Method for the Standard Density Fractionation of Soil
See Excel file “DensityFractionationWkst” for data entry sheets.
This method has been developed for soils with 0.3 to 5% C content. If you have a soil organic
matter content over 5% or less than 0.3%, you will need to experiment with the soil starting
mass.
PART 1: Pre-Fractionation steps (To be done prior to Part II to V)
1. Label the 50 mL plastic centrifuge tubes 1 to 16 (future runs are labeled 17 to 32 and 33 to
48). Weigh 4 to 6 g of soil to five decimal places into each tube. Record sample id along
with sample mass on the “Density Fractionation Worksheet” (see example worksheet, Table
1).
a. Rule of thumb: 4 g sample for 2 to 5 % soil organic C content; 5 g sample for 1 to
2% soil organic C content; and 6 g sample for <1% soil organic C content. You will
need to experiment with the starting mass to make best use of this method for your
samples.
b. Try to weigh sample as close to 4, 5, or 6 g. Duration of sonication (in min.sec) is
calculated using these exact weights.
2. Each run of 16 samples requires 48 tins (three sets of tins, one set for each fraction; see
example worksheet, Table 1). Make sure that the tins are weighed to 5 decimal places.
3. Check the density of the 1.800 g/cm3 NaI solution (hereon referred to as NaI).
PART II: Pre-Fractionation steps
1. Double check the density of NaI.
2. Gently spray soil sample with NaI solution (in spray bottle) to get soil to approximately field
capacity. Pre-wetting soil insures that aggregates will not be broken open by escaping air
from pores. This aggregate breakdown occurs when dry soil is placed in an aqueous
solution.
3. Once samples are at field-capacity, slowly add NaI to the 35 to 37 mL mark on the
centrifuge tube.
4. Gently tip tube up and down until the soil sample is in solution. Once the soil is in solution,
gently tip tube up and down three additional times.
5. Run samples for 15 minutes on Stahl’s centrifuge (Lab 1) at the 40 mark or at 75% max
speed (This probably corresponds to 2,000 rpm). When placing samples in the centrifuge,
match tubes with similar soil masses and total volumes of solution.
6. While centrifuge is running:
a. set-up collection apparatus ( Table 2, 3) and turn on vacuum;
b. organize tins;
c. turn on ovens to 65 degrees C;
d. fill a large wash bin with ice;
e. set-up lab lift and stir plate in the sound proof Sonicator box;
f. power-up Sonicator and enter in Preset number based on mass of sample (Table 4).
7. Once the centrifuge has finished, remove the tubes and begin fractionating.
Page 1 of 9
PART III: Collecting the Free Light Fraction (F)
1. The inside of the centrifuge tubes may need to be gently sprayed with NaI (in the spray
bottle) to rinse organic materials into the NaI.
2. Gently tilt tube 1 and, using the aspirator, remove the NaI along with any material which is
floating on or suspended in the NaI. Gently rotate the bottle once while collecting any
material round the side of the bottle. Take care not to collect the heavy fraction as it resuspends in the NaI. You do not have to vacuum all of the NaI in the bottle, just enough
to remove the F.
3. Transfer vacuum to the rinse flask. Then transfer the filtration apparatus (with the filter in
place) to the rinse flask.
4. Pour NaI from the NaI collection flask back into its corresponding centrifuge tube. Add
NaI to the 37 mL mark on the tube and screw cap back on. Shake tube to break-up the soil
pellet. Place tube in the bin of ice.
5. With the vacuum on, place aspirator in a beaker of DI water to rinse aspirator. Wash out the
apparatus with DI water to remove all organic matter and pour this water through the filter.
Wash the organic matter down the sides of the reservoir onto the filter paper. Once all of
the F is collected on the filter, leave the vacuum on and rinse the excess NaI off the F and
filter with a lot of DI water. Rinse F at least 3x with DI.
6. Remove the vacuum from the rinse flask. Remove the reservoir and rinse all of the F off of
the filter into the appropriate tin using a DI squirt bottle.
7. Once the F is collected, rinse the filter with DI water. This filter is to be reused throughout
the fractionation.
