Measuring pH Protocol with 5 cm cuvette
General Tips:
Be sure that you don’t touch the ends of the cuvette with your fingers. They need to stay very clean.
Don’t forget to record the temperature of the cuvette in the dilution factor field of your first (and ideally last) measurements.
Make sure you check the baseline for each new sample, and clean if it is high. If that does not bring it down, then blank the instrument. (Only when no dye added to that sample yet.)
Check the 730 wavelength for each dye addition and be sure that it matches the original baseline closely. If it does not, then clean the cuvette ends. If this does not work, do not blank. Try to clean again, and if no luck then just keep going but be aware that data from that sample may not be good.
The ratio of the other two wavelengths is the main thing that we use to determine pH. They are the absorbance of the two states of the dye. It has a red color when it is in the acidified state, and a purple color when in the base state. The ratio of these two form, and therefore the two peaks is a very exact measure of the pH.
When doing your calculations, make sure that the temperature and salinity are listed correctly for each of your samples in the dye addition tabs of the spreadsheet. The temperature to use here it the one you measure in the cuvette.
Turn off the spec, drain the water bath and disconnect and rinse the cuvette when you are done.
Step by Step:
One hour before taking measurements turn on water bath to bring to 25°C.
The main power switch is in back, then push red power button in front. Make sure you hear a whirring noise as bath starts up, otherwise screen may be on but in standby mode and you need to press red power button again.
-Get your cuvette, and fill it with deionized water.
-Connect your cuvette to the two small tubing fittings and open the valves to allow water to flow into the water jacket. Tip the cuvette to expel bubbles. Place it back in the v shaped cuvette holder.
-Take your m-cresol dye from the fridge and allow it to come to room temperature. Check to be sure that it is less than one month old.
-Place your sample vials in the water bath. 15 ml vials are about the right size. They should be filled carefully, overflowed and sealed with no headspace. Be sure that there is no label tape that will come off and cause problems.
30 min before taking measurements
-Turnturn on the Agilent spectrophotometer (button labeled #2 on spec).
-Log into the computer using SPMC log in. Password: !spmclab1
-‘CAG Bootp Server’ will be open/minimized (pops up when you log on). It will talk to the spec, this may take a minute, then light on the upper right corner of the spec will turn green.
-Once the green light has appeared, open ‘Instrument 1 online’ icon. Log in with your name and no password (to track user time.)
-If a dialog box pops up warning that lamp settings will be changed, Click ‘Use Current’, then click on yellow lamp icon in lower left (deuterium lamp) and turn off.
-If the method pH.m does not open automatically, then open it. (File, Load Method, choose pH.m)
-Click on the tungsten lamp icon in the lower left (small red bulb icon) and turn on if not on already. Do not turn on the other deuterium lamp. You only need the tungsten lamp and it should warm up for at least 20 minutes.
-Turn on the Fluke reference thermometer and place the tip in one of the necks of the cuvette. Set it to graph the temperature then zoom in so that you can see the small variations in the temperature that it is recording. Be sure it is stable before you begin your measurements.
Filling the cuvette
-Carefully pour contents of cuvette into a waste container. You can let it dangle by its tubes in the waste container while you take the next steps.
-Rinse the cuvette two times from the DI squirt bottle.
-Eject any remaining fluid from the last sample in the syringe tubing by drawing in some air then expelling it. Draw up a few ml of your sample. Turn the syringe upwards and aim the tube into the waste and expel that water as a rinse, along with any bubble. (Rinsing with the sample is better than rinsing with DI in this case.)
-Fill the syringe with most of the remaining water from your sample vial.
-Fill the cuvette through one neck, and empty it once.
-Place the syringe tube into one neck and carfully fill the cuvette again, taking care to expel any bubbles through the other neck. Overflow the cuvette until the syringe is empty.
-Place the stoppers in the cuvette and rinse the outside with DI water over the waste container.
-Blot dry the body with a paper towel. Clean both ends with alcohol on a small square of lens paper, then polish with a dry piece of lens paper. ( I find dipping a corner of a piece of lense paper into a scintillation vial of alcohol works well.)
