The Theory of HPLC

Introduction to Ion Chromatography

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Aims and Objectives

Aims and Objectives

Aims

To introduce some of the working principles of ion chromatography and related

techniques
To highlight the various approaches to analyzing ions in solution
To explain the equipment / eluent modifiers necessary for ion chromatography

Objectives

At the end of this Section you should be able to:

Recognise ion chromatography as a set of chromatographic techniques, suitable

for analyzing ionic species
Describe the correct approach for analysing ions in solution using a variety of
eluent additives and instrumentation
Recognise application types where ion chromatography approaches may be
required
Content

Introduction 3
The HPLC Process 4
Types of Ion Chromatography 5
Acid-Base Chemistry 6
Ion Exchange Chromatography Overview 7
General Ion Exchange Considerations 8
Ion Exchange Mechanisms 12
Ion Exchange Elution Considerations 13
Ion Exclusion Chromatography Overview 15
Ion Exclusion Chromatography Process 16
Ion Pair Chromatography 17
Ion Suppression 18
The Ion Chromatographic System 19
Ion Chromatography Columns 21
Detection 24
Hyphenated Ion Chromatography 26
Applications 27
References 34

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Introduction

The Russian botanist Mikhail Tswett first used the term chromatography (Greek for
coloured writing) in 1906. He used the term to describe the separation that occurred
when solutions of plant pigments were passed through columns of calcium carbonate or
alumina using petroleum ether.[1]

Ion chromatography, a form of liquid chromatography, describes the efficient

chromatographic separation of ionic species using any number of automatic detection
techniques.[2,3,4]

Ion chromatography is the technology of choice for the analysis of ionic (or ionisable)
species in solution from a variety of different application types including food analysis,
pharmaceutical development, corrosion studies, oil exploration, nuclear power plant water
quality control and many more.

The primary objective of this module is to describe in a clear and concise manner the
principles, methods and different chemical and instrument based approaches to ion
chromatography currently used in the modern analytical lab.

A typical Liquid Chromatograph

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Where:

1: The mobile phase composition (usually water and an organic solvent plus other
additives) needs to be optimised in order to gain good separation.
2: Degassers are often used to remove air from the mobile phase leading to better
chromatographic baselines.
3: The detector conditions are chosen to give the best response to the analytes of interest
and achieving good sensitivity.
4: The column dimensions and stationary phase chemistry are chosen and optimised to
give separations of the quality required.
5: The autosampler introduces a plug of sample into the mobile phase flow which is then
swept onto the column.
6: Dual reciprocating pumps are used to deliver the mobile phase at back pressures of up
to 400Bar. As steady stream of liquid delivered at constant flow rate is important.

The HPLC Process

The HPLC process requires a continuous flow of mobile phase, a column that shows
affinity for the sample and a detection system capable of detecting the separated sample
components.

The HPLC pumping system must provide a constant flow of mobile phase and should be
capable of dynamic mixing of the eluent system components where required.

Because the mobile phase flow should not be interrupted during analysis or air introduced,
especially designed HPLC injectors are commonly used to introduce a plug of sample into
the HPLC system.

Liquid chromatographic process

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Once the sample is introduced, the mixture of components is carried in a narrow band to
the top of the column (where the chromatographic separation will begin).

Some compounds in the sample mixture will have a greater preference for the stationary
phase than for the mobile phase and will be retained in the column longer. The longer the
selected column, the more opportunities for interaction with the stationary phase and the
greater the separation within certain limiting factors.

Once the separation is performed, a detection system is then used to respond to a

physico-chemical property of the analyte. This response is digitally amplified and sent to a
data system where it is recorded as the chromatogram.[1]

Types of Ion Chromatography

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:[5]

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.

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Acid-Base Chemistry

The Brnsted-Lowry theory states that acids are substances that donate protons; in a
similar way bases are substances that accept protons.[26]

Polyprotic acids can lose more than one proton (acquiring more than one negative
charge); similarly polyprotic bases can gain more than one proton (acquiring more than
one positive charge).

