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The CHROMacademy Essential Guide - On Demand webcast

Introduction to Ion Chromatography

The Essential Guide from LCGC’s CHROMacademy present an educational webcast on the fundamental
principles of ion chromatography. In this session, Dr. Ken Cook (Bio - Separations Manager, Dionex)
and Tony Taylor (Technical Director, Crawford Scientific), present a definitive guide to the fundamental theory
and instrumentation of ion chromatography.

This guide considers the underlying theory of the technique, column and eluent selection and key separation variables as well as the various techniques used for ion suppression and detection. We also consider various applications of ion chromatography together with recent advances in the technique including capillary ion chromatography, fast and ultra high pressure ion chromatography as well as hyphenation of ion chromatography to mass spectrometric detectors.

A must see for everyone using or considering ion chromatography.

Tony Taylor
Technical Director
Crawford Scientific

Dr. Ken Cook
EU Separations Science Support Manager
Dionex / ThermoFisher, UK

 

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Topics include:

Basic theory of ion chromatography
  • Separation and detection
The ion chromatography system
  • Post column suppression
  • Detector types
  • Eluent preparation strategies
  • Detection strategies
Column choice and basic applications of ion chromatography
  • Column loading
  • Temperature effects
  Eluents for ion chromatography
  • Typical buffers
  • Altering elution times and selectivity
  • Quantitative aspects
Advances ion chromatography
  • Fast ion chromatography
  • Capillary ion chromatography
Hyphenating ion chromatography to mass spectrometry

Ask questions, share knowledge, join your colleagues in the Big Chromatography Conversations - Join us on //chrommunity.chromacademy.com

 


The CHROMacademy Essential Guide
Introduction to Ion Chromatography – Tutorial 31st August 2011
Ion chromatography is a generic term that applies to any method for chromatographic separation of ionic or ionisable species in solution.

The term ion chromatography (IC) encompasses a range of different techniques; however, the most important forms of IC are based on each of the following four separation mechanisms: [1]

  • Ion-exchange chromatography
  • Ion-exclusion chromatography
  • Ion-pair chromatography
  • Ion-suppression chromatography

Although some of the above mechanisms (like ion-suppression) do not involve traditional ‘ion exchange’ separation mechanisms, they are still considered forms of ion chromatography and are critical concepts within many ion chromatographic separations.

Based on ionic interactions between analyte ions and polar functional groups in the stationary phase, ion exchange chromatography is the most widely used of all forms of ion chromatography . [2-9]

 

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Applicability of selected forms of liquid chromatography

 

IC - Ion Chromatography
NPC - Normal Phase Chromatography
IPC - Ion Pair Chromatography
RPC - Reversed Phase Chromatography

 

 

 

« Figure 1: 

Applicability of selected forms of liquid chromatography

 
 
Nowadays the vast majority of ion chromatographic separations are dominated by ion exchange mechanisms using stationary phases with charged functional groups.[2]  These types of mechanisms dominate the separation of analytes that permanently hold electrostatic charges (i.e. weak or strong acidic/basic species or inorganic ions).
 

Analytes typically analysed with suppressed conductivity detection Ion Chromatography (IC)

Figure 2:  Species typically analysed with suppressed conductivity detection Ion Chromatography (IC)

Ion-exchange chromatography (IEC) is based on the different affinities of the analyte ions for oppositely charged ionic functional groups in the stationary phase or adsorbed counter ions.[2, 3]

 
 

Nowadays the vast majority of ion chromatographic separations are dominated by ion exchange mechanisms using stationary phases with charged functional groups.[1]  These mechanisms dominate the separation of analytes that hold electrostatic charges (i.e. weak and strong acidic/basic species or inorganic ions) and are used in a wide variety of applications as indicated in Figure 3.

 
 
  • Environmental Testing Labs
  • Water providers
  • Wastewater treatment facilities
  • Pharmaceutical companies
  • Chemical/petrochemical companies
  • Food producers and processors
  • Power utilities
  • Electronics manufacturers
  • Mining/metals/plating companies
  • Government agencies
  • Universities
  • and more…
 
Who uses Ion Chromatography
 

Figure 3:  Example of industry / application area in which ion chromatography is used

 

Ion-exchange chromatography (IEC) is based on the different affinities of the analyte ions for the oppositely charged ionic functional groups in the stationary phase or adsorbed counterions.[2, 3] When analysing inorganic ions this form of chromatography is often generically referred to as Ion Chromatography.

Depending on the charge of the exchange centres on the surface, the resin could be either an anion-exchanger (positive ionic functional groups on the surface) or cation-exchanger (negative functional groups on the surface).  The process for the retention and separation of both anionic and cationic species are shown in Figure 4, a) and b).

 
 

Anion exchange chromatography

Figure 4a: Anion exchange chromatography in which the stationary phase is a strongly cationic resin / polymer and analyte (chloride ion, Cl-) retention is controlled by altering the ionic strength of the eluent species, typically using the anionic counter ions shown, which are listed by counter ion ‘strength’

 
 
Cation exchange chromatographyFigure 4b:Cation exchange chromatography, undertaken using a anionic polymer surface and using cationic counter ions as shown to selectively elute the analyte (sodium, Na+)
 
 

In ion-exchange chromatography, retention is based on the affinity of different analyte and counter ions for the charged site on the stationary phase surface and on a number of other solution parameters such as counter ion type (strength) and concentration. 

