Theory of Ion Chromatography from Metrohm

There are many different ways of determining ions qualitatively and quantitatively. One such technique that is widely used is ion chromatography.

Ion chromatography is one member of the large family of chromatographic methods. It is used to determine all ions which carry one or two charges. In the past ion chromatography (IC) was a very expensive method but today it is much more favorably priced, thanks to the quality Compact IC's available from Metrohm.

Which Ion Chromatography Column?

There are many important fields of application today for ion chromatography such as:

1. the routine investigation of aqueous systems such as drinking water, rivers, effluents and rain water.

2. for the analysis of ions in chemical products, foods, cosmetics, pharmaceuticals etc

3. ultratrace analysis such as in the semi-conductor and power industry.

Ion Chromatography can be used for the analysis of anions, cations, organic acids and amines plus analytes such as carbohyrates

Schematic of an Ion Chromatography System

The above schematic represents a non-suppressed ion chromatography system. The sample is introduced onto the system via a sample loop on the injector. When in the inject position the sample is pumped onto the column by the eluent and the sample ions are then attracted to the charged stationary phase of the column. The charged eluent elutes the retained ions which then go through the detector (which is most commonly conductivity) and are depicted as peaks on a chromatogram.

Three main modes of Ion Chromatography Columns.

The different modes of chromatography (anion exchange, cation exchange and ion exclusion) simply relate to the different types of columns used to achieve the separation of the ions. The eluent used

depends on the column type and also the mode of detection - however unless stated the following is all based on conductivity detection.

Ion exchange

Ion exchange chromatography (IC) is based on a stoichiometric chemical reaction between ions in a solution and the oppositely charges groups functional groups on the column resin. In the simplest case in cation chromatography these are sulfonic acid groups or carboxylic acid groups (such as maleic acid) and in anion chromatography quaternary ammonium groups.

Anion exchange

Anion exchange chromatography forms the largest group of IC methods mainly because there are few alternatives with such simplicity, sensitivity or selectivity - particularly for sulphate. The two forms are anion exchange with or without suppression and of these two suppressed methods are the most widely used. Eluents for suppressed chemistries tend to be either carbonate based or hydroxide.

chromatogram showing an anion exchange separation followed by direct conductivity detection (non-suppressed)

Suppression in Ion Chromatograph

Often, a device called a suppressor is used and is placed between the column and detector as shown above. When suppression is used the detector is almost certainly conductivity.The chromatogram below shows a sample with a suppressor unit placed between the column and detector. The greatest achievment of suppression is to increase the sensitivity of the anion, however at the same time the background conductivity of the eluent is greatly reduced. The same

suppressor units can also be used to increase the sensitivity of organic acids.

Chromatogram showing an anion exchange separation followed by suppression and then conductivity detection

The suppressor used in anion chromatography is simply a cation exchanger and its job is to remove cations and replace them with an H+. So a sodium carbonate eluent (~800uS) would be converted to carbonic acid (~18uS) by the suppressor and the analyte, for example NaCl (~126uS without suppression) would become HCl (~426uS with suppression). The ways in which this can be done are varied but the two common ways are as follows:

1: The Metrohm MSM (Metrohm Suppressor Module) contains 3 separate suppressor units. At any one time, one will be in-line with the eluent and conductivity detector, one will be in-line with dilute sulphuric acid (replacing the removed cations with H+) and the third is washed with water. The benefits of this technique are lack of baseline noise and a ruggedness that is reflected in the fact that the MSM comes with a ten year warranty and is not, for example, adversely affected by the transition metals which can cause precipitation problems for other types of suppressor technology.

This suppressor module is present in all the Metrohm Compact ICs (792 Basic, 790 Personal, and 861 Compact) and is available in the modular system as the 833 Advanced IC Liquid Handling Unit.

