Updated: August 7, 1998

LADWP protocol for sample preparation:

Files for LADWP are archived in the following directories:

The preparation of the samples from the LADWP should be done as follows:

We have found that the Nucleopore Polycarbonate Filters (we are suggesting 0.4um polycarbonate filters) give the best results when it comes to running soluble samples(As). The soluble samples should also be collected in Quorpak Amber bottles, caps should be either the polyseal or the Teflon-Lined. GF/F filters should be used for the collection of the Ap's and Ad's.

Preparing the As samples:

Preparing the Ap samples:

The Quorpak Amber bottles should be washed and rinsed with distilled water first and then combusted in a muffle furnace. Remove the caps before combusting the bottles because they will melt. Wash the caps with soap and water. Then rinse the caps well with distilled water. Cut small pieces of square aluminum and wraps them over the mouth of each Quorpak Amber to prevent contamination of the bottles during and after the combustion.

On receiving samples from LADWP :

The samples will be kept in a refrigerator for no more than 24 hours upon being received. And it is recommended that the samples should be sent to SIO within 24 hours after sampling. It is not recommended that the samples be sitting in the refrigerator for more than a couple of days.


Measurement of Particulate and Detrital Absorption

Synopsis of method

The "Quantitative Filter Technique" (QFT) is designed to measure the spectral absorption by aquatic particulates. Particles are first collected by filtration on to the surface of a glass-fiber filter. The absorbance by all particles (= ap) is determined by measuring the sample filters within a standard laboratory spectrophotometer. After measurement, the sample filters are extracted in methanol to remove phytoplankton pigments ( "Kishino" method), and then remeasured in the spectrophotometer. This yields the "detrital" (actually non-pigmented) absorption component (=ad). The phytoplankton-specific absorption coefficient (aph) is then calculated as the difference ap – ad.


Ap Sample Preparation

Materials and supplies

25mm Filtration setup Forceps GF/F filters (25 mm) pasteur pipets
Eyedroppers Fresh Milli-Q water 100% MeOH (for ad extraction) Labeling tape
Petri dishes (for running fresh) Histopreps (for freezing) Sharpies or pencils  


1) Collect water samples, and maintain in the dark.

2) For each sample, place a GF/F filter onto the filtration rig. If you will be running the samples fresh, also prepare two blank GF/F filters by soaking them in fresh milli-Q water during the sample filtration. One method is to put two blank filters on the filtration rack and add 25 +/- ml of milli-Q, and keep the valves closed while your samples filter. When the samples are done, open valves to let water pass through the blanks.

3) Filter samples on to GF/F filters under low vaccuum (~5 in. Hg) in dim light. Filter enough material so that the color of the GF/F is light to moderate green/brown, the actual amount required will depend on the concentration of your sample. Be certain to keep track of the volume filtered for each sample!

Do not let the preps run dry on the filter rig. Turn off the vacuum to each position as it completes filtering. If you are busy with other samples, turn off the valve just before it goes dry, and then when ready re-open the valve and complete filtration.

4) After filtration, place samples in the appropriate container depending on how they will be stored.


4a) FRESH: If you will be running the samples fresh (within 24 hrs), store each filter in a labeled petri dish. First put a small drop of milli-Q into each petri dish (too little water and the sample dries out, too much and the sample smears), then place the filter on top. Wrap samples with aluminum foil to keep dark, and store in refrigerator (~4 deg. C) until analysis.

4b) LONG-TERM: If you will be running the samples at a later date, sample filters may be placed in histopreps and frozen in liquid nitrogen. Label histopreps with all relevant information (e.g. Location of Reservoir, depth, ) and put into a liquid nitrogen dewar. In this case, you do not need to prepare blanks every time you prepare samples; just soak and freeze 1 pair of blank filters from each box of GF/F’s you use. Samples can be stored in liquid nitrogen up to a period of 1 year.


Kishino Method for Obtaining Detrital Aborption

Only do this procedure AFTER the ap filters have already been scanned and saved on the spectrophotometer! After preparing an ap sample with standard procedure and analyzing it on the spectrophotometer.

1) Place sample and blank filters back onto a 25 mm Gelman filtration tower. Treat blank filters exactly as if they were sample filters.

2) Add 5-10 ml (or fill to the level of the bottom rim of the Gelman filtration tower) of 100% methanol to each filter by gently pouring it down the sides of the filter funnel so as not to resuspend any of the sample.

3) Allow sample to extract in methanol for 1 hour. Do not allow the methanol to go dry during the extraction period.

4) After extraction, turn on the vacuum and draw the methanol through the filter. Rinse the sides of the filter tower twice with small amounts of methanol. Last, rinse the sides of the filter tower two times with ~20 ml of fresh milli-Q. When rinsing, be careful not to re-suspend the particles.

