IC ANALYSIS FOR VEOLIA/DELTA

Contents:

1. Sample preparation.
2. Standards and controls preparation.
3. Eluent preparation.
4. IC purging and hardware overview.
5. Method selection and software setup.

Sample preparation

Overview: Samples need to be prepared prior to any IC analysis in order to get rid of as many solid
residues as possible. To achieve this, samples need to be centrifuged and filtered. A 1:10 dilution is then
performed and samples are properly stored until analysis.

1.1 Centrifugation:

1. Retrieve samples from designated locations using appropriate PPE (glasses, lab coat, and
gloves). For more information about safe handling of samples refer to the guidelines document.

2. Label 50 ml centrifuge tubes as needed. Remember to include name of the sample,

concentration (if applicable), date, and initials.

3. Transfer approximately 20 ml of sample into each tube.

4. Go to one of the centrifuges in the lab and make sure nobody is using it.

5. Change the rotor so 50 ml tubes can be placed. The correct rotor is shown in figure 1. Use key
on the left side of the centrifuge to unscrew the seal.

6. In case no 50ml centrifuge tubes are available, 15ml ones can also be used on the big centrifuge.

7. Ensure samples are always balanced so there is an even distribution in the overall mass on the
rotor. Figure 2 shows a balanced disposition of centrifuge tubes. Here is an interesting video on
the topic.

8. Once samples are loaded, place the cap on the rotor and twist the red screw until a “click”
sound is heard. In the case of 15 ml rotor, check that the black safe is pointing towards the
“closed” end of the cap.

9. Manually spin the rotor once until a notification displaying “new rotor detected” pops up. This
only needs to be done if the rotor is changed.

10. Set the temperature to 21°C.

11. Set the timer to 20 minutes.

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12. Set rotating speed to 8400 RPM using the “speed” button.

13. Close lid and press “start/stop”.

14. Stand by the centrifuge until it reaches the set speed. Listen to the centrifuge and ensure no
loud or sudden noises are being produced. If so, press “stop” and check that the samples are
properly balanced.

Figure 1.

Figure 2.
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1.2 Filtration/Dilution:

1. While the centrifuge is running, take a sample rack and label the IC vials as needed.

2. Label beakers as needed. The beakers will be used to contain the filtered sample.

3. Retrieve the 22µm filters from the LC shelf (large yellow filters).

4. Get 10 ml syringes as needed.

5. When samples are ready, take them out of the centrifuge and put them in your workspace.

6. Measure approximately 5 ml of each sample and filter them using the yellow filters. The filtrate
should be placed in the corresponding beaker.

7. Get a 100-1000µL micropipette (blue) and pipette 1000µ of each FILTERED sample into its
corresponding IC vial.

NOTE: Follow the micropipette indications expanded on the “Standards preparation” section.

8. Get a 0.5-5 ml pipette (green) and add 9 ml of water into each IC vial.

9. Cap each vial and rotate them upside down multiple times to ensure samples are properly
mixed.

10. Samples should be now ready for analysis.

NOTE: Veolia and delta samples are time sensitive. If analysis cannot be completed within the first
24-hour period, they need to be properly stored in a refrigerator. Use the water lab refrigerator for
this purpose.

Standards and controls preparation

Overview: For both projects, samples are expected to have a nitrate-nitrite concentration in the range
of 0.5-200ppm. To make the results fall within the linearity of the calibration curve, samples are diluted
as previously described. Taking this into account, the selected standards for this method are 0.5, 10, 15,
20, and 25 ppm. Standards are usually prepared every Monday; however, the validity of the calibration
curve is assessed with the controls daily.
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2.1 Standards calculations: using the C1 * V1= C2 *V2 equation for dilutions, we obtain the following
results:
VOLUME REQUIRED (ml)
STANDARD THAT NEEDS TO

0.5 5 10 15 20 25 50 RO
BE PREPARED

0.5 1 9
5 5 5
10 5 5
15 6 4
20 4 6
25 5 5

1. Retrieve and label 7 IC vials respectively.

2. Label two 10 ml beakers and transfer a SMALL quantity of the 1000ppm nitrite/nitrate standards
respectively.

NOTE: Nitrite/nitrate standards do not need to be manipulated under a fume hood.

