376: June 2018, VPP
EM
Introduction
The shipboard ion chromatograph (IC) is used to determine concentrations of major cations (Na+, K+, Mg2+, and Ca2+) and anions (Cl-, Br-, SO42-) in pore water. Separation and detection of each ion set occurs in two separate flow paths. Sample is directed from a sample loop into a chromatographic column in which dissolved ions absorb to a stationary phase resin. A cation or anion eluent following the sample is directed through the column and competes with the 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 (influencing factors include ionic radius and ionic charge). The separated analyte bands are eventually carried off by the eluent from the column and into a conductivity detector, which integrates each analyte’s signal over a short time interval. Concentrations in the original samples are calculated from calibration curves constructed from a set of known standards and blanks analyzed in the same fashion as the samples.
The columns are kept within a temperature-controlled chamber and the eluent flowrate is kept constant to maintain a consistent retention time for each ion. The main difference between the anions and cations flowpaths is that the anions employs a suppressor and CO2 exchange membrane between the column and the detector to remove sodium and bicarbonate (as CO2) from the eluent solution, and convert anions to their acid counterparts (which have higher conductivites), thereby boosting the analyte signal and lowering the background due to the eluent.
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 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:
- The MSM 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 flush the path flowing connecting 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 into through the bottom of the ultrafiltration membrane housing, is filtered, exits the top, and 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
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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. This may involve using a stir plate with a stir bar in the flask. Pour into the carboy and add three more 2 L flasks filled with DI water.
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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 the screwed shut. Verify that the Teflon tubing coming from the columns are 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. The instrument will begin 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.
- The anions baseline should stabilize around ~1.4 µS/cm. The cations baseline should stabilize to ~900 µS/cm.
- The anions pump pressure should stabilize between 6.5-7 MPa, the cations pump pressure should stabilize between 4-4.5 MPa.
- The column temperature thermostat should stabilize at 45°C.
- While the instrument is equilibrating check to ensure there are no leaks in the eluent flow paths. 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. To prime or rinse the flowpath if the autosampler probe; any tubing, filters or fittings along the sample flowpath; or the ultrafiltration membrane have been changed, 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.
- At this point, the instrument is ready to run. Once the Method has been loaded, the sample sequence created and the vials placed in the appropriate positions of the autosampler rack, select the Equilibration tab > green Start HW button. To shut down the instrument. A display of the current aspiration will be shown on the Live Display Window.
Shutting Down the IC
To shut down the IC, navigate to the Equilibration tab and select the red Stop HW button. Eluent uptake will terminate. Disengage the tubing from the peristaltic pumps on the IC and on the autosampler. Leave the instrument on.
Loading and Running a Method
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.
- Select METHOD icon in left panel. Select File -> Open -> select the method. 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)
Select DATABASE icon in the left panel. Select File -> Database Manager. Select Edit pulldown then New. Type in a new name (e.g. x376).
Here you can also make a database backup. Select Database Manager again and Backup, giving a unique Expedition name.
Go back to the METHOD panel. In the Evaluation window, click on Results icon. Select the Database tab. Double-click on the Name Database field and select your database name for the Expedition.
- 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).
Preparing the Calibration Standards and Samples
Samples are normally diluted 1:100. 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 vials. Make sure to switch the pipette tips between each sample. Vortex mix the samples and standards before analysis.
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 | standard 2 | standard 3 | standard 4 | standard 6 | standard 7 (DF50) | |
IAPSO (µL) | 100 | 66.7 | 50 | 28.6 | 125 | 150 |
DI water (µL) | 9900 | 9933.3 | 9950 | 9971.4 | 9875 | 9850 |
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.
A note on calibrations:
MagIC Net deals with calibration curves slightly different from other types of analytical chemistry 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 for 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.
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.
Figure 1 : Sample/sequence table example
- 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 and then the samples with checks every ten samples or so.
- 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.