A Brnsted-Lowry acid DONATES A PROTON

A Brnsted-Lowry base ACCEPTS A PROTON

Strong acids are substances that ionise completely in an aqueous solution (by losing one
proton), therefore they will always be ionised, at least in some part, over the entire pH
range. Ion exchange functional groups based on strong acidic functional groups (like
sulfonic functional groups) are known as strong cation exchangers and are denoted by
SCX. In a similar way, Ion exchange functional groups based on weak acidic (like
carboxylic acids), acidic compounds that do not fully ionise in an aqueous solutions and
as such can be fully ion suppressed below certain pH values are known as weak cation
exchangers and are denoted by WCX.

Strong bases are chemical compounds able to deprotonate very weak acids and will
remain ionised, to some extent, over the entire pH range. Ion exchange functional groups
based on quaternary amines are known as strong anion exchangers and are denoted by
SAX. Weak anion exchangers and are denoted by WAX and usually present primary,
secondary or tertiary amino functional groups and are capable of being fully ion-
suppressed at a sufficiently high pH.

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Ion Exchange Chromatography Overview

Nowadays the vast majority of ion chromatographic separations are dominated by ion
exchange mechanisms using stationary phases with charged functional groups.[6] These
types of mechanisms dominate the separation of analytes that permanently hold
electrostatic charges (i.e. strongly acidic/basic species or inorganic ions).

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.[7,8]

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).

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 like pH, ionic strength, counterion type, etc.

Ion-exchange chromatography is used for the separation of both organic and inorganic
ions.

Anion exchange Cation exchange

Because the hydrophobic moiety of the charged species dont strongly contribute to
analyte retention, selectivity in ion-exchange chromatography is limited.

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General Ion Exchange Considerations

In ion exchange chromatography, separation is mainly dependent upon the different

degrees of interaction with the exchanger. As expected, these interactions can be
controlled by altering the charge state of either analytes or ionic functional groups from the
stationary phase.[27,28]

The pKa of any ionic functional group determine the conditions of pH at which it will hold
charge:

For a cation to be charged, the pH must be kept around two units above its pKa
For an anion to be charged, the pH must be kept around two units below its pKa

The above considerations for ionic functional groups are valid not only in the case of the
analytes but in the case of stationary phases.

As expected, the charge state of functional groups within a molecule will determine the
correct chromatographic conditions for its separation to take place. As a consequence,
analyte retention in ion chromatography can be controlled through correct modification of
pH.

Degree of dissociation of an acidic compound as a function of pH

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 i

Degree of dissociation of a basic compound as a function of pH

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 Effect of pH in analyte retention

Effect of pH in analyte retention

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Ion Exchange Mechanisms

+ +
Consider the exchange of two ions A and B between the solution and exchange resin

R :

A R + B + B R + A+
The equilibrium constant for this process is:

[ A + ][ B R ]
K= +
[ B ][ A R ]

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 :

C E + D D E + C
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).

Oversimplified separation mechanism of cationic analytes (K+ in this example) on a cation-

exchange resin column

Re sin SO3 H + + K + Re sin SO3 K + + H +

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Ion Exchange Elution Considerations

The ionic strength of the mobile phase plays a major role in the retentive conditions of any
ion exchange separation. Eluent systems presenting high ionic strength facilitate analyte
desorption and are used to elute species from the column.

Low ionic strength buffer no analyte elution

a b
i

Analyte elution facilitated by high ionic strength buffer

As previously explained, analyte retention in ion chromatography can be controlled

through correct pH modification. This is a common practice in ion exchange
chromatography where the charge state of both analyte molecules and stationary phase
dominate the chromatographic process.

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 Analyte elution through analyte neutralisation

Analyte elution through surface neutralisation

In ion exchange chromatography, analyte retention can also be controlled by altering the
concentration of counterions present in the eluting system. As expected, the nature
(strength) of the counterion will also affect analyte retention.
i

Analyte elution using strong counter ion

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Ion Exclusion Chromatography Overview

Ion-exclusion chromatography (IEC), a very useful chromatographic technique, has been

used for the separation of relatively small weak acids (like carbonic and carboxylic acids),
weak bases (like ammonia) and hydrophilic molecular species (such as carbohydrates).
This technique uses strong anion or cation exchange resins for the separation of ionic
solutes.

Ion exclusion chromatography is based mainly on exclusion effects such as differences in

molecular size, shape and charge. The term size-exclusion chromatography may be used
when separation is based solely on molecular size. The term ion-exclusion
chromatography is specifically used for the separation of ions in an aqueous phase.[9,10]

Ion exclusion chromatography is an analytical technique that actually involves the

separation of molecular species rather than ions.[10]

In the representation opposite, only molecules of certain size can actually interact with the
resin to be absorbed. The resin, as is going to be explained on the next page, contains an
occluded liquid which acts as the medium in which molecules absorb.

