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Agricultural Service Laboratory
Clemson University
Soil Analysis Procedures

April 28, 2000

TABLE OF CONTENTS

Sample Preparation
Lab Numbers
Sample Check-In
Sample Extraction -  Mehlich 1
Soil pH
Buffer pH
Sulfur (Sulfates)
Saturated Extract (SE)
Nitrate-Nitrogen ISE
Nitrate-Nitrogen Cd. Rd.
Organic Matter
Soluble Salts
Quality Control for Soil Testing
References

Sample Preparation

1. Arrange samples by date received, county and grower in sets of 100.

2. Assign a 7 digit lab number to each sample. The 2 left hand digits correspond to
the month. The remaining 5 digits are assigned consecutively within the month as the
samples are arranged in the sets. The 3 right hand digits designate the
position of the sample within the set of 100 samples.

3. Place samples into the set. Sample information on the boxes and record sheets
should be compared for agreement. Any special analyses should be noted by lab number on
a separate sheet of paper as well as other special requests.

4. Samples received in a damaged condition, missing samples, or duplicated sample
numbers should be annotated on the record sheet. Samples received without paperwork
should have a record sheet completed from information on the sample box if possible.

5. Within each set, place Check sample boxes at approximately positions 40, 60,
and 80 and note the position number shown on a blue Check soil record sheet.

6. After a complete set is arranged, box tops should be removed with the exception
of 1 flap of the first sample in the set. The date received and lab number of the
first sample should be written on this flap.

7. Place samples in drying racks and allow to dry.

8. After drying, crush and screen samples through a 10 mesh screen. Collect screened
samples and return to the original box. Samples are now ready for analysis.

9. Following analysis, place samples into storage racks for 1 month from the date of
analysis to allow for recheck if necessary. After that time, samples may be discarded.

10. Soil from Marion and Horry must be treated by heating at 121oC for 2 hr.


Stamping Papers with Lab Numbers

1. Collect papers and start stamping with beginning lab number.

2. Write beginning lab number on raw data sheet. Note position of checks and blank on
raw data sheet.

3. Be careful to check for any special analyses. Note the special analyses with a
check mark on the raw data sheet.

4. Make labels for pH and extracted samples.

5. Turn papers in to data processing.


Sample Check-In

Sample order must be rechecked prior to any analyses.

1. Retrieve papers for individual sets from data processing with lab numbers already
assigned and stamped.

2. Check through each sheet to make sure samples are in the proper order in the
trays.

3. Return sheets to data processing.


Sample Extraction Procedure (Mehlich 1)

After samples are checked for proper order, labeled with Check and Blank locations
noted, samples are ready for extraction.

1. Using a 4 mL volumetric scoop (assume 5 g), measure an amount of soil from each
sample box using the following method:

1) Dip scoop with sweeping motion and fill to overflowing

2) Hold scoop over box and firmly tap handle three times to settle

3) Strike off excess soil with leveling rod and transfer

2. Measure samples into extraction racks containing 10 polyethylene cups each. A
Check sample is scooped at the appropriate location from a separate Check sample box or
no sample is scooped at the blank location.

3. Extract fifty samples at a time. Add twenty milliliters of Mehlich 1 extracting solution
(0.05N HCl + 0.025N H2S04) by automatic pipette to each sample.

4. Shake samples on a mechanical reciprocating shaker, adjusted to 180 oscillations
per minute with a 4 cm stroke, for 5 minutes.

5. Place prefolded, high quality filter paper, moistened with deionized water into
funnel tubes in racks that correspond with the extraction racks.

6. After shaking, immediately filter and save the collected extract for mineral
analysis (P, K, Ca, Mg, Na, Zn, Mn, Cu, B). Transfer to test tubes for ICP.

7. All glassware and cups should be thoroughly rinsed between samples with deionized
water. Weekly wash glassware using a minimum of detergent and rinse thoroughly.

Mehlich 1 Extracting Solution (0.05 N HCl + 0.025 N H2S04)
To prepare 18 liters: add 77 mL concentrated HCl and 13 mL concentrated H2S04 to
approximately 15 liters of deionized water in 20 liter carboy. Bring to 18 liters with
deionized water and mix thoroughly.


