i-SECTION: TUTORIAL REVIEW

www.rsc.org/analyst | The Analyst

Novel ion chromatographic stationary phases for the analysis of complex matrices
Brett Paull*a and Pavel N. Nesterenkob
Received 27th April 2004, Accepted 16th August 2004 First published as an Advance Article on the web 21st October 2004 DOI: 10.1039/b406355b Ion chromatography (IC) has a proven track record in the determination of inorganic and organic anions and cations in complex matrices. Recently, application of IC to the separation and determination of bio-molecules such as amino acids, carbohydrates, nucleotides, proteins and peptides has also received much attention. The key to the determination of all of the above species in the most analytically challenging complex matrices is the ability to manipulate selectivity through control of stationary phase chemistry, mobile phase chemistry and the choice of detection method. This Tutorial Review summarises some of the most significant recent advances made in IC stationary phase technology. In particular, the review details stationary phases specifically designed for ion analysis in complex sample matrices, and considers in which direction future stationary phase development might proceed.

1 Recent reviews
Reviews detailing various advances in IC technology have been fairly numerous in recent years and can be categorised as those detailing general advances in IC as a whole1,2 and those which focus on specific aspects of IC technology, such as new stationary phases,3,4 advances in suppressor technology,5 and advances in detection methods in general.6 Added to these volumes we also have reviews of particular fields of IC application, including drinking water,7 food stuffs,8 biological liquids,9 saline solutions,10 the semi-conductor industry,11 environmental samples,1215 and last but not least, a review of sample treatment techniques and methodologies for IC.16 Finally, we can include a number of personal perspectives on the historical progress of various aspects of IC1720 and the odd miscellaneous item such as a review of IC methods for simultaneous separations of anions and cations.21 Obviously
*Brett.Paull@dcu.ie

this is only a selection of such reviews and many more can be easily obtained, however the depth of material does act to highlight the importance of the technique in the vast field of inorganic analysis, and its status as the dominant method for anion analysis in particular shows no sign of abating. One area that still poses a significant challenge to the ion chromatographer is the application of IC to complex sample matrices. When using the phrase complex matrix in this context, one predominantly means solutions of high ionic strength or samples containing large disparities between the concentrations of the analyte ions and other species present within the sample. We can also bracket biological solutions as so-called complex matrices, as they often contain high concentrations of large bio-molecules such as peptides and proteins and lower levels of small inorganic and organic ions. For the sake of this review we will also classify non-aqueous solutions as complex matrices. What we are not talking about here are samples that simply require sample pretreatment, such as those containing high levels of particulate matter or

Brett Paull

Brett Paull is a Lecturer and researcher within the School of Chemical Sciences and National Centre for Sensor Research, Dublin City University (DCU), Ireland. Dr Paull obtained a PhD in analytical chemistry from Plymouth University, UK. Prior to joining DCU, he was an Associate Lecturer at the University of Tasmania, Australia. Dr Paulls research at DCU is focussed on the varied field of separation science, in particular ion chromatography.

Pavel N. Nesterenko

Pavel N. Nesterenko is a Professor within the Chemistry Department of Lomonosov Moscow State University, Moscow, Russian Federation. Prof. Nesterenko obtained a PhD (1982) and DSc (1999) in analytical chemistry from Lomonosov Moscow State University. His research interests are in the development and design of new stationary phases for various separation modes in chemical analysis.

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semi-solids for example, unless of course the pre-treatment step results in a strongly acidic or basic or otherwise concentrated extract. The key to the success of all chromatographic techniques is the ability to control and manipulate selectivity. In liquid chromatography this includes the selectivity exhibited by the stationary phase, the mobile phase and the type of detection method employed. In reality, the analyst will carefully control selectivity in each of the above areas, and it is the combination of these selectivities that results in the ability to analyse some of the most complex samples. In IC the nature of the stationary phase in particular plays a significant role in controlling selectivity due to the large range of stationary phase chemistries available, this being in contrast to reversedphase liquid chromatography, which is dominated by similar octadecylsilica based stationary phases. Therefore, this review will focus on the singular aspect of recent advances in stationary phase technology for ion analysis, and the attempts being made to improve the range of stationary phases available to apply to these complex sample types. This involves both the development of new stationary phase materials and column capacities, and perhaps most importantly, the development of new stationary phase chemistries.