3. Retrieve the appropriate micropipettes. Remember that the volume of the sample should be as
close as possible to the upper limit of the pipette.

4. Gently push the pipette tip into the micropipette (Do not use excessive force as it can damage
the equipment).

5. Push the plunger to the first stop and submerge the tip into the standard. Make sure the tip is
positioned low enough to avoid air contamination but also high enough to accurately aspire the
liquid. The pipette tip should be as perpendicular as possible during aspiration as shown in figure
3-a.

6. It is recommended to rinse the pipette tip 1 to 3 times before the final aspiration. Rinse the
pipette tip an equal number of times for every liquid aspirated.

7. After aspiration, wait 1 second before taking the tip out of the liquid. Check that there are no air
gaps in the tip.

8. To dispense the liquid, place the tip of the pipette against the wall of the IC vial creating a 45-
degree angle (see figure 3-b). Slowly push the plunger to the SECOND stop. Make sure no visible
droplets of liquid are left inside the tip.
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Figure 3.

9. After the right amount of solution/RO is placed in each vial, cap the samples and take them to
the vortex.

10. Place them on the vortex for about 20 seconds and turn them upside down 3-17 times.

11. Follow the exact same procedure for controls. This time, prepare a 6 ppm control from the 1000
ppm standards.

NOTE: Controls need to be prepared DAILY. It is also recommended that a different person prepares
them.

Eluent Preparation

Overview: A solution of carbonate/bicarbonate is used as the mobile phase. This solution comes in the
form of small sample bags that need to be diluted before running them through the system.
Additionally, the container used to keep the eluent needs to be properly sterilized, cleaned, and dried
before usage.

3.1 Eluent attenuation:

1. Retrieve a 1000 ml polypropylene volumetric flask from the LC shelf. Rinse it 3 times with tap
water and 3 times with RO.
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2. Add some RO to the volumetric flask. The eluent will be added to this small amount of water.

3. Get one eluent bag from the IC shelf and a pair of scissors.

NOTE: Look that the box containing the eluent bags is labeled “A supp 5”.

4. Carefully cut the tip of the eluent bag making sure all the liquid is at the bottom.

5. Gently dump the content of the bag into the volumetric flask.

6. Use a wash bottle to rinse the inside of the bag.

7. Add RO to the flask up to the filling mark. Remember to check your meniscus.

8. Cap the volumetric flask.

3.2 Container preparation:

1. Grab a 2-liter bottle from the glassware section.

NOTE: There should be a pre-prepared 500 ml bottle of eluent on the IC shelf. Place the aspiration
filter inside the bottle while the eluent is being changed.

2. Rinse it 3 times with RO and take it under the fume hood.

3. Get a methanol bottle from the flammable cabinet and a 250 ml beaker.

4. Measure approximately 50 ml of RO and add to the bottle. Using the same beaker, measure 50
ml of methanol and add to the bottle. This whole process is done UNDER THE FUME HOOD.

5. Cap the bottle and swirl it to properly rinse the inside of the container.

6. Transfer the liquid inside of the bottle to the appropriate methanol waste bottle.

7. Take bottle to the sink and rinse with RO 3 consecutive times.

8. Retrieve a pump tube from the microbiology section.

9. Connect the tube to the air dispenser in the fume hood and open the valve.

10. Use the tube to air dry the inside of the bottle.
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11. After the bottle and cap are completely dry, use one of the eluent stickers to label the bottle (IC
eluent, Date, and initials).

12. Carefully transfer the eluent from the volumetric flask into the bottle.

13. Put the cap on the bottle but DO NOT tighten it all the way.

14. Take the bottle to the large sonicator. Make sure the water level is within the acceptable range
inside.

15. Place the eluent bottle inside the sonicator and use the arrows to set the timer at 15 minutes.

16. Eluent should be ready after sonication.

IC Purging and Hardware

Overview: Ion chromatography is a technique which utilizes the retention time of various ions to
determine their concentration. For this purpose, it utilizes a resin column which shows higher affinity to
the analyte.

