Protocol for biogenic silica extraction
Modified by M-C Fortin, February 2007
Sarah Finkelstein and Brandi Podritske, September 2005
Use only plasticware for this analysis. Use only acids that came in plastic containers. No glass whatsoever can be used at any time during this analysis (including pipettes). Use only deionized (DI) water throughout the analysis.
This must be started 2 days prior to beginning silica extractions.
1. Select samples to be run (current set up allows 26 samples per batch, 1 replicate and 1 tester included).
2. Place approx. 0.5 cc into crucible (we have used 0.25 cc and have had enough sediment). Volume and weight do not need to be known.
3. Dry samples for at least 24 hours at 105 C.
4. Grind up samples individually using mortar and pestle until the sample is finely ground.
5. Using the electronic balance and weighing paper,
0.02 g (+/- 0.002 g) of each sample and transfer to labeled 125- ml flat-bottomed Nalgene bottle.
6. Record mass with sample codes and bottle numbers clearly in lab book. 7. Clean mortar and pestle between samples using kimwipe/DI water.
8. Always run at least one tester sample of known BSi concentration with a duplicate (we have four of these sent to us by Dan Conley - firstname.lastname@example.org).
9. Always run at least one set of duplicate samples within each batch. It is also preferable to run a sample from a previous batch.
Reagents to be made
Read MSDS for all chemicals. When weighing out molybdate and oxalic acid powders on the electronic balance (which is not situated in the fume hood and cannot be moved), wear a dust mask as inhaling these powders is dangerous.
1% anhydrous sodium carbonate Na2CO3
Dissolve 30 g Na2CO3 into 3 L DI water. Store in base cupboard.
REMEMBER, if you add to an existing volume of 1% Na2C03 or make a new batch of 1% Na2CO3, you need to replace the S0 by this newly made reagent.
We are presently using 1g/L stock solution. These can be stored long-term in the fridge.
S0: 1% Na2CO3
S1: 10 mg/L
S2: 20 mg/L
S3: 30 mg/L
S4: 40 mg/L
S5: 50 mg/L
S6: 60 mg/L
S7: 80 mg/L
S8: 100 ml/L
S9: 125 mg/L
S10: 150 mg/L
S11: 200 mg/L
Ammonium molybdate (make this in 2 L Nalgene stock bottle).
Dissolve 10 g into 1 L of 0.1 N H2SO4
To make 0.1 N H2SO4: 2.8 ml H2SO4 (conc) diluted to 1 L with DI water. Refrigerate. Can be kept indefinitely.
** This reagent must be shaken EXTREMELY well.**
Oxalic acid (make this in 2 L Nalgene stock bottle).
Dissolve 50 g into 1 L DI water. Shake well.
Refrigerate. Can be kept indefinitely.
Ascorbic acid (make this in 500 ml Nalgene stock bottle)
This reagent needs to be made up fresh every time the analysis is run.
For 26 samples, 150 ml ascorbic acid reagent is sufficient. To make this:
Combine 7.5 ml acetone and 142.5 ml DI water and dissolve in this 2.7 g ascorbic acid. Shake well.
It only keeps for 2 days – if you have to store it, do so in the fridge.
5 ml HCl (conc, in plastic container) diluted to 1 L with DI water. Remember: add acid to water, not the other way around.
Watch out for the molybdate reagent turning blue, indicating contamination. If this occurs, remake it. However you may only use a single batch of molybdate, oxalic acid or ascorbic acid reagents on a given day of analysis. Using another batch within a given day will invalidate the standards. Each batch is different and requires its own standardization.
1. Turn on water bath (heat only), and wait until temperature reaches approx. 85°C. If set to 87°C the water temperature stays constant at 85°C. This will take 3 hours.
2. Using the dispenser on stock bottle, add 40 ml 1% Na2CO3 to each sample bottle.
3. Cap bottles and place in water bath (place elastics around samples in groups of four-this keeps them from tipping over).
4. Turn on shaker. Record time.
5. After 2, 3, 4 and 5 hours, remove the samples and place them in an ice water bath. Follow these steps:
i. Transfer 1 ml of each sample using the micropipette, to clean labeled 15-ml graduated Nalgene centrifuge tube.
ii. Neutralize each sample with 3.2 ml 0.06N HCl (use dispenser on stock bottle)
iii. Dilute each sample with 10 ml DI water (use dispenser on stock bottle)
iv. Mix each sample well by inverting tubes 3 times.
6. Keep water bath hot while you are processing subsamples by placing the lid back on. Once samples are back in the water bath, start timing for the next hour. Make sure temperature stays around 85°C.
7. At some point during the extraction, follow steps i-iv above for each of the 12 standards (S0 through to S11). Use 1 ml standard in place of 1 ml sample.
8. Store all centrifuge tubes in the fridge for measurements on the spectrophotometer the following day. Samples cannot be left longer than 1 day.
It is CRITICAL to maintain stock bottles UPRIGHT throughout the ENTIRE experiment to avoid losing sediment on the inner walls of the bottles.
1. For each standard and sample, follow these steps:
Use micropipette to transfer to cuvette:
i. 0.64 ml sample/standard
ii. 0.84 NH4 molybdate reagent
iii. 0.64 oxalic acid reagent
iv. 0.84 ascorbic acid
** It is crucial to shake the reagents vigorously in between each group of samples you are preparing and to stir reagents with the pipette tip before pipetting.**
** Also, invert each tube at least twice before removing the sub-sample.**
6. Start with S0. This should read zero in the spectrophotometer.
7. Continue with the other standards then move onto samples. Readings for the standards should make sense – e.g. upward progression with increasing Si concentration
8. Measure and record absorbance at 660 nm.
9. Wash all dishes and rinse with DI water.
** Note about pipetting. Practise beforehand if you have not done this before. Even small air bubbles or an unsteady intake will affect the analysis. The analysis is very sensitive and requires exact volumes of reagents and sample. Hold pipette steady in an upright position. Make sure you have the right tips.
1. Compute [SiO2] for each subsample using calibration of Si standards.
i. Calculate the regression equation (slope + intercept) using a least squares regression of [SiO2} of each of the standards (in mg/L) (independent variable, x) vs. Abs660 (dependent variable, y). Do two regressions, one for low- and one for high-range.
ii. Use the slope and intercept determined from the equations to calculate [SiO2] for each of the Si standards using the formula:
[SiO2] = (Abs660-intercept)/slope
iii. Use the appropriate regression equation to calculate the [SiO2] for each of the subsamples (2, 3, 4 and 5 hour) from Abs660 readings. The cut-off point is the mid-point in Abs660 between S5 and S6.
** Excel functions SLOPE and INTERCEPT can be used for this.
2. Determine wt% (bBSi/100g dry sediment) in each sample:
i. Compute wt% BSi for each sample (after 2, 3, 4 and 5 hours) using this formula:
Wt% = [SiO2(mg/L)]/25/(wt dry sediment extracted(g)*10)
This formula is derived based on an extraction with 40 ml Na2CO3 and converting to wt% (mass of BSi per 100 g of dry sediment).
ii. Do a regression of wt%SiO2 vs time and compute the y-intercept which represents the wt% BSi of the sample.
3. Look at your results. Is the tester sample within one SD of the mean value reported in Conley (1998)?