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Ion Exclusion Chromatography Process

In the ion exclusion process shown opposite, a mixture consisting of CH3COOH (weak
acid) and HCl (strong acid) is subjected to exchange process on a cation exchange resin
using water as an eluent system.

CH 3COOH CH 3COO
Fully dissociated species are excluded from the stationary phase, anions are repelled
from the negatively charged surface (chlorine and acetate anions cannot approach the
surface of the resin) and they do not undergo any chromatographic separation. The
retention volume for these species is the so-called exclusion volume Ve.

By using a dilute solution of a strong acid as the mobile phase, a perimeter of water
molecules (occluded liquid phase) will be established a short distance from the surface of
the stationary phase. This perimeter is known as the Donnan membrane.

Molecules in their neutral state can approach and pass through the Donnan membrane to
finally move into the occluded liquid phase. The size of each molecule will determine
whether or not it absorbs into the occluded liquid phase.[11]

Bear in mind that the ion exclusion process on an anion exchange resin would be similar
to the one already presented.

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Ion Pair Chromatography

The use of salt modifiers and ionic liquids to enhance strongly acidic or basic analyte
retention has been firmly established for many years in reversed phase HPLC. This
approach has fallen out of favour in recent years due to the limiting factorings of running
gradients, suppressing MS signals and irreversibly modifying and reducing column
lifetimes and has been superseded by reversed phases capable of retaining ionisable
analytes.

Because ion-pairing reagents can be used to suppress charge, the ionic equilibrium of
certain analytes can be altered to increase analyte retention under reversed phase
conditions:

A+ + B A B
Where
A+ is the cationic analyte of interest
B is the anionic ion pairing reagent
A B is the neutral ion pair formed

If the analyte ion of interest is anionic, then a similar analysis can be performed (note how
the required ion paring reagent would be of cationic nature).

Ion pair chromatography is performed on standard reversed phase columns. The mobile
phase consists of modifier(s) and a buffer solution, to which an ion pair reagent is added
at low concentration.[12]

Ion pairs are neutral species formed by electrostatic attraction between oppositely
charged ions in solution. The ion pair formation is dependent on the ions size, solvent,
and temperature.

Ion Pair Chromatography (IPC) is used for ionic compounds which are difficult to separate
on a covalently bonded ion-exchange resins and for samples with widely different
components, e.g mixtures of acidic and basic analytes or zwitterions.

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Ion Suppression

The technique of using pH to suppress ionisation and therefore gaining retention for
ionisable analytes in reverse phase HPLC is termed Ion suppression and is applicable to
weakly acidic or basic compounds only.

The pH of a solution will influence the charge state of an acidic or basic analyte. For
example, addition of an acid to an aqueous solution of a basic analyte will increase the
concentration of charged analyte in solution. Conversely, raising the pH by addition of a
base will increase the concentration of the neutral form of the basic analyte. This principle
was first described by Le Chatelier and the converse applies to acidic analyte species.

It is important to realise that the two forms of ionisable analyte molecules give different
retention characteristics. The ionised form is much more polar, and its retention in reverse
phase HPLC is much lower (shorter retention time (tR), smaller retention factor (k)). This
behaviour is expected, as the more polar (charged) analyte form has a higher affinity for
the more polar mobile phase and moves more quickly through the column. The converse
is true of the non-ionised form as it is much more hydrophobic, relative to the ionised
form.[13]

If a separation of mixtures of weak acids and bases (or amphoteric analytes) is required,
then ion suppression by pH control is of limited use. The ionisation of acidic functional
groups can be suppressed at the same conditions of pH at which basic functional groups
are ionised. This situation will result in a non-robust methodology. In practice, a
combination of ion-suppression and ion-pair chromatography is used.

In the case of strong acids and bases, where effective ion suppression is achieved at
extreme conditions of pH, ion suppression is not recommended as the optimum pH may
lie outside the working range of traditional HPLC columns.

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The Ion Chromatographic System

In general terms there are only a few additions to a traditional HPLC system in order to
achieve ion 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).[14]

As in traditional HPLC, ion chromatography pumping systems use reciprocating pumps

which cope with the pressure and volumetric needs of most ion chromatography
applications.