2 Anion analysis
There is a significant range of commercial anion exchange columns currently available, albeit produced by a small number of manufacturers and based upon a limited range of stationary phase technologies and chemistries. The main players in the IC industry are the Dionex Corporation, Metrohm AG and Alltech Associates Inc., and one or two smaller specialist column manufacturers, such as Transgenomic Inc. The majority of anion exchange columns produced and/or supplied by the above companies are polymer rather than silica based, these being substituted polystyrene divinylbenzene resins (PS-DVB) (e.g. the Hamilton PRPX range, the Metrohm Metrosep range and the Phenomenex Star Ion A300 IC columns), or methacrylate based resins (e.g. Alltech Allsep and Novasep ranges). The largest single producer of polymer based IC stationary phases is the Dionex Corporation (IonPac range of columns), who produce ethylvinylbenzene divinylbenzene (EVB-DVB) based resins with a variety of differing structural designs, including surface functionalised, agglomerated and grafted resins. For most of the above producers, the ion exchange functional group used remains the standard strong quarternary ammonium group, with some weak anion exchangers based upon tertiary amine groups. Table 1 shows some examples of new anion exchange columns on the market, together with some of the complex applications to which they have been applied. 2.1 New selectivity in commercial anion exchangers The above mentioned IC companies are continuously striving to explore and develop new selectivities to overcome increasingly complex sample matrices. The Dionex Corporation have in the past few years developed a number of new products specifically designed for certain applications.2224,55,56,147,148,150155 The company recently released the
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so-called hydroxide selective IonPac AS16 column, specifically for the determination of polarisable anions such as perchlorate, iodide, thiocyanate and thiosulfate, using NaOH or KOH only eluents. The resin itself is a high capacity macroporous 9 mm diameter EVB-DVB substrate with a surface coverage of 65 nm diameter latex particles functionalised with very hydrophilic alkanol quarternary ammonium groups. This results in a stationary phase that exhibits ultralow hydrophobicity, which Dionex describe as being optimised for the determination of the above anions in scrubber solutions, process streams, and brines. However, the biggest application of this new column will be the monitoring of ultra-trace levels of perchlorate in drinking and ground waters, whereby the high capacity of the phase allows for large sample injection whilst still maintaining resolution of the analyte from excess matrix anions, and the selectivity results in reduced run times and improved peaks shapes for perchlorate compared to alternative columns.23 A second hydroxide selective anion exchanger new to the market is the Dionex IonPac AS19 column. This high capacity resin (160 meq column21), according to the manufacturer, exhibits optimised selectivity for bromate and bromide, and is therefore particularly applicable to the determination of bromate in drinking water. The stationary phase itself is based upon a hyper-branched anion-exchange condensation polymer, electrostatically attached to a macro-porous surface sulfonated EVB-DVB resin. The high capacity of the column once again allows large sample volume injection (up to 500 mL), with which bromate detection limits in drinking water samples of approximately 1 mg L21 can be obtained with suppressed conductivity detection.24 2.2 Adjustable-capacity anion exchangers An interesting development in stationary phase technology, which has direct implications for the analysis of complex sample matrices, is the commercial availability of so-called adjustable capacity anion exchangers (not an ideal name, given that any weak anion exchanger has an adjustable capacity). These anion exchangers are based upon immobilised macrocyclic ligands (either coated or chemically bonded), which exhibit varying selectivities towards anionic species depending upon the nature of a central coordinated metal cation. Changing the coordinated cation during a chromatographic run results in a change in column capacity, resulting in what is effectively a capacity gradient. A number of early investigations illustrated the potential advantages of this approach,2530 including short column regeneration time after capacity gradient separations, low baseline drifts during gradient runs and the use of eluents that are simple in composition and low in ionic strength. The commercial product that has evolved from these early studies is the Dionex IonPac Cryptand A1 column, which is based on a cryptand molecule, covalently attached to a macroporous, EVB-DVB resin. A cryptand is a bi-cyclic compound capable of complexing metal cations such as sodium, lithium and potassium, thus forming the anion exchange site. Each metal produces a specific capacity range, which is directly related to
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Table 1

New stationary phases designed for IC of inorganic species in complex matricesanion exchangers Column properties

Stationary phase IonPac AS9-HC

Bonded groups N+R2R9OH

Column dp/mm size/mm 9.0 250 6 4.0

Capacity/meq column21 Matrix 190

Applications to analysis of complex matrices

Ref. 56, 150

IonPac AS11-HC

N+R2R9OH

9.0

250 6 4.0

290

IonPac AS15

N+R2R9OH

8.5

250 6 2.0

56

IonPac AS16

N+R2R9OH

5.0

250 6 2.0

9.0

250 6 4.0

170

IonPac AS17

N+R2R9OH

10.5

250 6 4.0

30

IonPac AS19 Novosep A-2

N+R2R9OH N+R3

7.5 5.0

250 6 4.0 250 6 4.0

160

Metrosep A Supp 2, N+R2R9OH identical to Transgenomic AN1 Metrosep A Supp 4 N+R3 Metrosep A Supp 5, N+R3 identical to Shodex IC SI-50 4E Metrosep A Supp 7 N+R3 IonPac Cryptand A1 2,2,1 cryptand

8.010 250 6 4.0

35

Macroporous 200 nm Determination of trace anions in pores; EVB-DVB, concentrated weak acids with 55%; 90 nm latex ion-exclusion pretreatment, beads with 15% organic solvents cross-linking Macroporous 200 nm Determination of trace anions in pores; EVB-DVB, methanesulfonic and phosphoric 55%; 70 nm latex acids with ion-exclusion beads with 6% pre-treatment. Determination cross-linking of ClO42 in fertilizers EVB-DVB, 55%; Determination of trace anions 10 nm pores (CO322, Cl2, SO422 and PO432) in 7 g L21 solution of NaNO3 and CO322, Cl2 in 0.7% nitric acid Macroporous Simultaneous determination of EVB-DVB, 55%; F2, CO322, SO422, glycerol, sorbitol, saccharin, 80 nm latex monofluorophosphate, PO432, beads with 1% pyrophosphate and crosslinking tripolyphosphate in toothpaste Determination of trace ClO42 (5 ppb) in drinking water in presence of 200 ppm Cl2, in fertilizers Microporous The determination of trace level EVB-DVB, 55%; phosphorus in purified quartz, 75 nm latex beads trace anions in boric acid with 6% crosslinking Macroporous The determination of trace EVB-DVB, 55% bromate (5ppb) in drinking water Polyvinylalcohol Determination of common inorganic anions and oxyhalides (EPA Method 300.1) PS-DVB, 8 nm pores; Analysis of total fluoride content surface area in dentifrices 415 m2 g21 Polyvinylalcohol Polyvinylalcohol Suitable for all routine tasks in water analysis Determination of trace level ClO42 by IC-MS

55, 56, 151

148

147

23, 152, 153

154, 155

24

156

9.0 5.0

250 6 4.0 100 6 4.0

46 39

157

5.0 5.0

250 6 4.0 150 6 3.0

73a

Determination of water disinfectant by-products, bromate in particular Macroporous, 100 nm Determination of polyvalent pores; EVB-DVB, anions including polyphosphates 45:55% and polysulfonates; alkanesulfonic acids in a chromic acid plating bath