-Visually check both ends for smears. If clean, place in the cuvette holder.
-Be sure the arrange the tubing such that it sits firmly in the cradle, and it not pulled at an angle. The position should be the same from one sample to another.
Measuring samples (2 dye shots / 1 dye shot)
-Choose your first few samples to represent the range of pH you expect in your run. You will use them to establish how the pH of the sample changes when you add the dye, so that effect can be removed for other samples.
-Take a blank by clicking blank. The baseline on the graph should be pretty flat between 350nm and 750nm.
-Now take your first measurement by hitting sample. You will be prompted for a sample name. Something with the date and sample ID is a good idea. Do not include spaces in your sample name. If name is too long, it will not export fully to your file.15 characters or fewer will work. Ie2_5_17SampleID.
-Record the temperature measured during setup in the dilution factor field so that it will be in your data file.
-Look at the data generated for your sample. All three of the wavelengths should have very small absorbance, on the order of 10-3 or 10-4 or less. Positive or negative values are fine but you want them all to be of the same sign. The idea is you want a nice flat baseline that is very close to the zero mark. When you get into high 10-3 or 10-2 range this usually means there is something blocking the light, or that your blank was not very good.
-If absorbances are higher than that, then clean the ends of the cuvette again. If that does not work, then take another blank. Add “AB” to the sample id, or some other indication that you have blanked the instrument. Only take a blank when there is sample but no dye in the cuvette.
-Draw up 20µl of m-cresol dye, making sure there are no bubbles in the tip or droplets on the outside. Do not set down the pipette with liquid in the tip. Keep it vertical in one hand.
-Add 20µl of m-cresol dye by opening one stopper, and holding the cuvette at a steep angle (at least 45 degrees) so that when the dye is expelled and floats upwards, it will go into the corner of the barrel, not back up into the neck of the cuvette. Remember not to touch the ends.
-Replace the stopper. Holding a chimwipe around the stopper absorbs the small amount of sample displaced by the dye addition.
-Insert stopper. Lift the cuvette and mix the dye into the water. Holding it by the tubes with the cuvette upwards and gently flopping backwards and forwards is effective, but do be very careful with the cuvette. It’s not easily replaceable.
-Put back into spec and hit sample to take another reading. This time name your sample the same name but add a + to show that indicator dye has been added.
-Check the 730 wavelength. It should be very close to the baseline value for that sample. (Less than 5x10-3 different, ideally.) If the 730 absorbance is high (10-2) or very different from baseline, then clean the ends of the cuvette again. Do not blank with dye in the cuvette. If 730 remains high, data from that sample may not be usable. The idea here is that this a wavelength where the dye has no absorbance, so after the blank has been taken, there should be no differences from the baseline.
**
-Add another 30µl of m-cresol dye, repeating steps above to mix and take measurement. This time name it with ++ to show two dye additions.
-Proceed to the next sample, changing your pipette regularly.
-Measure 3 to 6 samples with 2 shots of dye. The rest of the samples can be done with only one dye addition by following these step up to the ** then proceeding to the next sample.
Saving data file in Chem32/1/Data/YourFolder
-Periodically as you work, save your samples by clicking File—Save—Samples As…to save as .SD file.
-When you are finished, export results to a text file by clicking File –Print to File—Results…to save as a text file. Be sure to change Report name.
-Save under Chem32/1/Data/… and a folder specific to you or your project.
-Open the text file in excel, change file type to‘all files’, choose delimited, space delimited. You may need to rearrange the file to get things all in one set of rows and columns.
-Select your data starting with the row containing your first sample or buffer (just the columns with the data)
-Paste the data for sample with two dye additions into the two additions dye tab in your spreadsheet.
-Make sure that the correct temperature and salinity are entered for each sample. Update any cell references as needed.
-Make sure that your data line up correctly with the calculation rows.
