Note: Page under construction, not updated to new Metrohm Model 940 Professional IC Vario TWO ion chromatograph

Introduction

The shipboard ion chromatograph (IC) is used to determine concentrations of major cations (Na⁺, K⁺, Mg²⁺, and Ca²⁺) and anions (Cl^-, Br^-, SO₄^2-) in pore water. Each ion set is separated and detected in its own flow path. Sample is directed from the anions and cations sample loops into their respective chromatographic columns in which dissolved ions absorb to a stationary phase resin. A unique eluent flows through each column and competes with sample ions for adsorption sites. This causes a gradual separation of sample ions into discrete bands within the column—ordered by each ion’s strength of adsorption (influenced by ionic radius and ionic charge). The separated analyte bands are carried off by the eluent from the column into a conductivity detector, which integrates each analyte’s signal over a short time interval. Analyte concentrations in the samples are calculated based on calibration curves created prior to each sample sequence by analyzing a set of known standards and blanks.

In order to maintain a consistent retention time for each ion, the columns are housed in a temperature-controlled chamber and the eluent flowrate is kept constant. For reduced background conductivity and better detection of the analytes, the anions flowpath employs a chemical suppressor and a CO₂ exchange membrane between the column and the detector. These suppressors remove sodium and bicarbonate (as CO₂) from the eluent solution and convert anions to their more conductive acid counterparts.

Safety Considerations

The ion chromatograph is a fairly safe and straightforward instrument to use. The following are a succinct overview of several important safety precautions that are described in more detail within this user guide.

Concentrated nitric acid and sulfuric acid are necessary for the preparation of the cations eluent and suppressor regenerant solution, respectively. Use proper PPE (nitrile/isoprene gloves and eye protection) when handling acids. Always add acid to water. Be aware of acid spill control and neutralization kits.
Be aware of the autosampler position and movements to avoid pinching a finger.

Instrument Diagram and Operation

Figure 1: Metrohm 850 Professional Ion Chromatograph

Figure 2: Metrohm 850 Professional IC Autosampler

The Instrument Flow Path

The following is an overview of the sequence of events which occur when a sample is analyzed. Consult IC Flow Path and Function.pptx for greater detail about the instrument flowpath.

The MSM (Metrohm Suppressor Module) switches to a new clean channel. The peristaltic pumps on the IC and AS initiate. Suppressor regenerant and rinse flow through and clean the two alternative channels of the MSM. The eluent pumps continuously uptake eluent via the bottles and degassers, and then flushes the path which connects the pump purge valves, pulsation dampeners, columns and detectors (the anions eluent also flows through the MSM and MCS before reaching the detector).
Meanwhile, the autosampler moves the rack, injects the probe into a sample vial and begins uptake of the sample.
Sample flows in through the bottom of the ultrafiltration membrane housing, is filtered, exits the top, and is sent directly to the cations sample loop, the anions sample loop and then to the waste container on the back of the instrument. The waste line from the ultrafiltration membrane empties into a drain on the countertop.
After the sample has been drawn for a predetermined time necessary to purge the sample loops, the sample loop valves switch and sample is forced into both columns. Eluent trails the sample pulse and maintains the flow. MagIC NET begins data acquisition.
The IC system maintains this configuration for the 28 minutes necessary for all the analytes to elute and the acquisition to finish. During this time, the AS directs the probe to a waste vessel. The Dosino sends DI water to the vessel to clean the outside of the probe, then switches ports and backflushes DI through the probe. The Dosino switches ports once more and flushes DI water through the sample lines which go from the AS to the IC sample loop valves and exit to the waste container on the back of the instrument.
Once the acquisition is finished the system returns to its regular state in which only eluent, MSM regenerant and MSM rinse are pumped through the IC. The MSM switches channels automatically every 10 minutes.