Polyvinylalcohol

31, 32, 149

Variable capacity depending on eluents used.

the metalligand binding constant. Several publications have emerged recently which detail the use of this new anion exchanger for the analysis of complex mixtures of inorganic anions. For example, Woodruff et al. have utilised hydroxide gradients, varying both hydroxide concentration and the nature of the subsequent metal ion (LiOH, NaOH and KOH), to control column capacity in order to initially retain, and then rapidly elute, large concentrations of matrix anions.3133 Woodruff et al. applied this technique to the
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determination of trace anions in 2% sulfuric acid,31 the simultaneous determination of inorganic, organic and polarisable anions in industrial wastewater,32 and the determination of chloride and sulfate in semi-conductor-grade etchants, comprised of acetic acid, nitric acid and phosphoric acid.33 Fig. 1 shows the ion chromatogram of an alkaline (pH 14) industrial wastewater sample from a light hydrocarbon plant, run on a IonPac Cryptand A1 column using a KOH and LiOH combined capacity and concentration gradient.
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Fig. 1 Alkaline (pH 14) industrial wastewater from light hydrocarbon plant. Column 5 IonPac Cryptand A1 150 mm 6 3 mm id, 5 mm. Eluent 5 10 mM KOH, at 5 min step to 22 mM LiOH, at 10 min step to 40 mM LiOH. Flow rate 5 0.5 mL min21. Column temp. 5 35 uC. Injection vol. 5 5 mL. Peaks 5 1fluoride, 2acetate, 3formate, 4propionate, 5chloride, 6carbonate, 7sulfate, 8thiosulfate. Reproduced with permission from ref. 32.

2.3 Zwitterionic stationary phases The potential advantages to be gained from the use of zwitterionic stationary phases in assorted modes of LC are now reasonably well understood.34 Neutral hydrophilic stationary phases show increased retention for polar and hydrophilic compounds relative to reversed-phase substrates. Recently we have seen the commercial availability of a silica based covalently bonded zwitterionic column, the SeQuant ZIC-HILIC column, especially designed for so-called hydrophilic interaction chromatography of nucleotides, amino acids amines, phenols, carbohydrates and other polar analytes.35 This product came directly off the back of the work of Jiang and Irgum, who covalently functionalised both polymer36,37 and silica substrates38 with zwitterionic functional groups, and showed how these phases could then be applied to the simultaneous separation of inorganic anions and cations. The use of coated zwitterionic phases for the separation of inorganic and organic ions, (these being reversed-phase substrates coated with zwitterionic surfactants) was first proposed by Hu et al. in the early 1990s.39 Since that time, the technique, which was christened at the time electrostatic ion chromatography (EIC), has been extensively investigated and applied to the analysis of many complex samples, particularly biological fluids and seawater matrices.4048 A short review on the technique was published in 1998.49 Due to the nature of the zwitterionic phases developed, ions within a sample experience both attractive and repulsive electrostatic forces as they travel through the stationary phase. The positioning and nature of the anionic and cationic groups within the immobilised zwitterion governs the relative strength of these forces, which determines whether the stationary phase shows a general selectivity towards anionic or cationic species. Recently, Cook et al. considered a detailed retention mechanism for this mode of IC, which proposed that the terminal ionic group (in this study sulfonate) of the adsorbed zwitterion forms a charged layer on the surface of the stationary phase, which acts as a Donnan membrane. The effective charge on
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this membrane is then dependent upon the nature of the oppositely charged eluent cations, which in turn affects the ability of analyte anions to pass through and interact with the inner ionic site (in this case quarternary ammonium).50 Interestingly, it has been shown that with certain zwitterionic stationary phases the effect of eluent concentration has a much smaller effect upon analyte retention than simple ion exchange,42 and in certain configurations it has been shown that the interaction of the analyte ions with the zwitterionic phase increased with an increase in eluent concentration (up to a certain concentration, after which retention remains constant). This effect, together with a low affinity for sulfate and chloride ions shown by most of the zwitterionic surfactants investigated (see Fig. 2(a) for a Zwittergent 314 coated column), means the direct analysis of high ionic strength samples, particularly highly saline samples is possible using this approach. Specific complex applications demonstrated using EIC include the direct determination of UV absorbing anions in biological fluid including serum, urine and saliva,39,40 the determination of UV-absorbing anions (nitrite, bromide, iodide and nitrate) in seawater,42,43 the rapid determination of iodide in seawater,45 and the determination of iodide in iodised table salt.46 Fig. 2 shows the determination of iodide in solutions of iodised table salt containing 20 g L21 of NaCl using a zwitterion coated ODS column. 2.4 New phases for organic acids Usually organic acids can be separated by either ion-exclusion on strong cation-exchange columns, as recently reviewed by Fischer for environmental samples,51 or by ion exchange on

Fig. 2 Chromatograms showing; (a) the standard separation of sulfate, chloride, nitrite, nitrate, chlorate, iodide and thiocyanate. (b) A sample of iodised table salt (20 g L21) spiked with between 0.8 and 8 mM iodide. Column 5 ODS coated with zwittergent 314. Eluent 5 2 mM zwittergent 314. Reproduced with permission from ref. 46.

