Ion chromatography is a form of liquid chromatography that separates and quantifies ions and ionizable molecules. It utilizes different stationary phases and separation techniques including ion exchange, ion exclusion, ion pairing, and ion suppression chromatography. Ion exchange chromatography separates ions based on their affinity for oppositely charged groups on the stationary phase. Sample preparation techniques such as dialysis and solid phase extraction are used to reduce matrix effects. Ion chromatography is widely used in environmental analysis due to its ability to detect ions at trace levels. It has been used to detect perchlorate in drinking water within 7 minutes with a detection limit of 20 μg/L.

1. Ion Chromatography
An Instrumental Method
Jordan Sedlock | CHEM 310 | November 25, 2014

2. 2
Methods of quantifying ions and polar molecules have been developed and used since the
mid 19th century.1 Ion chromatography (IC) was developed around the 1950s and is used to
separate and quantify ions or ionizable molecules as well as large proteins and small nucleotides
in solution at very low levels.2 It is a form of liquid chromatography and describes efficient
chromatographic peak separation amongst ionic species.2 Ion chromatography has proven to be
very effective at quantifying ionic species in many fields including, but not limited to: food
analysis, water quality treatment, and pharmaceuticals.
Ion chromatography refers to a group of liquid-chromatographic methods that are used to
quantify certain groups of analytes.3 Analytes can include low-molecular-mass acids and bases,
also, inorganic anions and cations.3 Different methods of IC exist and include: ion-exchange,
ion-exclusion, ion-pair, and ion-suppression chromatography.2 Ion-exchange chromatography
(IEC) is one of the most applicable areas of IC, used to separate inorganic anions and cations.3
Ion-exclusion chromatography (also abbreviated IEC) is commonly used to separate weak acids
such as carboxylic acids, weak bases such as ammonia, and carbohydrates as well as other
hydrophilic biomolecules.2 Ion-pair chromatography (IPC) is commonly used to separate
analytes with widely different components as would be the case in mixtures of acidic and basic
analytes as well as compounds that are very difficult to separate, possibly due to presence of
covalent bonds.2 Ion-suppression chromatography (ISC) is generally used to separate polar, weak
acids and weak bases.4
Ion-exchange chromatography (IEC) separation techniques are based on the affinities of
the analytes of interest and the oppositely charged ionic functional groups within the stationary
phase of an ion-exchange column.2 There are two types of exchangers used in IEC, anion and
cation exchangers.5 Anion exchangers contain positively charged functional groups on the

3. 3
stationary phase to attract solute anions.5 Cation exchangers contain negatively charged
functional groups on the stationary phase to attract solute cations.5 Of these two types of
exchangers, there are three different classes used for ion-exchange based on particle size. There
are resins, which are used for applications involving small molecules, gels, which are used for
applications involving macromolecules, and inorganic exchangers, which are used for
separations involving extreme chemical conditions such as powerful oxidizing agents or high
temperatures.5 Anion exchanger resins are known to be either strongly basic, containing
quaternary ammonium groups attached to styrene and divinylbenzene copolymers or weakly
basic containing polyalkylamine groups attached to styrene and divinylbenzene copolymers.5
Cation exchanger resins are known to be either strongly acidic, containing sulfonic acid groups
attached to styrene and divinylbenzene copolymers or weakly acidic containing carboxylic acid
groups attached to styrene and divinylbenzene copolymers.5 Gels used in ion-exchange are
commonly made from cellulose and dextran, which are softer materials than polystyrene resins.5
Ion-exclusion chromatography (IEC), another type of IC, is especially useful for
separating organic acids as well as small, neutral molecules and weak bases.6 This technique
involves the separation of molecular species, rather than ions but can be used to separate analytes
by differences in molecular size, shape, or charge.2 This technique also involves what is known
as the Donnan Exclusion mechanism, wherein, certain molecules (depending on size, shape, or
charge) can pass through a semipermeable membrane and onto the stationary phase (or resin
phase) after absorbing into an occluded liquid phase surrounding it and other molecules cannot,
which are therefore, not retained.2 This is useful when separating weak organic acids because
such compounds can easily adsorb onto the resin phase while strong, anionic compounds are
unable.6

