US Long-Term Ecological Research Network
EQUIPMENT
 
  • Radiometer pHM84 pH Meter
  • Fisher Series 5000 Strip Chart Recorder
  • Radiometer Model GK2401C Combination Electrode
 
SAMPLE COLLECTION
 
1) Rinse bottle 3 times with small volumes of the water to be sampled.
2) Collect pH and alkalinity samples in separate 20-ml scintillation vials with displacement caps. Collect a second pH sample in a 125-ml wide mouth bottle. Fill the 20-ml vials with a minimum of splashing and air entrainment. Fill to overflowing and cap, EXCLUDING ALL AIR BUBBLES FROM THE SAMPLE. It is important to ensure that no atmospheric gas exchange occurs between the time of sample collection and analysis. The 125-ml bottles are for air equilibrated pH determination, and may have air in them.
3) Keep samples cold and dark to minimize biological activity.
 
pH MEASUREMENT
(used for Biocomplexity Cross Lake Comparison Samples from 2001-2003)
 
1) pH samples should be analyzed as soon as possible after collection, and MUST be analyzed the same day they are collected.
2) Before measuring pH:
a) Warm samples to room temperature in a dark container. The container may be filled with room temperature water.
b) Suspend the electrode in the syringe barrel setup if it is not already there. Seal the top of the syringe barrel around the electrode with teflon tape.
c) Fill the electrode syringe barrel with an extra sample of the lake water to be analyzed. Leave the electrode to soak in this sample at least 15 minutes. The longer the electrode is "conditioned" before pH measurement, the faster it will respond during measurement.
d) Bubble air through the samples in 125-ml bottles for at least 5 minutes before analysis using a vacuum pump and the gang valve assembly. The intake hose should be pulling air from outside the lab. Cap samples tightly until analysis.
3) pH meter and chart recorder settings before beginning measurement are:
a) pH meter - HOLD button depressed
- 1500 mV button depressed
- small toggle switch on back panel down (this inactivates the Buffer, Temp., and IsopH knobs found on the front of the meter).
b) chart recorder
- CHART SPEED set at 2 in/min.
- DIVIDER switch at 10
- CHART paper drive off
- voltage input selector on CHECK
4) Uncover the electrode filling solution hole. Remove the conditioning solution from the electrode syringe barrel using the three way valve and aspiration system. Pull air through after the solution is drained to remove as much of the soak solution as possible.
5) With a clean 5-ml syringe take an aliquot of the sample to be measured and inject about 2 ml into the electrode chamber. Drain chamber.
6) Inject another 2 ml and flush in and out of the chamber several times. Drain.
7) Refill syringe with another aliquot of the sample and inject 2 ml into the electrode chamber. There should be enough sample in the chamber to cover the electrode junction. Close the 3-way valve to keep the sample in the electrode chamber.
8) Release the HOLD button on the pH meter, turn on the chart recorder paper drive and set the voltage knob to 1 volt.
9) When the pen reaches a plateau, depress the HOLD button, turn off the paper drive, and turn the voltage knob to CHECK. Record the millivolt value from the LED display of the pH meter.
10) Continue to run 2-ml aliquots of the same sample (steps 7-10) until two consecutive millivolt readings are within 1 millivolt.
11) Repeat steps 4-11 for each pH sample. Run the samples in 20-ml vials before the air equilibrated samples. Measure and record the temperature of each air equilibrated sample AFTER you are done measuring pH of the sample.
12) When all samples have been run, repeat steps 4-9 with the 3 buffer solutions: pH 3.557, 4.01, 6.84. Only one 2-ml aliquot of each buffer must be run.
13) Rinse the electrode chamber with Trout Lake tap water and store the electrode in Trout Lake tap water.
14) Turn off chart recorder, remove and cap the pen, cover electrode filling solution hole.
 
