‘Chromatography’ is the general term for a variety of physico-chemical separation techniques, in which
the common property is that the distribution of analyte component between the mobile phase and
stationary phase. Based on these distribution properties and the physical state of mobile phase and
stationary phase, the chromatographic techniques are subdivided into various classes. In these
subdivided classes, if the component separation is based on ion-exchange processes, i.e., occurring
between the mobile phase and the Ion-exchange groups bonded to the stationary phase called “Ionexchange chromatography”. Ion-exchange chromatography is used for the separation of both inorganic
anions and cations. This newsletter focuses on the historical perspective of ion-exchange
chromatography and its mechanism of separations.
Ion exchange has been known from the time of Aristotle (330 BC), who has mentioned that sand filters
were used for the purification of sea water and impure drinking waters.(1) In 1623, Francis Bacon and
Hales described a method for removing salts by filtration and desalination from sea water(2). In 1790,
Lowritz purified sugar beet juice by passing through charcoal(2) . Considering these works, the practical
applications of ion exchange were well recognized before the 19th century, but the physical
phenomenon was not known at that time.
The identification of ion exchange phenomenon was attributed by two agricultural chemists, Thomson
and Way. In 1850, they reported the first systematic observation on ion exchange with soils. The soil
had a greater ability to absorb ammonia from fertilizers. They passed a fertilizer solution containing
ammonia through the column filled with soil. The ammonium ions were taken up by the soil and an
equivalent amount of calcium and magnesium ions were released. They have concluded that the soils
could retain cations such as NH4+, K+ and Na+ ions and equivalent amounts of Ca2+ and Mg + ions
were released.(3)
Earth Ca2+ + 2NH4+
Earth 2NH4+ +Ca2+
In 1850, Eichorn recognized the alumino silicates (zeolites) are responsible for the ion exchange
process in soil. The first synthetic aluminum based industrial ion exchanger was prepared in 1903 by
Harm and Rumpler to purify the beet syrup. In 1905, Gans succeeded in utilizing the synthetic
aluminum silicates ion exchangers for industrial purpose like water softening and sugar solution
treating. But it could not success in industries because it’s poor reproducibility and chemical
The real breakthrough in this field eclipsed in 1936, Adams and Holmes synthesized insoluble organic
ion exchangers by condensing the formaldehyde with polyhydric phenols / phenolsulphonic acids yield
cation exchangers and anion exchangers were prepared by condensation with amines. They synthesized
several organic ion exchangers, which had better properties (4).
IICMS Newsletter
Page 1 of 4
The ion exchange separation technique played a major role in the identification of trans-uranium elements during the
Second World War. This technique was required to separate and concentrate the radioactive elements needed to create
atom bomb. The next success of ion exchange chromatography was the separation of rare earth elements and separates
the trace level impurities.
In 1971, Dow chemical company was started the research with Small, Stevens and Baumann. They described a novel
ion- exchange chromatography method for the separation and conductometric detection of anionic and cationic
molecules. In the mean time, the term “Ion chromatography” has been coined by Dionex Corporation for this ionexchange chromatographic technique on 1975 (5). Further, Ion chromatography has been categorized into three major
types based on their separation mechanism. They are:
1) Ion exclusion chromatography (IC)
2) Ion pair chromatography (IPC)
3) Ion exchange chromatography (IEC)
Ion exclusion chromatography (IC)
Ion exclusion chromatography is a useful technique for the separation of ionic and non-ionic substances using an ion
exchange stationary phase in which ionic substances are rejected by the resin while non-ionic or partially ionized
substances are retained and separated by partition between the liquid inside the resin particles and the liquid outside of
the resin particles. The ionic substances are transitory quickly through the column but non-ionic or partially ionized
substances are eluted more slowly(6).
Fig-1 Schematic diagram of ion exclusion chromatography (7)
• The mechanism of Ion exclusion chromatography proposes the sulfonate groups are fixed on the surface of
the resin and form a negatively charged shield on the polymeric surface; often refer to as ‘Donnan
• The interior of the resin contains some occluded component, which is act as a stationary phase.
• The Donnan membrane separates moving fraction of eluent (mobile phase) from occluded component of the
stationary phase.
• The analyte molecules enter the column; they interact with the sulfonated groups. The dissociated fraction
(HCl) of the analyte is repelled from the vicinity of the Donnan membrane, while the non-dissociated
molecule (R-COOH) penetrates the membrane and enters the occluded fraction of the eluent and thus
effects the analyte separation.
IICMS Newsletter
Page 2 of 4
Ion pair chromatography (IPC)
Ion pair chromatography (IPC) is a modification of reverse phase chromatography for the separation of ionic samples.
The only difference in conditions of IPC is the addition of an ion pairing reagent (R+ or R-) to the mobile phase, which
can interact with ionized acids (A-) or bases (BH+) to achieve the separation.