Table 1: List and explanation of sample table fields
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 1–5, Blank or Check Standard |
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 : | Should be “1.” |
Info 1 : | Here you can enter a comment or Label ID for example. Use this field to enter a name that is interpretable by the user (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.” |
The IC will analyze samples according to the order in the sample sequence, not necessarily the position in the autosampler rack. You can insert samples into the sequence on fly by right clicking on the sequence table and selecting “Insert New Line”. Enter the sample info and double-check that the Position corresponds to where to put the vial in the autosampler rack (at the end, for instance.)
Evaluating the Calibration/Results
The calibration curve and sample chromatograms may be viewed by navigating to the Database panel and selecting a result of interest in the Determination Overview window. In the Curves 1 window are tabs for Anions and Cations. Select either, the window will populate with the sample Chromatogram. Click on the Calibration curve radio button. Select the element to evaluate from the pull-down menu to view its calibration curve and the calibration table (Figure 3). Several statistics and the curve equation are also displayed in this window.
Figure 3: Calibration curve display of a sample within the database tab.
The analysis results are also displayed under the Database tab within the windowpane entitled “Results.” Results are only shown if the software has identified a peak within the retention time window specified for the analyte. The analytes are distinguished by the type of ion (cation or anion) and then ordered by retention time. The result fields include the retention time, peak height, peak area, concentration (without accounting for dilution), and final concentrations (with accounting for dilution). Beneath the results table is a list of all the parameters and equations defined in software to reach the results displayed for the particular sample.
Information about the instrument configuration during the select sample acquisition may be found in the Information windowpane of the Database tab.
Uploading Data to LIMS
Use MUT to upload results to the LIMS.
1. | In the Database panel, select the result(s) to upload. Either click on a row or hold down the <Ctrl>-key and select multiple results. |
2. | In the top menu, select Determinations -> Export. Select All Selected Data Records and export template Exp368P. Click OK. The Excel csv files will be created in C:\Metrohm Export. |
3. | Copy these csv files to the MUT uploader directory (C:\DATA\in). |
| Run MUT. |
4. | Move the csv files in C:\Metrohm Export to an Expedition folder and at the end of the Expedition copy over this folder to the DATA1 volume for backup for the shore db. |
Helpful Hints and Tricks
The following hints will aid in monitoring and troubleshooting instrument performance while analyzing a sample sequence.
- At the beginning of the run note how much sample is being drawn from the vial. Compare the levels of the vials as the sequence proceeds to ensure that sample is consistently extracted. If it is not, the peristaltic pump tubing may be wearing or an inline filter is clogged.
- Similarly, note the flowrates of the lines draining into the waste container on the rear of the instrument. Count the number of drops per 5 secs as a rough estimate of flow. Monitor the rate during a sample sequence.
- Monitor the cations and anions baselines and pump pressures.
- Every time new eluent is added to the carboys the baselines and response for check standards changes slightly. This can be mitigated by creating a large volume of a single cations solution. Do the same for the anions solution only at the beginning of an analysis. During a run, fill up the eluent bottles fully at the beginning, and then refill once the level has gotten to the Teflon tip (don’t let the tip contact air). This ensures a maximum # of samples are analyzed under consistent conditions.
- Adding eluent to the reservoirs slightly alters the signal baseline, even if the eluent to be added is of the same batch as the eluent in the reservoirs. It is best to “Pause” the run, let the current sample finish, add the necessary eluents, wait for ~30 minutes, and then “Cont” (continue) the run. This is ample time for the new eluent mixture to saturate the flow path.
- Insert check standards into the sample table on the fly if necessary, or if something major changed mid-run (changed inline filter, pump, tubing, etc.)
Sometimes the autosampler ends at a position where some vials cannot be accessed. To get the autosampler to move, select the Manual tab on the main panel, then select 858 Professional Sample Processor. Select the Tower tab, change the rack position input, then click Start.
The business card for the Service rep is taped to the side of the instrument.
