UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Biogenic Silica (BSi) Analysis

Modified Mortlock and Froelich procedure
Purpose

To determine the percent biogenic silica in sediments using sodium hydroxide dissolution of silica and
molybdate blue spectrophotometry.

Principle

A freeze dried sediment sample is heated in sodium carbonate to dissolve the silica in the sample. The
dissolved silica comes from biogenic sources, i.e. diatoms, and non-biogenic sources, i.e. clays. One
timed aliquot is extracted from the sodium carbonate and dyed molybdate blue. Spectrophotography is
used to determine the samples absorption at 812 nm. The absorption percent is then used to calculate
the percent biogenic silica in the sample.
A more accurate measurement is made if multiple timed aliquots are removed from the sodium carbonate
solution, allowing for differentiation of rapidly soluble and slowly soluble silica.

Safety Precautions

When preparing reagents, take care to avoid spills, skin contact, eye contact, inhalation, or ingestion.

You will be working with strong acids and bases. Use protective gear, such as safety goggles, vinyl
gloves, lab coat, closed-toe footwear, long pants, etc.

When handling strong acids you must work in the fume hood.

During sample digestions, the water bath will be hot and will contain hot liquid. The digestion tubes will
contain hot Na2CO3. Wear protective eyewear and latex gloves.

Label and date everything! Do not pour reagents from their original bottles into unlabeled or mislabeled
bottles. Reagents prepared by you must be stored in properly labeled containers. Label water bottles
appropriately.

Notes

When using the microliter pipette, it is critical that the amount of sample you draw into the pipette and the
amount expelled from the pipette is always exactly the same. Pay attention to the level of liquid in the tip,
and start over if the amount of liquid doesn’t look quite right or if bubbles are present. Problems
commonly arise from improperly seating the tip on the pipetter and from failing to depress the plunger
completely.

Use polyethelene products unless otherwise noted. Use of glass may contaminate the samples.

Have all materials needed pre-labeled and set up ahead of time prior to beginning the digestions.

Use silica free water for all mixtures (“18.2 ΜΩ” water from the recirculating water purifier). This is often
referred to as ddH2O.

Always add the sample to the Molybdate (never the reverse). This will help to keep the mixture above a
certain pH.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Getting ready

1. Analyze 54 samples, 4 standards and 2 blanks simultaneously (60 50-mL tubes). About 5 samples
should be duplicates. 1 cm3 samples should provide plenty of samples for duplicate runs. When you
are proficient with the method, increase the number of samples to 80.
2. Record your observations (e.g., possible loss of sediment, overflow of solution, etc.) in the “Biogenic
Silica” notebook or your sample list. Enter data in spreadsheet as collected, and back up
spreadsheet onto the Paleo server daily.
3. Unless you have already measured sediment bulk density, BSi analysis should begin with volumetric
samples (e.g., 0.5 or 1.0 ml) for bulk-density calculation. Bulk density data are necessary for
calculating accumulation rates.

Part 1. Sample drying and weighing

1. Sample 1 cm3 of sediment and place in a ½ dram shell vial. Cover shell vial in tissue paper held on
with rubber band. To freeze dry without a freeze-dryer: place in automatically defrosting freezer for
several weeks. If samples are not completely dry, finish drying in a drying oven at 40°C. To freeze
dry with a freeze dryer: Put a small hole pushed through the plastic cap. Freeze the sediment
samples, and then freeze dry for at least 24 hours, until completely dry.
2. Gently grind sample within shell vial to mix sample.
3. Weigh total amount of sediment to determine bulk density of your sample (g/cm3). (Note: If samples
are stored in a cold room, they should be taken out 24 hours prior to extraction to equilibrate to room
temperature. It's usually helpful to weigh the samples the day before. Zero the scale and write down
the weight until the reading is stable. If weight continuously increases, it is absorbing moisture, and
the lowest weight should be used).
4. Weigh the appropriate amount (typically 0.06 ± 0.01 g) of 44 samples into auto-analyzer vial caps.
Make sure to add duplicates as shown in the spreadsheet. Do not touch the cap with your fingers,
use tweezers so that the oil from your fingers does not change the weight of the cap. For standards
(Eleanor Lake standard, ca. 40% BSi), use 40 mg. Note: scale is in units of grams, so 40 mg = 0.040
grams. Record core, depth, weight, and sample number (1-60) in the biogenic silica spreadsheet. It
is handy to mark the cap of the vial with a dot for each time you take a sample from it so that you
know which samples have been duplicated. This step should not be done more than a day or two in
advance. When done in advance, weighed samples must be kept in a desicator. Keep remainder of
sediment in ½ dram vials with caps for storage.
5. Label 50-ml test tubes 1-60 (or however many samples are run). Tubes should be labeled on caps as
well. Place samples in empty 50 ml test tubes, then grind gently with a glass stir rod to break up
clumps. Wipe stir rod with a dry kim-wipe between samples. Note: be sure to not lose any sample on
the stir rod.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Part 2. Extracting biogenic silica from bulk sediment

