Gradient analysis in ion chromatography

If different substances have very different retention times, changing the mobile phase during the separation procedure can reduce the analysis time for the entire separation.

A continuous change in the composition of the mobile phase is called gradient elution.

Isocratic separations are performed with a constant concentration of the eluent or mobile phase.

In the majority of cases, applications can be carried out isocratically and usually it is desirable to keep the chemistry of the methodology as simple as possible.

It is sometimes necessary to form a gradient of a weak to concentrated strong eluent over the course of the chromatographic run allowing separation of anions that may have a wide range of affinities towards the separation column.

Those anions weakly retained by the column elute first and as the eluent concentration is increased the more strongly adhering anions can then be eluted by the stronger eluent.

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 (IC) is a subdivision of high performance liquid chromatography (HPLC).

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.

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.

Types of gradient analysis.

Generally there are two different methods for forming gradients at low and high pressures.

The most popular and less expensive method is using a single pump and three to four micro-proportioning valves at the inlet of the pump.

At low pressure the gradients are formed from the combinations of the different solvents by metering a defined amount from the various eluent reservoirs into the pump.

The composition in the low pressure mixing chamber is controlled by timed proportioning valves, only one of which is open at given time.

The gradient is formed by the relative time that the valves are open; at the start of the chromatographic run, the valve connected to the weak eluent is open the longest time.

As the gradient progresses, the valve attached to the strong eluent is open for longer and longer times, while conversely the weak eluent valve is open shorter times.

Often the gradients are formed using just two of the valves, allowing the others to be used for cleaning solutions. For the second method, the flow from two high pressure pumps is directed into a high pressure-mixing chamber.

The first pump reservoir contains a weak eluent while the second contains the stronger eluent.

The different eluents are mixed in the chamber before the mobile phase flows through the injection valve and then onto the analytical column.

The total flow from the two pumps remains constant, and controlling the relative pump rates forms the gradient.

The gradient commences with a high flow of the weak eluent pump and a low flow of the strong eluent pump. Over the course of the chromatographic run, the flow of the strong eluent is increased while the flow of the weak eluent pump is decreased, keeping the total flow constant.

The advantage of a high pressure mixing system is a smaller dwell volume and the formation of faster gradients.

With a high pressure system one obtains passive mixing of the eluents as a result of diffusion in the capillary, meaning that the only moving parts of the mobile phase delivery system are the pistons present in the pump head. Some considerations for gradient techniques.

Historically the use of gradient systems in ion chromatography was restricted to the use of hydroxide eluents, as any change in the carbon dioxide concentration caused a marked rise in the baseline when a carbonate/bicarbonate eluent was used as the mobile phase.

The eluting power of hydroxide is extremely weak compared to carbonate, and means that high alkali concentrations often have to be utilised to achieve the desired separation.

The use of such solutions is often undesirable and can cause corrosion problems with metal pump delivery systems, as well as adversely affecting certain separation columns.

The use of a carbonate/bicarbonate system reduces the injection and system peak to an absolute minimum, and using a moderate concentration of carbonate benefits both the IC components and reduces the running costs of the IC system.

The advantages of a gradient system are that for complex samples they offer higher resolution of fast eluting analytes, an acceleration of later eluting analytes, and the option to clean up the analytical column after injection of heavily loaded sample matrices.

Conversely, the disadvantages are that the chromatography system is more complex and thus the handling more complicated, the cost of the initial system requires a higher outlay, and the associated running costs are also increased. Another important consideration that is often overlooked is the time losses due to the reconditioning of the analytical column. At the start of the run, the ion exchange sites of the column are in the weak eluent form, whereas at the end of the gradient run they are in the strong eluent form and have to be converted back to the weaker type before analysis of the next sample.

Depending upon the substrate material of the separation column, this can take an additional 10-15 minutes which has to be added to the analysis time.

For an automated system this is not an issue but for a manual, direct injection system then this factor should be considered.

Metrohm 828 IC dual suppressor.

The 828 IC dual suppressor is a continuous, regeneration-free solid phase suppressor that can be used to increase the sensitivity of the detection of anions in ion chromatography.

In addition to the usual chemical suppression of the carbonate/bicarbonate background, a secondary suppression occurs that removes the dissolved carbon dioxide to reduce the background conductivity of the mobile phase even further.

These produces an improved signal to noise ratio and peak areas in the mg l-1 range and above are increased by 30%.

The areas of the injection peak (water dip) and the system peak are reduced to an absolute minimum.

The reduction of the injection peak improves the chromatography of early eluted anions and similarly by reducing the size of the system peak one is able detect anions that would normally co-elute with this peak.

With the use of a carbonate/bicarbonate eluent, the baseline drift is minimised.

Within a Metrohm ion chromatography system the 828 IC dual suppressor can be controlled via a remote interface, and it can also be combined to other commercially available HPLC systems.

The 828 IC dual suppressor is used to increase the sensitivity of the anions when using conductivity measurements, and is installed between the separation column and the conductivity detector.

The 828 consists of a suppressor cell, a direct current source, and a degassing unit.