Suppressor

The IC employs the MSM to lower the background conductivity of the anions eluent in order to gain detector sensitivity while measuring anions. The MSM is composed of an acidic cation exchange resin which exchanges hydronium ions for the sodium ions originating from the sodium bicarbonate of the anion eluent. The bicarbonate is converted to carbonic acid which has a much lower specific conductivity (and is mostly removed in the MCS). In addition, the anion salts are converted into their mineral acid counterparts, eg NaCl becomes HCl, NaBr becomes HBr, etc, which have much higher specific conductivities.

The anions baseline is typically at 1.4 µS/cm. It is normal for the baseline to jump up to between 5-7 µS/cm when the MSM switches cartridges. The baseline eventually stabilizes.

The Method

A Metrohm representative created the default method for data acquisition during the initial instrument installation. The Method consists of a time program, device setup and type of chromatogram evaluation. The time program is a series of instrument events that control sample and eluent uptake and flow through the system, it should never need to be adjusted by technicians or scientists. The Devices menu enables the user to assign hardware and hardware parameters. The menu is particularly useful to assign variable names and locations to the hardware (pumps, columns, detectors, etc) that the method uses for control and data calculations. Finally, the Evaluation menu contains the parameters necessary for analyzing chromatograms, constructing a calibration curve and compiling results. Use this menu to add additional calibration standards, check standards, or to specify additional analytes and their retention times.

Reagents

Make sure all reservoirs are filled with the following reagents:

Dosino Reservoir	18 mega-ohm DI water
Suppressor Rinse Solution	0.1% methanol in DI water (1 mL methanol to 1 L DI water)
Suppressor Regenerant Solution	0.05 M sulfuric acid solution (2.7 mL sulfuric acid to 1 L DI water)
Anions Eluent	3.2 mM sodium carbonate/1.0 mM sodium bicarbonate solution (purchase concentrated packs from Metrohm, called “A Supp 5 Eluent Snips”) snip off tube with scissors and pour contents into a 1 L volumetric flask rinse the tube with DI water, adding to the flask bring flask to volume
Cations Eluent	1.7 mM nitric acid/1.7 mM PDCA (pyridine-2,6-dicarboxylic acid) dissolve 0.248 g PDCA in DI water in a 1 L flask add 106 µL concentrated trace metals grade nitric acid bring flask to volume with DI water. To make a carboy’s worth, in a 2 L volumetric flask, mix 872 µL nitric acid, 2.272 g PDCA with DI water and bring to volume. Pour into the carboy and add three more 2 L flasks filled with DI water. Place on stir plate and stir with a magnetic stir bar for at least 24 hrs.

Preparing the Calibration Standards and Samples

Standards

The dilution scheme for the standards from IAPSO standard seawater is displayed in the following table. Use a fresh bottle of IAPSO every expedition. To reduce contamination of the IAPSO bottle, decant a few mLs of IAPSO into a secondary container and use this volume to prepare standards. Parafilm the IAPSO lid after use to reduce evaporation. Additional calibration standards may need to be prepared to constrain the instrument response for low analyte levels.

Prepare a standard curve, 10 mL per level, in IC vials:

	Standard 1 DF67	Standard 2 DF80	Standard 3 DF100	Standard 4 DF133	Standard 5 DF200	Standard 6 DF400	Standard 7 DF1000 (From DF100)	Standard 8 DF2000 (From DF100)	Standard 9 DF10000 (From DF100)
IAPSO (µL)	150	125	100	75	50	25	1000	500	100
DI water (µL)	9850	9875	9900	9925	9950	9975	9000	9500	9900

To prepare "pourable" 100 ml standards, use the following recipe: (pipette IAPSO in a 100 ml volumetric flask and bring to volume with DI water)

	Standard 1 DF67	Standard 2 DF80	Standard 3 DF100	Standard 4 DF133	Standard 5 DF200	Standard 6 DF400	Standard 7 DF1000 (From DF100)	Standard 8 DF2000 (From DF100)	Standard 9 DF10000 (From DF100)
IAPSO (mL)	1.5	1.25	1	0.75	0.5	0.25	10	5	1
DI water (mL)	98.5	98.75	99	99.25	99.50	99.75	90	95	99

To add/edit standards, select METHOD panel and Standards in the Evaluation window. Here you can also add/edit check standards information. Standard concentrations of 0 for particular analytes are not considered by the software when calculating the analyte calibration curve.