The anion chemical suppression can be taken a stage further with the new 853 MCS instrument (fitted inline after the MSM) which removes carbon dioxide from the suppressor reaction and carbonate from the sample, which means that the sodium carbonate/sodium bicarbonate mobile phase is converted to water instead of carbonic

acid so a background conductivity approaching 1uS is achieved. The 853 MCS can be fitted to all Modular and 761/861 Compact IC’s and the benefits include no injection peak, no system peak, superb linearity and an enhancement in the peak areas allowing lower limits of detection to be achieved.

2. The Metrohm Dual Suppressor is a continuous suppression device which removes the cations and replaces them with H+ (which is provided by electrolysis of water) so with a carbonate eluent it forms carbonic acid. The Dual Suppressor then reduces the conductivity of the carbonic acid (~18uS) by removing it to leave water (~1uS). As the concentration of the eluent increases throughout the gradient, the baseline rise is a result of the increasing conductivity of the eluents suppression product. The importance of this is that carbonate eluents can now be used for gradient elution of anions which means more versatility, no system peak, less corrosive eluents and no gases required.

Schematic of the Metrohm Gradient System with two 818 Advanced IC Pumps forming the high pressure gradient

pump (P1 and P2) and the 828 IC Dual Suppressor.

Chromatogram showing gradient elution with carbonate eluents.

Cation Exchange

There is a variety of cation columns available, however the modern ones contain carboxylic acid functional groups. A large number of

applications for silica-gel-based ion exchangers exist. These columns allow simultaneous separation of alkali metals and alkaline earths plus the separation of transitional metal and heavy metal ions is also possible. Small amines can also be analysed using cation exchange columns.

Chromatograms showing cation exchangewith two different tartaric acid/dipicolinic acid eluents

The eluents used for non-suppressed cation exchange are weak acids with a complexing agent such as dipicolinic acid, the concentration of which can effect the elution of calcium and heavy metals such as iron, zinc and cobalt.

Cations become less sensitive when suppressed and so are analysed with direct conductivity detection which also allows heavy metals to be analysed as shown above.

Ion exclusion

Ion exclusion chromatography (IEC) is mainly used for the separation of weak acids or bases. The greatest importance of IEC is for the analysis of weak acids such as carboxylic acids, carbohydrates, phenols or amino acids.

Chromatogram showing ion exclusionwith an acid eluent

For a more detailed explanation of the theory of ion chromatography and detection see the Metrohm

Monograph 'Practical Ion Chromatography' available free of charge from Metrohm UK. HYPERLINK enquiry@metrohm.co.uk

For compact ion chromatography units containing all components see 792 Basic IC, 790 Personal IC and 861 Compact IC. For modular IC see 819 Advanced IC Detector, 818 Advanced IC Pump, 833 Advanced IC Liquid Handling Unit and 820 Advanced IC Separation Centre. For on-line IC see the 811 Online IC and the 821 Compact Online IC.

Other modes of Detection

Amperometric detection (see 791 IC-VA Detector and 817 Bioscan)

 

(Amperometric detection (see 791 IC-VA Detector and 817 Bioscan)

In principle voltammetric detectors can be used for all compounds which have functional groups which are easily reduced or oxidized. The amperometric detector is the most important version. Amperometry is very sensitive. Apart from a few cations (Fe3+, Co2+) it is chiefly anions such as nitrite, nitrate, thiosulfate as well as halogens and pseudo-halogens which can be determined in the ion analysis sector. The most important applications lie, however, in the analysis of sugars by anion chromatography and in clinical analysis.

Photometric detection (see UV-Vis Spectrophotometer, 844 Compact UV-Vis and Post Column Reactor)

Chromatogram showing analysis of 1ppb chromate using the 844 Compact UV-Vis

Because of its extremely wide range of application photometric or UV/VIS detection is the most important detection method used in

HPLC, as many organic molecules contain chromophore groups, or can have one introduced or added, which are able to absorb in the UV or VIS spectrum. In the field of inorganic ion analysis UV/VIS detection plays a smaller role. While of the simple anions only analytes such as nitrate, bromide or iodide absorb, important analytes such as fluoride, sulfate or phosphate can only be measured indirectly. Many cations do not absorb at all, but multivalent and transitional metals in particular can be converted in a post-column derivatization with chelate formers such as 4-(2-pyridylazo)-resorcinol (PAR) or Tiron to form colored complexes. Redox-active analytes such as bromate and other oxohalide ions can be analyzed by UV/VIS detection after undergoing a post-column reaction with an electrochemically active indicator.

