ap and ad scanning

a_p and a_d scanning (Particle absorption)

Supplies

Sample filters (kept in liquid nitrogen)

2 new 25mm GF/F filters (one blank and one reference)

1 beaker of Q-water

1 Pasteur pipette with bulb

2 pairs of forceps

Petri dishes (one for each sample filter + 1 for blank + 1 for reference)

Kimwipes (large and small)

Denaturated alcohol for cleaning integrating sphere and spec. windows

A dust brush

Setup

Clean the two integrating sphere ports and the spec. windows (on the right hand side of the spec. sample compartment). For the integrating sphere ports (marked ‘R” for reference and ‘S’ for sample), wet a small Kimwipe with Q-water and wipe each port. The spec. windows should be cleaned as follows: (a) fold a small Kimwipe in half, then in half once or twice more so you have a small square or rectangle, (b) wet the Kimwipe with denaturated alcohol and wipe one spec. window, (c) remove any remaining lint, streaks with a small dry Kimwipe. Use a dust brush to brush the all compartment, especially the mirrors and the compartment floor.

Power up the computer and attach the spec on the lower USB port of the computer. Turn on the spec. (it has to be turned on before the computer), wait about 30 seconds, then turn on the computer. There is no password. Just click on ‘OK’ when it asks for a password.

Go to ‘START’, ‘My computer’, ‘Local Disk (C:)’, ‘Program Files’, ‘Shimadzu’, ‘UV Probe’,‘Data’. Create a new folder if necessary to store your data files. Minimize the window.

On the desktop, click on the UVProbe software shortcut (the red square with a white circle, and a cross). On the bottom of the screen, click on ‘Connect’ to start the communication between the spec. and the computer. The spec. is going through its testing cycle (it takes about 5 to 10 minutes).

Meanwhile, put the blank and the reference filters each in a Petri dish. The concave side of the filters should face outward (this way, the filters stick better to the integrating sphere port). Saturate the filters with Q-water.

Take the samples out of the nitrogen dewar, leave the first sample to be run out and put the rest of the samples in the freezer.

Prepare some Kimwipes pads. Fold a small Kimwipe several times lengthwise to obtain a ½ inch wide band. Cut the band in half crosswise. You’ll about 1 pad for 2 samples. Prepare as many pads as you will need.

Spectrophotometric measurements

Once the spec. has gone through the testing cycle, click ‘OK’ on the testing screen. Wait a few seconds. Go to ‘Edit’, ‘Method’. Check the parameters. The wavelength should be starting at 850nm and ending at 350nm. The speed should be ‘fast’ and the Sampling interval (nm) should be ‘1’.

Check the acquisition screen. Make sure the wavelength display (i.e. the X axis) is 350 through 850. The Y axis should go from -0.05 through 0.6. This range can be changed during the measurements if needed.

The soaked blank and reference filters are used to set up the instrument baseline. A new instrument baseline scan should be measured each time the spectrophotometer is powered up. Place the two hydrated Q-water filters on the integrating sphere ports (one the reference port, one on the sample port). You may need to drip the edge of the filters on a paper towel before mounting them on the integrating sphere. The filters should be wet and shiny but not dripping. Close the sampling compartment door. Click on ‘baseline’ at the bottom of the screen. The baseline does not show on the screen and is not stored as a file. When the baseline correction is finished, and without touching the blank filter, run a blank sample scan by clicking on ‘Start’. The spectrum should be flat and close to zero. At the end of the scan, a saving screen will appear. Change the saving directory if necessary (that’s the folder you created at the beginning of the session), and name the file keeping the .spc extension. Click on OK to save it (I think that if you press ‘Enter’ instead, it does not save it properly). Then, you have to save the file in a text format. Go to ‘File’, “Save as’. Select the ‘Data Print Table (txt)’ format and enter the same filename with a .TXT extension. Click on ‘OK’ (not ‘Enter’) to save it.