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anion exchange columns, as reviewed earlier by Hajos and Nagy.52 For the improved separation of aliphatic organic acids and alcohols in complex or high-ionic strength samples, Dionex have recently designed the new moderately hydrophobic mixed sulfo/carboxylic acid functionalised IonPac ICE-AS6 ion-exclusion column. This column has been applied to the determination of a wide range of carboxylic acids in fruit juice, landfill and composting and fermentation plant effluents53,54 and in combination with a Dionex AS11HC anion-exchange column, was found useful for the determination of chloride, sulfate and nitrate in concentrated phosphoric acid, semiconductor grade 49% HF and 70% glycolic acids.55,56

3 Cation analysis
Although IC, as a technique for cation analysis of relatively simple sample matrices, competes with alternative technologies, such as atomic absorption spectroscopy, it still remains a potent analytical technique when it comes to the analysis of complex sample matrices (which can cause substantial interference problems with alternative spectroscopic and electrochemical based techniques).
Table 2

In cation exchange chromatography there are two main approaches that have been taken in the development of stationary phases for complex sample analyses. These are, (i) the development of high capacity phases which can handle concentrated samples and disproportionate concentration ratios of analytes, and (ii) the alteration of selectivity by incorporation of some form of selective analyte complexation within the stationary phase. In the latter case, what results is essentially a dual retention mechanism which gives the analyst added scope to selectively manipulate the retention of target analytes through control of various eluent conditions, such as eluent pH, and not just eluent concentration. Table 2 shows some examples of cation exchange columns currently available, which have been developed for complex sample analysis. 3.1 Grafted moderate and high capacity cation exchangers Recent years have seen the Dionex Corporation extend their range of cation columns to include two new carboxylate grafted cation exchangers. These are the IonPac CS16 and CS17 columns, which are of high (8.4 meq column21) and moderate capacity (1.45 meq column21), respectively. The

New stationary phases designed for IC of inorganic species in complex matricescation exchangers Column properties

Stationary phase IonPac CS12A IonPac CS15

Bonded groups COOH PO3H2 COOH PO3H2 18-crown-6 ether

dp/mm 8.5 5.0 8.5

Column size/mm 250 6 4.0 150 6 3.0 250 6 4.0

Capacity/meq column21 Matrix 2800 940 2800

Applications to analysis of complex matrices

Ref.

IonPac CS16

COOH

5.5

250 6 5.0

8400

IonPac CS17

COOH

7.0

250 6 5.0

1450

Universal cation Metrosep C 2

COOH COOH

3.0 or 7.0 100 6 4.6 5.0 250 6 4.0 194

LiChrosil IC CA Hamilton PRP-X800 Ammonia isotope column

COOH COOH SO32

5.0 5.0 5.0

100 6 4.6 150 6 4.0 3700 meq g21 300 6 4.0

EVB-DVB, 67 Trace-level quantification 55%; 15 nm of NH4+ (50 ppb) in pores; 450 m2 g21 NaCl brine (1000:1 ratio) Determination of trace-level 62, 63 EVB-DVB, Na+ (10 ppb) in amine-treated 55%; 15 nm 2 21 + (100 ppm NH4 ) cooling pores; 450 m g waters, trace-level NH4+ (25 ppb) in environmental waste water containing a high Na+ concentration (100 ppm), trace level NH4+ (10 ppb) in a KCl (100 ppm) soil extract EVB-DVB, Determination of trace level 58, 59 55%; 15 nm NH4+ (10 ppb) in high 2 21 + concentrations of Na pores; 450 m g (100 ppm); trace level Na+ (10 ppb) in high concentrations of NH4+ or amines (100 ppm), alkali and alkaline-earth metal ions in acid samples (up to 0.1M) without pH adjustment Simultaneous separation of alkali, 57 EVB-DVB, alkaline-earth cations and 55%; 15 nm boiler water amine additives pores; 450 m2 g21 with gradient elution. Silica based Determination of cations at trace 158 levels in ice core samples Silica based Determination of 7 mg L21 sodium in presence of 7 mg L21 monoethanolamine in boiler water Silica coated Simultaneous separation of alkali, with polymer alkaline-earth, transition metal cations, ammonia PS-DVB, Trace alkaline-earth and transition 70 macroporous metals in brines 71 PS-DVB, 12% Determination of 14NH4+ and 15 NH4+ ion ratios in sea water

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grafting technology used to produce the above resins results in a hydrophilic surface, which shields the hydrophobic EVBDVB core. This is achieved through first modifying the resin with a non-functional coating and subsequently grafting a polymeric ion exchanger to this coating. With the CS17 column, this results in improved peak shape for hydrophobic cations, without the need for the addition of organic solvents.57 This column has been applied to the simultaneous separation of Group I and II cations, ammonium and ethanolamines in boiler water, using a simple methanesulfonic acid gradient.57 The higher capacity CS16 column is particularly suited to the analysis of samples with large variations in cation content. By simply utilising its high capacity the column has been used for the determination of trace ammonium ions in excess of sodium (10 mg L21 ammonium in 100 mg L21 sodium)58 and has also been applied to the determination of trace ammonium (0.22 mg L21) in industrial wastewater.59 The column is also suitable for the analysis of acidic samples, containing up to 100 mM hydronium ion, and as such has also been applied to the determination of Group I and II cations in acidic soil extracts.59 3.2 Cation exchangers incorporating crown ethers The incorporation of crown ethers into eluents for cation exchange chromatography to improve resolution of closely eluting metal ions is well established and has been shown again recently by Ohta et al., who used crown ether containing eluents with silica gel columns for the simultaneous separation of Group I and II cations.60 For obvious reasons bonding crown ethers to the stationary phase itself is a more complex procedure, however, many workers have produced such phases, a review of which has been compiled by Lamb and Smith.61 A commercial end-product of this early research is the Dionex IonPac CS15 column, which is a polymeric macroporous resin functionalised with carboxylic acid groups, phosphonic acid groups and 18-crown-6 ether. The selectivity exhibited through the combination of the above functional groups sees greater resolution of sodium and ammonium than shown with alternative cation exchangers and also sees potassium elute after calcium and magnesium. Obviously, this selectivity is ideal for the analysis of samples with excessively high concentrations of one or more of these cations, and this has been notably demonstrated with the resolution of potassium and sodium62 and sodium and ammonium,63 both at concentration ratios of 10,000:1, on the IonPac CS15 column. Fig. 3 shows the determination of sodium, potassium, calcium and magnesium in an ammonium acetate extract of a soil sample, obtained using the above column. Crown ether containing stationary phases have also become available through a number of other specialist manufacturers. Although mainly developed for enantioselective separations of amino acids, companies such as USmac Corporation are now selling a silica based Opticrown range of columns and most (http://www.opticrown.com/products.html),64 recently, Alltech Associates have published results of work
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Fig. 3 Chromatograms showing an ammonium acetate extract of a soil sample. Column 5 IonPac CS15. Eluent 5 5 mM sulfuric acid, 9% acetonitrile. Column temp. 5 40 uC. Injection vol. 5 25 mL. Peaks 5 1sodium, 2ammonium, 3magnesium, 4calcium, 5potassium. Reproduced with permission from ref. 62.