 
Variables affecting retention

Figure 5:  Variables affecting retention in ion chromatography as exemplified by the separation of anions of various ‘size’ (hydrodynamic volume) and charge

 

In general, the elution (eluotropic) strength of the mobile phase is controlled via the nature of the counter ion chosen (some counter ions are more surface active than others and therefore displace analytes ions more readily) and the concentration of the counter ion (displacement by the law of mass action).

The selectivity in ion exchange chromatography is mainly based on differences in the material of column construction and the functional groups bonded to the polymer surface (stationary phase).

 
 

The main mechanisms for controlling retention and selectivity are shown in Figure 6.

Altering Selectivity in Ion Chromatography

 

Altering Selectivity

 

 

 

 

 

 

 

 

 

 

Figure 6: Principle variables for altering retention
and selectivity in ion chromatography

 

 
 

Mechanisms

Consider the exchange of two ions  and  between the solution and exchange resin R- :[1, 6]

The equilibrium constant for this process is:

K essentially determines the relative affinity of both cations to the exchange centres on the surface.  If the constant is equal to one, then no discriminating ability is expected for the system.

Similarly, the exchange of two ions C- and D-  between the solution and exchange resin E+ :

Depending on the charge state of the exchange functional groups on the surface, the resin could be either anion-exchanger (positive ionic functional groups on the surface) or cation-exchanger (negative functional groups on the surface).

 
 
 

Animation 1:  The cation exchange chromatography mechanism (oversimplified)

 

 

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Practical Ion Exchange Chromatography

In general terms, the ion exchange chromatographic process can consist of four main steps.

 

Step 1. Equilibration of the stationary phase to the desired conditions: 
When the equilibrium is reached all functional groups in the stationary phase are associated with exchangeable counterions.  See below:

 

 

Important pointers to consider:

  • In the case of anion exchangers, the exchangeable counter-ions would be of anionic nature (such hydroxide, carbonate, borate etc)
  • In the case of cation exchangers, the exchangeable counter-ions would be of cationic nature (H+ from HCl, methanesulfonic acid, tartaric acid etc)

 

Animation 2.  Stationary phase equilibration on a cation exchange column (oversimplified).

 
 

Step 2. Sample application and wash:
The objective is to bind analytes to the stationary phase while washing out unwanted constituents.  See below:

 

 

Where: is the Analyte of interest

 

Note that in many applications the optimum separation may be achieved by choosing conditions so that major and troublesome contaminants are bound to the exchanger while the substance of interest are eluted during the washing step. This procedure is sometimes referred to as “starting state elution”.

 

Animation 3. Sample application and stationary phase
wash on a cation exchange column (oversimplified).

 

 
 

Step 3. Elution of retained analytes from the column:

This step is accomplished by a change in the buffer composition, usually increasing the ionic strength of the mobile phase would be enough to elute all retained analytes.  See below:

 

 

Where: is the Analyte of interest

 

The highest ionic strength which permits binding of the analytes of interest and the lowest ionic strength that causes their elution should normally be used as the starting and final ionic strengths in subsequent analysis.  A higher ionic strength buffer is frequently used as a washing step before column regeneration.

 

Animation 4. 
Cation exchange sample elution (oversimplified).

 

 
 

Step 4. Column Regeneration:
All molecules that still remain bounded to the stationary phase must be removed to restore the full capacity of the stationary phase.  This step is usually accomplished by changing the eluent system or buffer composition.  See below:

 

 

Animation 5. 
Cation exchange column regeneration (oversimplified).

 

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In general terms there are only a few additions to a traditional HPLC system in order to achieve ion exchange chromatographic separations; primarily because the critical elements required for a good chromatographic separation remain the same (good mass transfer, low dead volume, suitable mobile and stationary phases).[9]

As in traditional HPLC, ion chromatography pumping systems use reciprocating pumps which cope with the pressure and volumetric needs of most ion chromatography applications.  As many of the eluent systems used in ion chromatography can be corrosive, ion chromatographs tend to be constructed from, or have hydraulic pathways which are constructed from polymer materials (PEEK, polyether ether ketone is typical)

 

Figure 7: » Typical reciprocating pump design with polymer components and/or hydraulic pathways used for corrosive eluent systems in ion chromatography

 

 

Columns packed with suitable packing materials have been developed to provide good separation performance in minimum time.  The working life of the column can be increased by using a filtering system (guard column or in-line filter) between the autosampler and the column.

The electrical conductivity detector is one of the most important detection types for ion chromatography.  It actually measures the conductivity of the mobile phase and therefore it is not a solute property detector but a bulk property detector.  The principles and working principles of detectors for ion chromatography will be given in another chapter.

 

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Typical reciprocating pump

 
 

Figure 8:  Schematic diagram of an Ion Exchange Chromatograph (Courtesy of Dionex, a Thermo Fisher Company)

Perhaps the most noteworthy differences between a standard HPLC system anda modern Ion Chromatograph are the  Eluent Generator and Electrolytic Eluent Suppressor. These components will be dealt with in greater detail in subsequent sections.