8. Rinse the NaI collection flask with DI water and dry the flask. If needed, discard the water
in the Rinse flask. Dry the filter holder, reservoir, and rubber stopper with Kimwipes and
reassemble with the NaI collecting flask and the filter. Drying is necessary so that you do
not dilute the NaI collected from the next sample.
9. You may need to rerun Part II (5-7) and Part III (1-8) if there is a lot of F.
10. Repeat steps 1-8 for the remaining samples.
PART IV: Collecting the Occluded (Intra-Aggregate) Light Fraction (O)
1. Place the centrifuge tube in a 4 cup Pyrex glass measuring cup. Pack ice around the tube,
add some water to the ice and take off the centrifuge lid making sure no ice/ice water falls
into bottle.
2. Add a small stir bar to the sample.
3. Place the glass measuring cup containing the sample on top of stir plate. Turn on the stir
plate to setting 1.5.
4. Raise the soil sample (using the lab lift) to the mark on the sonicator probe, so that
approximately 15 mm of the sonicator probe tip is in the soil solution. Close the soundproof door and begin sonication (the sonicator settings were determined in Part II, Step 6).
a. At the end of the day, record time and number of samples run. Sonicator needs
to be calibrated every 300 minutes of use.
b. Sonication time is significantly reduced when the samples are cooled to about 8
degrees C prior to running them.
5. When sonicating has finished, remove the probe from the bottles. Use a small amount of
NaI to remove any soil from the probe.
Page 2 of 9
6.
7.
8.
9.
10.
11.
a. Once all of the samples have been sonicated, rinse the probe with DI water and
gently dab with Kim-wipes, making sure not to scratch probe.
Repeat steps 1 to 5 for remaining samples. Replenish ice as needed.
a. To maximize your time, you should collect F (Part III, Steps 1 to 8) while
sonicating (Part IV, Steps 1 to 5).
Run samples for 15 minutes on Stahl’s centrifuge (Lab 1) at the 40 mark or 75% max speed
(This probably corresponds to 2,000 rpm). When placing samples in the centrifuge, match
tubes with similar soil masses and total volumes of solution.
When the centrifuge finishes, remove the tubes and begin fractionating.
Remove O as for the F, but this time collect the used NaI for recycling.
Dry the fractions at 65 degrees C (usually overnight, 12 hours, is suffice for F and O). Once
dry, record their weights to 5 decimal places on “Density Fractionation Worksheet” (see
example worksheet, Table 1).
Store the fractions in glass vials and re-dry just prior to milling (ideally, grind the soils just
after recording their weight).
PART V: Rinsing the Organomineral Fraction (OM)
Once both the F and O are removed from the sample, the remaining pellet is the OM. The goal
of PART V is to remove the NaI from the OM by rinsing the OM 3x with DI.
1. To each centrifuge tube, add DI to the 40 mL mark on the tube. Shake tube to break-up the
OM pellet and to mix well.
2. Run samples for 20 minutes on Stahl’s centrifuge (Lab 1) at the 40 mark.* When the
centrifuge finishes, remove the tubes and pour out DI water (careful not to discard any
sample).
3. To each centrifuge tube, add DI to the 30 mL mark on the tube. Shake tube to break-up the
OM pellet and to mix well.
4. Place the tubes horizontally on the shaker and shake on low for 30 minutes.
5. Once the 30 minutes has passed, run the samples for 40 minutes on Stahl’s centrifuge (Lab
1) at the 40 mark.* When the centrifuge finishes, remove the tubes and pour out DI water
(careful not to discard any sample).
6. Repeat Step 3 and then run samples for 60 minutes (or longer depending on the amount of
suspended clays) on Stahl’s centrifuge (Lab 1) at the 40 mark.* When the centrifuge finishes,
remove the tubes and pour out DI water (careful not to discard any sample).
7. Wash OM into a tin using as little water as possible, followed by drying in a 65 C oven for at
least 12 hours.
8. Once OM is dry, grind using the Retsch Grinder.
*This probably corresponds to 2,000 rpm. When placing samples in the centrifuge, match tubes
with similar soil masses and total volumes of solution.