Regardless of the provider, ion chromatography systems have 3 main components:

1. Sample Rack
2. Ion Chromatography module (includes pump, suppression module, valve, column, signal
detector etc.).
3. Control panel (CPU).

The main components are displayed in figure 4 from left to right respectively.

Figure 4.
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The details of how the whole process is carried out are not in the scope of this SOP. Nevertheless, a
diagram displaying a summary is shown below in figure 5.

Figure 5.

4.1 Hardware considerations BEFORE using the system:

1. Check that the waste bottle under the IC table is properly drained.

NOTE: It is encouraged that you check the waste bottle at least twice a week to keep it from
overfilling.

2. Check that RO bottle inside the sample rack has enough water. Water should be visible to the
user when looking down from the front. This can be seen in figure 6.

3. Check that the eluent bottle is full enough. This can be verified using the following equation:

E=( N∗t )∗0.7 ml /min

Being:
 IC ANALYSIS FOR VEOLIA/DELTA

 “E” the amount of eluent that will be used in ml.

 “N” the number of samples that need to be analyzed.
 “t” the sample run time. For this specific method, each sample takes 18 minutes.

4. Make sure there are no leaks in the system.

Figure 6.

4.2 Purging (retrieved from the original SOP):

Should be done before every run, especially after changing the eluent.

1. Open the purge valve counterclockwise.

2. In the software click on the manual icon in the bottom left corner of the screen

3. Under device selection, choose all devices, and click on the main IC unit in the devices list.

4. Select the pump tab and make sure it is off (the stop button is greyed out)

5. Change the flow input to 2mL/min (don’t click start!) and ensure the Pmin is 15 MPa as shown in
figure 7.
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Figure 7.

6. Connect the purge syringe to the purge line of the pump.

7. Pull on the syringe plunger a little bit until some liquid starts to enter the syringe – don't release
the pressure (note the position of the syringe in figure 8)

Figure 8.

8. Turn on the IC pump by clicking the start button in the manual control window.

9. With the pump running, pull on the plunger a little more so fluid flows freely into the syringe,
hold this pressure.

10. Release the hold on the syringe plunger and allow it to equalize, turn the syringe upside down,
the liquid should fill the syringe and push the plunger up.
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11. Tap on the front of the pump head lightly to knock free any air bubbles that may be trapped.

12. Repeat 8-11 until no air bubbles come out.

13. Click stop in the manual control window, close the purge valve.

Method selection and Software setup

Overview: Having completed the hardware checklist and assuming that the eluent is already prepared,
we can proceed to the actual analysis. After the samples are loaded and the adequate data is registered,
the system need to have a stable baseline. This is achieved by running just the eluent for 30-60 minutes
in a process referred to as “equilibration”. This needs to be done before starting any determination.
When a stable baseline is obtained, the system is ready to start the analysis.

5.1 Equilibration:

1. Click on the workplace tab located on the right of the screen. Select “equilibration” window and
use the dropdown menu to locate the right method (NO2 NO3 June 29).

2. Press the green “Start HW” button and leave the system running for 30 to 60 minutes.

3. After 30-60 minutes, check that the conductivity forms a stable baseline as seen in figure 9 (Top
right).

4. Press the red “stop HW” button.

NOTE: Continuously monitor the IC module for possible leaks and pump damages while the
equilibration is running. Make sure that the column pressure does not go above 11 MPa. This can be
monitored both on the top right window named “pressure” and in the “Watch window” at the
bottom.

Figure 9.
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5.2 Table of determinations and database:

1. To input the sample information, click on the “determination series” window. Here you can
modify the sample identity and start the analysis.

2. Double click on one of the lines and input the correct information as seen in figure 10. Change
identity to the name of the sample, select the sample type (sample, standard, and control or
check standard), select the position of the sample in the rack, set number of injections to 1,
dilution factor to 10, and select the batch that corresponds to the date in which the calibration
curve was prepared. Click on the “apply” button and select a new empty line.

NOTE: A new batch needs to be created every time new standards are prepared (preferably, on
Monday). The batch can be thought of as a “folder” which stores data. The system will then use the
calibration curve saved in that batch to process the samples.

3. After all the samples are loaded in the table, click “start” and let the system run the samples
normally.

Figure 10.