Ion chromatography

During the crops conducted in the i-RTG, samples of the nutrient solution and the leachates were collected and analysed using ion chromatography. The main goal of these analyses was to measure the quantity of nutrients supplied and leached and to adapt the nutrient solution to the needs of the plants. The results were used to adjust the nutrient supply during the crops and to study the nutrient balance in i-RTG crops.

The ion chromatography methodology had not been used in the Sostenipra research group previously. The equipment used was twelve years old and had been unused for a long period, for this reason it had to be prepared. The equipment was a Dionex™ ICS-1000 Ion Chromatography System (ICS-ICS-1000), which had been unused for years. A tune-up was done by Vertex Technics S.L., an external enterprise, checking all the elements and making the necessary repairs.

The main elements of the ion chromatography system (ICS) are described in Figure 2.2 and Table 2.4. In the operation of the system, a mobile phase or eluent is constantly pumped along the circuit while the system is running. The sample that must be measured is injected into the stream of eluent and goes through the ion separation columns, where the different ions of the solution are separated (each has a different speed while going through the resin of the column). The ions arrive at the conductivity cell one after the next, which enables identifying and quantifying its concentration in the sample according to the chromatogram (Figure 2.3).

Figure 2.2 Picture of the interior of the ion chromatography system, with each of the elements marked (names in Table 2.4).

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Table 2.3 Description of the elements of the ion chromatography system (picture in Figure 2.2).

Code Element Description

A Eluent bottle

Recipient that holds the eluent or mobile phase to feed the circuit of the ion chromatography system.

B Pump Device for pumping the mobile phase.

C Injection valve

This valve connects the following tubes: eluent entrance (from the eluent bottle), sample (autosampler), waste (to waste container) and columns (to guard column). It has two positions: charge and inject.

Charge: Eluent entrance and columns are directly connected, and the sample is connected to waste through the loop. If a sample is injected, it fills the loop.

Injection: Eluent entrance and columns are connected through the loop, sweeping the sample within it.

D Loop A section of tube in the injection valve that stores the sample when it is injected until it is swept by the eluent into the mobile phase.

E Guard

column

A tubular component similar to the separation column but shorter that has the function of protecting the separation column.

F Column Tubular component that separates the ions in the problem sample, which enables its identification and quantification.

G Suppressor

A component with prismatic shape that reduces the background noise of the conductivity detection and improves the signal of the ions being measured.

H Conductivity cell

A device that makes continuous measuring of the conductivity of the eluent and sample. The data generated allow defining the chromatograms.

I Waste

container Recipient that collects the waste liquid.

J Control panel

Panel that shows the state of the different components of the ion chromatography system (green LED on=running).

To obtain the concentration of each element in the sample from this chromatogram, a calibration must be done defining a quantification method. The quantification method is made up of one calibration curve for each of the elements analysed, which shows the relation between the concentration of the element and the value of conductivity measured in the cell. This method is obtained sampling standard solutions with known concentrations for each of the different ions (Figure 2.3). Although when new columns are used each ion has a characteristic speed, this speed through the columns is reduced over time and thus the quantification method must be updated (calibrated) periodically sampling new standards. In order to detect when a new calibration was needed, a standard was measured once per week to check the accuracy of the system and a new calibration was done if the error was higher than 10%. However, for the measurement of the samples carried out during the dissertation a calibration was done at least once per month even if the errors of the standards were lower than 10%.

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Figure 2.3 Chromatogram with anion measurement (top) and calibration curve with eight standards (bottom).

The equipment (defined in Figure 2.2 and Table 2.4) has two sets of columns and suppressor for the measurement of anions and cations. Regarding the anions, a Dionex™

IonPac™ AS9-HC column was used for the ion separation and a Dionex™ IonPac™ AG9-HC as guard column. The suppressor used was a Dionex™ Anion Self-Regenerating Suppressor ASRS® 300 (4-mm). For the cations, a Dionex™ IonPac™ CS16 column, a Dionex™ IonPac™ CG16 guard column and a suppressor Dionex™ Cation Electrolytically Regenerated Suppressor CERS 500 (4 mm) were used. After the second crop, the separation columns for anions and cations reached its end of life and were replaced (columns are consumable equipment). A more appropriate model for the conditions of the system was selected for the column of cations, improving the resolution and reducing the time required per sample. The new columns were a Dionex™ IonPac™

CS12A column and a Dionex™ IonPac™ CG12A guard column. Regarding the rest of the components, a Dionex™ injection valve (P/N 057968) with a 25 µL loop was used, along with a Dionex™ DS6 Heated Conductivity Cell (P/N 057985). A 2 L bottle was used to contain the eluent and a container to store the waste from the eluent circulation and the samples.

