Alkalinity Titrator_protocol

Standard Protocol Procedure for Using the Mass Lab Titrator

Created By: Silbiger Lab - 20190227

Revised: Putnam Lab - DM Becker - 20211005

Last revised: Tali Mass- 20260607

A detailed training video for this protocol can be found here.

To plan your titrations, keep in mind that the setup (i.e., purging the HCl, pH calibration, and running a CRM) takes ~45 minutes to 1 hour. Running a total of 20 samples (i.e., the full amount of samples available on this specific rondolino) or one titration run takes ~1 hour and 45 minutes. With about ~15 - 20 minutes of clean up and waste disposal.

Make sure to take your time for each step of the titration process to ensure accuracy and precision of measurements. While this protocol is tedious, all steps and specifications are necessary to ensure confident titration measurements.

Contents

Materials

  • Metrohm 855 Robotic Titrosampler

  • 110 ml dark glass sampling bottles with plastic screw caps (this volume is enough for 2 replicates)
  • Analytical Balance with an accuracy of 0.001 grams
  • [Tiamo 2.5 Software (connects titrator, balance, burettes, etc.)] (https://www.metrohm.com/en/products/8/1018/81018070.html)
  • pH electrode
  • KCl 3 mol/L solution (for titrator pH electrode) [Metrohm # 62307230] (https://www.fishersci.com/shop/products/storage-solution-6/NC0393232)
  • Nitrile gloves
  • Kimwipes
  • 250 mL glass beaker
  • 80 mL tall plastic beakers (titrator cups)
  • 0.45µm seringe filter
  • 50ml seringe Luer-Lock
  • Two thin silicone tubes for connecting to the filter
  • A computer capable with Tiamo program
  • [Metrohm Buffer solutions pH 4, 7 and 9 (25 °C) in single use sachets, colourless, #62307230 ] (https://www.metrohm.com/en/products/6/2307/62307230.html)
  • Type I DI water
  • Filtered seawater (for junk samples)
  • Two designated waste bottles
  • Certified Reference Material (CRM) from the Dickson Lab (standards)
  • [HCl Titrisol] (https://www.sigmaaldrich.com/IL/en/product/saj/131730?utm_source=google&utm_medium=cpc&utm_campaign=22359489047&utm_content=177707544898&gad_source=1&gad_campaignid=22359489047&gbraid=0AAAAAD8kLQRHgG8ETrpGwRAvZX60UVinh&gclid=Cj0KCQjwio_RBhDMARIsAJPveNPfj2QhuLieP5fbn9qmUwPGjlqzy1L9MOj_H-7AoaapsIYcGMTBkLYaAnpSEALw_wcB) 0.1 N
  1. Pre-Setup
    1. Since you are working with acid. You must wear a Lab Coat and Examination Gloves always, before starting the experiment. Safety Goggles are optional. No food or drink.
  2. Sampling
    1. Rinse the sampling bottle 3 times with the sample water.
    2. After rinsing, sampling will be done by inserting the tube into the bottom of the bottle and releasing the water through the tube in such a way that the water level in the bottle slowly rises. At the end of filling, the sample water should be allowed to flow out of the bottle, remove the tube while the water flows out and close the bottle with a stopper.
    3. The samples should be kept in a cool, shaded place until they reach the laboratory, then the samples should be stored in a refrigerator at 4 degrees Celsius.

  3. Setup

    1. Remove the samples from the refrigerator and wait until they reach room temperature.
    2. Make sure that the waste tank is empty and that at least one third of the DDW water tank is full (the large caps should be left slightly open).

    3. Ensure the 855 Robotic Titrosampler is properly connected to the laptop via USB.
      ##### Connecting and disconnecting the Disk on key must only be done when the device is turned off, otherwise the software will freeze.