Page 3 of 9
Part VI: Making NaI
1. Place a 1000 mL graduated cylinder on a stir plate. Secure cylinder with clamp to a stand.
2. Add 700 mL of DI H2O to the cylinder.
3. Gradually add approximately 1050 g of NaI to the water while water is being stirred by stir
bar to reach a density of 1.800 g/cm3.
Part VII: Recycling NaI
1. Filter the used NaI through the Whatman GF/A Glass Fiber Filters to remove any clay
from the NaI.
2. Place 1 L of NaI in a beaker or flask. Add approximately 20 g of activated charcoal and
approximately 15 g of NaI.
3. Stir on a magnetic stirrer for at least 35 minutes.
4. Filter the NaI slurry through a Whatman 2V folded filter.
5. Store the recycled NaI in a glass jar with a secure screw cap. NaI is light sensitive, so either
use an amber glass jar or wrap clear glass with Al-foil.
Page 4 of 9
4. Part VIII: Citations
This method is largely based on Sohi’s 2006 online method
(http://www.rothamsted.ac.uk/CarbonCylcing/pdf/fractionation_manual.pdf)
AND
Rasmussen, C., M.S. Torn, R.J. Southard. 2005. Mineral Assemblage and Aggregates Control
Carbon Dynamics in a California Conifer Forest. SSSAJ, 69: 1711-1721.
This method was modified with comments from
Sohi, S.P., N. Mahieu, J.R.M. Arah, D.S. Powlson, B. Madari, J.L. Gaunt. 2001. A Procedure
for Isolating Soil Organic Matter Fractions Suitable for Modeling. SSSAJ, 65:1121-1128.
AND
Sohi, S.P., N. Mahieu, D.S. Powlson, B. Madari, R.H. Smittenberg, J.L. Gaunt. 2005.
Investigating the Chemical Characteristics of Soil Organic Matter Fractions Suitable for
Modeling. SSSAJ, 69:1248-1255.
AND
Horwath Lab, UC Davis. SOP for Density Fractionation of Particulate Organic Matter.
Part IV: Selected differences between our methods and others
Horwath Lab Method:
Uses a 20um filter for faster filtration (he says the 2.5um is very slow)
Rasmussen et al ’05:
Centrifuged samples for 10 min at 1180 g (he used 1.6g/cm3 SPT solution)
Filter paper: 0.8um polycarbonate filters
Repeated the removal of the FLF 3x (shaking, centrifuging, filtering)
Ultrasonic probe tip was inserted 5-cm below the liquid surface during disruption (probe
diameter not given, but used a 450 like ours)
Sohi et al. “01
Centrifuged samples for 30 min at 8000 g.
Used 6 mm diameter tubing.
GF/A 47 mm diameter, 1.6 um retention
9.5 mm Ultrasonic probe tip inserted 15 mm into the soil suspension
Sohi et al. ‘05
42 mm filter paper diameter with 5-um retention
Page 5 of 9
Table 1. Example Worksheet for Density Fractionation.
Page 6 of 9
Table 2. Inventory of Items needed for Density Fractionation Procedure.
Items needed to run OM fractionation:
Chemicals
Quantity
2
1+
Unit Price
Sodium Iodide( 3 kg) ACS Grade 99.5% minimum by
anhydrous basis (VWR chemical)
*this amount should give us enough soln for 35 samples at
90mL per sample (6000g X 1cm3/1.83g X mL/cm3 X sample/90mL)
**Sodium polytungstate is twice as expensive
***There are several NaI grades to choose from. The one
above seemed the best for price and quality
Activated Charcoal; norit CA1 powder
Total Price Notes:
VWR: BDH0294-2.5KG
Sigma Aldrich: C3345
ordered by Linda at the RMMC (766.3696)
Vacuum Filtration Set-up
Quantity
48+
3
1
1
2
1-2
1
4
1
4
1
1
1+
1
Description
50 mL Fisherbrand centrifuge tubes PP FLTP w/ racks
1 L glass Buckner side arm flasks
Magnetic filter holder and reservoir (primary)
Millipore Sterifil 47mm Aseptic Vacuum Filter System & Holder (backup)
Millipore System is back -up for the Magnetic filter holder
250 mL glass beakers
Aspirator: 1 mL pipette tip attached to Nalgene plastic and then glass tubing.