investigating a 18-crown-6 bonded silica column which was used in series with their Universal Cation column, allowing the isocratic separation of sodium and ammonium at a concentration ratio of 5000:1.65 3.3 Novel weak acid cation exchangers The use of alternative functional groups to the standard sulfonated and carboxylated substrates, will naturally result in alternative stationary phase selectivities towards various cations. The above mentioned IonPac CS15 column with its mixed functional bed was a further development of the IonPac CS12A column, which also had a mixed carboxylate/ phosphonate functionality. The incorporation of the complexing phosphonate group improved resolution of certain metal ions, in particular allowing the isocratic separation of Group I and II cations together with manganese in under 12 min.66 The combination of this novel selectivity and the moderate/high capacity of the CS12A column have seen it applied to such complex challenges as the determination of trace calcium and magnesium in 30% sodium chloride brines.67 Interestingly, the complexing ability of the phosphonate groups within the CS12A column has been shown to result in unusual selectivity towards transition and heavy metal ions, with a strong affinity in particular for bismuth and the uranyl ion under acidic conditions.68,69 Recently, the Hamilton Company have also released a weak acid cation exchanger with complexing capabilities. The PRPX800 resin is PS-DVB based and functionalized with itaconic acid. Its intended application is the simultaneous separation of Group I and II cations, but due to the complexing nature of itaconic acid, the column also shows strong affinity for transition and heavy metals, particularly lead and copper.70 Under non-acidic eluent conditions, complexation exceeds simple ion exchange as the dominant retention mechanism for divalent metal ions, allowing potential application of this column to higher ionic strength samples. This potential was exploited recently with the determination of low mg L21 concentrations of magnesium, calcium, strontium and barium in a 2.3 g L21 sodium chloride solution.70
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Table 3

New stationary phases designed for IC of inorganic species in complex matriceschelating ion exchangers Column properties Applications to analysis of complex matrices

Stationary phase

Bonded groups

dp/mm 7.0 5.0

Column size/mm 250 6 4.0 250 6 4.0

Capacity/meq g21 130 100

Matrix Modified silica, 13 nm

Ref.

Diasorb IDA N(CH2COOH)2 APAS NHCH2PO3H2

N-methyl P13 N-methyl-c-aminobutyro-hydroxamate NTA-resin Nitrilotriacetate

63150 250 6 3.0 1.51.9 mmol g21 10 50 6 4.6 60 mmol g21

PDTA-resin

1-Aminobenzyl-1,2diaminopropaneN,N, N9,N9-tetraacetic acid

10

125 6 4.6

127

Determination of trace 96, 97 alkaline-earth metals in brines, seawater Modified silica, 10 nm Determination of 101, 102 beryllium in a stream sediment Poly(acrylonitrile), 8% Determination of 77 lanthanides in seawater 103 PS-DVB, 60% Determination of trace metals (Co, Ni, Cu, Zn, Cd, Mo, Sb, Pb, U) in sea-water at 1 mg L21 level by ICP-MS interfaced with an IC system Macroporous Isocratic separation of 104 glycidylmethacrylate gel fourteen REE

3.4 Chelating stationary phases When discussing the application of chelating stationary phases for cation determinations using IC, it is important to distinguish between those used simply for preconcentration and matrix elimination purposes, and those employed for actual high-performance analyte separations. The incorporation of short chelating columns within a modified IC system, for on-line analyte preconcentration and removal from complex matrices, prior to separation using ion exchange, is the basis of the Dionex chelation ion chromatography (CIC) system. This system has been successfully applied to the analysis of many complex samples over the past 14 years since its inception,7284 including trace metals in seawater,7274,77,83 geological digests,76,7880 biological samples,72,80,82,84 drinking water,81 fertilizers79 and reagent grade chemicals.82 However, here we will briefly focus on the application of chelating stationary phases for the direct analysis of complex sample matrices, so-called high-performance chelation ion chromatography (HPCIC), as last reviewed by Jones and Nesterenko in 1997.85 To-date the majority of such applications have been carried out using iminodiacetic acid (IDA) modified polymer and silica based substrates, either covalently bonded or dynamically coated onto the surface. The use of IDA-silica phases is particularly well documented,86100 predominantly due to the fact that IDA-silica columns are commercially available (Diasorb IDA, BioChemMack ST, Moscow, Russia). This column shows strong selectivity towards both alkaline earth metals and transition/heavy metal metals over alkali metal ions at eluent pHs greater than y45 and y23, respectively, and therefore the selectivity is ideally suited to the determination of these metals within samples containing high levels of alkali salts, such as seawater and industrial brines. Application of the IDA-silica column to the analysis of complex sample matrices include cobalt, zinc and cadmium spiked into seawater,86 zinc and lead in wastewater from a galvanic bath,87 beryllium in acidic rock digest,88 trace
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beryllium in natural waters and simulated seawater96 and trace alkaline earth metals in NaCl and KCl brines.97 Alternative covalently bonded chelating stationary phases investigated recently include aminophosphonate functionalised silica, which has also been applied to the determination of trace beryllium, on this occasion in stream sediments.101,102 There have also been a number of polymer based chelating phases developed, such as a N-methyl-c-aminobutyrohydroxamate resin, which was used for the determination of lanthanide metal ions in seawater,77 and a nitrilotriacetate PS-DVB resin used for the determination of transition/heavy metal ions including uranium in seawater (detection with ICP-MS).103 Fourteen REE can be isocratically separated on a IDA-silica column92,93 or on a methacrylate gel with attached 1-aminobenzyl-1,2-diaminopropane-N,N,N9,N9-tetraacetic acid, except for Eu and Gd which are co-eluted.104 Table 3 details properties and some complex applications of chelating ion exchange columns. Simply coating (either permanently or dynamically) reversed-phase substrates with hydrophobic chelating ligands is an alternative, and perhaps simpler, approach to covalent bonding. This approach has been extensively explored by Jones and co-workers105116 using polymeric substrates coated with chelating dyestuffs (several containing one or more IDA groups) and various heterocyclic carboxylic acids. Within this substantial body of work, these coated substrates have been applied to alkaline earth and transition/heavy metals in concentrated salt solutions,106 transition/heavy metals in coastal seawater,107 barium and strontium in excess calcium containing matrices such as milk powder,108 trace aluminium in seawater,109 traces of bismuth in lead metal,110 trace uranium in environmental waters,113 actinides in environmental and biological samples,114 and the determination of trace metals in highly mineralised waters.116 Fig. 4 shows an application of this approach, using a dipicolinic acid dynamically coated polymeric reversed-phase column for
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4 New phases for bio-analysis