IAPSO Concentrations

Constituent	Standard 1 150%	Standard 2 125%	Standard 3 100%	Standard 4 75%	Standard 5 50%	Standard 6 25%	Standard 7 10%	Standard 8 5%	Standard 9 1%
Sodium (mM)	721.0	600.9	480.7	360.5	240.3	120.2	48.07	24.03	4.807
Potassium (mM)	15.69	13.08	10.46	7.847	5.231	2.616	1.046	0.5231	0.1046
Calcium (mM)	15.81	13.18	10.54	7.906	5.271	2.635	1.054	0.5271	0.1054
Magnesium (mM)	81.21	67.67	54.14	40.60	27.07	13.53	5.414	2.707	0.5414
Chloride (mM)	839.3	699.4	559.5	419.6	279.8	139.9	55.95	27.98	5.595
Bromide (mM)	1.295	1.079	0.8632	0.6474	0.4316	0.2158	0.08632	0.04316	0.008632
Sulfate (mM)	43.41	36.18	28.94	21.71	14.47	7.235	2.894	1.447	0.2894

A note on calibrations:

MagIC Net deals with calibration curves slightly different from other types of laboratory instrumentation. When a new batch of vials are run, the calibration from the previous run (which is the active calibration) is gradually overwritten by the individual calibration points of standards in the new batch. For example, after STD 1 is analyzed the calibration curve will consist of STD 1 and standards from the previous run (less the previous STD 1). After STD 2 is analyzed, the calibration curve consists of STD1, STD2 and the other levels of standards from the previous analysis. Eventually, all standards are analyzed and the active calibration consists only of calibration points from the current analysis. It is then samples may be analyzed. It is important to recognize that classifying an aliquot in the sample sequence as a “Standard” will cause the software to incorporate it into an updated calibration curve. This is desirable at times, but if the user analyzes a standard simply to check analytical accuracy or monitor instrument drift, then they should classify the aliquot as a “Check Standard” or a “Sample”. MagIC Net has the functionality to edit and apply calibration curves post run. Consult the section about reprocessing samples for instructions.

Samples

All samples are diluted 1:100 (100 µL sample made up to 10 mL with DI water).

However, this dilution scheme may need to be adjusted for pore waters with high salinity relative to IAPSO. The Hamilton dilutor is quicker and more precise than using the Eppendorf pipettors for adding the DI water. Use the Eppendorf pipettors for adding sample to each vial. Make sure to switch the pipette tips between each sample. Vortex mix the samples and standards before analysis.

Instrument Operation

Method Setup

Load a method to configure the IC’s operational parameters. The method loaded will be the method used by MagIC Net for the instrument equilibration. Other saved methods may be specified individually for each sample in the sample sequence.

Opening and saving a method
1. Open MagIC Net 3.2, select METHOD icon in left panel.
2. Select File -> Open -> select the method.
3. You can then save the method with a new name for the current Expedition (File -> Save As).
Make a new database for the Expedition (saves results in a central Expedition database)
1. Select DATABASE icon in the left panel.
2. Select File -> Database Manager.
3. Select Edit pulldown then New.
4. Type in a new name (e.g. x376).
5. Here you can also make a database backup. Select Database Manager again and Backup, giving a unique Expedition name.
6. Go back to the METHOD panel. In the Evaluation window, click on Results icon.
7. Select the Database tab.
8. Double-click on the Name Database field and select your database name for the Expedition.
9. Save the method
Go back to the WORKPLACE icon in the left panel. Select the method that you want the instrument parameters to be set at (Equilibrium tab).