The eluent containing the sample ions is split proportionally towards the detector, anode and cathode.

Three simultaneous processes occur inside the 828 during its operation.

1.

The eluent and sample ions emerge from the separation column and an acid/base reaction converts them to their protonated forms.

The cell is packed with a strong cation exchanger present in the protonated form and exchange reactions occur.

The counter ions (Na+) of the eluent are exchanged for the hydrogen ions (H+) of the resin in the cell.

If NaOH is used as the eluent then water will be formed, and if NaHCO3/Na2CO3 is used as the mobile phase then carbon dioxide is produced.

At the same time the counter ions of the sample (M+ = metal cation) are exchanged for protons from the resin.

Along with the sample anions these form acids that have an increased conductivity eg, hydrochloric acid, nitric acid, etc, which give an increased signal to noise ratio and improves the sensitivity of detection.

2.

During operation of the 828 a direct current is constantly applied to the suppressor electrodes.

Water in the cell is electrolysed and the electrode reaction takes place.

At the anode, hydrogen ions and gaseous oxygen are produced, and at the cathode, hydroxide ions and gaseous hydrogen.

This allows the hydrogen ions produced at the anode to continuously regenerate the cation exchanger.

The sodium cations of the eluent and the cations of the sample head towards the cathode in the direct current field.

The eluate that leaves the suppressor cell on the cathode side contains the hydroxide salts of these cations, gaseous hydrogen and some sample anions in the form of their sodium salts.

The eluate at the anode side contains carbonic acid or water together with gaseous oxygen and sample ions in their protonated form.

The protonated sample anions are eluted towards the detector with carbonic acid or water.

3.

Upon leaving the suppressor cell, all eluates pass through a degassing unit where oxygen and hydrogen are removed from the eluate flows at the anode and cathode respectively, before reaching the waste container.

Before reaching the conductivity detector the eluate, containing sample anions and carbonic acid (using a carbonate/bicarbonate eluent) passes the degassing unit where the carbonic acid dissociates to form carbon dioxide and water.

The carbon dioxide is removed and the water remains resulting in a further reduction in the background conductivity providing an improved sensitivity and more stable baseline.

Some examples of Metrohm gradient applications.

The modular system used for gradient applications comprised the Metrohm modules two 709 IC pumps, 732 IC detector, 733 IC separation centre, 762 IC interface, and the 828 IC dual suppressor fitted with Metrosep A Trap 1 and gradient mixing spiral controlled through data acquisition software IC Net 2.1.

Using the Metrohm gradient system, different types of soft drink beverage can quickly be determined (after sample dilution) for their citrate content as well as the common anions using a carbonate/bicarbonate eluent with the Metrosep ASupp 5 column (100mm) as the subsequent chromatographs demonstrate.

Chromatograph for a diet cola drink. Using an isocratic separation, citrate is strongly retained by the exchange sites on the separation column, but with a gradient separation the citrate species is eluted at around 18 minutes. This illustrates perfectly the advantages of gradient applications when one wants to quantify what would otherwise be strongly retained analyte species with long retention times when performed isocratically.

Polyphosphate applications are of great commercial importance due to their use in a variety of industrial applications.

Polyphosphates have found uses as detergents ('Calgon') but disposal of the residual phosphate causes major problems.

They can be often be found in water treatment systems, hence it is useful to be able to easily quantify their presence.

Phosphates have also found importance as flame proofing agents.

Phosphorous can exist in the +5 and +3 oxidation states as well as sharing oxygens particularly between tetrahedral atoms giving a range of different phosphorous oxyacids.

With the Metrohm gradient system it is possible to quantify orthophosphate, metaphosphate (cyclic phosphates), pyrophosphate, and polyphosphate.

Conclusion.

Ion chromatography as an analytical technique has seen an enormous surge in popularity due partly to the simplicity of many of the methods as well as other factors such as market forces driving down the expenditure costs of the initial instrumentation and an 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 often within a matter of minutes.

The majority of applications can be performed isocratically and it is usually not necessary to adopt gradient separations.

However in the instance of complex sample matrices and where quicker quantification of strongly adhered analyte groups is required, then it is sometimes necessary to perform analysis with a gradient separation.

The advantages of gradient separation include higher resolution of fast eluting analytes and an acceleration of the late eluting species.

A major advantage of gradient chromatography is the option to clean up the separation column by washing the column with a strong concentration of the mobile phase at the end of the analytical run to effectively regenerate the anion exchange sites.

Thus any strongly retained species that would ordinarily remain on the column are removed by this clean-up theoretically prolonging the lifetime of the analytical column.

For current Metrohm modular users it is of course possible to upgrade an isocratic system to one with gradient capabilities, all that is required is the 828 IC dual suppressor (with associated accessories) along with an additional 709 IC pump.

With the Metrohm modular Ion Chromatography system it is easy to switch between isocratic and gradient chemistries by connecting an alternative system within the software and a simple re-plumbing of the mobile phase flow path capillaries.

The low running costs and flexibility of ion chromatography using Metrohm modular instruments really does mean that ion chromatography today is the method of choice for the analyst even with the most difficult and problematic sample matrices.