Ion Chromatography Paper

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Ion Chromatography An Instrumental Method Jordan Sedlock | CHEM 310 | November 25, 2014
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Ion Chromatography

Ion ChromatographyAn Instrumental Method

Methods of quantifying ions and polar molecules have been developed and used since the mid 19th century.1 Ion chromatography (IC) was developed around the 1950s and is used to separate and quantify ions or ionizable molecules as well as large proteins and small nucleotides in solution at very low levels.2 It is a form of liquid chromatography and describes efficient chromatographic peak separation amongst ionic species.2 Ion chromatography has proven to be very effective at quantifying ionic species in many fields including, but not limited to: food analysis, water quality treatment, and pharmaceuticals. Ion chromatography refers to a group of liquid-chromatographic methods that are used to quantify certain groups of analytes.3 Analytes can include low-molecular-mass acids and bases, also, inorganic anions and cations.3 Different methods of IC exist and include: ion-exchange, ion-exclusion, ion-pair, and ion-suppression chromatography.2 Ion-exchange chromatography (IEC) is one of the most applicable areas of IC, used to separate inorganic anions and cations.3 Ion-exclusion chromatography (also abbreviated IEC) is commonly used to separate weak acids such as carboxylic acids, weak bases such as ammonia, and carbohydrates as well as other hydrophilic biomolecules.2 Ion-pair chromatography (IPC) is commonly used to separate analytes with widely different components as would be the case in mixtures of acidic and basic analytes as well as compounds that are very difficult to separate, possibly due to presence of covalent bonds.2 Ion-suppression chromatography (ISC) is generally used to separate polar, weak acids and weak bases.4 Ion-exchange chromatography (IEC) separation techniques are based on the affinities of the analytes of interest and the oppositely charged ionic functional groups within the stationary phase of an ion-exchange column.2 There are two types of exchangers used in IEC, anion and cation exchangers.5 Anion exchangers contain positively charged functional groups on the stationary phase to attract solute anions.5 Cation exchangers contain negatively charged functional groups on the stationary phase to attract solute cations.5 Of these two types of exchangers, there are three different classes used for ion-exchange based on particle size. There are resins, which are used for applications involving small molecules, gels, which are used for applications involving macromolecules, and inorganic exchangers, which are used for separations involving extreme chemical conditions such as powerful oxidizing agents or high temperatures.5 Anion exchanger resins are known to be either strongly basic, containing quaternary ammonium groups attached to styrene and divinylbenzene copolymers or weakly basic containing polyalkylamine groups attached to styrene and divinylbenzene copolymers.5 Cation exchanger resins are known to be either strongly acidic, containing sulfonic acid groups attached to styrene and divinylbenzene copolymers or weakly acidic containing carboxylic acid groups attached to styrene and divinylbenzene copolymers.5 Gels used in ion-exchange are commonly made from cellulose and dextran, which are softer materials than polystyrene resins.5 Ion-exclusion chromatography (IEC), another type of IC, is especially useful for separating organic acids as well as small, neutral molecules and weak bases.6 This technique involves the separation of molecular species, rather than ions but can be used to separate analytes by differences in molecular size, shape, or charge.2 This technique also involves what is known as the Donnan Exclusion mechanism, wherein, certain molecules (depending on size, shape, or charge) can pass through a semipermeable membrane and onto the stationary phase (or resin phase) after absorbing into an occluded liquid phase surrounding it and other molecules cannot, which are therefore, not retained.2 This is useful when separating weak organic acids because such compounds can easily adsorb onto the resin phase while strong, anionic compounds are unable.6

Ion-pair chromatography (IPC) is another type of IC that separates polar and ionic compounds via a reverse-phase HPLC column rather than an ion-exchange column.5 This method is commonly detected using UV detectors, because analytes of interest contain chromaphores, as well as conductivity detectors, because they are universal.7 IPC relies heavily on the composition of the mobile phase in order to achieve separation.7 The mobile phase contains an organic solvent as well as an ion-pair reagent.7 The ion-pair reagent contains a large, ionic molecule whose charge is opposite that of the analyte trying to be analyzed.7 This large molecule is composed of two different regions, one wherein there is a strongly hydrophobic segment that interacts with the stationary phase and another region which contains the opposing charge of the analyte of interest.7 The stationary phase is commonly comprised of polystyrene/divinylbenzene (PS/DVB).7 The retention mechanism in this separation technique is not fully understood, however, three theories exist in attempt to explain this: ion pair formation, dynamic ion exchange, and ion interaction.7 The first theory is similar to reversed-phase chromatography in that the ion-pair reagent and the analyte of interest form a neutral pair and then partition between the mobile and stationary phases; retention times can be varied depending on concentration of organic solvent in the mobile phase.7 The second theory involves the hydrophobic