As an alternative to 1 hr. extraction, 3 successive ten minute extractions can be used. At the end of each 10 min. extraction, filter through the methanol, turn off the vacuum, and add another layer of fresh methanol. Do all rinses at the end of the third 10 min. extraction. I have found this procedure often yields better extraction efficiency than the standard 1 hr. method, so if your samples still appear to have significant pigment absorption after extraction try this method.


Ap/Ad Sample Analysis

After preparation, the sample filters are scanned in a spectrophotometer. The following procedure is written generically for any (double-beam) spectrophotometer, for details of operating particular SPG instruments.

Materials needed

Samples and Blanks GF/F filters Milli-Q Spectrophotometer
Pasteur pipettes Petri dishes Labeling tape
Forceps Ethanol  
Filter mounting plates w/ quartz windows    


1) Warm up spectrophotometer at least 10 min. (preferably 1 hr.) before running samples.

2) If using frozen samples, remove filters from histopreps and place into pre-labeled individual petri dishes that contain a drop of milli-Q. Allow to thaw (~5 min.) and store in refrigerator until analysis. If you do not have frozen blank filters, prepare 2 blanks as outlined above in sec.

3) Clean the quartz face plates of the mounting device with ethanol.

4) Set the appropriate instrument parameters. Nominally, we use the following settings:

Instrument mode: Absorbance

Scan range: 250 to 800 nm

Scan resolution: 1 nm

Spectral bandwidth (SBW): 4 nm

Scan speed: 600 nm/min

5) Mount two blank filters (one for sample beam, one for reference beam). First, place a small drop of milli-Q on the center of quartz window, then slowly place the blank filter directly onto the drop. Make sure no air bubbles are trapped between the filter and the window. Place mounts into the spectrophotometer.

6) Run instrument baseline (background) correction using the two blanks. This stores the baseline into memory, and it will be automatically subtracted from subsequent scans. Immediately after baseline correction is finished and without touching the filters, run the two blanks as a sample scan to get a measure of the ‘true’ baseline. This spectra should be almost flat spectrally, and the magnitude should be less than +/- 0.01 OD. Save this scan as your baseline.

7) Remove the blank filter from the sample mount, and replace it with a sample filter (again, use a drop of milli-Q on the quartz plate to moisten the filter and hold it in place). Run it as a sample scan, and save the file. Keep track of which computer files correspond to which sample. Repeat this process until all your samples have been analyzed.
8) For processing of raw spectrophotometer data files.



1. It is important to keep your sample moist with fresh milli-Q. The test for proper saturation is to lift the filter and confirm that there is a drop of milli-Q left on the mounting plate. Upon placing the filter on the mounting plate, there should be a sheen on the surface of the sample, and a very slight "halo" of water around the edges of the filter. Be careful not to have too much water, as the sample may wash away. Saturated, but not swimming!

2. No bubbles should be between the filter and the glass on the sample holder. Test by examining the back of the filter on the mounting plate. There should be a uniform layer of water between the filter and quartz mounting plate. Bubbles will be obvious. If they are present, pick up the filter with forceps, and place back on the plate with a slight dragging of filter across mount and milli-Q drop. Check again. Repeat until no bubbles.

3. The blank reference filter will dry out over time, and needs to be regularly re-wetted with drops of milli-Q. If your absorption signal gets significantly greater than zero in the infrared (750-800 nm), this often indicates a dry reference filter. Get into the habit of checking the reference filter after every 2-3 scans, and moistening it as needed. Note that ad samples/blanks tend to dry out more quickly because of the MeOH treatment.

4. How files are stored and named differs between instruments (for example, all Cary scans are numbered sequentially). Just keep track of which files corresponds to which sample, and in comments line put as much information as possible. For cruises, we have a specific naming scheme which needs to be followed to allow convenient processing.

5. After running ap samples, the filters can then be extracted with methanol to determine detrital absorption.


Dissolved Material Absorption (as)

Synopsis of method

This technique measures the spectral absorption by soluble material (as) dissolved in milli-Q. Samples are collected and particulate material is removed by filtration. The absorption of the filtrate is measured, relative to pure water, using a spectrophotometer. Because water absorption is temperature-dependent, the optical cuvettes should be jacketed and connected to a running waterbath to ensure that both reference and sample cells are maintained at the same temperature.


Our experience has been that samples cannot be stored for considerable lengths of time (1-2 weeks max), and therefore should be run as fresh as possible to avoid potential artifacts.


The Qorpak bottles used to collect sample filtrate need to be rigorously cleaned in advance to remove any potential organic contaminants.