You need to run a second and third blank replicates. While the blank replicates are scanning, put a quarter size drop of Q-water in a Petri dish (labeled with the sample identification), unwrap the filter and place it in the Petri dish to get it hydrated. Put it by the spec., covered with a Kimwipe box to keep it from direct light exposure. Get the second sample out of the freezer and leave it on the bench to thaw.

To run the second blank replicate, pick up the blank filter with the forceps and move it a little bit on the port by rotation or shifting in order to scan a different surface area on the filter to get a good representation of the filter. Close the compartment and click on ‘Start’. Save the .spc file and the .TXT file. Move the blank filter again and do the third replicate the same way. The three replicates are conventionally named A, B and C. Wipe the Petri dish assigned to the blank filter with a large Kimwipe to dry it up. After the third replicate, remove the filter and put it in its Petri dish.

The reference will dry out over time and must be hydrated before each new sample filter. Put a Kimwipe pad on the integrating floor just under the reference filter. Put a few drops of Q-water on the reference filter using a Pasteur pipette with bulb.

Put the first sample filter on the sample port of the integrating sphere. All sample filters will be scanned with the material (the yellow-brownish side) facing the integrating sphere port.

Scan the first sample filter the same way you scanned the blank filter (3 replicates moving the filter between each replicate), save the data files in .spc and .TXT formats.

While the sample is scanning, put 30ml of 100% methanol in the Petri dish containing the blank filter (30 ml correspond to two squirts of the automatic pipette mounted on the methanol bottle). Set the Petri dish in the fume hood in dark (put a box over it) to extract.

When the three replicates of the sample are done, the sample should be processed the same way to remove its pigments. Wipe the sample Petri dish, place the sample filter back in it, put 30 ml of methanol in the Petri dish (not directly on the filter). Set it in dark in the fume hood. The filters will take anywhere from 24-48 hours to extract depending on the filter load and phytoplankton species composition.

When all the samples have been run, go the ‘File’, ‘Save All’ to save the .spc files on the disk.

Then, click on ‘Disconnect’ and turn the spec. off.

Check on the disk that your files are there. The size of the .spc file should be 19KB (or so) and the size of the .TXT file should be 7 or 8 KB. If you find a .TXT file with a size of 19KB (or 1KB), it means that it has not been properly save. You need to rename it with a .spc extension (click yes to confirm the name change). Then go back to the UVProbe software, open that .spc file and save it as a .TXT file making sure that you select ‘Data Print txt’ format.

De-pigmented spectra

Set up the spectrophotometer as for the particle absorption spectra. Change the Y axis maximum to 0.3 (the absorption will be lower as the pigments are gone).

Place the reference filter on the filtration system connected to the vacuum pump making sure that the filter funnel is tightly attached to the base. Pour the methanol in the filter funnel. Turn the vacuum pump on, filter the methanol and rinse the Petri dish 2 times with Q-water, pouring the water in the filter funnel but keeping a few drops in the Petri dish.

The part of the procedure is similar when filtering a sample. When the filter is dry (after rinsing the Petri dish), add 15 ml of 100% methanol (one squirt) into the filter funnel. Let it stand a 5 to 10 minutes (the time to finish the scanning of the previous filter). Then filter the methanol and rinse the sides of the filter tower with Q-water. This short extraction can improve the pigment extraction. This step can be skipped for the reference and the blank as they do not contain pigments. It also may be not necessary for very light samples.

The de-pigmented samples should be measured in the spectrophotometer as described above for the particle absorption (baseline with reference and blank, 3 replicates for the blank and for each sample). Once the 3 replicates are obtained, the filter can be discarded.

Glassware cleaning

Fill a tub with warm water. Add a small amount of Liquinox (less than half of the cap because it’s very bubbly). Wash the Petri dishes in this soapy water with a cleaning brush. Rinse with Purified Water. Let dry in the dish drainer. Wash and rinse the different parts of the filtration system the same way.