CDOM measurements
Instructions to measure CDOM absorbance (CDOM) and fluorescence (FDOM) from filtered water samples.
Sampling
Filter water samples with 0.2 µm membrane filters OR combusted GF/F glassfiber filters.
Analyse right away or store at 4 °C up to 4 weeks. If longer storage is needed, then freezing should be used. Note: do not use two different storage techniques with your batch of samples, i.e. if freezing is needed for some samples, all samples should be frozen.
Storage in combusted scintillation vials or acid washed HDPE vials.
Equipment and consumables
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Spectrophotometer (Shimadzu).
Spectrofluorometer (Varian).
Quartz cuvettes (marked with a "Q")
- for absorbance: 1-10 cm cuvette (depending on the optical density of the sample), with two clear sides and two opaque sides
- for fluorescence: 1 cm cuvette, with all sides clear
100-200 ml 1-2 M HCl in a small plastic box for cleaning cuvettes.
Kimtech wipes.
Preparations
Day before measurement
Place frozen samples into the fridge (4°C) for thawing overnight.
Measuring day
Turn on Shimadzu for warming up at least 30 minutes before starting measurements. 1-2 hours warm-up time is better.
Take samples from fridge into the measuring room, they need to have stable room temperature when measuring.
Have fresh MQ in a clean spray bottle.
Soak the cuvettes in acid for 15 minutes. Rinse thoroughly, first with warm tap water, then with lots of MQ. Use a Q-tip to brush the cuvettes carefully.
If not in place, put the cuvette holder into the Varian. Instrument can not be turned on during this operation, it will break.
Turn on Varian, and turn on both computers.
In both computers, make a folder with your name/project and a sub-folder for each measuring day (not sampling day): yyyy-mm-dd.
In Varian computer: open “Scan” program from desktop; in Shimadzu computer: open “UVProbe” from desktop.
Measurement
General
You will measure the same sample both first in Varian (for fluorescence) and then in Shimadzu (for the spectrum)
MQ serves as blank, standard and internal standard, thus it should be measured at the beginning, end and after every ~10 samples
Sediment pore water samples need to be diluted: start with 1:10 dilution (1 part sample : 9 parts MQ) and if that is too dilute, gradually decrease the dilution factor (trial and error). Adjust the dilution in such way, that the FIRST peaks (Varian), i.e. the peaks at low excitation range, are >100 and <1000, at best btwn 400 - 800. Cuvette volume: 3.5 ml (i.e. 1:10 à 0.35 ml sample to 3.15 ml MQ). First add sample, then MQ, then pump up the entire volume with the pipette to mix (3x).
- Note down the exact dilution volumes.
- Measure the samples ordered by location, as those might always have the same dilution factor.
Gently turn sample up-site-down before use (no shaking which might introduce bubbles)
After filling a sample into a cuvette, check for bubbles and snip them away if necessary; no bubbles wanted.
When changing samples, tap cuvette gently onto fresh tablecloth to remove most sample and then clean cuvette 3x with MQ, tap again gently to remove most MQ and let additionally stand up-site-down for a little while, before gently filling in new sample (again: no bubbles).
For all measurements, put the cuvettes with the same direction into the instruments; if they might have a scratch etc that might be detectable as regular noise and thus can be taken care of. Touch the 1-cm Varian cuvette only at the top, wipe sides carefully before inserting into instrument; 5-cm Shimadzu can be touched at the frosted sides.
Very important: file-names need to be identical between Varian and Shimadzu; MQ samples need to have “MQ” in the file-name. This is critical for efficient data post-processing.
Start-up
- Start at Varian: load method file (“Asmala FDOM 2015”: has all the right excitation and emission ranges, which are: excitation: 220, emission start 280 nm, end 600 nm). Then fill MQ into the 1-cm cuvette, wipe sides and check whether the glass is clean, put it in, and press start. A safe-data window will pop up: do not have dots (like in a date) in the file name, otherwise the program thinks this is a file-ending and data will not be saved as scan-file. Also, have continuous numbering as the first entry, otherwise csv files might be overwritten (program doesn’t seem to look further than the first entries in the file-name). After naming the file, Varian will start measuring, going through all excitation wavelengths up to 450 nm. This MQ-measurement is both blank and standard. When ready, press the rainbow-button to get a colored contour graph, which should look like this:
These two patterns are always there; as long as the area between those patterns is blue, everything is OK (= no CDOM contamination) and samples can be measured subsequently. Always check the MQ sample with the contour graph as shown in the figure before starting to run samples. If there are other smudges etc, re-run with fresh MQ. If smudge doesn't go away, cuvette needs to be cleaned in acid.
Remove all graph windows until only a grey surface remains. Then start measuring samples: press start and input sample name. Then it will start measuring producing a plot like this:
NOTE: the FIRST peaks (the peaks at low excitation range) should be >100 and <1000. If they are lower, your sample is very dilute and detector voltage must be increased in the method file. If they are higher, your sample is very concentrated and it needs to be diluted. Subsequent peaks at higher excitation ranges will quite often be very high and overshoot, but they are from the water, not the sample - this is OK.
In the meanwhile of the first Varian MQ-run, start the Shimadzu process: press “connect” in the program, then it will go through all necessary parameters and show a green light if all ok. If a parameter is not turning green, eg / especially the RAM, then turn off both computer and instrument, turn on and connect again (should do the trick). All checked parameters need to be green.
Press ok, weird sounds start; push button “baseline” and put 800-200 nm into the opening dialog box, numbers will start running at the left-hand corner. The baseline is the alignment of the two light sources that are used in Shimadzu: the visible and the UV light. Do the baseline without a cuvette.
When baseline is done, fill the 5-cm cuvette with MQ, put it into the slit closest to you and the window, close lid and adjust the program: it should be the “spectrum”-surface, adjust the windows in a way that the middle is the largest and the right one the second largest. Press “method” (M) and adjust wavelength: 800-200 nm; interval: 1.0; speed: slow; press “ok”. OBS: method will not be saved, thus check before starting a measurement. Click: “overlay” graphs. Press “start”. Storage window will pop-up: locate the folder where the file should be saved, file-format: spectrum, file-name: the same as in Varian. “Ok” and measurements will start, drawing spectra like this:
Adjust the y-axis to 0-2.
Safe after each measurement, as the program will not safe automatically “safe all”
Do Varian and Shimadzu interlaced at once.
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