Apart from the ICS, a Thermo Scientific™ Dionex™ AS-DV Autosampler was used to run the samples and both the ICS and the autosampler were operated using the software Dionex™ Chromeleon™ 6.8. Figure 2.4 shows these three main elements of ion chromatography ion chromatography (ICS, autosampler, software).

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Figure 2.4 Autosampler and Ion chromatography system (left), interior of the autosampler (centre), screenshot of the software Chromeleon (right).

During the operation of the system, only anions (Cl^-, NO^2- , NO^3-, SO^4-, PO^4-) or cations (Ca⁺, K⁺, Mg⁺) can be measured at a time because only one set of columns and suppressor can be installed in the equipment. Changing from one to the other involved uninstalling one set and installing the other one. A different eluent is used for anions and cations, it is important to use the adequate eluent and not to mix them because it would spoil the resin of the column, with a risk of breakage. When a set of columns and suppressor (for anions or cations) is installed, eluent must be circulated at least once per week, even if the equipment is not used, to avoid breakage (the column can dry). The procedure to change from one set to another includes the cleaning of the tubes that connect the different elements and the cell circulating deionized water to avoid mixing eluents and the hydration of the suppressors (the one being removed to store it and the one being installed to activate it). The cleaning of the tubes and the cell must also be done when the equipment is uninstalled and will not be used for more than a week.

To start using the equipment, a purge must be done first to remove any bubble of air in the tubes of the circuit. A basic purge is required when the eluent bottle has been changed and air has entered the tube (purging the tube from the bottle to the pump) and a complete purge when the set of columns and suppressor has been installed (purging each section of tube between the elements installed). Then, eluent must be circulated until stabilization, reached when the second decimal place of the value measured in the conductivity cell (in µS) do not vary for 1minute (when the baseline of the chromatogram is completely flat). Table 2.5 shows the values that the system should hold when it is stable.

Table 2.4 Values of the parameters in the ion chromatography system when it is stabilized.

CATIONS ANIONS

Temperature of the conductivity cell: 40°C Suppressor current: 88 mA Conductivity stabilized*: 0.6-1.2 µS

Pressure stabilized*: 1,150 psi

Temperature of the conductivity cell: 35°C Suppressor current: 45 mA Conductivity stabilized*: 20-30 µS

Pressure stabilized*: 2,070 psi

*These values can vary depending on the components and the age of the equipment.

The eluent used for cations was methanesulfonic acid with a concentration of 20 mM, which is a slightly lower concentration than indicated by the manufacturer to compensate the lack of heating recommended for that column model to improve resolution (this adaptation is common practice and the results obtained were

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acceptable). However, it was changed to 25 mM methanesulfonic acid after the second crop, according to the specification for the new model of separation column. For anions, the eluent used was sodium carbonate (Na2CO3) with a concentration of 9 mM. To ease the preparation of the eluent, a stock solution of Na2CO3 with a concentration of 900 mM was done.

The standard solutions used for the calibration of the ion chromatography system included all the ions measured. Various standard solutions were prepared for each calibration, holding each a different concentration of the ions contained. A stock solution was prepared to ease its preparation, with a concentration of 500 ppm for all the elements included. The following solid reagents were used for anions: NaCl, NaNO2, NaNO3, Na2SO4, KH2PO4; and the following ones for cations: CaCl2 · 2 H2O, MgCl2 · 6 H2O, KCl. Between six and eight standard solutions were measured to obtain the curves of the quantification method, each with a different concentration.

The samples measured were diluted 1:5 to improve the accuracy of the measurements because usually samples held high concentrations of nitrogen, which were close to the upper measurement limit of the ion chromatography system.

All the water used for the preparation of eluent, standard solutions and samples must be deionized and the eluent must be degasified before being used to avoid air in the circuit of the ICS. For the measurement conducted in this dissertation, all the water used was deionized with a Milli-Q® Integral Water Purification System and the final eluent was degassed using an ultrasonic degassing equipment. Moreover, the samples analysed and the stock solutions were filtered using disposable nylon filters of 0.2 µm for syringe.

In order to transmit the know-how to future users of the ICS in the group, a guide was written in the framework of the dissertation including all the procedures and frequently asked questions affecting the equipment. This guide can be found in the Appendix I.II of the chapter.