The help function in the software is good. Accessed from the top menu.
Maintenance
For maintenance, there are a couple of great guides, located in the Metrohm_backup\METROHM MANUALS folder on the desktop.
850_IC.pdf main instrument guide
IC_maintenance_guide.pdf Outlines maintenance schedules to follow
: changing autosampler ultra-filtration membrane
: changing H2O and CO2 scrubbers
: replacing peristaltic pump tubing
: changing the guard/inline filters
: pump maintenance
Autosampler.pdf autosampler guide
Dosino.pdf Dosino guide
850_pump_maintenance.pdf anion/cation pump maintenance
Metrohm_parts_guide.pdf Parts information to send in requisitions
Metrohm_ultra_filtration.pdf More on the autosampler’s ultra-filtration
MagICNet_tutorial.pdf Software guide
IC | Pumps | Once an expedition |
| Column | If the retention times are changing |
| Anions, Cations inline-filteres | Every several runs |
| Guard Columns | Every several runs |
| H2O Scrubber | Check every run, replace if the beads start to fade from orange to transparent |
| CO2 Air Scubber | Check every run, replace if the material within begins to clump. |
| MSM Peristalitic Pump tubing | Every Several Runs |
| Any tubing | On an as needed basis, if there is a lot of sample carryover. |
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Autosampler | Probe | Every several runs |
| Peristaltic Pump Tubing | Every several runs, or monthly |
| Ultrafiltration membrane | Inspect every run, replace if discolored. |
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Solutions | Cations Solution | Replace if the bottle has been sitting around for a week or so |
| Anions | Make fresh every several days |
| Water | As needed |
| Methanol | As needed |
| Sulfuric | As needed |
| Bottle air scrubbers | When the dierite color changes from blue to purple, or when the gray/brown ascarite starts to clump. |
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Troubleshooting
Consult Metrohm’s IC Troublshooting guide for information concerning troubleshooting commons IC problems. Below are additional issues the technicians have encountered over the years:
Chromatograms
Check standards are coming out low:
- Dilution error: The percent accuracy of all peaks should be nearly the same
- Guard columns or eluent inline filters are contaminated. Replace them with new parts
- A/S has not drawn up enough sample due to blockage. Check level of fluid in the aspirated vial and note if less sample was extracted. On the subsequent samples note the flow rate of fluid entering the waste container on the rear of the instrument.
- Peak shouldering for anions. Due to contamination of the anions guard column. Replace the guard column.
Column
- Retention times for peaks are shifting
Detector:
Detector signal is highly variable. Usually means that air has gotten into the conductivity meter. Remove the line from the end of the chromatographic column and attach it to a syringe filled with DI. Slowly force water through the line and detector. If the bubble does not dislodge, attach the syringe to the line coming out of the back of the detector, and backflush DI through.
Reprocessing calibration curves, retention times, peak identification, or adding additional analytes
Pump:
- Pressure is high: blockage
- Pressure is low: bad pump, or a leak
- Pump pressure is variable. Usually means there is blockage or air in the eluent flowpaths.
Leak is present.
Autosampler:
- Autosampler collided with test tube projecting from the rack and needs to be reconfigured.
Software issues:
MagIC Net misidentified an analyte: This usually happens when two peaks are within the same retention time window. Sulfate is the worst offender. Reprocess the sample by adjusting the time window to identify to the correct analyte.
Interpreting the Ion Chromatograms (Needs to be edited)
Cations
Figures (x-x) are typical chromatograms collected when measuring an aliquot of IAPSO diluted 1:100 (DF100). The order cations elute the ion exchange column is governed by first by the strength of the ionic charge and second, by cation size, where smaller, highly charged analytes are the last to elute.
Changing Inline Filters
Figure 4: Frits within the guard columns. The left hand frit has a buildup of iron oxides filtered from the sample to protect the anions column.
Figure 5: Example of a clean inline filter membrane (left) and dirty membrane (right).
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