 

 

The quality of the eluent system is of overriding importance to ion chromatography, especially the level of potentially interfering background ions, as one might expect.  Solutions should be freshly prepared with high quality water and additives.  Please bear in mind that eluent systems for ion chromatography should always be.[10]

  • Microfiltered: use 0.45 μm filter
  • Degassed: use helium or vacuum
  • Continuously stirred: use magnetic stirrers
  • Freshly prepared: ideally on a daily base
  • Prepared at the correct standard: use high quality water and chemicals

It is highly advisable to use carbon dioxide absorbers, especially when dealing with either alkaline or low buffering capacity eluent systems.

In order to avoid CO2 absorption, eluent reservoirs must be well closed with a head pressure of Helium.  Remember that carbonate absorption onto low concentration OH eluents can lead to drifting problems early in the gradient.

Note that precipitation should always be avoided; this is of particular importance when changing eluent system.  Solutions used in succession should be miscible.  If the system has to be rinsed with an organic solution, then use solvents with increasing or decreasing lipophilic character (e.g. chloroform ↔ acetone ↔ water).

 
 
 

Buffers

In ion chromatography, the required initial buffer concentration will vary depending on the nature of the buffering species (i.e. buffer ion ‘strength’). 

Relative affinities of counter ion for the stationary phase versus the analyte ion will vary depending upon the ion-exchanger type and the analytical conditions.  However it is possible to give an approximate ‘relative’ series in terms of counter ions, although modern resins are often designed to operate with simple H+ and OH- counter ion gradients.

As a rule, an increase of the charge-density (charge to solvated ion ratio) of the solute ion will increase retention due to increased coulombic interactions as the analyte and surface charges move closer together.  This trend becomes more pronounced in more diluted mobile phases.

The order of affinities of commonly available cations relative to strong acid cation exchange stationary phases are generally in the following order:

Ba2+ >  Ca2+ >  Mg2+ > Cs+ >  K+ >  NH4+ >   Na+ >  H+
Cation-exchange mobile phases of 0.1 M KCl are stronger then those containing 0.1M NaCl, provided that all other parameters are identical.

And similarly the relative affinity of anions can be approximated as:
citrate> salicylate> ClO4 > S2O32- > HPO42- > NO3- > Br- > NO2- > CN- > Cl- > HCO3- > H2PO4- > CH3COO- > HCOO- > BrO3- > ClO3- > F- > OH-

Higher degrees of cross linking result in ion-exclusion effects, i.e., exclusion of ions with higher solvated radii from the stationary phases pores.  Since these ions are also less retained, they elute more quickly than ions with a smaller hydrodynamic radius, which can enter the small pores.  More highly charged ions and small radius are polarizable are therefore retained longer.  The effects of the last two properties in difficult to predict, making the relative series above tentative, rather than absolute.

 

 

Eluent Generation / Re-generation

Most modern instruments are equipped with eluent re-generation or generation devices which function to either re-generate the eluent system post detection OR generate simple (H+ and OH-) eluent  systems ‘in-situ’, which significantly improve the ‘convenience’ of the system and serve to reduce operator error.   There are many different strategies to generate high quality IC eluent systems and therefore, it would be unpractical to try to cover all of them.[11, 12]

In Figure 9, after the conductivity cell, the eluent system is recycled as a source of water for the electrolytic suppressor.  Note that there is an ion trap column between the conductivity cell and the suppressor to guarantee that analyte and impurity ions will not reach the electrolytic suppressor.  A fraction of the eluent system exiting the electrolytic suppressor is degassed prior to regeneration (an appropriate eluent purification column should be used) and collection into the eluent reservoir.  See below.[13, 14]

 
IC system with eluent recycle
 
Figure 9: IC system with eluent recycle (Courtesy of Dionex, a ThermoFisher company)
 
 
 
Figure 10: IC system with eluent generation (Courtesy of Dionex, a ThermoFisher company)
 
 

Eluent generation systems based on resins are currently used in many modern IC systems.  In essence, two separate chambers form part of an electrochemical cell [15, 16]

Water is oxidized to form H+ ions and oxygen at the anode:

These protons interact with a resin and displace K+ ions from its surface:

Water is reduced to form OH- ions and hydrogen at the cathode:

 
 
As a consequence, a flow of KOH is produced.  Figure 11 below illustrates the working principle of this device.
 
IC eluent generation device
 

Figure 11:  IC eluent generation device (Courtesy of Dionex, a ThermoFisher company)

Note that other designs are available.

 
 
Figure 12 below shows a 3D schematic view of the previous device.
 
3D schematic
 
Figure 12:  Hydroxide Eluent Generation for Anion Analysis
 
 

Separation and Linearity of detector response

Figure 13:  Separation and Linearity of detector response of sulfate with KOH eluents.  Conditions: 10cm ID × 20 cm length (1.7 meq/g) K+ ion supply column, will generate a flow of KOH (20.0 mM) that at 1.0 mL/min will last for up to 2225 hours (considering 100% efficiency) or 1300 hours (considering 60% efficiency).
 
 
High-resolution anion analysis
 

Figure 14:  High-resolution anion analysis with eluent generation (conditions shown)

 
 
Electronically generated eluents
 

Figure 15:  Electronically generated eluents provide highly repeatable gradients

 
 

The column stationary phase is responsible for the ion exchange separation and its properties are of primary importance for successful separations (retention and selectivity).

The lifetime of the ion chromatography column is maximized through the use of stable bonding chemistry, and optimal proprietary packing procedures.