Columns packed with suitable 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 and/or in-line filter) between the autosampler and the
column.

In terms of detection, ion chromatography implements similar types of detectors

traditionally used in HPLC separations.

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.

Pre column
Heat exchanger

The IC system.

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Where:

Solvent: Choose a mobile phase composition and gradient ramp rate that elutes the
analyte with the narrowest peak possible.

Solvent Degasser: Removing dissolved air and other gases from incoming solvent
streams is critical to insure proper functioning of the pump check valves and to avoid
outgassing of dissolved gases in detector flow cells.

Pump: Pump performance is critical to ensuring good chromatography and a poorly

performing pump will cause baseline disturbance, retention time drift and poor
reproducibility (both qualitative and quantitative). Select Binary pumps when rapid and
accurate gradients are required and quaternary pumps where more than two mobile
phase components need to be mixed simultaneously. HPLC pumps are designed to
eliminate pulsation (multiple action pumps, in-damper built, etc) providing uniform flow
over a wide range of pressures.

Injector: The function of the injector is to place the sample into the high-pressure flow in
as narrow a band as possible (to maintain high efficiency) so that the sample enters the
column as a homogeneus, low-volume plug. To minimize spreading of the injected volume
during transport to the column, the shortest possible length of tubing should be used from
the injector to the column, with the minimum number of zero dead volume connections.

Filter: A major cause of column deterioration and damage is the build up of particulate
and chemical contamination at the head of the column. This can lead to increased back
pressure and anomalous chromatographic results. HPLC Columns normally contain
stainless steel inlet and outlet frits (acting as filters) and retain the column packing. The
pore size of the frit must be smaller than the particle diameter of the packing, e.g., a 0.5
m frit for 1.8 m packing. HPLC filters are designed for maximum filtration of particulate
matter with minimal dead volume or back pressure.

Pre-Column Heat Exchanger: Temperature impacts on peak efficiency, if the

temperature difference between the column and the incoming mobile phase is too large,
band broadening results. For the best column temperature control and most uniform
results, it is often necessary to pre-heat or pre-cool the mobile phase before it enters the
chromatographic column

Guard Column: A guard column is inserted between the injector and analytical column to
protect the latter from damage or loss of efficiency due to the presence of particulate
matter or strongly adsorbed impurities from analytical samples. For maximum protection
against contaminants and particulate matter, the guard column can be placed between a
set of frits (that act as filters).

Colum: HPLC columns are designed considering a variety of factors such as separation
performance, durability and column pressure. These columns achieve good balance
between separation efficiency and pressure.

Thermostatted Compartment: The ability to accurately and reproducibly control the

column temperature is critical to promoting the sample diffusion rates required to achieve
ionic separations. This device eliminates thermal gradients across the column, resulting
in better column performance and precise retention times.

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Post-Column Heat Exchanger: The columns effluent is delivered at high temperatures.
When the detection system is affected by the columns effluent temperature (for example
when measuring the refractive index) then a post-column heat exchanger is required to
cool down the eluent.

Electrolytic Suppressor: The coupling of IC to certain detection types (like MS) can be
done by implementing post-column ion suppressors; they act to selectively reduce the
ionic strength of the columns effluent.

Detector: 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 columns effluent.

Ion Chromatography Columns

The column is the only device in ion chromatography which actually separates an injected
mixture. The stationary phase is responsible for the separation and its properties are of
primary importance for successful separations.

The lifetime of the ion chromatography column is maximized through the use of stable
bonding chemistry, high purity silica 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 expected, different packing materials have been developed to speed up the ion
chromatography separation process.[7, 10, 11]

Where:

IC: Ion Chromatography

IPC: Ion Pair Chromatography
NPC: Normal Phase Chromatography
RPC: Reversed Phase Chromatography

Mobile phase versus stationary phase polarity for selected types of chromatography.