Sample Preparation Techniques for Ion Chromatography

Introduction to Sample Preparation Techniques

Quite often with problematic ion chromatography applications, the matrix of the sample makes it difficult to accurately quantify the species of interest with the standard ion chromatography set-up and some form of sample preparation then becomes necessary.

Schematic Diagram of Standard Ion Chromatography Set-up

The sample preparation may be as straightforward as simply diluting the sample with deionised water or can involve injection of the sample through a solid phase extraction cartridge to remove the

interference. In the case of more difficult forms of sample matrices it may be necessary to add additional dedicated sample preparation modules to the standard ion chromatography configuration.

What is Ion Chromatography?

Chromatography is a method for separating mixtures of substances using two phases, one of which is stationary and the other mobile moving in a particular direction. Chromatography techniques are divided up according to the physical states of the two participating phases. The term Ion Exchange Chromatography or Ion Chromatography (I.C) is a subdivision of High Performance Liquid Chromatography (H.P.L.C).

A general definition of ion chromatography can be applied as follows;” ion chromatography includes all rapid liquid chromatography separations of ions in columns coupled online with detection and quantification in a flow-through detector”.

A stoichiometric chemical reaction occurs between ions in a solution and a solid substance carrying functional groups that can fix ions as a result of electrostatic forces. For anion chromatography these are quaternary ammonium groups and for cation chromatography sulphonic acid groups. In theory ions with the same charge can be exchanged completely reversibly between the two phases. The process of ion exchange leads to a condition of equilibrium, the side to which the equilibrium lies depends on the affinity of the participating ions to the functional groups of the stationary phases.

Different Types of Sample Preparation Techniques Employed

Dilution of the Sample

Dilution of the sample is performed when the concentration of the analytes of interest either exceed the working capacity of the separation column chosen, or there are sample matrix effects that can often be minimised by a dilution usually with water but eluent can also be used.

Filtration of the Sample

It is recommended to filter all samples prior to injection with 0.45mm filters to ensure that any particulate material from the samples don’t make their way onto the injection valve or the analytical column where they can cause blockages and considerably reduce the lifetime of the column(s).

Solid Phase Extraction Cartridges

Passage of the sample through one or more solid phase extraction cartridges prior to injection will often retain selectively certain species within the homogeneous sample. Quite often the retained species are substances that would interfere with the chromatography had they not been previously removed. There are a number of different cartridges whose suitability depends upon the type of chemistry undertaken.

For anion analysis, the sample can be treated with a cation exchanger in the H+ form that removes divalent cations that can mask any fast eluting anions. This type of exchange cartridge removes carbonate/bicarbonate and is also useful for the removal of cations from samples being determined by ion exclusion chromatography. Another option is the use of a cation exchanger in the Ag+ form for the removal of any halides present in the sample.

Similarly for cation analysis, one can employ an anion exchanger in the OH- form to remove any interfering anions present in the sample.Another common type is the non-polar exchange cartridge (reversed phase) that often utilises C18 groups to remove organic substances that would otherwise interfere with the chromatography.

Digestion of the Sample

If digestion techniques are to be employed then analyte content should be changed as little as possible and any organic matter present should ideally be destroyed completely. One can obtain analytical inaccuracies due to an exaggerated blank value as a result of the chemicals used during the digestion. Different types of digestion include wet, microwave and UV, the suitability of each depends on both the sample matrix and the analytes of interest being determined.