Soil pH

Soil pH is determined on all samples using a 1:1 (weight:volume) soil to water ratio.

pH 6.86 buffer standard - purchased at pH 7.0 (0.5M Phosphate)
Dissolve 3.40 + 0.01 g of dried reagent-grade potassium dihydrogen phosphate (KH2PO4)
and 3.55 + 0.01 g of dried reagent-grade anhydrous disodium hydrogen phosphate
(Na2HPO4) in sufficient deionized water to make 1000 mL.

pH 4.01 buffer standard - purchased at pH 4.0 (0.05M Potassium hydrogen phthalate)
Dissolve 10.21 + 0.05 g of dried reagent-grade potassium hydrogen phthalate (KHC8H4O4)
in sufficient deionized water to make 1000 mL.

Procedure

1. Transfer 12 mL scoop of soil (assume 15 g) to paper cups arranged in numbered
trays. After 100 samples (1 set) is transferred, scoop every 20th sample an additional
time as rechecks and include at the beginning of the set.

2. Add 15.0 mL of deionized water by automatic pipette with enough force to mix
thoroughly and allow to stand at least 1 hour.

3. An AS-3000 Dual pH Analyser is used to measure pH. Check the filling solution level of
the electrodes daily.

4. Follow instrument procedures for calibrating with buffers;
pH 7.0 and pH 4.0.

5.  Set lab number parameters accordingly.

6. Allow the meter to stabilize at least 15 minutes.

7. Measure the pH of the check soils. The pH should be read to 1 decimal place (0.1)
and should be +0.2 pH units from the standard Check value. If the Check soil is not
within tolerance, recalibrate the meter and/or seek assistance.  Make sure both checks
read properly before leaving the analyser.

8. After the set is completed, compare the additional recheck readings with the
original readings and record on notebook.

9. Store electrodes in storage solution or buffer standard solution with meter in
Standby mode.

10. Clean electrodes weekly.


Buffer pH (Lime requirement)

A routine buffer pH or lime requirement pH is made on all samples. The Moore-
Sikora buffer method is used to calculate the lime requirement of ground, agricultural
limestone required to raise the soil pH of the surface 8 inches to the target pH
suggested in EC 476 (Nutrient Management for South Carolina Based on Soil-Test
Results). 

Components of the Moore-Sikora Buffer  

For every liter of solution, the following quantities of chemicals are dissolved.
MES hydrate, 99% (4-morpholinoethanesulfonic acid (C6H13NO4S.xH2O, fw without
water=195.23)):  7.43 g
MOPS (3-(N-Morpholino) propanesulfonic acid (C7H15NO4S fw=209.26)):  27.40 g
Boric Acid (H3BO3, fw=61.83):  13.10 g
Potassium Chloride (KCl fw=74.56):  74.00 g
Potassium Hydroxide (KOH fw=56.11):  11.07 g 

Quantitatively add each component above to a container that will hold the appropriate
volume.  Use deionized water to rinse out the weighing containers.  Add deionized water
 to 80% of the final intended volume and stir the solution overnight.  Dilute the solution
with deionized water to the intended final volume and stir thoroughly.   

Calibrate a pH meter to pH buffers of 7.00 and 4.00.  After calibration, a pH 10.00 buffer
is checked to ensure the electrodes are accurately measuring pH above 7.00.  The pH
10.00 buffer should read 10.00 +- 0.01.   

Fifteen mL deionized water is added to a 15 mL aliquot of the solution and the pH of the
1:1 mixture is measured.  If the pH of the 1:1 mixture is not 8.00 +- 0.01, the pH of the
original solution is adjusted using dropwise additions of concentrated KOH or HCl. 
The pH of the full strength buffer should be 7.92+-0.01 to ensure a pH of 8.00+-0.01 for
a 1:1 mix of buffer with water.

Procedure

1. To each soil-water mixture from the pH determination (15 g soil + 15 mL H20) add
15 mL of the buffer solution by automatic pipette.

2. Stir thoroughly and allow to stand a minimum of 30 minutes.

3. Standardize pH meter with buffer 7.0 and 4.00.  Read buffer blank  (buffer blank = 15 mL buffer +
15 mL deionized water) and set buffer offset.

4. Verify values of check samples before leaving analyser.

5. If the Check soil readings are not within tolerance, check standardization
and/or seek assistance.  Verify all check values at end of run.