Most commonly used ion-pair reagents are alkylsulfonates (R–SO3-), tetraalkylammonium salts (R4N+) and strong
carboxylic acids like Trifluoroacetic acid (TFA), Heptafluorobutyric acid (HFBA), etc.,(8)
Ionized solute
A- + R+
Ion pair
BH+ + R-
Schematic diagram of ion-pair chromatography (7)
Ion pair chromatography mechanism is completely different from Ion exclusion chromatography. Highly nonpolar stationary phases are used in reversed phase chromatography. The anionic or cationic surfactants containing
ion pair reagent (lipophilic counter ion) is added to the eluent (mobile phase), which is responsible for regulating
the retention of ionic compounds. The opppositively charged analyte ion and ion pair reagent together with form
non polar molecule. Molecules can be retarded at the stationary phase by hydrophobic interaction. These induced
interaction leads to the retention of analyte.
Ion exchange chromatography (IEC)
Ion exchange chromatography technique separates the ionic molecules based on their charge properties. Ion
exchange occurs between the analyte ions and counter ions (ions in the mobile phase) interaction with the
stationary phase ionic groups of opposite charge. The strength of interaction depends upon the charge of
molecules and the stationary phase (resin) due to the difference in their charges, charge density and distribution
charge on their surfaces. The interaction can be controlled by adjusting the ionic strength and pH of the mobile
The stationary phase used in Ion exchange chromatography are displays the functional groups having positive
(+ve) or negative (-ve) charge properties and are termed as anion and cation respectively.
1) Anion exchanger - The analyte ions interact with the basic ion exchanger sites of the stationary phase.
2) Cation exchanger - The analyte ions interact with the acidic ion exchanger sites of the stationary phase.
Steps involved in the mechanism of ion exchange chromatography:
1) Equilibration
2) Adsorption
3) Desorption
4) Regeneration
IICMS Newsletter
Page 3 of 4
End of desorption
Fig-3 Schematic diagram of ion exchange chromatography(10)
Prior to the sample injection, column must be equilibrated with the mobile phase (eluent). The equilibration
of the stationary phase depends on the pH and ionic strength of mobile phase. The stationary phase charged
groups are associated with mobile phase counter ions called Equilibration. Most commonly used counter
ions are chloride and sodium.
• The ionic strength of the analyte must be equal to the mobile phase ionic strength, to get the good analyte
separation. If the ionic strength of mobile phase is too low than analyte, it allows the analyte to strongly bind
with stationary phase and retains the analyte molecule called Adsorption. If the ionic strength of the mobile
phase is too high than analyte, it does not allow the analyte to bind with stationary phase and the analyte
molecule quickly pass through the column called Desorption.
• After the molecules are eluted, the column is regenerated by high concentration of counter ions. High
concentration of counter ions, removes the fouling material adsorbed to the stationary phase called
Regeneration. After regeneration, the stationary phase can be equilibrated with the mobile phase for the next
To summarize,
Now we know, the mechanism behind the separation of
1) Ion exclusion chromatography (IC)
2) Ion pair chromatography (IPC)
3) Ion exchange chromatography (IEC)
Vassilis J. Inglezakis (2006) Adsorption, ion exchange and catalysis, first edition, Elsevier inc; chapter 2, Page 38.
Mu.Naushad, Ion exchange letters; Inorganic and composite ion exchange materials and their applications; 2009,
page 1-14.
3. Yeshajahu Pomeranz, Clifton E. Meloan (1994), Food Analysis: Theory and Practice: Third edition; Aspen
publication; chapter 20; page 313-317.
4. Zdeněk Deyl, Karel Macek; (1975) Liquid column chromatography: a survey of modern techniques and
applications; Journal of chromatography library -volume 3; Elsevier inc ;chapter 9; Page 205.
Paul R. Haddad, Peter E. Jackson (1990); Ion chromatography: principles and applications; Journal of
chromatography library - volume 46; Elsevier inc; chapter 1; page 6-7
6. Lokesh Bhattacharyya, Jeffrey S. Rohrer; Application of Ion chromatography in the analysis of pharmaceutical
and biological products; Wiley publication; chapter 2.
7. Maximilian Kolb, Andreas Seubert; practical ion chromatography-An introduction; Metrohm publication; page
8. Lloyd R. Snyder, Joseph J. Kirkland and John W.Dolan (2010) Introduction to Modern Liquid Chromatography,
Third Edition A John Wiley & Sons Inc., chapter 7 page 331-337.
9.4 of Jan-Christer
Janson (2011); Protein purification; Third edition; John Wiley & Sons Inc; chapter 4; 93-125.
10. www.amersham biosciences.com
U. Rampriya - IICMS
IICMS Newsletter
Page 4 of 4