Sodium carbonate filled digestion tubes Samples

Hot water bath 50 mL glass repipet

NOTE: This is a 6-hour procedure, with lots of free time available between tasks. Plan your day
accordingly.
NOTE: Timing is very important. When loading samples or extracting aliquots you need to spend the
same amount of time on each sample. Use a timer to keep track of elapsed time.
NOTE: If samples are high in carbonate, dissolve carbonates in 10% HCl, centrifuge at highest setting
possible (5300 rpm) for 15 minutes, and fully decant liquid.

1. Turn on the water bath (85° C) and allow to warm up to 85°C. (about 1 hour).
2. Start timer (or note time on accurate clock). Add exactly 40.0 mL of 10% Na2CO3 solution using the
50 ml glass Repipet to the first 20 tubes. Work fairly quickly but accurately. (Be sure air intake is
uncapped on the side of the glass repipet). Cap tubes and shakes vigorously (be sure to shake all the
test tubes the same amount of time). Unscrew the caps on the test tubes just enough so air can
escape during the dissolving process, and place tubes in the racks inside water bath at 85° C.
3. At 15 minutes, add 10% Na2CO3 solution to tubes 21-40. Repeat as in step 2. At 30 minutes, add
10% Na2CO3 solution to tubes 41-60. Etc. etc. etc.
4. After 100 minutes, screw on cap tightly and shake test tubes 1-20. Uncap loosely and place back in
water bath. At 115 mins repeat this step for test tubes 21-40. At 130 mins repeat his step for test
tubes 41-60, etc.
5. After 200 minutes, screw on cap tightly and shake test tubes 1-20. Uncap loosely and place back in
water bath. At 215 mins repeat this step for test tubes 21-40. At 230 mins repeat his step for test
tubes 41-60, etc.
6. At 300 minutes, remove samples 1-20 from bath, cap tightly. Centrifuge for 10 minutes at highest
setting.
7. At 315 minutes, remove samples 21-40 from bath, cap tightly. Centrifuge for 10 minutes at highest
setting.
8. While samples 21-40 are centrifuging, use a plastic syringe (we have 80+ of them) to remove ca. 20
mL from each 50 mL tube and add this sample to a 20 mL (labeled) scintillation vial. Work relatively
quickly through the samples, because you have about 10 mins to do this step.
9. At 330 minutes, remove samples 41-60 from bath, cap tightly. Centrifuge for 10 minutes at highest
setting.
10. Repeat removing 20 mL from each 50 mL tube after centrifuging until all are done.
11. Store 20 mL scintillation vials in cold room.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Part 3: Preparing samples for determination of BSi concentrations

30-mL reaction bottles (n=202).