region of the ion-pair reagent adsorbing to the stationary phase and the analyte of interest interacting with the ionic region of the ion-pair reagent like it would in ion-exchange chromatography.7 The ion interaction model involves an electrical double layer that is formed from the permeation of the stationary phase by the ion-pair reagent which carries with it a counter-ion; retention is dependent upon factors described in the first two models.7 Ion-suppression chromatography (ISC), the last type of IC being discussed, is comprised of two sub-categories: suppressed-ion anion chromatography and suppressed-ion cation chromatography.5 In ion-suppression chromatography, an unwanted electrolyte is removed before the analyte of interest undergoes detection via electrical conductivity.5 In the suppressed anion technique, a mixture of anions is separated using an anion separator column and strong base as an eluent.5 Anions and eluent are then passed through a suppressor where the strong base, which possesses high conductivity is converted into H2O, which has low conductivity.5 This process allows analytes of interest to be detected without interference of eluate.5 In the suppressed cation technique, a mixture of cations is separated using a cation separator column and strong acid as an eluent.5 Cations and eluent are then passed through a suppressor where the strong acid is converted into water, reducing conductivity of the eluate and allowing for accurate detection of the analytes of interest.5 This technique essentially neutralizes weak acids and bases which is what causes the suppression in ISC.4 ISC is commonly used with reversed-phase HPLC columns, however, the silica material is not preferred as much as PS/DVB is due to the small pH range that the silica material possesses (2.5 7.5).4 The mobile phase for this technique contains an organic modifier and a buffer and the retention mechanism is similar to that on a reversed-phase HPLC column when separating weak acids and bases or neutral molecules.4 Based on the types of samples that are analyzed using IC, sample preparation steps must be taken in order to ensure sufficient resolution. The most basic rule of sample preparation is that all samples must be in the aqueous phase as IC is a form of liquid chromatography.8 It is particularly difficult to quantify compounds that exist at trace levels in matrices with extreme conditions such as pH or high ionic strength.3 To reduce the amount of an interfering matrix, a simple solution is to inject a smaller volume onto a column as well as using a selective detection method, however, this is problematic because it also reduces the amount of analyte of interest that is present.3 A second method, which involves handling matrices with extreme pH is to perform active dialysis (Donnan dialysis), a commonly employed sample cleanup technique which involves transferring ions of a specific charge across a membrane, for example, with highly alkaline samples.3 This type of sample cleanup helps reduce peak distortion, baseline noise, and prolong column life.3 This method can be further refined by adding an electrical current across the membrane, known as electrodialysis, which only differs from Donnan dialysis due to the presence of the electrical current.3 Electrodialysis is also helpful when analyzing alkaline samples which contain trace amounts of inorganic anions as well as neutralizing acids and bases prior to analysis of trace metals such as magnesium(II) and calcium(II).3 Samples with matrices of high, ionic strength must be prepared in order to account for matrix effects, which greatly affect resolution and noise in the final chromatogram. A technique to do this is referred to as matrix elimination IC.3 This technique, simply put, involves using the dominant ion in the matrix as the eluent in a separation which causes sufficient separation of the analyte of interest and no retainment of the matrix on the column.3 This method greatly reduces band broadening in the final chromatogram.3 Another method of reducing matrix effects is to perform sample cleanup and a matrix eliminating step via solid-phase extraction (SPE).9 Environmental samples, particularly water samples need very little sample preparation in order to be separated and quantified via IC.9 Drinking water generally only needs to be filtered through a 0.45 m filter to remove particulates and is then ready for analysis.9 Wastewater samples generally only need dilution (to range of 2 100 g/l) and filtering through a 0.45 m filter and are then prepared fully.9 Solid samples such as soil can go through very simple aqueous extractions before being prepared fully.9 Known amounts of sample are mixed with water, preferably so as not to introduce extraneous peaks into final chromatogram, but can be mixed with other solvents such as methanol or weak acids, and filtered through a 0.45 m filter and are then ready for analysis.9 Acid digestion in a strongly concentrated acid, such as nitric acid, can be employed for solid samples such as rock.9 This method is not favorable for IC analysis of anions because the co-anions that are present from the acid digestion can cause large, distorted peaks in a final chromatogram.9 This sample preparation method can be employed for analysis of cyanide, sulfide, and fluoride, however, when coupled with amperometric detection, which involves applying a voltage between two electrodes positioned in column effluent and measuring the difference between them.9 Acid digestion is used frequently when using IC to determine cations; examples include nitrogen, transition metals, and rare earth metals.9 Alkali fusion is an alternative to acid digestion which requires mixing a sample composed of geological material with an alkaline flux and