1. Combust bottles without caps at 500 degree C for 4-6 hrs. (cap bottles with aluminum foil)

2. Rinse caps with 10% HCl, twice with Alpha-Q, then dry at 70 degree C for 4-6 hrs.

3. Assemble the combusted bottles and clean caps.

4. Fill clean, combusted bottles with fresh Alpha-Q (directly from tap).


Sample Collection

Materials needed

Plexiglass filtration rig setup 25mm 0.2 um Nucleopore filters Jar of 10% HCl in Alpha-Q
Forceps Fresh Alpha-Q water  
Clean 118 ml Qorpak amber bottles (small mouth, polyseal lined caps; Fisher Catalog 03-320-6B)    




Nucleopore filters leach out colored material which can contaminate the samples, so the filters must be thoroughly rinsed in advance before using them for filtration. Plan ahead and do step #1 before collecting water samples. Remember to reserve one space on the rack for your blank (Alpha-Q).


1. Pre-soak filters for 15-20 minutes in 10% HCl. Rinse filters thoroughly with Alpha-Q water. Mount filters on rack with clean pre-labeled bottles below and filter ~20 mls of Alpha-Q through filter into sample bottles. Shake bottles, discard water (pour discard over inside of caps to rinse them), then repeat the procedure. Cover filtration cups with aluminum foil until ready to filter sample.

2. Collect ~25 ml of sample. Wash hands with soap and water first to avoid contaminating the samples.

3. Filter 5-10 ml of sample into bottles at low vacuum (5-10 in. Hg). For the blank, use clean Alpha-Q water. Shake bottles, discard water, and repeat.

4. Filter ~20 ml of sample into bottles. For the blank, filter ~25 ml of Alpha-Q water. When finished, cap bottles and store until analysis.

a) For samples that will be run within 24 hrs., store in the dark at room temperature.

b) For samples that will be run within a few days, refrigerate samples in the dark.

c) Longer storage is not recommended, as there are artifacts of undocumented magnitude.



1. Do not use the black Nucleopore filters for this prep, only the non-colored (white) ones!

2. The Corpus bottles can be re-used at sea. After analysis, thoroughly rinse bottles (and caps) 3x with fresh Alpha-Q water.

Sample Analysis

Materials needed

Spectrophotometer Waterbath 10cm quartz cuvettes
Kimwipes Ethanol Alpha-Q water




1. If samples have been refrigerated, allow them to warm up to room temperature.

2. Allow spectrophotometer to warm up for 1 hr. Turn on waterbath and set temperature control to room temperature (see Note #1). Nominal spectrophotometer settings we use are:

Mode: Absorbance

Scan range: 250 to 800 nm

Scan resolution: 1 nm

Spectral bandwidth: 4 nm

Scan speed: 600 nm/min

3. Wash hands with soap and water to avoid contamination

4. Clean the 1cm cuvettes. Discard the Alpha-Q water stored inside, rinse inside and outside of cuvettes 2x with ethanol, then rinse inside 3x with Alpha-Q water. Use Kimwipes when handling the cuvettes, do not touch with your bare-hands (to avoid fingerprints).

5. Fill both cuvettes with your Alpha-Q blank. Make sure there are no bubbles present.

6. Carefully dry the cuvettes. Bulk dry with paper towels but dry the quartz end windows with Kimwipes only. Look through the long path of the cuvettes to be sure there is no dust or smudges on windows.

7. Place cuvettes in spectrophotometer, and wait a few minutes so that temperature will equilibrate. (see Note #2)

8. Run baseline (background) correction with your Alpha-Q blank in both cuvettes. After baseline complete, make sure baseline correction is on and run your two blanks as a sample. This spectra should be spectrally flat, with magnitude less than +/- .001. Save this spectra as your baseline.

9. Remove sample cuvette and discard liquid. Rinse inside of cuvette 2x with Alpha-Q, twice with sample, then fill with sample.

10. Place sample cuvette back into spectrophotometer, wait a few minutes for temperature equilibration, then run scan. Repeat steps 9-10 until all samples have been run.


Note :

1. Water absorption is temperature dependent. These effects are most pronounced in the 700- 750 nm range. To avoid temperature effects, sample and blank should be maintained at a constant temperature with a circulating water bath. If this is not possible, the sample compartment to the spectrophotometer should not be closed during sample preparation. In other words, don’t close the compartment unless you are running a sample. It becomes more and more difficult to avoid the "warming up" of the spectrophotometer as time passes. Remove reference from compartment between runs. If necessary, immerse sample and reference in a room temperature water bath and dry thoroughly with Kimwipes before running spectrum.

2. When placing a sample cuvette into the spectrophotometer, it will take a few minutes to temperature equilibrate. You can monitor this by watching the OD display on your spec; it will change rapidly at first then stabilize over time.


