‘Separation of sample components after their distribution between two phases.’’ - IUPAC definition Ion Chromatography (IC) was introduced in 1975 by Small, Stevens and Baumann as a new analytical method for sensitive detection of ions via their electrical conductance. Chromatography is a separation technique that is used for separation a sample mixture into its constituents or components.

1. MPEP-1206 : EIA and Monitoring
Ion
Chromatography
Hrishikesh Satpute
(2019MEP012)

2. 1. Definition
2. Environmental Significance
3. Analytical method – Working and Principle
4. Operational Procedure
5. Procedure of Sample Procurement
6. Applications in Environmental Monitoring
Contents

3. Definition - Ion Chromatography
▪ Ion Chromatography (IC) was
introduced in 1975 by Small,
Stevens and Baumann as a new
analytical method for a sensitive
detection of ions via their
electrical conductance.
▪ Chromatography is a separation
technique which is used for
separation a sample mixture into
its constituents or components.
3
Ion Chromatography is a process that separates ions and polar molecules
based on their affinity to the ion exchanger.
Ion Chromatography System

4. ▪ Ion exchange chromatography is
the chromatography process that
separates ions and polar
molecules from a given sample.
▪ The negatively charged ions are
known as Anions and the positively
charged ions are known as
Cations.
▪ This process is used for the
detection of inorganic components
in a given sample.
Introduction
• Lithium
• Sodium
• Ammonia
• Potassium
• Calcium
• Magnesium
• Fluorides
• Bromides
• Chlorides
• Nitrates
• Sulphates
• Phosphates
Anions Cations
4
+
-

5. 5
Environmental Significance
▪ Ion Chromatography (IC) is a popular method to
analyze materials in a number of industries such as
electronic, clinical, biological, pharmaceutical,
chemical and food.
▪ Due to the flexibility of IC, it is also used for
Environmental Testing – specifically in water
analysis, measuring pollutants and contaminants.
▪ Ensuring safe drinking water is an important
environmental factor and as industrial pollutants
increase, it is even more vital to understand how to
test water for toxic chemicals such as cyanide.
▪ IC has been accepted world-wide as a reference
method for analyzing anions and cations in water and
wastewater . Ion Chromatography System

6. Ion Analysis
6
Ion Chromatography Schematic
Ion Analysis
Chromatography Electrophoresis
Ion
Exchange
RP
Methods
IE IC Ion
Suppression
Ion Pairing
▪ Conventional
▪ Capillary
Source: Ion Exchange Chromatography Handbook (Principles and Methods)

7. Ion Chromatography System
7
The main components of the ion chromatography
system are -
▪ A high pressure pump with pressure and flow
indicator, to deliver the eluent/solvent.
▪ An injector for introducing the sample into the
eluent stream and onto the column.
▪ A column, to separate the sample mixture into
the individual components.
▪ A detector, to measure the analyte peaks as
eluent from the column.
▪ A data system for collecting and organizing the
chromatograms and data.
Solvent
Reservoir
Pump
+
Gradient
System
Pressure and
Flow Indicator
Injection
Unit
Column
Detector
Data Processing
Time
mV
Thermostatted
Housing
(optional)
Block Diagram of a Basic Ion Chromatograph. From Fritz and Gjerde, 2000

8. Principle
8
▪ Ion chromatography is based on
the interaction between the
Stationary Phase (stagnant/fixed)
and the Mobile Phase (in motion).
▪ The sample or solution in the
reservoir acts as the mobile
phase.
▪ This solution is made to run
through the column. The column
is fitted with a solid adsorbent
material (resin) which acts as the
stationary phase.
▪ The interaction between stationary phase and mobile phase varies with different
molecules and this varying nature of different molecules will help in detecting
them as some molecules will separate faster and some will separate slower.
Ion-Exchange Chromatography
(Anion Exchange)

9. Types of Ion Chromatography
9
Biomolecules are purified using chromatography techniques that separate them
according to differences in their specific properties. On the basis of the charge carried by
resin, ion chromatography can be of two types:
a) Cation Exchanger – In this, the resin carries Negative Charge and the functional group
attached to it carries positive charge.
b) Anion Exchanger - In this, the resin carries Positive Charge and the functional group
attached to it carries positive charge.

10. Working - Analytical method
10
An Ion Chromatography medium comprises a matrix of spherical particles
substituted with ionic groups that are negatively or positively charged. The matrix is
usually porous to give a high internal surface area. The medium is packed into a column
to form a packed bed. The bed is then equilibrated with buffer which fills the pores of the
matrix and the space in between the particles.
There are four main steps in the process of Ion Chromatography:
Equilibration
Loading
Washing
Elution
The stationary phase is equilibrated with specific requirements as per the experiment.
The sample is run through the column.
The column is washed to remove impurities/molecules that do not bind to the resin.
The ions or charged molecules that got bound to the resin are eluted out of the column.
1
2
3
4
Source: Ion Exchange Chromatography Handbook (Principles and Methods)

11. Process of Ion Chromatography
11
▪ Equilibration buffer is taken in the reservoir and
made to run through the column at a particular
rate of flow.
▪ When equilibrium is reached, all stationary
phase charged groups are associated with
exchangeable counter ions, such as chloride or
sodium.
▪ The solution containing the unbound particles
is released from the column to the detector
and is collected in a collecting tube.
▪ We’ll observe that the graph on the computer screen comes out as a straight line.
The straight line indicates that the column is properly packed and further analysis
can be done.
STEP 1 : Equilibration
Source: Ion Exchange Chromatography Handbook (Principles and Methods)