In addition to the need for robust IC methods for the determination of small ions in biological matrices,121 there is now also a strong growth in interest from IC manufacturers in the application of IC systems to the sensitive and selective determination of low and moderate molecular weight biomolecules, particularly those that are charged species under IC separation conditions. In terms of the types of biological samples being analysed for the presence of these bio-molecules, it is safe to say practically all can be considered as complex due to the very large number and varied nature of species present. The list of bio-molecules, which can be readily separated through ion-exchange and, hence determined using IC, includes organic acids, amino acids, amino sugars, nucleotides and carbohydrates. Of course, corresponding oligomeric molecules, such as peptides, oligonucleotides and oligosaccharides, can also be efficiently separated using ion exchange stationary phases, although often additional retention effects are observed such as hydrophobic interactions and size-exclusion, which can detrimentally affect peak shapes. Table 5 shows a range of new stationary phases designed for bioanalytical applications of IC. 4.1 Amino acid columns For many years the standard practice for amino acid analysis was based upon cation exchange chromatography with a step gradient of eluent concentration and pH, combined with ninhydrine post-column reaction. More recently, an alternative approach proposed by Dionex involves an anion-exchange based separation, with electrolytic eluent generation of an hydroxide gradient and detection using pulsed amperometry.122 The novel polymeric pellicular anion exchange resin specifically developed for this, is commercially available as the AminoPac PA10 column, and has been applied to amino acid analysis of cell culture media and fermentation broths.122124

Fig. 4 Chromatograms showing the separation of 239Pu from uranium, and consequently the 238U1H+ interference in NIST 4351 human lung reference material. Column 5 100 mm Polymer Laboratories PLRP-S coated with dipicolinic acid. Eluent 5 0.1 mM dipicolinic acid and 0.75 M HNO3. Detection 5 HR-ICP-MS. Reproduced with permission from ref. 114.

the separation of actinide species within the acidic digest of a human lung tissue reference material. The selectivity of the chelating phase combined with the selective detection method of high-resolution inductively coupled plasma mass spectrometry (HR-ICP-MS) proved a very powerful combination. Table 4 shows details of some miscellaneous stationary phases, which have been applied to complex analytical tasks. This table includes novel phases that have been used for simultaneous anion and cation separations.

Table 4 New stationary phases designed for IC of inorganic species in complex matricesmiscellaneous ion exchangers Column properties Stationary phase ZIC-HILIC Bonded groups N+(CH3)2(CH2)3SO32 dp/mm 5.0 Column size/mm Capacity Matrix Silica, 20 nm pores Applications to analysis of complex matrices Ref. 35

PolyCat A

Poly(aspartic)acid

5.0

200 6 4.6

TSKgel COOH SuperIC-A/C

3.04.0 150 6 6.0 0.2 meq mL21

IonPac ICE-Borate

SO32

7.5

250 6 9.0 27 meq column21

Determination of nucleotides, amino acids, amines, phenols, carbohydrates, peptides, glucuronated/ glycosylated and phosphorylated compounds Silica, 6 nm Simultaneous determination pores of inorganic anions (Cl2, NO32) and cations (Na+, K+, Mg2+, and Ca2+ in water Polymethacrylate Improved version of Tosoh TSKgel OApak-A designed for simultaneous determination of inorganic anions (SO422, Cl2, NO32) and cations (Na+, NH4+, K+, Mg2+, and Ca2+ in acid rain water Microporous, Trace borate analysis in DVB, 8% deionized water

117, 118

119

120

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Analyst, 2005, 130, 134146 | 141

Table 5

New stationary phases designed for bioanalytical applications of IC (including organic acids) Bonded groups dp/ mm Dpore/nm Capacity, column/mm Matrix PS-acrylate-DVB, 8% crosslinking, intermediate hydrophobic resin Polymethacrylate Hydrophilic resin Application Ion-exclusion determination of organic acids in fruit juice, effluents of landfills, composting and fermentation plants Vacancy chromatography of aliphatic carboxylic acids Formate in methanogenic degradation of butyrate Analysis of protein hydrolysates and complex mammalian cell cultures Determination of amino acids in samples with high content of carbohydrates Mono and disaccharides with amperometric detection Separation of polysaccharides with length of up to 30 glucose units Fast separation of monosaccharides and disaccharides, glycoproteins Ref. 53, 54