When selecting a column for a particular separation, the chromatographer should be able to decide whether a packed, capillary, or monolithic column is needed and what the desired characteristics of the packing material should be.

As with pumps, due to the corrosive nature of the eluent system, IC columns are usually based on alternative materials (such as PEEK) rather than stainless steel.

 

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Supports

The separation properties of IC columns are basically determined by the functional groups bonded to the support material.  Certain analytes, however, can interact with the support material.  The pdf below lists selected support materials for IC columns.[1, 2, 3, 5, 6, 17 - 22]

Ion Chromatography resins have to withstand extremes of pH as the eluents can often be KOH or Methane Sulfonic acid. The most common support material is polystyrene with divinyl benzene cross links [PSDVB].


Click here to view the - Selected-Support-Materials-for-IC-Columns.pdf

 

Examples of column packing morphologies are given below:

 

column packing morphologies

Figure 16:  Particle morphologies for modern ion chromatography columns

 

 

Functional Groups

Ion exchangers are prepared by attaching ionic functional groups to the supporting matrix.  Here the functional groups are attached to a largely polar matrix and the dominant forces that control retention will be ionic and polar.

The most common functional groups in commercially available ion exchangers are sulfonic and carboxylic acids for cation exchangers and tetramethylamino functional groups for anion exchangers. .[1, 2, 3, 10, 20, 21, 22]

Click here to view the - Selected-Ion-Exchangers.pdf

 

 
 

Anion Exchangers

The following pdf will help you to identify the stationary phase types and applications for various commercially available phases.

Click here to view the - Anion-Exchange-Columns

 

 
 

Cation Exchangers

Cation exchange column selection.[1, 2, 10, 17 - 23]

Click here to view the - Cation-Exchange-Columns

 
 

The Exchange Capacity Q

The total exchange capacity of an ion exchange resin (Q) is a quantitative parameter which defines the total number of chemical equivalents available for exchange per some (nominal) unit weight or unit volume of resin. The capacity may be expressed in terms of milliequivalents per gram of resin or in terms of milliequivalents per gram of resin (mmol/g or in meq/g). 

The unit equivalent (eq) can be defined as the amount of substance which will either:

  • react with one mole of hydronium ions (H3O+) or hydroxyl (OH-) ions in an acid-base reaction
  • react with or supply one mole of electrons in a redox reaction

As a matter of fact, the ion exchange capacity is usually expressed in meq (1000 meq = 1 eq) per gram

According to their exchange capacity, there are roughly three types of ion exchangers:

  • low-capacity ion exchangers: Q < 0.1 mmol/g
  • medium-capacity ion exchangers: 0.1 < Q < 0.2 mmol/g
  • high-capacity ion exchangers: Q > 0.2 mmol/g
 

Ion exchanger capacity classification

Figure 17:  Ion exchanger capacity classification

 
 

Effect of Stationary Phase Capacity

The higher the capacity, the greater the retention but the more difficult to suppress the strong eluents required for elution from the column.

  • Most ion exchange column have low capacity to improve detection and sensitivity
  • Complex samples and measurement of trace levels in the presence of high level matrix ions require higher capacities
 
Separation of anions using resins  

These principles are demonstrated in Figure 18 in which a high capacity resin is required to gain enough retention in order to effectively separate a range of anions.

 

Analyte Ions:

1.  Fluoride 
2.  Chloride 
3.  Nitrite
4.  Phosphate 
5.  Bromide 
6.  Nitrate 
7.  Sulfate

 

« Figure 18:  Separation of anions using resins of differing capacity (HC indicates High Capacity!)

 
 

Suppressor systems are used to increase sensitivity of the technique by lowering the column’s effluent background conductivity before it enters the detector.  As a consequence, ion suppressors may be considered integral parts of the detection system.  Different approaches had been implemented to achieve such aim.

Ion suppressors are designed to lower the ionic strength of the column’s effluent, allowing the use of conductivity detectors.

There are two ways of ion suppression

  • Continuous (columns)
  • Discontinuous (regenerating devices)

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Column Suppressors

Columns containing strongly acidic cation exchange resins have been used to decrease conductivity background in anion exchange chromatography.

The principle of column ion suppression relies on the use of weakly dissociating species in the eluent system.  Let’s consider an eluent system that contains the strongly conducting sodium bicarbonate.  In this case, conductivity can be reduced by using a strong cation exchange resin.

 

Resin - SO3H + NaHCO3 Resin - SO3Na + (CO2 + H2O)

 

The weakly dissociated carbonic acid (CO2 + H2O)  is formed from the strongly conducting sodium bicarbonate (NaHCO3 ).  The depletion of sodium bicarbonate will be reflected in a reduction of eluent conductivity.

 

Inorganic salts (such as sodium chloride or sodium bromide) can undergo a similar process:

 

Resin - SO3H + NaCl Resin - SO3Na + HCl

 

The inorganic acid (HCl) thus produced has a higher conductivity than the original salt (NaCl).  The signal to be measured is the conductivity of HCl (high) against a low background conductivity.

Low column capacity and high dead volume are the major drawbacks of suppression columns. In fact, the need for periodic regeneration, has limit the use of suppression columns. 