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Anion Exchangers:

Diethylaminoethyl (weak anion exchanger)

Quaternary aminoethyl (strong anion exchanger)

Quaternary ammonium (strong anion exchanger)

Cation Exchangers:

Carboxymethyl (weak cation exchanger)

Methyl sulphonate (strong cation exchanger)

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Detection

The ion chromatography detection system is used to monitor the passage of the
components as they emerge from the column.[5]

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 columns effluent. The most common detection
types currently used for IC separations are as follows (click to get more information):

Electrochemical Detection
Conductivity
Amperometry
Coulometry
Voltammetry
Optical Detection
UV-Vis
Fluorescence
Refractive Index
Others
Mass Spectroscopy

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Because electrolytic suppressors are designed to reduce ionic strength, the columns
effluent can be detected by any traditional HPLC detection system.

The conductivity detector.

In the upper part of the diagram, no ions are passing through the detector and despite the
applied voltage, no current is measured. Ions in solution help to transport current (bottom)

Conductivity 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.

Selected detection types in ion chromatography

Detector Selectivity Sensitivity
Refractive Index Low 1 5 g
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

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Hyphenated Ion Chromatography

Ion chromatography has been hyphenated to a range of 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.

Ion suppressors are designed to lower the ionic strength of the columns effluent, allowing
the use of the full range of traditional HPLC detectors.

The ion suppressor device shown below,[14] 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 charges.

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Applications

The determination of ionic species is a classical analytical problem that can be found in
many different application areas.

Aqueous or water-miscible samples can be directly analyzed by IC. Water-immiscible

liquids, solids and gases must be extracted into or dissolved in aqueous solution before
analysis.

To list the full range of IC application areas is prohibitive since its flexibility makes it
suitable to a multitude of application types. Examples of some interesting applications are
shown below:

Agrochemistry

Mono-chlorophenols (MCPs) and di-chlorophenols (DCPs) are used as disinfectant

agents and as the base for different pesticides; however, due to new environmental
regulations their use has been restricted.

IC-MS trace of a river water sample preconcentrated by SPE for gradient elution.

Sample: 1= 2-CP; 2= 4-CP; 3= 3-CP; 4= 2,6-DCP; 5= 2,3-DCP; 6= 2,5-DCP; 7= 2,4-DCP;

8= 3,4-DCP; 9= 3,5-DCP

Column: anion exchange column 250mm4.0mm.

Eluent system: 04.5 min, 20 mM KOH; 4.510.0 min, 2040 mM KOH (linear gradient);
10.012.0 min, 40 mM KOH.

Eluent flow rate: 1.0 mL/min

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Biotechnology

An enormous effort in providing new methods of detection and analysis of gene

sequences has been made and Ion Chromatography has played a central role.

High-performance ion-exchange chromatographic analysis of a mixture of peptide nucleic

acids (UV detection at 260nm)

Sample: PNA1= H-AGAGTCAGCTT-NH2; DNA3= 5-AAGCTGACTCT-3; DNA5= 5-

AGAGTCAGCTT-3.

Column: polystyrenedivinylbenzene column 50mm7.5mm.

Eluent system: linear gradient from 100% A (0.05 M trisHCl in water, pH 8) to 100% B
(0.05 M trisHCl, 0.5 M NaCl in water, pH 8) in 60 min. Where tris = tris(hydroxymethyl)
aminomethane

Eluent flow rate: 1.0 mL/min

Cements

In the production of cements, the levels of chlorides and sulphates determine the quality
of the final product.

Column: anion exchange column

250mm4.0mm.

Eluent system: deionised water

Eluent flow rate: 1.0 mL/min

IC determination of chlorine and sulphur species in a commercial Portland cement sample

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Clinical Chemistry

The determination of metal ions in physiological fluids is of considerable diagnostic

interest in clinical chemistry.

Separation of heavy metal ions with spectrophotometric detection (530nm) after post
column derivatization. (1) standard solution, (2) serum sample.

Column: ethylvinylbenzene functionalized with ammonium and sulfonate functional

groups 250mm4.6mm, 9m.

Eluent system: 1.4 mM pyridine-2,6-dicarboxylicacid + 13.2 mM potassium hydroxide +

1.1 mM potassium hydroxide + 14.8 mM formic acid (pH = 4.2 0.1)

Detection: postcolumn reagent 0.5mM (4-(2-pyridylazo) resorcinol) + 1.0 M 2-

dimethylaminoethanol + 0.5 M ammonium hydroxide + 0.3 M sodium bicarbonate (pH =
10.4 0.2)

Eluent flow rate: 0.3mL/min

Environmental Chemistry

Fresh water shortages and new findings on multiple toxicity of several arsenic species
have intensified the As remediation problem in recent years.