Stationary phase Organic acids IonPac ICE-AS6

8.0 Microporous 27 meq column21, Mixed SO32 and COOH 250 6 9.0 mm groups 5.0 0.1 meq mL21, 150 6 7.8 mm 150 6 4.6 mm

Tosoh TSKgel COOH OApak-A Shim-pack N+R3 IC-A3 Amino acids AminoPac PA 10 N+R3

126 130

5.0

250 6 2.0 mm

EVB-DVB substrate 55% crosslinking agglomerated with 80 nm functionalized latex with 1% crosslinking PS-DVB substrate PS-DVB substrate

122, 123 124

Carbohydrates ESA Sucrebead I MetroSep Carb 1 CarboPac PA-20

N+R3 N R3 N+R3 difunctional

+

7.0 5.0 6.5

N/a

250 6 2.0 mm 1530 meq, 250 6 4.6 mm

127 128, 129

CarboPac PA-200

N+R3

Dynamically loaded EVB-DVB resins Nucleotides Shodex IEC DNApak Hydrocell QA NP10 DNAPac PA100

Decyl-2,2,2 cryptand

EVB-DVB substrate 55% crosslinking agglomerated with 130 nm functionalised latex with 6% crosslinking 5.5 Microporous 90 meq, EVB-DVB substrate Separation of oligosaccharides 250 6 3.0 mm 55% crosslinking based on fine structural agglomerated with differences 34 nm functionalized latex with 6% crosslinking Various types of EVB-DVB substrates Mono-, di- and oligosaccharides

,1.0 nm

65 meq, 150 6 3.0 mm

29

DEAE N+R3 N+R3

2.5 Non-porous Non-porous 13 Non-porous

0.70 meq g21, 50 6 6.0 mm

Polyhydroxymethacrylate PS-DVB beads

Separation of oligonucleotides

250 6 4.0 mm

Hydrocell NS 1500 Zorbax Oligo

N+R3

10

50

150 6 2.1 mm

Primesep 200

Mixed 5.0 hydrophobic and ionexchange Alkylsilica with 5.0 embedded COOH groups 5.0

15

125 6 4.0 mm

High speed analysis of small nucleotides, oligonucleotides and DNA fragments EVB-DVB substrate Resolution of oligonucleotides 55% crosslinking up to 60 bases, agglomerated with oligonucleotides with 100 nm functionalized secondary structures, latex with 5% analysis phosphorothioatecrosslinking based clinical samples Highly cross-linked Separation of nucleotides from PS-DVB 2 to 40 residues and DNA restriction fragments of up to 1000 base pairs Oligonucleotides from 3 to Silica stabilized with 48 residues zirconia layer, 140 m2 g21 Separation of nucleosides

131

10

250 6 4.6 mm

Silica

132

Proteins and peptides BioBasic AX Polyethyleneimine

30

0.22 meq g21, 300 6 4.6 mm

Silica, 100 m2 g21

Separations of peptides, proteins, nucleic acids

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Table 5 New stationary phases designed for bioanalytical applications of IC (including organic acids) (continued) Stationary phase ProPac SAX-10, WAX-10, WCX-10, SCX-10 Bonded groups N+R3, NR2, COOH, SO32 dp/ mm Dpore/nm 10 ,1.0 Capacity, column/mm 250 6 4.0 mm Matrix EVB-DVB, 55% crosslinking Application High resolution of proteins with small differences in charge Ref.

Other Alkion SO32 Pickering Laboratories

10

Non-porous

Low capacity

PS-DVB substrate

Determination of biogenic and polyamines in foods, beverages and medicines, aminoglycosides, antibiotics in feeds, aminecontaining herbicides

Fig. 5 shows the impressive separation of amino acids and selected carbohydrates possible with the AminoPac column in a diluted fermentation broth sample. In the application shown a novel bi-modal integrated amperometric detection method was used to improve detector selectivity. The two chromatograms shown are of the same sample, determined under two differing detector modes. 4.2 Carbohydrate columns There has also been significant progress in the development of new anion-exchangers designed for the separation of carbohydrates. As with amino acid analysis, the IC determination of carbohydrates can be achieved through ion-exchange chromatography, following by amperometric detection. The CarboPac range of new columns from Dionex (Carbo-Pac PA20, and PA200) offers a range of capacities and resin structures to suit particular applications. The CarboPac PA200 is the newest of the range, designed for mono- and disaccharide determinations. The column is based upon a 5.5 mm EVB-DVB agglomerated resin and is the recommended column for separation of oligosaccharides having fine structural differences such as the composition and the sequence of the oligo-saccharides,

linkage isomerism, degree of sialylation, and degree of branching. Examples of complex applications of these CarboPac phases include the analysis of starch and the determination of complex novel food carbohydrates125 and glycoprotein monosaccharides.128,129 Methrom have also moved into the bio-analysis market with the release of their Metrosep Carb 1 carbohydrate column range, which are low and medium capacity polystyrene based anion exchangers, designed for the separation of mono- and disaccharides, although also suitable for the separation of poly- and oligosaccharides. Example applications include the carbohydrate analysis of vodka, amino aid solutions and plant extracts. Recently, one more company Shiseido introduced ESA Sucrebead I anion-exchange column for carbohydrate determination with amperometric detection. 4.3 Nucleotides and oligonucleosides Two types of anion-exchanger are seen to be suitable for the separation of nucleotides, these being small particle size nonporous substrates, such as those found in the Shodex IEC DNApak and Hydrocell QA NP10 columns, and alternatively larger porous particles, like those within the Hydrocell NS 1500 column (both from BioChrom Labs). The DNAPac PA100 column produced by Dionex is a pellicular agglomerated anion exchange resin specifically designed to obtain resolution of synthetic oligonucleosides to 60 bases and beyond, and the resin based column is also compatible with strongly denaturing conditions such as high pH and temperature conditions.131 Taking into account the zwitterionic nature of nucleotides and oligonucleosides, various interactions and their combinations can be realised for chromatographic resolution. Both cationand anion-exchangers,131 zwitterionic ion-exchangers,35 mixed mode ion-exchange and reversed-phase bonded phases like Zorbax Oligo produced by Agilent, or alkylsilica with embedded ion-exchange groups like Primesep 200132 were found useful for separation of these charged molecules. 4.4 Proteins and peptides