 
Column suppression

« Figure 19:  Column suppression
use to reduce KOH background
signal in ion
chromatography

 

 

Dual suppression column systemThe use of switching valves and suppression columns in parallel have been used to decrease system dead time during re-generation.  Of course, this requires the addition of an extra pump to the system for the off line re-generation of the supprerssor.

 

 

 

 

 

« Figure 20:  Dual suppression column system, allows off-line re-generation which speeds up analysis with this type of suppressor system

 

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Hollow Fiber Suppressors

A hollow fiber suppressor consists of a semi-permeable membrane that is wrapped around a cylindrically shaped body.  While the eluent system flows through the fiber, a dilute solution (either acidic or basic) flows countercurrent to the eluent, in contact with the exterior of the fiber.

Sulfuric acid based hollow fiber device

 

 

 

 

 

 

 

Figure 21.  Sulfuric acid based hollow fiber device

 
 
In the animation below, the ionic strength of an eluent system composed of NaHCO3 is chemically reduced by using a hollow fiber suppressor that uses a dilute solution of sulfuric acid (H2SO4).
 

Animation 6:  Hollow fiber suppressor operation (sodium hydrogen carbonate buffer strength reduced using sulfuric acid)

 
 

 

Continuous chemical suppression Figure 22:  Continuous chemical suppression used to reduce a sodium carbonate background

 

Hollow fiber suppressors permit continuous IC operation; however, the effects of bandbroadening due to increased extra column volume and the relatively low capacity made their use obsolete by instrument manufacturers in favor of flat screen membrane suppressors.

 
 

Flat Membrane Suppressors (Micro-Membrane Suppressors)

With a much higher capacity and lower dead volume than any of its previous counterparts, flat membrane suppressors (also called ‘micro-membrane’ suppressors) are capable of continuous operation with minimal attention and minimum extra column volume.

In the same manner as hollow fiber suppressors, flat membrane suppressors use the principle of continuous regeneration; however, in comparison, feature a much higher exchange capacity.  Strong ion exchange suppressor screens and ion exchange membranes are placed in alternating order, separated by ion-exchange membranes which allow very close contact of the eluent and regenerant flows.  The basic design is shown in Figure 23 below.

 
Self regenerating continuous suppression device principle

Figure 23:  Self regenerating continuous suppression device principle

 
 

Electrochemical Suppressors

The ion suppressor device shown in Figure 24,[24] uses platinum electrodes for the hydrolysis of water to produce H+/OH- ions and semi-permeable ion exchange membranes to selectively reduce the ionic strength of the eluent system.  Organic solvent may be added as a makeup flow to aid the desolvation process in the electrospray interface but this is often not required.

Post-column anion suppressors work in a similar manner to cation suppressors but with opposite charge.

 

Electrochemical self regenerating continuous suppression device

 

 

 

 

 

 

 

 

 

 

 

Figure 24:  Electrochemical self regenerating continuous suppression device

 
 
 

Animation 7:  Self regenerating continuous suppression process

 

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Electrochemical suppression schematic

Figure 25:  Electrochemical suppression schematic

 
The invention of the high capacity, continuous suppression system allowed the use of higher capacity ion exchange columns and gradient elution for more difficult sample analysis and much higher resolution. The suppression of strong eluents can be regarded as part of the detection system and is strongly linked with the wide choice of columns now available for ion chromatography
 
 

Overview

The ion chromatography detection system is used to monitor the passage of the components as they emerge from the column.[1]

As with any chromatographic technique, the detector measures some physico-chemical property of the mobile phase/analyte as it elutes from the column.  The response of the detector will change due to changes in the column’s effluent.

Because electrolytic suppressors are designed to reduce ionic strength, the column’s effluent can be detected by conductivity.

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Others detection systems

  • Mass Spectroscopy
  • Evaporative Light-Scattering Detector (ELSD). [25, 26]

The conductivity detector is by far the most commonly used of all IC detectors.

 

Detector

Selectivity

Sensitivity

Refractive Index

Low

1 – 5 μg

Conductivity

Low

10 – 50 ng

UV/Visible

Medium

0.5 – 1.0 ng

Electrochemical

High

50 – 500 pg

Fluorescence

High

10 – 100 pg

Mass Spectrometer

High

10 – 100 pg

LSD

High

10 – 20 ng

Table 1:  Selected detection types in ion chromatography

 
 

Direct Ion Chromatography

Direct ion chromatography refers to the form of IC where the column’s effluent is directly fed to the detector without any ion suppressive treatment.

The major challenge in this mode is to select an eluent system which does not produce a detector response in order to maintain required sensitivity.

Due to the drawbacks this is now rarely used

 

Indirect Ion Chromatography

Indirect ion chromatography refers to the form of IC where the column’s effluent is directly fed to the detector with a deliberate high background eluent system and the ions are detected as negative dips in the high background. The polarity of the signal is usually reversed to simulate a positive peek in the trace which analysts and chromatography management systems are more used to. The high background in this system does give noisy and unstable baselines and could only be used reliably for high levels.

Due to its high sensitivity and reduced background noise, the most common form of conductivity detection implements the use of ion suppression.

It has been reported that when using conductivity detectors, the use of ion suppression will increase sensitivity when analyzing cations; however, when dealing with anions, the use of ion suppression will decrease sensitivity.  The table below summarizes selectivity experimental observations with conductivity detectors.[2, 27, 28]

 

Determination of

With Ion Suppression

Without Ion Suppression

Cations

Higher Selectivity

Lower Selectivity

Anions

Lower Selectivity

Higher Selectivity

Table 2:  Selectivity experimental results with conductivity detection

The table above lists suitable and unsuitable IC detection strategies for selected compounds.