Instrumental Sample Preparation Modules

Often with more complex sample matrices, one has to add additional dedicated sample preparation modules to the standard ion chromatography configuration. There are a number of different instrument options available within the Metrohm range depending on the type of sample treatment required prior to analysis. Metrohm has actually been a pioneer of inline sample preparation modules with the release of the 754 IC Dialysis Unit in 1997, since then the technology has been optimised and considerably improved so that today the Metrohm IC portfolio contains many different sample preparation instruments.

833 Advanced IC Liquid Handling Dialysis Unit (2.833.0040)

The Metrohm 833 Dialysis Unit is a module for online sample preparation in ion chromatography permitting the use of automatic sample dialysis directly before sample injection. It consists of a dual channel peristaltic pump for conveying the sample and acceptor solutions and the actual dialysis cell in which the ions from the flowing sample solution are enriched in the

resting acceptor solution. The 833 Dialysis Unit is strongly recommended for demanding applications that contain strongly loaded samples. Dialysis is the diffusion of ions from a sample solution into an acceptor solution to achieve a concentration equilibrium. The sample solution is constantly being renewed resulting in no depletion of the sample. The ions pass through the membrane without hindrance but larger sample particles – the sample matrix - are transported past the membrane to waste reducing matrix effects to an absolute minimum. Calibration of the standards is easily done requiring no extra outlay. The reported dialysis recovery rates have been found to be in excess of 98% using the patented stopped flow method.

838 Advanced IC Ultra-Filtration Sample Processor (2.838.0210)

The aim of sample filtration is to protect the separation columns from contamination and blockage from particulates that may be present in the sample. The 838 IC Ultra-Filtration sample processor combines inline filtration with automatic sample injection through the use of an ultra-filtration cell. It is eminently suitable for those samples with a light to medium load such as surface waters and digestion solutions.

The samples are placed onto the sample carousel before being processed automatically. Sample filtration and introduction to the injection valve is achieved by means of an integrated double channel

peristaltic pump meaning that it is possible to aspirate slightly viscous samples.

The sample is conveyed by one channel of the pump through the ultra-filtration cell passing the membrane. At the same time the filtrate is aspirated off from the rear of this membrane and transferred to the sample loop by the second channel of the pump. Only a small fraction of the sample is removed as filtrate so the contaminants remain mainly in the sample stream preventing the regenerated cellulose membrane from becoming blocked too quickly.

 

 

833 Advanced IC Liquid Handling Sample Preparation Unit (2.833.0030)

Picture of Metrohm 833 Advanced IC Liquid Handling Sample Preparation Unit

Inline sample preparation is rapidly becoming the method of choice for eliminating difficult sample matrices in ion chromatography and Metrohm has developed the 833 Advanced IC Liquid Handling Sample Preparation Unit based upon its Metrohm Suppressor Module (M.S.M) for difficult anion analyses such as those found in concentrated alkaline solutions. The modules consists of a reactor block that houses the cation exchangers with a control unit that contains a two channel peristaltic pump that conveys the regenerant and rinse solutions.

Schematic Diagram of Inline Sample Preparation

The matrix elimination occurs inline whilst the regeneration and rinsing of the packed bed suppressor occur simultaneously offline. A fresh suppressor channel is used for each new analysis and because the rinse and regeneration occurs after each determination, the capacity of the 833 is unlimited.

The sample solution is transferred to the 833 module from an autosampler via a loop injection and rinsed with deionised water. The sample cations are exchanged against protons (H+). If sodium hydroxide constitutes the sample matrix, water is formed by neutralisation. The sample solution then passes onto the preconcentration column where the trace anions to be determined are retained and then eluted by the eluent flowing in a counter flow direction. The analyte anions are then separated on the analytical column before quantification using chemical suppression with conductivity detection.

Example Chromatogram Showing Matrix Elimination upon a Sample of Caustic Soda for the Determination of Chloride, Chlorate and Sulphate

Metrohm Inline Sample Preparation (MISP)

With Metrohm it is possible to perform the time consuming sample preparation inline using the 838 Advanced IC Sample Processor equipped with MISP technology. The 838 comes in a number of different variants that mean it is possible to dialyse, ultra-filtrate or even dilute the sample automatically inline.