Fig. 5 Chromatograms showing the separation of amino acids and carbohydrates within a fermentation sample (diluted 1:250). Column 5 Dionex AminoPac PA10. Eluent 5 NaOH gradient. Detection 5 Bi-modal integrated amperometric detection. Reproduced with permission from ref. 122.

Ion-exchange chromatography has seen significant growth in interest for use in the multidimensional chromatographic fractionation of complex protein digests in proteomics studies. Although polymeric ion-exchangers have long been used for protein separations, in the past they could not compete with
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the resolution capabilities of reversed-phase sorbents. Recently, specialist protein columns based on pellicular ionexchangers with a hydrophilic charged outer layer have been introduced (ProPac series from Dionex), which offer much better separation performance than traditional columns. For example, a number of different C-termini Lys variants have been separated and collected using the ProPac WCX-10 column.133 Other stationary phases designed for protein separations are based upon the introduction of different ionexchange groups in alkyl chains, this approach has been used in many new silica based phases, like for example, Kaseisorb ODS-SAX or Primesep 200.132

5 Future directions
At this late stage of this review it seems appropriate to touch on some specific recent directions in IC stationary phase technology which are attracting much attention and which show potential for the analysis of complex matrices. 5.1 Monolithic phases Monolithic polymer and silica based anion and cation exchangers are beginning to become available commercially and will no doubt gain in popularity over the next few years. On the polymer side a number of manufacturers are now producing ion exchange functionalised short monolithic columns for fast separations of bio-molecules from biological matrices (Isco Swift columns and BIA Separations Convective Interaction Media or CIM disks).134 There have also been work carried out on zwitterionic functionalised polymer monolithic columns, again primarily designed for the rapid separation of large bio-molecules such as proteins.135 On the inorganic side, silica based reversed-phase monoliths (Merck Chromolith range) have been coated with cationic and anionic ion exchangers and applied to rapid anion and cation separations, albeit in relatively simple sample matrices.136139 The production of a covalently bonded IDA silica monolith for the determination of alkaline earth cations in high ionic strength samples has recently been reported by Sugrue et al.140,141 This work combined the selectivity of the chelating IDA group with selective post-column reaction detection, and, with the aid of elevated flow rates achievable through the use of the monolithic silica support, was able to determine sub-mg L21 concentrations of calcium and magnesium in 2 M KCl and NaCl brines in 40 s. Fig. 6 shows this impressive separation and illustrates the future potential such phases have for rapid and even on-line analysis of complex matrices. Rybalko and Nesterenko142 have also demonstrated an interesting application of a linear flow gradient to the separation of inorganic anions on 3 mm CIM-disk functionalised with L-hydroxyproline. 5.2 New phases for IC-MS The recent combination of IC with MS detection is ideally suited to complex sample analysis and so also deserves specific mention. To facilitate the coupling of the two techniques, new low capacity stationary phases are required which can be used with MS compatible eluents. Suppression of hydroxide eluents
144 | Analyst, 2005, 130, 134146
Fig. 6 Shows the separation of 10 mg L21 Mg(II) and 10 mg L21 Ca(II) in 2 M KCl (15% w/w) solution in under 40 s. Column 5 10 cm IDA silica monolith. Eluent 5 1 M KCl, pH 4.85, flow rate 5 5 mL min21. Peaks; 1 5 Mg(II), 2 5 Ca(II). From ref. 140 Reproduced by permission of The Royal Society of Chemistry.

is the approach being promoted by Dionex, who now supply several hydroxide selective phases specifically packed in microbore columns for use with MS detection. The application of IC-MS to the trace ion analysis of a number of complex sample matrices has already been shown, including trace oxyhalides in various water samples,143,144 ionic species within agricultural chemicals145 and a range of other ionic contaminants, including ultra-trace perchlorate in environmental water samples.146,156

6 Conclusions
It is clear from this review that over the past ten years IC has moved into whole new areas of complex application, and a major part of this is due to developments into new and more selective stationary phases. Much of this progression has been gradual, with small refinements in selectivity and efficiency being tailor made for specific complex applications. This will of course continue on apace, and it is clear that at this point in time, and for the immediate future, IC holds a dominant position in the field of ion analysis. However, analytical techniques need to evolve or they will soon be superseded. The movement of IC into the field of bio-analysis over the past few years has seen a substantial increase in new and challenging applications. It is likely that increasing numbers of novel columns and stationary phases will be developed to tackle these new and complex problems and this alone means research into the development and application of IC is still an exciting place to be.
Brett Paull*a and Pavel N. Nesterenkob a National Centre for Sensor Research, School of Chemical Sciences, Dublin City University, Glasnevin, Dublin 9, Ireland. E-mail: Brett.Paull@dcu.ie; Fax: +353 (0)1 7005503; Tel: +353 (0)1 7005060 b Department of Analytical Chemistry, Moscow State University, Moscow, 119899, Russian Federation. E-mail: PavelN@analyt.chem.msu.ru; Fax: +7 (095) 939 46 75; Tel: +7 (095) 939 44 16

This journal is The Royal Society of Chemistry 2005

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