Glass tubing is fit through a rubber stopper (size 13) that fits reservoir.
Nalgene tubing
Nalgene stopcock PP/TFE (size 4 mm)
3-prong clamp
Stirbar, Octagonal1 1/2 x 5/16"
Spray Bottle
Wash Bottle LDPE 500 mL
2 L Glass Flask or Beaker
Al Weigh Dishes (ID=2.64")
Nylon Net Filter 20um 47mm 100/P (for OM collection)
Pyrex 4 cup measuring cup
Page 7 of 9
Unit Price Total Price Notes:
500 in a case
Fisher: 05 539 8
Each/$82.00
$82.00
Stockroom
Stockroom: Nalgene # 6460-0004
Stockroom: Fisher # 5-769-7Q
Fisher: 14 513 60
Ace Hardware
Fisher: 03 409 10CC
Each
6/pk
100/$43.04
100/$109
PALL
Fisher: XX11-047-00
$43.04
Fisher: 08-732-103
Millipore NY2004700
Bed, Bath and Beyond
Table 3. Inventory of Items needed for Density Fractionation Procedure.
NaI solution: making stock solution and collection of, storage of & recycling solution
Quantity
1
1
1-2
1
1-2
1
1-2
1
1
1
Description
VWR specific gravity hydrometer (1.500 to 2.000)
Tuff-cote evoh safety coated bottles, graduated; case of 4
Al Foil to wrap clear glass bottles
1L glass flask
VWR 1000 mL Glass vol. cylinder, BMP, SC
100 mL volumetric measuring cylinder
Spinbar, OCT PTFE 7/8x3/8in
Large plastic funnel
Whatman 2V folded filters (24 cm dia); pack of 100 (for NaI Recycling)
Glass Microfiber Filters, Whatman GF/A, 4.7 cm (for clay/silt removal)
Millipore Membrane Filter Forcep
Unit Price
Total Price Notes:
Each/$25.78
$25.78
VWR: 346-22-250
Case/$230.95
$230.95 VWR: KT321000-2000
VWR: 89000-260
VWR: 58947-124
Pack/$77.43
100/$31.13
Each/$12.00
$77.43
$31.13
$24.00
Fisher: 09-832D
Fisher: 09-874-14A
Fisher: XX62-000-06
Sonication Set-up and Calibration
Quantity
1
1
1
1
Description
Digital Sonifier 450 with converter and horn (see Sonifier file)
Omega Temperature Probe for Sonicator
Fisher Stnd Lab Jack 6x6"
Stir Plate
Flask Shallow Dewar Cyl, Pope Scientific, 350 mL
Unit Price
Each/$70.00
Total Price Notes:
Branson Ultrasonics Corporation
$70.00
Omega.com
Fisher: S63081
VWR: 63884-971
Sample Grinding
Quantity
1
1
2
2
1
Description
Coors Mortar_50mL
Coors Pestle for s33761-1cr
Dia-cut Stirrer for glass rods (3/16in) pack of 12
Glass Stir Rods, Pack of 10 (5x200mm)
Retsch Grinder with agate mortar and pestle :)
Unit Price
~$12/each
~$12/each
Pack/$2.09
Page 8 of 9
Total Price Notes:
Fisher: s337611cr
Fisher: s337611crm
VWR: 53801-008
$2.09
VWR: WLA5730-A
Table 4. Time of Sonication based on Sonicator Displayed Energy and Measured Total Energy.
Based on Sonicator Calibration Table, the Sonicator energy display at 74 - 75 W corresponds to an actual energy output of 82 W.
To get the Sonicator energy display at 74-75W while running density soln samples for the Fractionation procedure, you need to input
an amplitude of 63 to 64 %.
This input amplitude may vary, so you may need to play around with this input to get the correct displayed energy in W.
Amplitude
%
63-64
63-64
63-64
63-64
Displayed Energy
W
74-75
74-75
74-75
74-75
Actual Energy
W
82
82
82
82
Time
s
55
73
91
110
Sonication Time
min.s
55
1.13
1.31
1.50
Page 9 of 9
J
4500
6000
7500
9000
J/g
1500
1500
1500
1500
Soil
g
3
4
5
6
Sonicator
Preset
1
2
3
4