    4. Open Tiamo software on the Laptop. It will turn on the titrator.
    5. Make sure the HCl bottle is full, and that the wastewater gerican on the floor is empty.
    6. You can adjust the electrode position in Manual -> Tower 1 -> Move to move your samples safely and prevent collisions with containers.
    7. Position an empty container at Position 22 and a container with DDW at Position 21.
    8. Remove the temperature probe from its slot and set it to the side while you purge the HCl.
    9. PURGING (RINSING): Using the Tiamo software, tap “Manual”, “Dosing Device”, “Prepare”, “Start”.
    10. YOU MUST PARK THE AUTOSAMPLER FIRST (AT POSITION 22), OTHERWISE THE ACID WILL SPILL ALL OVER THE AUTOSAMPLER.
    11. If it ever occurs, you must use the chemical wipes with DI water to soak up the acid.
    • Note: Rinse is used to purge any air bubbles that may occur in the acid line. If there are more air bubbles, flick both tubes until they disappear. DO NOT start titrations if there are still bubbles, this will affect the amount of acid the titrator gives the sample.
    1. After Purging/Rinsing, click “OK” to bring the titrator screen back to home.
    2. If you still see bubbles in the lines, redo the purging step!
    3. Place the temperature probe back in its original slot before next steps.
    4. Place the pH sensor in the position next to the propelle.
    5. Open the plastic cap on the pH sensor and leave open throughout use. pH will not be measured otherwise.
      1. Before calibrations can be made, look at the top of the Sensor for KCl, make sure the KCl is filled to fully cover the metal inside by the grey cap, skip to [19]. If not, follow the step below.
      2. If KCl reservoir is low, open the cap on the sensor’s reservoir and add a few drops of KCl on the opening.
    6. Make sure that the cap is open at all times, so it can measure the pH.
    7. Make sure the probe rinse reservoir is filled with KCl solution (a plastic tube on the right side of the instrument).
    8. Once the pH probe is placed in its slot next to the propeller, fill a DI Only cup 2/3 full with DI water and place this cup in poistion 21. On the Titrator screen, “Park” the autosampler. The pH probe should always be submerged in a liquid (DI or its storage solution).
  4. pH_Calibration

Things to check and know about the pH probe to ensure proper handling, usage, and storage:

  • The reference KCl electrolyte solution should be replaced in the glass electrode reservoir ~every month.
  • Always check to make sure the pH probe is in KCl electrolyte solution in its holding tube when stored and the internal buffer solution (3) is covering the inside of the glass membrane (1). If you see bubbles in the membrane, gently shake the probe in a vertical direction.
  • Calibration must be performed before starting work.
  • Note: Do not expose the electrode to air for more than a few minutes. If you take a long break (over 10 minutes) from work, immerse the electrode in undiluted seawater and make sure the level in the beaker is high enough.

  • Check the level of the electrolyte solution inside the electrode. The electrolyte level should reach 0.5 cm below the hole. If the level is lower, add 3M KCl storage solution.
  • External rinsing: Rinse the electrode with DDW water and gently absorb the excess with kimwipes without rubbing and move it to its designated place in the titration position.
    1. Fill 3 beakers with DDW and set them on the carousel in positions 1, 2, 3. Fill 3 beakers with buffer solutions: 4.00, 7.00, 9.00 pH and place them on the carousel in positions 4, 5, 6.
    2. Ensure the electrode is positioned at Position 21 (DDW) before starting.

    3. In the application, go to Workplace -> Sample Table -> New -> Method -> Calibration pH Electrode -> Start. Wait for the calibration to finish. Verify the slope in the Results Window: The slope should be >95%. If not, recalibrate.

    4. Once finished, the graph should be a Flat Line for individual samples or a downward graph if all three points are displayed. When the calibration is complete, the zero pH and slope values will appear on the device screen:

pH[0] 7.443
Slope 983%
Variance 1.729 Calibration temperature (manual) 22.0 ◦C

Buffer 1 pH= 3.994 U (mV)=198.2 T(◦C)= 22.0 T(s)= 140

Buffer 2 pH= 7.012 U (mV)=25.9 T(◦C)= 22.0 T(s)= 87

Buffer 3 pH= 9.024 U(mV)= -91.7 T(◦C)= 22.0 T(s)= 37

  • If the results are not as described above, replace buffers and redo calibration.
  • If the slope values are between 96-103% and the gap between pH 3 to pH 7 is 170 mV then the device is ready for samples’ measurements.
  1. Remove the cups containing the buffers and close them with the lids.
  2. Prepare the first sample in duplicates.
  3. The buffers will be replaced at least once a month.