6.  Save data to a text file and print.

7. Dump cups then autoclave samples from Marion and Horry.

8. Store electrodes in storage solution or buffer standard solution with meter in
the Standby mode.


Lime requirement for fish pond samples

The same procedure for buffer pH on fish pond and shrimp pond samples is followed.


Sulfur (Sulfates)

Extractable total sulfur is determined on request and on subsoil samples where the
argillic horizon is less than 20 inches from the surface if requested.
Sulfate Extracting Solution (0.5N NH4OAC in 0.25N CH3COOH)

1. Dissolve 38.5 g NH4OAC in deionized water in a 1 liter volumetric flask. Add
14.2 mL of glacial acetic acid (CH3COOH) and bring to volume with deionized water.
Or, add 66.5 mL of concentrated NH4OH to approximately 1000 mL of deionized water in a
2 liter volumetric flask. Add 86.0 mL of glacial acetic acid (CH3COOH) and bring to
volume with
deionized water.

2. To prepare 18 liters, add 600 mL of concentrated NH4OH to approximately 10 liters
of deionized water in a 20 liter carboy. Add 770 mL of glacial acetic acid (CH3COOH)
and dilute to 18 liters with deionized water.

Procedure

1. Samples are extracted using sulfate free glassware, with sulfate extracting
solution, following the same procedure used in the Mehlich I soil extraction.

2. After shaking, samples are filtered through number 42 Whatman filter paper or
equivalent, and the extract saved for S analysis on the ICP. Record results in lb/A
with no decimal.


Saturated Extract (SE)

Samples should be logged in the log book.

Procedure

1. Fill 600 mL plastic beakers with sample mix. Bring to field capacity with
distilled water, stirring gently to avoid breaking encapsulated fertilizer pellets and
allow to equilibrate for 1 h.

Note:  At saturation, the sample will flow slightly when tipped and can be easily stirred
with a spatula.  The saturated sample should have no appreciable water on the surface,
nor should it have stiffened.  If additional water needs to be added, allow to equilibrate an
additional 1/2 h.  

2. Vacuum filter through # 4 filter paper and collect at least 110 mL of the extract.

3. Pour an aliquot into a test tube to determine extractable P, K, Ca, and Mg
routinely in the laboratory, using the ICP.

4. Save 100 mL to use for the rest of the analyses.

5. Determine soluble salt content on the solubridge and convert to mmhos/cm by
moving decimal three (3) places to the left. Record value with 2 decimal places.

(See Plant and Feed Manual for meter instructions.)

6. Determine soil pH by glass electrode in a separate subsample by routine lab
procedure.

7. Determine NO3-N by FIAlab nitrate analyzer.

8. Saturated extract results are reported on the Special Analyses Report sheet.
Lab number should be the log book number for that sample as
recorded. Method of extraction is SE. Nutrient analyses should be reported in ppm.
These values may be calculated by dividing soil test lb/A values by 8 (now done
automatically by the computer). NO3-N values do not require calculation.

Nitrate-Nitrogen

Nitrate-Nitrogen (NO3-N) is determined routinely on saturated extracts and mineral
soils when requested.

Nitrate Extracting Solution

Dissolve 173.2 g AL2(SO4)3.18H20, 12.8 g H3BO3, and 0.7222 g KNO3 in 8 liter of
deionized water. Dilute to 9.5 L and check pH. Adjust to pH 3.0 with NaOH. Dilute final
volume to 10 liter.

Boric Acid Preservation Solution -

Dissolve 6.2 g boric acid in 100 mL hot distilled water.
Ammonium Sulfate Ionic Strength Adjustor (ISA)-
Prepare a 2 M ammonium sulfate solution by placing 26.42 g reagent-grade (NH4)2SO4 in a
100 mL volumetric flask. Dissolve and dilute to the mark with distilled water.


Standard Stock Solution (1000 mg/l NO3-N) -

Dissolve 0.7218 g KNO3 (dry) in deionized water in a 100 mL volumetric flask. Add 1 mL
preservation solution then dilute to 100 mL.

Silver sulfate - Reagent grade Ag2SO4.