 
ALKALINITY MEASUREMENT
(used for Biocomplexity Cross Lake Comparison Samples from 2001-2002)
 
1) Before beginning titration:
a) Warm samples to room temperature in a dark container.
b) Move the electrode to the side of the ring stand where the magnetic stirrer is, if necessary, and uncover the electrode filling solution hole. Place the electrode in a sample of water from the lake that the alkalinity samples are from.
c) Turn the stirrer on 15 minutes before beginning. Do not change stirring speed during titrations and buffers. A change in stirring rate introduces a millivoltage error called the 'streaming potential'.
d) Fill the Gilmont buret with standard 0.01N HCl as follows. Place the delivery tip of the buret in a waste beaker and deliver acid until there are no air bubbles in the tubing. Rinse with milli-Q water, wipe dry, and deliver a few more drops of acid. Place the tip in a beaker of standard HCl and draw acid into the buret. Rinse and wipe tip again, and deliver acid into the waste beaker until the buret is zeroed. (If there is air in the buret, remove it from the clamp, invert, and deliver the air out the tip.)
e) Run a 'dummy' alkalinity titration on a sample of conditioning water from the lake before running any samples. This is done because the first alkalinity titration often gives a suspicious value.
2) The pH meter and chart recorder settings before beginning titration are as in step 3 of PH MEASUREMENT.
3) Rinse a small stir bar with milli-Q water, dry with a kimwipe, and place in a clean, dry 2-dram vial. Do not touch the stir bar with your fingers; handle with a magnet and forceps only.
4) Place 3 or 4 ml of sample (depending on the alkalinity of the sample) into the vial using the Eppendorf pipet with a 1-ml tip. Draw up and deliver sample from the pipet slowly so that no air bubbles are drawn into the tip, or sample droplets splashed onto the side of the vial.
THIS STEP IS PARTICULARLY IMPORTANT!
Because of the small volume titrated, any loss of sample by spattering or any sample forming a droplet on the side of the vial will not be titrated, thus giving low values. If you make an error, start over with a clean, dry vial.
5) Turn off the stirrer and remove the electrode from the conditioning lake water vial. Slide the magnetic stirrer out from under the vial and loosen the clamp to free the vial rather than raising and lowering the electrode each time.
6) Rinse the electrode with milli-Q water and carefully blot it dry with a kimwipe.
7) Place the electrode in the vial containing the sample, slide the magnetic stirrer below the vial, and center it on the 'x' on top of the stirrer. Turn stirrer on.
8) Zero the acid buret, wipe the delivery tip, and insert it into the vial.
9) Release the HOLD button on the pH meter. Turn on the paper drive on the chart recorder and set the voltage knob to 1 volt.
10) Note the initial millivolt reading and add small increments of acid until the meter reads >115 mV. When the pen levels off, record the volume of acid added and the mV reading as your first increment.
11) Titrate the sample from +115 to approximately 160 mV using 10 EQUAL INCREMENTS of acid. Generally 0.010 ml increments are required. Record the volume of acid added and the equilibrium millivolts after each increment.
12) Depress the HOLD button, turn off the paper drive, and set the voltage knob to CHECK.
13) Remove the buret delivery tip from the vial, rinse with milli-Q water, and dry. Deliver some excess acid from the tip to flush the tip.
14) Repeat steps 3-13 for each sample. It should require about 20 minutes per sample for the titration.
15) After completing sample titrations, standardize the electrode using fresh buffer solutions (pH 3.557 and 6.84). Place a few mls of buffer and a stir bar in a vial. Insert electrode, and set meter and chart recorder settings as for a titration. Do not place the acid buret tip in the buffer vials. When the pen levels off, record the mV value on the data sheet.
16) Turn off chart recorder and magnetic stirrer, remove and cap the pen, cover electrode filling solution hole. Rinse electrode and store in Trout Lake tap water.
 
revised 8/89
LTER Keywords
Protocol Format
Process
Protocol ID
biocom_ph_alkalinity1
Protocol Type
laboratory