Located on the side of the tower is the relevant sample preparation technology for example an

ultra-filtration cell or an injection valve which is utilised for dilution along with the proven Dosino™ liquid handling technology.

 

 

 

The new Range of Anion Columns from Metrohm include:-

 

The disinfection by-products generated in water processing plants are suspected to be not only a health hazard but could even be carcinogenic. For this reason the oxo halides, above all bromate that is generated from bromide during the ozone-treatment of drinking water, have become the object of many investigations and standard methods (e.g., EPA 300.1 Part B, EPA

317.0). Metrohm now presents a high-performance separation column for the simultaneous determination of the standard anions, the oxo halides and dichloroacetic acid.

With the Metrosep A Supp 7 these ions can be determined reliably and precisely down to lower ppb range. The outstanding detection sensitivity is obtained by applying the 5-µm polyalcohol polymer, which yields extremely high plate numbers and accordingly excellent separation and detection characteristics. The separations require a temperature of 45 °C.

The high-capacity Metrosep A Supp 8 – 150 allows the determination of nitrite, bromide and nitrate in concentrated salt solutions. UV detection at 215 nm opens up the determination of concentrations in the one-digit ppb range. A special sodium chloride eluent is used for these applications.

The Metrosep Dual 4columns contain an entirely novel carrier material, namely a functionalized monolith based on silica. This monolith allows flow rates of up to 5 mL/min. Even at these high flow rates, the column's counter-pressure remains small. Compared to conventional materials, the monolith

with its structure made up of macro- and mesopores has a much larger surface area. This contributes to the high capacity of the Metrosep Dual 4 column, whose dead volume is very low.The high-capacity Metrosep Dual 4 – 100 column is the separation column of choice for the detection of very small amounts of toxic perchlorate. It allows to determine 0.5 ppb perchlorate in the presence of a total of 3 g/L of chloride, carbonate and sulfate. The Dual 4 – 100 also easily achieves the base-line separation of chloride and nitrite present at a concentration ratio of 1000 to 1.

 

The Metrosep A Supp 10 – 100 separation column is based on a high-capacity polystyrene/divinylbenzene copolymer having a particle size of only 4.6 µm. Metrohm has optimized this time-tested column concept, which is characterized by its robustness, high selectivity and excellent separation performance. By variation of temperature, flow rate and eluent composition, the column characteristics can be adapted to the application at hand.The A Supp 10 – 100 is the column of choice for routine applications. Thanks to its robustness, excellent price-performance ratio and very good separation performance, combined with moderate run times, the A Supp 10 – 100 is a universally applicable anion separation column.

Dialysis – An Overview

Dialysis is a successful method for the separation of low molecular substances from high molecular ones by means of a semi-permeable membrane and is used on patients with kidney deficiencies.

Low molecular substances in the blood refer to ions that disturb the electrolyte balance. As the kidneys can not functioning correctly, then the concentrations of these ions increases impairing the metabolic functions. The concentrations of these ions must be reduced at frequent intervals and this is achieved by continuous flow dialysis.

An acceptor solution of a low ionic strength (usually deionised water) is pumped along the semi-permeable membrane with the blood flowing past on the other side. As the ions pass through the membrane virtually unhindered they diffuse from the high molecular strength blood into the low ionic strength acceptor solution. The acceptor solution is permanently renewed in its continuous flow ensuring a steep concentration gradient with relatively large efficiency. Deliberate care is taken to ensure that no concentration equilibrium can be established between the two solutions.

Metrohm has developed the patented stopped flow method for dialysis where the sample solution is continuously pumped past a semi-permeable membrane but the acceptor solution lies at rest and here lies the inherent difference from continuous flow dialysis. This ensures that equilibrium is attained between the sample solution and the acceptor solution usually in less than 10 minutes. Once the equilibrium has been set up the dialysed acceptor solution is transferred to the sample loop and injected onto the separation column.