4. 4
Ion-pair chromatography (IPC) is another type of IC that separates polar and ionic
compounds via a reverse-phase HPLC column rather than an ion-exchange column.5 This
method is commonly detected using UV detectors, because analytes of interest contain
chromaphores, as well as conductivity detectors, because they are universal.7 IPC relies heavily
on the composition of the mobile phase in order to achieve separation.7 The mobile phase
contains an organic solvent as well as an ion-pair reagent.7 The ion-pair reagent contains a large,
ionic molecule whose charge is opposite that of the analyte trying to be analyzed.7 This large
molecule is composed of two different regions, one wherein there is a strongly hydrophobic
segment that interacts with the stationary phase and another region which contains the opposing
charge of the analyte of interest.7 The stationary phase is commonly comprised of
polystyrene/divinylbenzene (PS/DVB).7 The retention mechanism in this separation technique is
not fully understood, however, three theories exist in attempt to explain this: ion pair formation,
dynamic ion exchange, and ion interaction.7 The first theory is similar to reversed-phase
chromatography in that the ion-pair reagent and the analyte of interest form a neutral pair and
then partition between the mobile and stationary phases; retention times can be varied depending
on concentration of organic solvent in the mobile phase.7 The second theory involves the
hydrophobic region of the ion-pair reagent adsorbing to the stationary phase and the analyte of
interest interacting with the ionic region of the ion-pair reagent like it would in ion-exchange
chromatography.7 The ion interaction model involves an electrical double layer that is formed
from the permeation of the stationary phase by the ion-pair reagent which carries with it a
counter-ion; retention is dependent upon factors described in the first two models.7

5. 5
Ion-suppression chromatography (ISC), the last type of IC being discussed, is comprised
of two sub-categories: suppressed-ion anion chromatography and suppressed-ion cation
chromatography.5 In ion-suppression chromatography, an unwanted electrolyte is removed
before the analyte of interest undergoes detection via electrical conductivity.5 In the suppressed
anion technique, a mixture of anions is separated using an anion separator column and strong
base as an eluent.5 Anions and eluent are then passed through a suppressor where the strong base,
which possesses high conductivity is converted into H2O, which has low conductivity.5 This
process allows analytes of interest to be detected without interference of eluate.5 In the
suppressed cation technique, a mixture of cations is separated using a cation separator column
and strong acid as an eluent.5 Cations and eluent are then passed through a suppressor where the
strong acid is converted into water, reducing conductivity of the eluate and allowing for accurate
detection of the analytes of interest.5 This technique essentially neutralizes weak acids and bases
which is what causes the “suppression” in ISC.4 ISC is commonly used with reversed-phase
HPLC columns, however, the silica material is not preferred as much as PS/DVB is due to the
small pH range that the silica material possesses (2.5 – 7.5).4 The mobile phase for this technique
contains an organic modifier and a buffer and the retention mechanism is similar to that on a
reversed-phase HPLC column when separating weak acids and bases or neutral molecules.4
Based on the types of samples that are analyzed using IC, sample preparation steps must
be taken in order to ensure sufficient resolution. The most basic rule of sample preparation is that
all samples must be in the aqueous phase as IC is a form of liquid chromatography.8 It is
particularly difficult to quantify compounds that exist at trace levels in matrices with extreme
conditions such as pH or high ionic strength.3 To reduce the amount of an interfering matrix, a
simple solution is to inject a smaller volume onto a column as well as using a selective detection

6. 6
method, however, this is problematic because it also reduces the amount of analyte of interest
that is present.3 A second method, which involves handling matrices with extreme pH is to
perform active dialysis (Donnan dialysis), a commonly employed sample cleanup technique
which involves transferring ions of a specific charge across a membrane, for example, with
highly alkaline samples.3 This type of sample cleanup helps reduce peak distortion, baseline
noise, and prolong column life.3 This method can be further refined by adding an electrical
current across the membrane, known as electrodialysis, which only differs from Donnan dialysis
due to the presence of the electrical current.3 Electrodialysis is also helpful when analyzing
alkaline samples which contain trace amounts of inorganic anions as well as neutralizing acids
and bases prior to analysis of trace metals such as magnesium(II) and calcium(II).3
Samples with matrices of high, ionic strength must be prepared in order to account for
matrix effects, which greatly affect resolution and noise in the final chromatogram. A technique
to do this is referred to as matrix elimination IC.3 This technique, simply put, involves using the
dominant ion in the matrix as the eluent in a separation which causes sufficient separation of the
analyte of interest and no retainment of the matrix on the column.3 This method greatly reduces
band broadening in the final chromatogram.3 Another method of reducing matrix effects is to
perform sample cleanup and a matrix eliminating step via solid-phase extraction (SPE).9
Environmental samples, particularly water samples need very little sample preparation in
order to be separated and quantified via IC.9 Drinking water generally only needs to be filtered
through a 0.45 µm filter to remove particulates and is then ready for analysis.9 Wastewater
samples generally only need dilution (to range of 2 – 100 µg/l) and filtering through a 0.45 µm
filter and are then prepared fully.9 Solid samples such as soil can go through very simple aqueous
extractions before being prepared fully.9 Known amounts of sample are mixed with water,