Molybdate working solution in 4L plastic bottle
Reducing working solution in 2L plastic bottle
200 μL pipetter
50 mL glass re-pipetter
10 mL pipetter (Oxford Benchmate)

NOTE: Check the solutions needed. Make new solution if needed (refer to end of the protocol).
NOTE: When using the microliter pipette, it is critical that the amount of sample you draw into the pipette
and the amount expelled from the pipette is always exactly the same. Pay attention to the level of liquid in
the tip, and start over if the amount of liquid doesn’t look quite right or if bubbles are present. Problems
commonly arise from improperly seating the tip on the pipette and from failing to depress the plunger
completely, or depressing it too far.
1. Arrange 80 of the 30 mL reaction bottles in trays as done for 5 mL vials. Bottles do not need to be
labeled if only one bottle is removed from the tray at a time. Uncap bottles.
2. Dispense 17.5 mL of molybdate working solution in each reaction vessel (i.e., 30 ml plastic bottle).
Use 50 mL glass pipetter.
3. At 30-second intervals, add 125 μL of sample (or standard or blank) using the 200 μL pipetter. Swirl
reaction vessel to mix solution inside. Do not reuse pipet tips. If working alone, it is best to only do 40
samples at a time.
4. Cap vessel and allow reaction to continue for 20 minutes.
5. At 30-second intervals, add 7.5 ml of reducing working solution to reaction vessel using glass pipette
tips. Cap and mix the vessel (note: do not let the solution touch cap).
6. Allow reaction to continue overnight (over 12 hours).

Note: This part can be completed right after Part 2.

Part 4: Measuring absorbance with a spectrophotometer

NOTE: Cuvettes are delicate and fairly expensive. Handle gently and do not insert anything but kim-
wipes and cut sponges within the cuvette to clean it. Use only lens cleaning paper or fine cloth to wipe the
optical surfaces, most paper products contain wood fibres which may scratch or damage the cell face or
surface.
19. Turn on the power switch of the spectrophotometer (GENESYS 8). Allow to warm up for at least 5
minutes. Display should read “Abs”
20. Using glass cuvettes (hold only on frosted sides), fill one with ddH2O and cap.
21. Place in spectrophotometer. Press “Zero”
22. Fill four remaining glass cuvettes using glass pipettes with bulb. Cap, take readings, and enter in
notebook. Rinse pipettes as needed (we have ~40 of them). Note: Some days the spectrophotometer
is more finicky than others. To make sure that the zero has not drifted between sample readings,
always check the zero (cuvette with ddH2O) between each sample reading.
23. Empty cuvettes into beaker, and when done pour into labeled waste container.
24. Rinse cuvette using squirt bottle of ddH2O, be sure exterior is clean. Dry inside of cuvette gently with
compressed air. Fill next four samples.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

• Input data into Excel spreadsheet and plot the data. If you have multiple batches, adjust the
sample values according to standards.
• Clean up utilities used (tubes, racks, bottles, cuvettes, graduated cylinders, repipets etc.)
• Check all the chemicals and refill or order new items if necessary.
• After spectrophotometer readings are entered and backed-up, discard reagents (working
solutions) into a labeled container. Have EHS take away. Clean out reaction bottles. Save
the 1 mL extractions (5 mL vials) in the cold room until several batches from same are run and
the data appears robust.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Preparing stock solutions

Measure large volumes of water using tall graduated cylinder. Read off of the bottom of the
meniscus.
Wear safety glasses, gloves, lab coat, closed-toe footwear.

Stock solution

1. Molybdate reagent
• Dissolve 16.0 g of ammonium paramolybdate – (NH4)6Mo7O24 • H2O in 1000 mL of ddH2O.
• Store solution in a tightly capped polyethylene bottle out of direct sunlight. The solution is
stable indefinitely, but should be discarded if it forms a white precipitate or turns faintly blue.

2. Metol-sulfite reagent
• 6 g of Na2SO3 (sodium sulfite) in 500 mL of ddH2O.
• Add 10 g of metol (4-(Methylamino) phenol sulfate)
• Dissolve everything
• Filtering through no.1 whatman filter paper
• Store in an amber-glass stoppered bottle
• Stable for 1 – 2months

3. Oxalic acid reagent

• Dissolve 60 g of oxalic acid dihydrate – (COOH)2 • 2H2O in 1000 ml of ddH2O
• Store in polyethlene. The solution is stable indefinitely

4. Sulfuric acid reagent

• Slowly add 300 ml of concentrated sulfuric acid into 770 ml of ddH2O. Work in the fume
hood.
• Cool to room temperature and store in polyethylene

5. Hydrochloric acid (for Molybdate Working solution)

• Add 100 mL of concentrated (12N) HCl to 1100 mL of ddH2O à1200 ml 1N HCl
Work in the fume hood.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Preparing working solutions from stock solutions

Estimate the amount of solution needed.