12. Process of Ion Chromatography
12
▪ In this, the sample is mixed with the buffer (of a specific pH ) and is run through the
column. The flow rate is kept low so that the ions in the sample get enough time to
replace the functional group attached to the resin.
▪ So, in this way, the sample gets loaded in the column.
STEP 2 : Loading
▪ Certain molecules/ions don’t get bound with the resins in the column.
▪ Hence, washing is done in which buffer solution is made to run through the column
so that the unbound molecules are washed out.
▪ As this solution passes through the detector, it leads to the formation of peak in the
graph.
STEP 3 : Washing
Source: Ion Exchange Chromatography Handbook (Principles and Methods)

13. Process of Ion Chromatography
13
▪ When all the sample has been loaded and the column washed with start buffer so
that all nonbinding proteins have passed through the column, conditions are altered
in order to elute the bound proteins.
▪ In this step, the ions/charged molecules that got bound with the resin are eluted out
of the column.
▪ An elution buffer is used for this. It breaks the bonds between the resin and the
attached ions. The solution that is released from the column is made to pass through
the detector, resulting in the formation of a second peak in the graph.
STEP 4 : Elution
Regeneration
▪ A final wash with high ionic strength buffer regenerates the column and removes any
molecules still bound.
▪ This ensures that the full capacity of the stationary phase is available for the next
run. The column is then re-equilibrated in start buffer before starting the next run.
Source: Ion Exchange Chromatography Handbook (Principles and Methods)

14. 14
Chromatogram
Graph obtained after Data Processing
▪ Each peak represents a separate
ion from the sample solution. The
elution time, or time it takes for the
ion to move through the column,
varies for each ion species as they
elute from the column separately
as the pH and/or ionic strength of
the eluent is increased.
▪ The concentration of ions moving through the column at a particular time is
represented by the height and the breadth of the peaks and can be correlated to the
concentration of a particular species in the sample solution.
▪ Ion concentrations can be calculated using the area under each peak, where a larger
area correlates with a higher concentration of a particular ion species.

15. 15
Operational Procedure of the Instrument
1. Pre-analysis Procedures
▪ Turn ON ‘ICS-1000’ (for eg.), Autosampler and PC.
▪ Open the Chromeleon (for eg.) software and Connect to Chromelon Server.
▪ Check the eluent reservoir located on top of the ICS-1000. Approximately 1 L
of eluent is required per analysis.
▪ Ensure that the “Connected” field within the ICS-1000 System and then ‘Start
Up’.
2. Set System Operating Conditions
▪ Click (Acquisition ON/OF) within the main window toolbar to start the data
acquisition.
▪ Click Autozero, ensure that the instrument has truly autozeroed.
▪ Check the waste bottle for free board volume.
Source: Standard Operating Procedure for: Dionex ICS-1000 Ion Chromatography System

16. 16
Operational Procedure of the Instrument
3. Sample Preparation
▪ Organize the project sample bottles to minimize confusion when preparing
samples and entering sample information into the template.
▪ Place the 5 ml plastic vials into the autosampler cassettes.
▪ Use filter syringes to filter samples directly into each vial and Transfer
approximately 5 ml from the sample container to the syringe.
▪ Place the caps on the vials and initiate the autosampler.
4. Sample Analysis
▪ After the instrument self-check, the Chromeleon-Check Results window will be
displayed and then START.
▪ Analysis progression can be checked by noting that the sample being analyzed
is highlighted green. Close the window after peak retention updation.
Source: Standard Operating Procedure for: Dionex ICS-1000 Ion Chromatography System
5. Export and Save Data

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Procedure of Sample Procurement
Sample Collection, Preservation,
Shipment and Storage
▪ Samples are collected in 500 mL
plastic bottles.
▪ Sample bottles are stored on ice
for transport to the laboratory.
▪ Samples are stored in the
refrigerator prior to analysis.
▪ Samples will be analyzed within
48 hours of Collection.
Source: Standard Operating Procedure for: Dionex ICS-1000 Ion Chromatography System
Sample Preparation for Ion
Chromatography

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Practical Applications
▪ Softening of Hard Water and Separation of similar ions.
▪ It is useful for pre-concentration of trace components of a solution to obtain
enough for analysis
▪ Water polishing equipment used in many laboratories uses several ion
exchange cartridges.
▪ Ion exchange chromatography is used to convert one salt to other.
▪ Ion exchange is used to prepare de-ionized water.
▪ Removal of interfering radicals.
▪ Complete demineralization of water.
▪ To concentrate metal ions in the sample.
Source: Ion Chromatography in Environmental Analysis Article - 2012

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Conclusions
▪ The most significant part of ion chromatography is a broad range of application, well
developed hardware, many detection options, reliability with good accuracy and
precision, high selectivity, high speed, high separation efficiency, good tolerance to
sample matrices, and low cost of consumables.
▪ With this included, ion chromatography is widely accepted as the standard reference
methodology not only for water and waste water analysis, but also for many other
matrices.
▪ The use of ion chromatography over conventional methods of inorganic determination
was a crucial progression in the field.
▪ The development of ion chromatography allows the determination of ionic
contaminants in samples with very low detection limits and expands the range of
determined analytes. Ion chromatography will continue to develop as more and more
ionic contaminants become regulated at increasingly lower limits in the future.

20. 20
Thank You!