heated to high temperatures ranging from 800 1100 C.9 The flux becomes molten which is then cooled and digested in suitable solution prior to IC analysis.9 This method works well for analyzing fluoride and chloride in materials of geological origin.9 Another method used for solid samples includes combustion, wherein, the entire sample is burned in an oxygen flame which leads to conversion of nonmetallic elements into volatile and gaseous compounds that are collected in an absorbing solution and then analyzed via IC.9 This method of sample preparation works well for determining total nitrogen, sulfur, and phosphorous in plant material and different geological, rocky material.9 After sample preparation, analysis per sample is generally dependent upon which type of sample is being analyzed, as well as other general parameters such as mobile phase composition, stationary phase composition, column length, column diameter, flow rate, etc. Analysis is also dependent upon which form of IC is used. As previously stated, the four forms of IC are each used to detect different forms and classes of analytes. Sea water samples can be analyzed for transition metals in approximately 12 minutes.9 Lake water samples can be analyzed for anions in approximately 10 minutes.9 Wastewater samples, which are of particular environmental concern, can be analyzed for anionic and cationic nutrients in approximately 6.5 minutes.10 Weak acid determination, used in food chemistry, can be performed in approximately 18 minutes.6 Determination of toxic elements such as arsenic, in different types of cereal-based food can be completed in as little as 7 minutes.11 Separation of proteins from complex biological mixtures, such as human plasma, can be completed in approximately 20 minutes.12 Ion chromatography analysis is performed on an HPIC instrument that generally costs approximately $35,000 dollars.13 Thermo Scientific has manufactured an HPIC system designed to maximize resolution, speed, and sensitivity.13 All chromatography systems consist of the same basic components: eluent, pump, injection valve, columns, suppressor (which is specific to ion chromatography), detector, and some sort of data collection system.14 The eluent establishes basic ionic conditions, stabilizes sample in solution, and promotes progression of sample through the system.14 Pumps are used to move eluent and samples throughout the system.14 There are different types of pumps that can be used including serial and parallel, gradient and isocratic.14 Continuous, pulse-free flow is required so as not to introduce noise into the chromatogram.14 Eluent flows from the pump, into the injection site which contains a 2-position valve used to direct flow of eluent and to introduce the sample into the system.14 Leaving the injection site, the sample and eluent flow into the column where separation occurs based on the stationary phase within the column.14 Following the column, the sample will enter a suppressor (selected based on application).14 There are three types of suppressors: self regenerating suppressor (SRS), Atlas Electrolytic Suppressor (AES), and micromembrane suppressor (MMS).14 Suppression is required so that the ions being analyzed can be detected via one of three different methods: conductivity, amperometry, or absorbance.14 Readings from the detector appear in the data collecting system and final chromatograms are produced.14 Many applications have been pursued using instruments and separation techniques of this nature. IC has proven to be particularly useful in environmental analysis of water samples due to its ability to detect trace amounts of compounds with high accuracy. Perchlorate, an essential ingredient in rocket fuel, poses serious threats to human health as it is contaminating drinking water.15 Perchlorate causes interference of iodide uptake by the thyroid gland and therefore greatly affects proper thyroid functions.16 An anion exchange column and ion exchange chromatography can be employed to detect levels of perchlorate in drinking water.16 Separation of all ions can be completed within 7 minutes.16 It is concluded that this method provides valid results for determining trace-level perchlorate in drinking water.16 Other methods of determining trace level perchlorate in drinking water have been employed and compared to IC.17 One such method includes electrospray ionization mass spectrometry (ESI-MS).18 This technique involves creating ions by applying high voltage to liquid to create an aerosol.18 It is appropriate to use this method in analysis of permanently ionized or polar, ionizable compounds.18 Ions are then quantified via mass spectrometric methods. Unlike IC, ESI-MS requires the employment of a standard addition method to correct for severe matrix effects in the samples being analyzed and therefore requires more care during sample preparation.18 ESI-MS has linearity of 10-5 M and a detection limit in reagent water below 1 g/l, however, it is almost impossible to detect trace levels of perchlorate in wastewater samples.18 The IC method has a linear range of 2 100 g/l and a limit of detection of 20 g/l.16 High ionic strength of chloride greatly interferes with chromatogram signal, however, this can be greatly reduced with solid-phase extraction before analysis.16 More training would be required to perform the ESI-MS method of determining perchlorate in drinking water. Ion chromatography is a relatively simple technique overall. It is very important in environmental chemistry and will continue to be as it is so effective at determining trace elements as well as inorganic anions and cations. Ion chromatography has progressed a long way from development and has been proven to be a very dependable and accurate analytical separation technique.