Molybdate working solution: 17.5 mL/sample. So, 3500 mL for 200 samples.
Reducing working solution: 7.5 mL/sample (so, 1500 mL for 200 samples).

1. Molybdate Working Solution :

Molybdate Stock Solution : ddH2O : HCl (HCl stock) = 1 : 5 : 1 (Add in order listed)

To make 3500 mL of molybdate working solution in a 4 L plastic bottle:

a. Add 500 mL Molybdate Stock Solution
b. Add 2500 mL of ddH2O (measure in 1 L volumetric flask and 250 mL graduated cylinder.
c. Add 500 mL of HCl stock solution.

To use solution, pour in increments into small plastic bottle (use a funnel) in the fume hood.
Use pipetter out of small bottle.

2. Reducing Working Solution

Metol-Sulfate Stock Reagent : Oxalic Acid : Sulfuric Acid Stock = 1 : 1 : 1
(Add in order listed)

To make 1500 mL of reducing working solution in a 2 L plastic bottle:

a. add 500 mL Metol-Sulfate Stock Reagent
b. add 500 mL Oxalic Acid
c. add 500 mL Sulfuric Acid Stock

Materials for Biogenic Silica

• Sodium carbonate solution 10% W/V (weight by volume). Fisher Catalog 7160-32. Order in
10L volumes via Clarice in Chem Stores.
• 30 mL bottles (we have a large supply, please re-use. Fisher 03-700-30.)
• 20 ml scintillation vials. Fisher 03-337-24B
• 5 mL snap cap vials in trays. USA Scientific. Part 1505-7005. 5 trays of 200 have been
ordered. Please re-use.
• ½ dram glass shell vials. Fisher 03-339-26A
• Plastic vial cartons (holds 100 ½ dram vials). USA Scientific.

Dry chemicals:
• 4-(methylamino) phenol hemisulfate salt 99% ACS reagent (100 g quantities is sufficient)
• Ammonium molybdate (para) tetrahydrate (NH4)6Mo7O24 • 4H2O (99%)
(250 g quantities is sufficient, order from www.alfa.com, item A13766)
Note that Ammonium molybdate (para) hydrate puratronic (NH4)6Mo7O24 • H2O was used in
recent past (2008-2010). This is much more expensive (highly pure, >99.99%) and has a
different molecular weight. In 2011, switching to tetrahydrate form (less pure, as well).
• Sodium sulfite anyhydrous granular (500 g quantities is sufficient)
• Oxalic acid (H2C2O2•2H2O) crystals (500 g quantities is sufficient)

Mixing 10% sodium carbonate solution (10% weight/volume).

Mix cold…it may take an hour or more to dissolve.

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UO Paleoecology Lab Biogenic Silica SOP 25 April 2019

Divide total volume desired by 9. This is the weight in grams of Na2CO3 crystals to use.

To make 4 liters:
4000/9=444.4 grams.

Clean carboy by rinsing with ddH2O.

Add 4 L of ddH2O
Add 444.4 grams of Na2CO3 crystals. Mix until dissolved.

Waste disposal

Store Na2CO3 extracts in refrigerator until project is done (and published).

Pour extra Na2CO3 down drain with abundant tap water

Reaction vessel chemicals should be poured into large waste bottles. (Large empty bottles can
be found under the sink in PAC on the left hand side. Be sure to label what wast is being
placed into the container. Obtain empty waste bottles from environmental health and safety
services if needed).

Hazardous waste tag:

Ammonium paramolybdate: 0.2%
Hydrochloric acid: 1%
Sodium sulfite: 0.1%
Oxalic acid: 0.6%
Sulfuric acid: 2.8%
metol (4-(Methylamino) phenol sulfate: 0.3%

back side of tag: Check corrosive acid