##Running a titration

  1. You should start each day by running a Certified Reference Material (CRM) from the Dickson Lab. These are the standards.
    The procedure for running any titration, whether a CRM or unknowns are as follows. It is also advisable to start with a “junk” sample (filtered seawater that you don’t care about) at the beginning of each run to ensure there are no air bubbles in the acid line.
  2. Remove the samples from the refrigerator and wait until they reach room temperature.
  3. Mixing the sample.
  4. Weigh each sample on the scale by placing the cup on the scales and zeroing it.
  5. Filter the sample with 45 micron spinner filter
  6. Ensure each sample weighs approximately 52 grams. Make sure that there are no drops on the side of the beaker and if they have to be collected, these drops will be included in the sample weight but will not participate in the titration.
  7. Record the bottle number and sample weight.
  8. Include Dickson water samples at the start and end of your run to monitor drift. Use a serological pipette for the CRM sample. Weigh the Dickson water samples the same way as regular samples.
  9. In Tiamo: Go to Workplace under the Run tab in Single determination at the sample data choose Method -> Alkalinity_Ahmad.
  10. Under the determination series go to the sample table, new; method Alkalinity_Ahmad
  11. A window of the table should pop up, click on the line
  12. On the left, look for the “ID 1” column and label the sample. Put in all your sample IDs into this column.
  13. Next to the “ID 1” column, there should be a column named “sample size” enter the Weight [g].
  14. Record all sample IDs and corresponding weights in your notebook.
  15. Place the measured cups in the Autosampler, starting from position 1, then position 2, 3, etc. If more than one samples are used.
  16. Begin Titration
  17. After the data are set into the system, and all cups are placed in their slots in the autosampler, press Start on the bottom right of the “Task Editor” window.
  18. Once started, each titration will take ~10.5 minutes, unless there is a change of the method from the instructor.
  19. Save the Data: After the titration finishes, go to Sample Table -> Save/New Data File and name the file appropriately.

  20. Export Results: Navigate to Database -> Determination -> Export Template. Select your saved data file to export results into an Excel file. RS01-Value in the results file, represents the TA

  21. CRM_Titration

    Find the TA and salinity information for your specific CRM batch number here

    1. Skip to Data_Analysis to calculate total alkalinity of the CRM. Then check the accuracy of the data.
    2. In every CRM Titration, it is necessary to test the accuracy. It must never be more than 1% off. Accuracy must be tested before running any samples for the day.

  22. SAMPLE_Titration

    1. Repeat step [4.]. However, in step [4.iv], replace to [the sample name given on the sample bottle].

  23. Measure_Salinity

    1. While the samples are running on the titrator, you will measure salinity of each sample from the original sample vial.
    2. Measure the salinity of every sample and write down the value in the csv file you will make below. For CRMs, the salinity corresponds to its batch number, for junks just use salinity of 35.

  24. Data_Analysis

    1. After the titration is complete, you must gather the data and export it to the current folder. Make a file called “Mass_date” Ex: Mass_20210221 in your folder of the day that includes “Sample ID1”, “Weight”, and “Salinity”. fill in your corresponding label IDs, weights, and salinities into this file. Keep all labeling consistent from titration to analysis
    2. Calculate drift: Drift=Measured Dickson Average−Certified Dickson Value. If the drift is positive, subtract it from all sample TA values. If the drift is negative, add it to all sample TA values.
    3. Duplicates should be +-5 but up to 70 is acceptable
    4. Corrected alkalinity by the Dickson: TA corrected = TAdevice - (CRM1measured - CRM certified)-[N/k+1 * (CRM2measured – CRM dcertificate)-(CRM1 measured-CRM certificate))] CRM1- the first Dickson of the day CRM certified is the value of the batch number of the Dickson that can be found [here] (https://www.ncei.noaa.gov/access/ocean-carbon-acidification-data-system/oceans/Dickson_CRM/batches.html) N is the number of the sample run (1= the first one after the Dickson) K number of samples not include the Dickson CRM2 is the end of the day Dickson. After correcting all the alkalinity values, we average the 2 duplicates

  25. Clean_Up

    1. When the titrations are complete, remove the cups from the Autosampler, and dispose the samples into their corresponding labeled waste containers in the cabinet to the left of the laptop and titrator table. [note: all CRMs and nearly all samples in this lab have HgCl2].
    2. Clean the cups using Type I DI water. Then, dry them on KimWipes and drying pads nect to the titrator
    3. Using the Titrator, move the Autosampler arm into position 00 (which should have a cup with DI water in it) by pressing Park on the tablet.
    4. Return the sensor of the autosampler to its holder on the left of the InMotion arm. Double-check that it holder is filled with KCl.
    5. Shut off both Titrator and Autosampler, by disconecting the USB from the computer.
    6. Wipe down Sample Changer carefully with DI water.
Written on June 7, 2026