Diagram Illustrating Stopped Flow Dialysis Technique

Applications of Dialysis

The benefit of dialysis is that there are no complicated, time consuming sample preparation steps such as digestion that can potentially destroy the analytes of interest. This is particularly applicable to foodstuffs and other complex matrices that carry high organic loads such as waste waters or soil eluates. 

In the food industry the ionic contents of milk and other diary products can easily be determined using an ion chromatography system incorporating a dialysis module. It is no longer necessary to separate the proteins from the milk using Carrez precipitation ensuring that the sample preparation is reduced to a simple dilution step. Other difficult matrices include the analysis of fruit juices that contain fruit pulp, cutting oil emulsions and inks, with dialysis these types of samples no longer represent a problem for the ion chromatography user.

Method for Analysis of Milk Samples

The modular system used for the determination of anions present in samples of milk comprised the Metrohm modules 818 Advanced IC Pump, 819 Advanced IC Detector, 820 Advanced IC Separation Center, 833 IC Suppressor Module, 830 IC interface and 838 Advanced IC Dialysis Sample Processor.

Picture Showing Modular System Configuration for Dialysis of Milk Samples

The milk sample was diluted 1:5 with deionised water and placed in the sample vials upon the rack of the 838 Advanced IC Sample Processor. The subsequent dialysis of the sample and injection of the dialysed sample onto the separation column was fully automated and the response for the peaks recorded using a mobile phase eluent of sodium carbonate/sodium bicarbonate. The

calculation was carried out automatically using integration software IC Net 2.3 against a previously prepared calibration plot.

Chromatograph of Anions Found in Milk Samples after Dialysis

Ion chromatography as an analytical technique has seen an enormous surge in popularity due partly to the simplicity of the method as well as other factors such as market forces driving down the expenditure costs of the initial instrumentation and improved reliability and power. For a sample in a homogeneous, ionic form then very little sample preparation is required and quantified results can be obtained within a matter of minutes.

As in any industry, the consumer places ever more stringent demands and requirements upon the manufacturer and the world of ion chromatography is no different. Because of the ease of use at which ion chromatography as a method can be manipulated, the end user today wishes to analyse ions within increasingly complicated sample matrices which until recently would not have been possible.

Metrohm has developed a range of instrumental sample preparation modules that can be added to standard ion chromatography configurations to allow quantification even with the most difficult sample matrices. The analysis is fully automated so once the sample is loaded, the analyst is then free to carry out other functions within the laboratory affording an increase in efficiency of the employed manpower.Even for those samples requiring sample preparation by dialysis or ultra-filtration, still only a relatively small volume of sample is required. This coupled with the low running costs of ion chromatography using Metrohm instruments really does mean that ion chromatography is the method of choice for the analyst even with the most difficult and problematic sample matrices.

References

1. Fundamentals of Analytical Chemistry, (1992) 6th edition, D.A Skoog, D.M West and F.J Holler, Saunders College Publishing, ISBN 0-03-075397-X.

2. Principles and Practice of Analytical Chemistry, (1992) 3rd edition, F.W Fifield and D. Kealey, Blackie Academic & Professional, ISBN 0-216-92920-2.

3. The Essential Guide to Analytical Chemistry, (1999) 2nd edition, G. Schwedt, John Wiley & Sons, ISBN 0-471-97412-9.

The following internet site was also used extensively as a reference and can be used to obtain further information:-

www.metrohm.com

Article written by Jonathan Bruce, Product Application Manager (IC/VA Division) for Metrohm UK Ltd.

 

Coupling techniques

So-called coupling techniques represent the link-up of a chromatography system with an independent analytical method, such as mass spectroscopy

Ion chromatography with Metrohm meansHigh sensitivityAccurate and reproducible resultsLow acquisition costAll instruments backed by the COOL guarantee (Cost of Ownership is Lower)Very low service expenditureAnd don't forget:Swiss quality from over 60 years as a pioneer of Ion AnalysisMetrohm – First Class Ion Chromatography