Outer filling solution - Dilute 2 mL ISA to 100 mL with distilled water

Inner filling solution - Orion cat. 900002

Procedure (Soils):

1. Measure 20 g soil (16 mL scoop) into a paper cup.

2. Add 40 mL NO3 extracting solution, mix thoroughly, and allow to equilibrate
for 1 (one) hour.

3. Filter through # 4 filter paper and collect filtrate in 50 mL beakers.

4. Working Standards (2, 10, 100 mg/l NO3-N)

To prepare 2, 10, and 100 ppm working standards from commercial 1000 ppm
NO3-N stock solution, pipette 0.2, 1, and 10 mL respectively of the 1000 ppm stock
into 100 mL volumetric flasks. Bring to volume with NO3 extracting solution.
(Prepare fresh standards every two weeks.)

5. Determine ppm NO3-N by specific ion electrode.

6. Instrument calibration and operation for soils:

a) Remove electrodes from storage solution and rinse thoroughly.

b) Place electrodes in 2 ppm standard. Press calibration then enter 3-yes for
number of standards. Wait for beep then enter 4.0-yes.

c) Rinse electrodes then place in 10 ppm standard. Wait for beep then enter
20.0-yes.

d) Rinse electrodes then place in 100 ppm standard. Wait for beep then enter
200-yes. Slope should read 54-60.

e) To read samples press measure. Red check sample should read 15-19. Brown
check sample should read 7-9.

7. Record as ppm NO3-N (no decimals) on lab result sheets for special analyses.
Recheck standards periodically and recalibrate as necessary.

8. After analyses are completed, rinse electrodes, return to storage solution, turn
off stirrer, and return instrument to Standby.

Procedure (Saturated Extract):

1. Working Standards

a) To prepare 2, 10, and 100 ppm working standards, add 0.2, 1, and 10 mL
respectively standard stock solution (1000 mg/l NO3-N) to 100 mL volumetric flasks and
bring to volume with distilled water.

b) Prepare fresh standards every two weeks.

2. Add 1 mL preservation solution to each sample and standards.

3. Add 2 mL ISA to the standards and the samples.

4. Check levels of inner and outer filling solutions in reference electrode
(see reference electrode manual for specifications).

5. Instrument calibration for SE: (see NO3-N instrument calibration and operation
for soils - page 7 except enter exact value for standards, i.e.., 2, 10 and 100).

6. Rinse electrodes and place into sample. Record reading directly as mg/l NO3-N
(no decimal) on Report on Special Soils or Artificial Mixes form.

7. Recheck standards frequently.

8. When finished, place meter in Standby mode and submerge electrodes in Blank.


Nitrate-Nitrogen by Cadmium Reduction using Flow Injection FIALab 2500
(See water procedures for instrument set-up)

Extraction reagent:  0.04 M (NH4)2SO4

             To make 10 L:  Add 6 L DI water to a 20 L carboy.  Weigh 52.80 g
(NH4)2SO4 and add to carboy.  Dilute to 10 L mark and stir with a magnetic
stirrer.

Procedure:

1.  Use 4 mL (approximate 5 g) mineral scoop and scoop soil into an extraction cup.
2.  Add 25 mL extraction reagent and shake for 5 minutes, then let stand for 30 minutes.
3.  Filter the extract through Whatman 42 filter paper into nitrate analyzer test tubes.
4.  Analyze according to flow injection FIALab 2500 instrument procedures.

Reference:  J. Benton Jones, Jr., Laboratory Guide for Conducting Soil Tests and Plant
Analysis, p 123, CRC 2001.
 

Organic Matter

Loss on Ignition for Organic Matter.

Apparatus

1. Muffle Furnace

2. "High Form" Porcelain Crucibles

Procedure

1. Number crucibles with wax pencil.

2. Weigh crucibles to 4 decimal places.

3. Scoop soil samples into crucibles using 5 g scoop.

4. Put crucibles plus samples on tray in left-hand furnace.

5. Set dial to 100. (This corresponds to a temperature of 105o +/- 5oC.)

6. Ash at this temperature for 2 h. (It takes 30 min for the furnace to reach
105oC, so set the dial at 100 for a total of 2 h and 30 min.)

7. Remove samples from furnace with tongs, place in desiccator, and weigh to 4
decimal places immediately while still warm so moisture won't be absorbed.