IC-MS separation of As-species at pH = 8.2.

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Analytes: monomethylarsonate (MMA), dimethylarsonate (DM), arsenobetaine (AsB).

Column: low hydrophobic anion exchange column 250mm2.0mm, 13m.

Eluent system: solution a (0.5 mM ammonia/HNO3 pH = 8.2) and solution b (100

ammonia/HNO3 pH = 8.2). Gradient: 100% A for 2.0 mins, gradient step to 30% A in 5.0
min abd then 2.0 mins at this concentration.

Eluent flow rate: 0.44 mL/min

Food Analysis

Sulfites have been widely used as preservatives and blanching agents for many years in a
large variety of foodstuffs and beverages. Before 1986 sulfites were incorrectly
considered harmless but asthmatic reactions and food intolerance symptoms are related
with high consumtion of these species.

IC analysis of not-compliant cow fresh meat sample, with an observed SO2 content of
121.7mg/kg.

Column: high-capacity carbonate eluent anion-exchange column 250mm4.0mm, 9m.

Eluent system: solution a (8.0 mM Na2CO3 + 2.3 mM NaOH) and solution b (24 mM
NaOH). Gradient: 100% A for 15 mins, gradient step to 50% A in 1.0 min abd then 4.0
mins at this concentration.

Eluent flow rate: 1.0 mL/min

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Industrial Waste Analysis

Antimony has been extensively used in various industrial applications ,however, it is a

toxic cumulative element with similar chemical and toxicological properties to arsenic.

Isocratic IC separation of Sb-species present in waste water

Column: polystyrenedivinylbenzene-based anion-exchange column 250mm4.1mm,

10m.

Eluent system: 12mM tetramethylammonium hydroxide (TMAOH) at pH = 12.

Petroleum Exploration

The chemical analysis of oilfield waters has an important role in the exploration and
production of oil.

Isocratic IC analysis of oilfield water

Column: anion exchange column 250mm4.0mm.

Eluent system: 20.0 mM methane sulfonic acid solution

Eluent flow rate: 1.0 mL/min

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Pharmaceuticals

Alkylsulfonic acids are typically used as catalysts, solvents and blocking agents in the
synthesis of many organic compounds and pharmaceutical drugs.

IC separation of alkylsulfonic acids

Sample: 1= methanesulfonic acid; 2= ethanesulfonic acid; 3= propanesulfonic acid; 4=

butanesulfonic acid; 5= pentanesulfonic acid; 6= hexanesulfonic acid; 7= heptanesulfonic
acid.

Column: mixed anion-exchange and polymeric reversed-phase retention column

250mm4.0mm.

Eluent system: A (0.5 mM sodium carbonate + 1.0% acetonitrile), B (10 mM sodium

carbonate + 40.0% acetonitrile) linear gradient from 100% A for 15 mins, then increase B
to 100% in 20 mins.

Eluent flow rate: 1.0 mL/min

Polymers

Epichlorohydrin is an organic liquid with a garlic-like odour. It is mainly used in the

production of glycerine, certain plastics and polymers. Exposure to epichlorohydrin for
relatively short periods of time can damage skin, liver, kidneys and the central nervous
system.

Reaction between sulfur(IV) and epichlorohydrin.

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 Column: anion exchange column
250mm4.0mm.

Eluent system: 5 mM NaOH

Eluent flow rate: 1.0 mL/min

IC analysis of derivatized epichlorohydrin with sulphur (IV)

Power Generation

Power plants use different additives to decrease the corrosive effects of water.

IC separation of some inorganic cations and commonly used corrosion inhibitor additives
to power industry waters.

Sample: 1= Lithium; 2= sodium; 3= 2-diethylaminoethanol; 4= morpholine; 5=

ethanolamine; 6= ammonium; 7= 5-amino-1-pentanol; 8= magnesium; 9= calcium; 10= 3-
dimethylaminopropylamine; 11= potassium; 12= cyclohexylamine.

Column: anion exchange column 250mm4.0mm.

Eluent system: 9 mM methanesulfonic acid + 10.7% MEK, gradient from 6 to 9 min to 27

mM methanesulfonic acid + 10% MEK

Eluent flow rate: 1.0 mL/min

Crawford Scientific www.chromacademy.com 33

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