7. 7
preferably so as not to introduce extraneous peaks into final chromatogram, but can be mixed
with other solvents such as methanol or weak acids, and filtered through a 0.45 µm filter and are
then ready for analysis.9 Acid digestion in a strongly concentrated acid, such as nitric acid, can
be employed for solid samples such as rock.9 This method is not favorable for IC analysis of
anions because the co-anions that are present from the acid digestion can cause large, distorted
peaks in a final chromatogram.9 This sample preparation method can be employed for analysis of
cyanide, sulfide, and fluoride, however, when coupled with amperometric detection, which
involves applying a voltage between two electrodes positioned in column effluent and measuring
the difference between them.9 Acid digestion is used frequently when using IC to determine
cations; examples include nitrogen, transition metals, and rare earth metals.9
Alkali fusion is an alternative to acid digestion which requires mixing a sample
composed of geological material with an alkaline flux and heated to high temperatures ranging
from 800 – 1100 °C.9 The flux becomes molten which is then cooled and digested in suitable
solution prior to IC analysis.9 This method works well for analyzing fluoride and chloride in
materials of geological origin.9 Another method used for solid samples includes combustion,
wherein, the entire sample is burned in an oxygen flame which leads to conversion of
nonmetallic elements into volatile and gaseous compounds that are collected in an absorbing
solution and then analyzed via IC.9 This method of sample preparation works well for
determining total nitrogen, sulfur, and phosphorous in plant material and different geological,
rocky material.9
After sample preparation, analysis per sample is generally dependent upon which type of
sample is being analyzed, as well as other general parameters such as mobile phase composition,
stationary phase composition, column length, column diameter, flow rate, etc. Analysis is also

8. 8
dependent upon which form of IC is used. As previously stated, the four forms of IC are each
used to detect different forms and classes of analytes. Sea water samples can be analyzed for
transition metals in approximately 12 minutes.9 Lake water samples can be analyzed for anions
in approximately 10 minutes.9 Wastewater samples, which are of particular environmental
concern, can be analyzed for anionic and cationic nutrients in approximately 6.5 minutes.10 Weak
acid determination, used in food chemistry, can be performed in approximately 18 minutes.6
Determination of toxic elements such as arsenic, in different types of cereal-based food can be
completed in as little as 7 minutes.11 Separation of proteins from complex biological mixtures,
such as human plasma, can be completed in approximately 20 minutes.12
Ion chromatography analysis is performed on an HPIC instrument that generally costs
approximately $35,000 dollars.13 Thermo Scientific has manufactured an HPIC system designed
to maximize resolution, speed, and sensitivity.13 All chromatography systems consist of the same
basic components: eluent, pump, injection valve, columns, suppressor (which is specific to ion
chromatography), detector, and some sort of data collection system.14 The eluent establishes
basic ionic conditions, stabilizes sample in solution, and promotes progression of sample through
the system.14 Pumps are used to move eluent and samples throughout the system.14 There are
different types of pumps that can be used including serial and parallel, gradient and isocratic.14
Continuous, pulse-free flow is required so as not to introduce noise into the chromatogram.14
Eluent flows from the pump, into the injection site which contains a 2-position valve used to
direct flow of eluent and to introduce the sample into the system.14 Leaving the injection site, the
sample and eluent flow into the column where separation occurs based on the stationary phase
within the column.14 Following the column, the sample will enter a suppressor (selected based
on application).14 There are three types of suppressors: self regenerating suppressor (SRS), Atlas

9. 9
Electrolytic Suppressor (AES), and micromembrane suppressor (MMS).14 Suppression is
required so that the ions being analyzed can be detected via one of three different methods:
conductivity, amperometry, or absorbance.14 Readings from the detector appear in the data
collecting system and final chromatograms are produced.14
Many applications have been pursued using instruments and separation techniques of this
nature. IC has proven to be particularly useful in environmental analysis of water samples due to
its ability to detect trace amounts of compounds with high accuracy. Perchlorate, an essential
ingredient in rocket fuel, poses serious threats to human health as it is contaminating drinking
water.15 Perchlorate causes interference of iodide uptake by the thyroid gland and therefore
greatly affects proper thyroid functions.16 An anion exchange column and ion exchange
chromatography can be employed to detect levels of perchlorate in drinking water.16 Separation
of all ions can be completed within 7 minutes.16 It is concluded that this method provides valid
results for determining trace-level perchlorate in drinking water.16
Other methods of determining trace level perchlorate in drinking water have been
employed and compared to IC.17 One such method includes electrospray ionization mass
spectrometry (ESI-MS).18 This technique involves creating ions by applying high voltage to
liquid to create an aerosol.18 It is appropriate to use this method in analysis of permanently
ionized or polar, ionizable compounds.18 Ions are then quantified via mass spectrometric
methods. Unlike IC, ESI-MS requires the employment of a standard addition method to correct
for severe matrix effects in the samples being analyzed and therefore requires more care during
sample preparation.18 ESI-MS has linearity of 10-5 M and a detection limit in reagent water
below 1 µg/l, however, it is almost impossible to detect trace levels of perchlorate in wastewater
samples.18 The IC method has a linear range of 2 – 100 µg/l and a limit of detection of 20 µg/l.16

10. 10
High ionic strength of chloride greatly interferes with chromatogram signal, however, this can be
greatly reduced with solid-phase extraction before analysis.16 More training would be required to
perform the ESI-MS method of determining perchlorate in drinking water.
Ion chromatography is a relatively simple technique overall. It is very important in
environmental chemistry and will continue to be as it is so effective at determining trace
elements as well as inorganic anions and cations. Ion chromatography has progressed a long way
from development and has been proven to be a very dependable and accurate analytical
separation technique.