1. Ion Chromatography. (accessed Oct 15, 2014).2. Chromacademy. (accessed Oct 15, 2014).3. Haddad, P. R.; Doble, P.; & Macka, M. Developments in sample preparation and separation techniques for the determination of inorganic ions by ion chromatography and capillary electrophoresis. Journ. Of Chrom. A. 1999, 856, 145-177. 4. Chromedia Analytical Sciences. (accessed Oct 15, 2014).5. Harris, D. C. Chromatograpic Methods and Capillary Electrophoresis. In Quantitative Chemical Analysis; W. H. Freeman and Company: New York; 2010; 635-646.6. Milagres, M. P.; Brandao, S. C. C.; Malgalhaes, M. A.; Minim, V. P. R.; & Minim, L. A. Development and validation of high-perfomance liquid chromatography-ion exclusion method for detection of lactic acid in milk. Food Chem. 2012, 135, 1078-1082. 7. Dionex. (accessed Oct 16, 2014). 8. Koch, W. F. Sample preparation in ion chromatography. Journ. Of Res. 1979, 84, 241-245.9. Jackson, P. E. Ion Chromatography in Environmental Analysis. In Encyclopedia of Analytical Chemistry; Meyers, R. A.; Ed.; John Wiley & Sons Ltd: Chichester; 2000; 2779-2801. 10. Karmarkar, S. V. Analysis of wasterwater for anionic and cationic nutrients by ion chromatography in a single run with sequential flow injection analysis. Journ. Of Chrom. A. 1999, 850, 303-309. 11. Llorente-Mirandes, T.; Calderon, J.; Centrich, F.; Rubio, R.; & Lopez-Sanchez, J. F. A need for determination of arsenic species at low levels in cereal-based food and infant cereals. Validation of a method by IC-ICPMS. Food Chem. 2014, 147, 377-385.12. Gajdoski, M. S.; Kovac, S.; Malatesti, N.; Muller, E.; & Josic, D. Ion-exchange sample displacement chromatography as a method for fast and simple isolation of low- and high-abundance proteins from complex biological mixtures. Food Technol. Biotechnol. 2014, 52(1), 58-63. 13. Thermo Scientific. (accessed Oct 27, 2014). 14. Dionex. Principles and Troubleshooting Techniques in Ion Chromatography. Dionex Corporation: 2002; Document No. 034461. 15. Jackson, P. E.; Gokhale, S.; Streib, T.; Rohrer, J. S.; & Pohl, C. A. Improved method for the determination of trace perchlorate in ground and drinking waters by ion chromatography. Journ. Of Chem. A. 2000, 888, 151-158. 16. Jiang, S.; Li, Y.; & Sun, B. Determination of trace level perchlorate in Antarctic snow and ice by ion chromatography coupled with tandem mass spectrometry using an automated sample on-line preconcentration method. Chin. Chem. Lett. 2013, 24, 311-314.17. Li, Y.; Whitaker, J. S.; & McCarty, C. L. Analysis of iodinated haloacetic acids in drinking water by reversed-phase liquid chromatography/electrospray ionization/tandem mass spectrometry with large volume direct aqueous injection. Journ. Of Chrom. A. 2012, 1245, 75-82.18. Roehl, R.; Slingsby, R.; Avdolovic, N.; & Jackson, P. E. Applications of ion chromatography with electrospray mass spectrometric detection to the determination of environmental contaminants in water. Journ. Of Chrom. A. 2002, 956, 245-254.