 
 

Conductivity Detectors

Belonging to the electrochemical group, conductivity detectors, are by far the most widely used of all detectors for ion chromatography.  Here, the conductivity of the column’s effluent is measured by a detection system consisting of two electrodes to which an alternating potential is applied. The corresponding current is proportional to the conductivity of the ionic solution in which the cell is dipped.

The conductivity detector can be used either with or without a suppressor system. The main function of the suppressor system is to reduce the high background conductivity of the column’s effluent, without suppressing or adversely affecting the signal of analyte ions.

Animation 8:  Schematic and operating principle of a typical conductivity detector

 
 
Conductivity detection principleFigure 26: 
Conductivity detection principle
 
 

The conductivity of a solution is measured by applying an alternating voltage between two electrodes in a conductivity cell.

At any instant in time, negatively charged anions migrate toward the positive electrode and positively charged cations migrate toward the negative electrode.

The measured electrical conductivity  (measured in Siemens per cm) is given by:

 

 

Where:

 Equivalent conductivity [S cm2 mol-1]
 Equivalent concentration of the electrolyte [equivalents/1000cm3]

As can be seen from the previous expression, the electrical conductivity is proportional to the electrolyte concentration; this linear relationship can be expected only for dilute solutions.  However, the linear association between  and  is adversely affect by the following factors:

  • The equivalent conductivity ( ) is dependent upon electrolyte concentration
  • The electrical conductivity ( ) is dependent upon temperature and solvent polarity

The sensitivity of the conductivity measurement depends upon the difference between equivalent conductivities of analyte (subscript a) and eluent ions (subscript e):

 

Relation of Conductivities Resulting peak
Positive peak
No peak
Negative peak

 

Table 3.  Relation between equivalent conductivities of analyte and eluent ions and resulting peaks

 

 

The dependence between electrical conductivity and temperature is sometimes, very important.  As a consequence, temperature control should be implemented (ideally, constancy should be kept within 0.01 oC of target temperature). This is particularly important if suppression is not used as the background conductance will then be high and so variable to temperature changes.

 

Conductivity detection calibration curves

 

Figure 27:  Conductivity detection calibration curves for nitrate determination with and without suppression

 

 

 

 

 

In cases where chemical suppression is used, curved calibration functions can be found and is dependent on the eluent and suppression system used (see the figure above).  In such cases, it is recommended that calibration concentrations as close as possible to the concentration of the sample, (although modern data systems are able to model non-linear calibration functions).  This approach will render the most accurate results.  OH eluent systems especially those generated electrochemically reduce the background signal to bellow 1µs. This results in an essentially linear calibration curve for most analytes. With carbonate eluent systems  the background signal following suppression can be between 10 to 30 µs depending on the concentration used. This can result in curved or quadratic calibration curves.

 
 

IC-MS is a hyphenated technique, which combines the separating power of ion chromatography with the detection advantages of mass spectrometry.

Ion chromatography has been hyphenated to a range of detection techniques including mass spectrometry and atomic absorption.  The coupling of IC to these techniques can be accomplished with the implementation of post-column ion suppressors.

Note that MS renders more confidence in analyte identity than other IC detection types; this is because, IC-MS not only provides the analyte’s retention time but also it’s mass to charge ratio.  This confidence could be even enhanced by the use of tandem MS techniques.  Mass spectrometry detection can help to address situations where:

  • Specific compound identification is required
  • Very low limits of detection are required
  • Analytes co-elute

Post-column ion suppression provides the means to effectively reduce the high ionic strength of IC eluents to make them compatible with MS systems.

The conductivity of the ion suppressor’s effluent is constantly monitored and is kept below certain value (usually 1.0 μS cm-1) before infusion into the MS detector. To achieve this OH based eluent systems are used with eluent generation systems and electronic suppression to give high purity eluents and the lowest ion conductance delivered to the MS system.

These systems are particularly important in cellular metabolism studies as a significant number of analytes in central metabolism are charged or highly polar. This gives them poor retention on reverse phase columns but excellent chromatography characteristics on ion exchange. For many isobaric metabolic intermediates, it would be essential to have chromatographic separation in addition to mass / charge ratio to positively identify.

 

MS compatible Ion Exchange

  • Continuous Suppression IC system

Electrolytic gradient generation

  • isocratic deionised water used from the pump
  • gradient generated pre-column electrolytically

Electrolytic suppression

  • Exchange of post-column K+ with H3O+ using electronic suppressor
 
 
The figure below illustrates the coupling of ion chromatography with MS detection system.  Please bear in mind that temperature control and solvent degassing are of overriding importance to ion chromatography.
 

IC-MS schematic

Figure 28:  IC-MS schematic

IC Capillary Columns

There is an increasing interest in the development of capillary ion chromatography.  The use of small-bore columns with reduced diameters (usually lower than 1.0 mm id) present several significant advantages including:

  • Small sample volumes
  • Lower eluent consumption
  • Faster separations

There are many situations where resolution is more than adequate to separate sample components and in such situations, it is possible to optimize column dimensions and eluent flow rates in order to decrease analysis times while maintaining sufficient resolution.  Capillary ion chromatography columns provide the means for speeding up separations (usually up to four times) whilst maintaining acceptable resolution.