-If the baseline diff column is red, approach that measurement with caution. If it’s yellow, you are probably OK. If it’s green, good job. This indicates a check on the reading for 730nm for your sample with and without the dye. They should match closely. If they are off by more than about 0.001 there could be a problem with the cuvette getting dirty or you may need to filter your samples or some other issue.
-Your pH is listed in column N. This is the pH at the water bath temperature of close to 25 degrees.
-Paste the one dye addition data into the one dye addition tab. They can be interpreted in the same way. However, you should be aware that the correction for the addition of dye in not automatic in these samples. At the top is a spot where the slope (m) and intercept (b) of a regression of the change in the peak ratio upon the addition of the second dye shot (y axis) is regressed against the ratio before the dye was added (x). You should keep a running file which has all your 2 shot measurements from your experiment in it where you do this regression and keep the m and b in the one shot tab up to date on the best data for this correction.
-In general you will want to combine these date with DIC values and put them into CO2 sys. There are tabs for all of this on the master spreadsheet.
-Save your data to the dropbox, or whatever file management system you are using.
-Don’t overwrite Master specpH file – always ‘save as’ before inputting raw data.
Clean-up
-Disconnect the cuvette and rinse both the water jacket space and the sample volume space with RO water and set aside in foam boxes to dry.
-Use the cuvette cleaner liquid every few days as well.
-Turn off tungsten lamp by clicking on the icon
-Click File—close chem station. Don’t save method (unclick check box)
-Turn off spec by pressing #2 power button
-Close CAGbootp and log off
-Turn off red power button on water bath (may need to press twice. Once to wake up, then again to turn off)
-Then turn off power switch behind water bath
-Thermometer can be turned off and left sitting on lab bench if being used frequently. There is a black storage case for it under the bench, otherwise.
-The water bath should be drained (valve on the front panel) and water in the bottom sponged up. (If you are going to do several runs in a row over a few days you can leave the water in, but do not leave it in there if you are not making a run the very next day.)
-Make sure to leave the area in an orderly state. This is a common use area.
Making new m-cresol dye
Dye does not keep well and should be made fresh every 2 to 3 weeks or so.
-Add 20ml nanopure water to 18.1mg of m-cresol salt.
-M-cresol salt is in fridge #2 on the door, weigh 18.1 mg on balance and add to vial with nanopure water. Make sure nanopure is reading at least 18.0 ohms.
-Replace the 5 cm cuvette holder (v shaped channel) with the 1 cm cuvette holder.
-Load 1mm cuvette with nanopure water for blank reading using p200 pipette. (1mm cuvette lives in drawer labeled ‘Spectrophotometer parts’ below computer, in white foam box labeled ‘Precision cells’), It is delicate, and be sure not to touch the front and back of it with your finger either.
-Place cuvette into metal bracket with spring and put into spec. This is not that easy. I find it’s best to hold back the spring/holder piece with my thumb nail while I hold the cuvetter and cover slip by the edges and slide them in.
-Run blank by clicking blank.
-Slide out of bracket when done, again while holding the spring back, then slide apart cover and dry.
-Now load dye into same 1mm cuvette, load into metal bracket, place in spec and hit sample.
-Name something ie. pHdye0drops_date
-Look at the ratio – the target ratio is 1.61 for regular seawater samples. If you think your samples will be a range from pH 8 to 7.5 or less, then you might choose a lower value. We used a value of 0.9 in that case. This way it was between our expected sample values so was not exerting a much bigger effect on one treatment than on all the others.
-If the ratio is too low, add 2 drops of 0.02N NaOH to dye vial. This is in a vial above on shelf.
-Load cuvette again and test ratio.
-Keep adding drops of NaOH in 2 drop increments to 20 ml vial of dye and loading 1mm cuvette to test in spec. As ratio gets close you can add just one drop. Rinse cuvette with RO each time. Name each test with # drops ie. pHdye2drops_date
-If the ratio is already too low, then drops of dilute HCl can be added instead.
-Make sure all new dye is labeled with new expiration date 4 weeks from now.
4.10.14 njc