Setting up the instrument

Prepare the reagents specified above.
Ensure all instrument consumables are functioning properly; see the Maintenance schedule for when to replace a part.
Engage the pump tubing on the autosampler and MSM peristaltic pumps. Align the marks on the latches with those on the plastic frames then tighten by one additional click.
Ensure the door to the column housing is closed and screwed shut. Verify that the Teflon tubing coming from the columns is passing through notches on the side or on the top of the door. The door must not pinch the tubing.
Open MagIC Net 3.2, click on the Workplace button on the left-hand side of the application window. Within the Equilibration tab, click Start HW.
1. To shut down the instrument, click Stop HW.
The instrument will begin the uptake of eluent through both flow paths. Allow the system to equilibrate for an hour. Every ten minutes the MSM will switch channels and small pulse in conductivity (~6 µS/cm) will appear in the anions signal.
1. The anions baseline should stabilize around ~1 µS/cm. The cations baseline should stabilize to ~900 µS/cm.
2. The anions pump pressure should stabilize between 6.5-8 MPa, the cations pump pressure should stabilize between 4-4.5 MPa.
3. The column temperature thermostat should stabilize at 45°C.
While the instrument is equilibrating check to ensure there are no leaks in the eluent flowpaths. Typically, leaks form around the pump fittings, the inline filters and the guard columns. If a leak is present, untighten the connection, resituate it with the fluids still flowing, and then wipe off any mess. This ensures that a minimal amount of air enters the flow path.
The first vial analyzed usually yields spurious concentrations due to air being in the sample path. If the autosampler probe, any tubing, filters or fittings along the sample flowpath or the ultrafiltration membrane have been changed, the flowpath will need to be rinsed. To prime or rinse the flowpath, navigate to MANUAL, select the Dilution Dosino, change the port to “2” and the input volume to 10 mL, ensure the Dosing radio button is selected, then press Start. Verify the Dosino is priming the flowpath by watching fluid elute from the sample line into the waste container on the back of the instrument.

Setting up a Sequence

The sample sequence consists of a Sample Table created in MagIC Net workplace. The Sample Table contains information on how a sample will be analyzed (Method), how the sample is identified (Ident and Info 1), its position in the AS (Position), how the sample is classified by MagicNet for calibrations (Sample Type), and dilution factors (Dilution) to be incorporated into the final calculation of concentrations.

To set up a sequence, select the WORKPLACE panel and Determination Series tab.
To create a new sample table, use the dropdown menu Sample Table -> New.
Go to Sample Table drop-down and select Properties. In the Display tab, make sure that the Value 1 radio button is checked. Now go to the Edit tab and make sure that the Value 1 radio button is checked.
Double click the first row to start populating the sample/sequence table. The table should follow a basic guideline as follows (see Figure 1).
- The calibration standards will come first, followed by the blank (DI used as a sample)
- a check standard
- then the samples
- checks every ten samples or so

Figure 1 : Sample/sequence table example

Method	369 (expedition number)
Ident	Text_ID of the sample. Standards are not yet entered into the LIMS so use something like DF100 or DF100 CHECK (for a check standard). The TextID must be entered here for MUT to upload the data to LIMS properly.
Sample Type	Sample, Standard 1–9, Blank or Check Standard (check standard number refers to standard number)
Position	Autosampler vial position
Injections	1
Volume	10 µL
Dilution	Select “1.” Dilution is supposed to be the dilution factor for manual or hand dilutions performed by the analyst prior to the sample being placed in the system; however, the standards are built around 1:100 being baseline, so we don’t want the software to calculate dilutions. Note: MagIC does not seem to identify updated dilution factors where it reports “Final Concentrations” when “reprocessing” samples after a run. In these cases, use the values reported as “Concentrations”
Sample Amount	1
Info 1	Here you can enter a comment or Label ID for example. Use this field to enter a name that the user may interpret (in contrast to the TextID used in the Ident field).
Value 1	Dosino Dilution Factor, the dilution factor performed by the automated dilution system. For hand dilution, set this to “1.”

After the sample/sequence table is complete, go to the Sample Table pull-down, select Save As, and give a name.
Place the vials in the appropriate position of the autosampler rack according to the sequence table.

GCR Metrohm Ion Chromatograph (IC) User Guide