 

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Conventional IC

Capillary IC

Column  i.d.

4 mm

0.40 mm

Flow Rate

1.0 mL/min

10 µL/min

Injection Loop

25 µL

0.4 µL

Suppressor Dead Volume

60 µL

0.6 µL

EG Current (50 mM KOH)

80.4 mA

0.804 mA

K+ Consumption/Year

26.3 Moles
(50 mM KOH)

0.263 Moles
(50 mM KOH)

H2O Consumption/Year

525.6 L

5.256 L

 

Table 4:  Typical Conventional and Capillary IC Operating Parameters

 
 

Separation of common anions

 

Figure 29:  Separation of common anions using a capillary AS19 column

 
 

Fast IC inorganic capillary anion

 

Figure 30:  Fast IC inorganic capillary anion analysis at different flow rates

 
 
 

Figure 31:  Selected applications of ion chromatography.

 

References:
 
  1. Joachim Weiss.  “Handbook of Ion Chromatography”  Third Edition, @ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, ISBN 3-527-28701-9. Chapters 1 and 9.  Germany, 2001
  2. SPE Mechanisms.  Solid Phase Extraction from “Sample Preparation” from CHROMacademy.
  3. Helwig Schäfer, Markus Läubli and Roland Dörig.  “Ion Chromatography” Chapter 1.  Metrohm Ltd. CH-9101 Herisau Switzerland
  4. James S. Fritz and Douglas T. Gjerde.  ”Ion Chromatography” Chapter 1. 4th Ed. Copyright © 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 978-3-527-32052-3
  5. Pavel N. Nesterenko. ‘Simultaneous Separation and Detection of Anions and Cations in Ion Chromatography’, Trends in Analytical Chemistry, vol. 20, nos. 6-7, Pp 311-319, 2001
  6. Chi-san Wu “Handbook of Size Exclusion Chromatography” Chapter 1. ISBN: 0-8247-9288-2. Copyright © 1995 by Marcel Dekker, Inc. All Rights Reserved. New York
  7. Joachim Weiss.  “Handbook of Ion Chromatography”  Third Edition, @ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, ISBN 3-527-28701-9. Pp 359-361.  Germany, 2001
  8. Ion-Pair Chromatography Module from “The theory of HPLC”.
  9. Controlling the Extent of Ionisation.  Reverse Phase (Partition) Chromatography from “The theory of HPLC”.  LC/HPLC Channel from CHROMacademy.
  10. A. Seubert, W. Frenzel, H. Schäfer, G. Bogenschütz and J. Schäfer.  “Probenvorbereitungstechniken Für Die Ionenchromatographie”  Gedruckt in der Schweiz bei Metrohm AG, CH-9101 Herisau .2001
  11. Hamish Small, John M. Riviello, Yang Liu and Nebojsa Avdalovic.  “Ion Chromatographic Method and Apparatus Using a Combined Suppressor and Eluent Generator”  USA Patent # 6027643.  Feb 22, 2000.
  12. Yang Liu and Nebojsa Avdalovic.  “Electrolytic Eluent Generator and Method of Use”  USA Patent # 7767462 B2.  Aug 3, 2010.
  13. Yang Liu and Nebojsa Avdalovic.  “Electrolytic Eluent Generator”  USA Patent # 0211125 A1.  Sep 21, 2006.
  14. Kannan Srinivasan, Rong Lin, Sheetal Bhardwaj and Christopher A. Pohl.  “Ion Chromatographic System With Eluent Recycle”  USA Patent # 0211979 A1.  August 27, 2009.
  15. Yang Liu, Hamish Small and Nebojsa Avdalovic.  “Large Capacity Acid or Base Generator Apparatus”  USA Patent # 7153476 B2.  Dec 26, 2006.
  16. Yang Liu, Kannan Srinivasan, Christopher A. Pohl, Sheetal Bhardwaj and Zhongqing Lu.  “Ion Chromatographic Systems With Flow-Delay Eluent Recycle”  USA Patent # 0174737 A1.  Jul 21, 2011.
  17. Marvin C. McMaster.  “HPLC A Practical User’s Guide”  Second Edition.  Pp 56, 84-86.  Copyright © 2007 by John Wiley & Sons, Inc. Hoboken, New Jersey.
  18. Yuri Kazakevich and Rosario LoBruto.  “HPLC For Pharmaceutical Scientists”  Pp 75-115. Copyright © 2007 by John Wiley & Sons.  Hoboken, New Jersey
  19. Xiaodong Liu and Christopher Pohl.  “New Weak Cation-Exchange/Reversed-Phase Mixed-Mode Column for Pharmaceutical Applications”  Dionex Corporation, Sunnyvale, CA USA LPN 2047-01 06/08 ©2008 Dionex Corporation.
  20. Krisztián Horváth, Péter Hajós.  “Retention profiles and mechanism of anion separation on latex-based pellicular ion exchanger in ion chromatography”  Journal of Chromatography A, 1104 (2006) 75–81
  21. Paul F. Holmes, Mike Bohrer, Joachim Kohn.  “Exploration of polymethacrylate structure–property correlations: Advances towards combinatorial and high-throughput methods for biomaterials discovery”  Progress in Polymer Science 33 (2008) 787–796
  22. Brad Busche, Robert Wiacek, Joseph Davidson, View Koonsiripaiboon, Wassana Yantasee, R. Shane Addleman, Glen E. Fryxell.  “Synthesis of nanoporous iminodiacetic acid sorbents for binding transition metals” Inorganic Chemistry Communications 12 (2009) 312–315