CalCOFI Chlorophyll Procedure



Safety Issues

Chlorophyll samples are analyzed by extracting the pigment in a 90% acetone solution. Acetone is a hazardous chemical. If you have any questions about the dangers of acetone read the Material Safety Data Sheet in our MSDS file. The greatest danger presented by acetone as used in our procedures is eye injury. Acetone splashed or sprayed in the eye is very harmful. When handling acetone you MUST wear protective eye wear. Ordinary eyeglasses fulfill this requirement. If you wear contact lenses you must wear protective eye wear over them. Wearing contact lenses while working with chemicals is not recommended.


Sample Filtration:

1. Check the filtration funnels to be sure they are tightly installed, and to be sure that the proper filters are in place.

2. Turn on the vacuum pump, pour the sample into the filter funnel, and open the valve. Work from left to right across the filtration rack with deepest (lowest #) sample at the left. Check the vacuum pressure to see that it does not exceed 5 p.s.i..

3. While the samples are filtering list the vial numbers you will be using, the volumes filtered, and the extract volume. Extract volume will be 10 ml unless some special experiment is being done. List each vial number, do not simply list the start and finish numbers with a line connecting them.

4. When the sample has finished filtering, turn off the valve, using forceps pick off the filter and place it in the appropriate numbered vial of 90% acetone.

5. Place the vials in the tray, in order, and mark the last sample with a red tag. Put the tray back in the refrigerator. Fill in the 'Filtered by' portions of the data sheet with your initial, date and time.

6. Prepare the filtration rack for the next station by installing new GF/F filters in the filter funnels. Be sure that the filters are centered on the filter frit. Off center filters will not catch all the sample. Also make sure the funnels are firmly attached to the bases. Remember to turn off the vacuum pump.


Reading Samples on the Fluorometer:

1. The fluorometer should be left with the power on throughout a cruise. If for some reason it has been turned off, it must be warmed up for 1/2 hour before using.

2. Readings are taken from the digital voltmeter attached to the fluorometer, not from the analog meter built into the fluorometer. Full scale on the fluorometer meter equals 5 volts on the voltmeter. It is okay to read values on the voltmeter beyond 5 volts, even though the fluorometer meter appears pegged.

The maximum reading that can be read varies with fluorometers. It should be noted on the front of the fluorometer.

3. Samples must extract in acetone for at least 24 hours prior to reading on the fluorometer. They should be read before 48 hours.

4. Samples must be at room temperature for accurate readings. Plan ahead and set out a batch of samples at least one hour prior to reading them so they can warm up. While warming, samples should be covered to protect them from light. It often works out well to set out a batch of samples to warm up as you approach a station and to run them immediately upon leaving station.

5. Turn on the sea water tap in the sink and let it run while processing samples. The running water carries away the acetone and greatly reduces the amount of fumes in the van.

6. Using a clean cuvette filled with 90% acetone from the dispenser jug, read the blank values on all sensitivity levels, entering them in the 'Station Blanks' spaces. You are now ready to start reading samples.

7. Shake the sample gently. Rinse a clean cuvette (the same cuvette you used above for blanks) with a small amount of sample, then fill the cuvette with sample to within about 2 cm of the top. Place the sample in the sample well and install the cap. When the reading has stabilized record the value, Rb. Choose a sensitivity and range such the 6.9>Rb>2. Do not use level 100-4 unless absolutely necessary.. While waiting for the value to stabilize, remove the filter from the vial, dump the remaining sample, rinse the vial 2 times with 90% acetone, and place the clean vial inverted in the empty vial tray.

8. Lift the fluorometer cap, add 2 drops of 10% HCl to the top of the sample, and replace the cap. Wait for the value to stabilize and record the second value, Ra, using the same sensitivity levels as for the Rb value. Please be careful not to drip acid anywhere except into the sample cuvette. Acid running down the side of the cuvette into the fluorometer causes damage to the instrument.

9. Rinse the cuvette 2 times with small rinses of 90% acetone, and proceed to the next sample.

10. When finished with a sample set fill in the 'Read by' and Fluorometer I.D.' spaces on the data sheet.

11. If you run more than one station of samples at a time, run a second set of blanks at the end of the run, and note in the comments box which stations were run as a group.

12. When finished reading a set of samples, refill the rinsed empty vials with 90% acetone from the dispenser. Pay close attention to the action of the dispenser, it sometimes misfires and dispenses too small a volume. Replace the caps, and replace the vials in order in the sample tray.

13. Be sure you have turned off the Voltmeter, turned of the water, closed the window, and secured the refrigerator door and the stool with their bungees before leaving the van. Do not leave the van door hanging open.