11. 11
References
1. Ion Chromatography. http://ionchromatography.co.uk/2011/12/25/the-history-of-ion-
exchange-chromatography/ (accessed Oct 15, 2014).
2. Chromacademy. http://www.chromacademy.com/lms/sco111/theory_of_hplc_ion-
chromatography.pdf (accessed Oct 15, 2014).
3. Haddad, P. R.; Doble, P.; & Macka, M. Developments in sample preparation and
separation techniques for the determination of inorganic ions by ion chromatography and
capillary electrophoresis. Journ. Of Chrom. A. 1999, 856, 145-177.
4. Chromedia Analytical Sciences.
http://www.chromedia.org/chromedia?waxtrapp=lcejiGsHqnOxmOlIEcCbCeHhHcC&su
bNav=xododDsHqnOxmOlIEcCbCeHhHcCfC (accessed Oct 15, 2014).
5. Harris, D. C. Chromatograpic Methods and Capillary Electrophoresis. In Quantitative
Chemical Analysis; W. H. Freeman and Company: New York; 2010; 635-646.
6. Milagres, M. P.; Brandao, S. C. C.; Malgalhaes, M. A.; Minim, V. P. R.; & Minim, L. A.
Development and validation of high-perfomance liquid chromatography-ion exclusion
method for detection of lactic acid in milk. Food Chem. 2012, 135, 1078-1082.
7. Dionex. http://www.dionex.com/en-us/webdocs/4696-TN12_LPN0705-01.pdf (accessed
Oct 16, 2014).
8. Koch, W. F. Sample preparation in ion chromatography. Journ. Of Res. 1979, 84, 241-
245.
9. Jackson, P. E. Ion Chromatography in Environmental Analysis. In Encyclopedia of
Analytical Chemistry; Meyers, R. A.; Ed.; John Wiley & Sons Ltd: Chichester; 2000;
2779-2801.
10. Karmarkar, S. V. Analysis of wasterwater for anionic and cationic nutrients by ion
chromatography in a single run with sequential flow injection analysis. Journ. Of Chrom.
A. 1999, 850, 303-309.
11. Llorente-Mirandes, T.; Calderon, J.; Centrich, F.; Rubio, R.; & Lopez-Sanchez, J. F. A
need for determination of arsenic species at low levels in cereal-based food and infant
cereals. Validation of a method by IC-ICPMS. Food Chem. 2014, 147, 377-385.
12. Gajdoski, M. S.; Kovac, S.; Malatesti, N.; Muller, E.; & Josic, D. Ion-exchange sample
displacement chromatography as a method for fast and simple isolation of low- and high-
abundance proteins from complex biological mixtures. Food Technol. Biotechnol. 2014,
52(1), 58-63.
13. Thermo Scientific. http://www.thermoscientific.com/en/product/dionex-ics-5000-
capillary-hpic-system.html (accessed Oct 27, 2014).
14. Dionex. Principles and Troubleshooting Techniques in Ion Chromatography. Dionex
Corporation: 2002; Document No. 034461.
15. Jackson, P. E.; Gokhale, S.; Streib, T.; Rohrer, J. S.; & Pohl, C. A. Improved method for
the determination of trace perchlorate in ground and drinking waters by ion
chromatography. Journ. Of Chem. A. 2000, 888, 151-158.
16. Jiang, S.; Li, Y.; & Sun, B. Determination of trace level perchlorate in Antarctic snow
and ice by ion chromatography coupled with tandem mass spectrometry using an
automated sample on-line preconcentration method. Chin. Chem. Lett. 2013, 24, 311-314.

12. 12
17. Li, Y.; Whitaker, J. S.; & McCarty, C. L. Analysis of iodinated haloacetic acids in
drinking water by reversed-phase liquid chromatography/electrospray ionization/tandem
mass spectrometry with large volume direct aqueous injection. Journ. Of Chrom. A.
2012, 1245, 75-82.
18. Roehl, R.; Slingsby, R.; Avdolovic, N.; & Jackson, P. E. Applications of ion
chromatography with electrospray mass spectrometric detection to the determination of
environmental contaminants in water. Journ. Of Chrom. A. 2002, 956, 245-254.