  23. “Ion Exchange Chromatography.  Principles and Methods”  Amersham Biosciences.  Chapters 2, 7 and 9. ISBN 91 970490-3-4
  24. John M. Riviello.  “Method of Ion Chromatography Wherein a Specialized Electrodeionization Apparatus is Used”  United States Patent.  US2006/0231404 A1.  USA Oct 19, 2006.
  25. Jing Li, Meilan Chen and Yan Zhu.  “Separation and determination of carbohydrates in drinks by ion chromatography with a self-regenerating suppressor and an evaporative light-scattering detector”  Journal of Chromatography A. Volume 1155, Issue 1, 29 June 2007, Pages 50-56
  26. Scott, Raymond P. W.  “Chromatographic Detectors.  Design, Function and Operation”  Pp 211-214.  Marcel Dekker Inc.  1996
  27. Helwig Schäfer, Markus Läubli and Roland Dörig.  “Ion Chromatography.  Theory, Columns and Eluents”  Metrohm Monograph. Pp 1-35. Switzerland. 2003
  28. Claudia Eith, Maximilian Kolb, Andreas Seubert and Kai Henning Viehweger.  “Practical Ion Chromatography.  An Introduction”  Metrohm Ltd.  Pp 7-50. Switzerland 2001.
  29. Joachim Weiss.“Handbook of Ion Chromatography” Third Edition, @ 2004 WILEY-VCH Verlag GmbH & Co. KGaA, ISBN 3-527-28701-9. Chapters 1 and 9. Germany, 2001
  30. Micong Jin, Yiwen Yang.“Simultaneous determination of nine trace mono- and di-chlorophenols in water by ion chromatography atmospheric pressure chemical ionization mass spectrometry” Analytica Chimica Acta 566 (2006) 193–199
  31. Francesca Lesignoli, Andrea Germini, Roberto Corradini, Stefano Sforza, Gianni Galaverna, Arnaldo Dossena, Rosangela Marchelli.
    “Recognition and strand displacement of DNA oligonucleotides by peptide nucleic acids (PNAs) High-performance ion-exchange chromatographic analysis” Journal of Chromatography A, 922 (2001) 177–185
  32. Marjeta Poznič, Roman Gabrovšek, Milko Novič. “Ion chromatography determination of chloride and sulphate in cement” Cement and Concrete Research 29 (1999) 441–443
  33. Anna Błażewicz, Grażyna Orlicz-Szczęsna, Andrzej Prystupa, Piotr Szczęsny. “Use of ion chromatography for the determination of selected metals in blood serum of patients with type 2 diabetes” Journal of Trace Elements in Medicine and Biology 24 (2010) 14–19
  34. Adrian A. Ammann. “Arsenic speciation by gradient anion exchange narrow bore ion chromatography and high resolution inductively coupled plasma mass spectrometry detection” Journal of Chromatography A, 1217 (2010) 2111–2116
  35. Marco Iammarino, Aurelia Di Taranto, Marilena Muscarella, Donatella Nardiello, Carmen Palermo, Diego Centonze. “Development of a new analytical method for the determination of sulfites in fresh meats and shrimps by ion-exchange chromatography with conductivity detection” Analytica Chimica Acta (2010), doi:10.1016/j.aca.2010.04.002
  36. Miravet, J.F. López-Sánchez, R. Rubio. “New considerations about the separation and quantification of antimony species by ion chromatography–hydride generation atomic fluorescence spectrometry” Journal of Chromatography A, 1052 (2004) 121–129
  37. Mona El-Said, Mahmoud Ramzi, Thanaa Abdel-Moghny. “Analysis of oilfield waters by ion chromatography to determine the composition of scale deposition” Desalination 249 (2009) 748–756
  38. P.L. Annable. “Determination of alkyl sulfonic acids in pharmaceuticals by ion chromatography” Journal of Chromatography A, 724 (1996) 199-206
  39. Corrado Sarzanini, Maria Concetta Bruzzoniti, Edoardo Mentasti. “Determination of epichlorohydrin by ion chromatography” Journal of Chromatography A, 884 (2000) 251–259
  40. Christopher Pohl, Maria Rey, Detlef Jensen, Jutta Kerth. “Determination of sodium and ammonium ions in disproportionate concentration ratios by ion chromatography” Journal of Chromatography A, 850 (1999) 239–245
 
Related Reference Materials from CHROMacademy:
 
Introduction to Ion Chromatography *** CHROMacademy Registered users only ***
 
Further reading and resources:
 

Ion Chromatography: An Overview and Recent Developments *** CHROMacademy Registered users only ***

Hyphenated Techniques in Ion Chromatography *** CHROMacademy Registered users only ***

Application of Multidimensional Matrix Elimination Ion Chromatography for Bromate Analysis *** CHROMacademy users only ***

Achievements and Trends in Ion Chromatography *** CHROMacademy Registered users only ***

 
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