8. Replace crucibles plus samples in left furnace.

9. Turn dial up to 360. (This corresponds to a temperature of 360o +/- 5oC.)

10. Ash at this temperature for 2 h. (It takes 1 h for the furnace to reach this
temperature, so set the dial at 360 for a total of 3 h.)

11. Turn furnace off and open door.

12. Place samples in desiccator with tongs and allow samples to cool.

13. Weigh samples to 4 decimal places.

14. Calculate OM % and record on data sheet with one decimal.

Calculation

crucible wt = a

crucible wt + sample wt after 105oC heating = b

crucible wt + sample wt after 360oC heating = c

%OM = ((b-a) - (c-a))(100)/(b-a)

Reference

E. E. Schulte, Recommended Soil Organic Matter Tests.


Walkley-Black procedure.

Potassium Dichromate Solution (1.0N K2Cr2O7)

Place 49.04 g of potassium dichromate (K2Cr2O7) (dried at 105oC) in a one-liter flask
and dilute to volume with demineralized water.

Ferrous Sulfate Solution (0.5N FeSO4.7H20)

Place 139.01 g of ferrous sulfate (hydrated) FeSO4.7H20 in a one liter flask. Add about
200 mL of demineralized water and 15 mL of concentrated sulfuric acid. Dilute to
volume. Indicator (if necessary):

Use 0.025 M O-phenanthroline ferrous sulfate (Ferroin) as an indicator (commercially
available).

0.5946 g/100 mL phenanthroline = 0.01N

0.2780 g/100 mL FeSO4.7H20 = 0.01N

Procedure:

1. Place 1.0 g of soil in a 250-mL Erlenmeyer flask and prepare a Blank by adding no
soil.

2. Add exactly 10 mL of 1.00N potassium dichromate and mix by gentle rotation of
flask.

3. Add 20 mL of concentrated sulfuric acid and mix for one minute by gentle rotation
of flask.

4. Allow to stand for 30 minutes.

5. Add 150 mL demineralized water.

6. Add 10 mL of concentrated phosphoric acid and allow to cool to room temperature.

7. Add 4-5 drops of indicator. (If solution turns dark green weigh 1/2 g and
multiply answer by 2 or weigh 1/4 g and multiply answer by 4.)

8. Titrate immediately with 0.5N ferrous sulfate solution until end point is
reached.

9. Determine % organic matter using given formula:

% O.M. = (1 - T/S) (6.8)

Where T = sample titration in mL of 0.5N ferrous sulfate

Where S = standard blank in mL of 0.5N ferrous sulfate

This formula is based on two important assumptions which may not be valid for all
soils:

(1) only 76% of the organic carbon present is oxidized to CO2, and (2) soil organic
matter contains 58% carbon.

10.Record on lab result sheets for special analyses as % O.M. (1 decimal place).


Soluble Salts

Soluble salts are run on samples as requested in mineral soils and on artificial mixes.

Samples should be logged in the log book.

Procedure:

1. Scoop 20 mL soil to a 250 Erlenmeyer flask.

2. Add 40 mL of distilled water with graduated cylinder, stir, and allow to
equilibrate
for 1 hour, shaking intermittently.

3. Decant liquid (through screen filter) and collect in small wide mouth bottle.

4. Read mmhos/cm on conductivity meter. (See Plant and Feed Manual for meter
instructions).

5. Record 2 decimal places, e.g., 0.200 = 0.20.

6. Report values on Report on lab sheet. Lab number should
be either the log book number if only soluble salts are to be reported, or the
laboratory number of the mineral soil received by the lab for routine soil analysis.

Method of extraction is 1:2.


Quality Control for Soil Testing

The large number of routine soil samples extracted and analyzed daily in the Soil
Testing Laboratory (100-400) requires that a precise system of quality control be used.
The system insures that samples remain in their proper order so no mix-up is made in
the sample sequence and the quality of the analyses will remain constant from day to
day and throughout the year. "Check" samples are purchased or established by collecting, drying,
homogenizing, and storing several kilograms of a soil that has varying levels of pH,
P, K, Mg, and Ca. A "standard" value for pH and extractable nutrients and a standard
deviation is established by analyzing this soil ten to twenty times on different days
and establishing a mean value. This soil is designated as a Check sample, given a
letter, and used daily in maintaining a quality control for soil samples.

The laboratory participates in the North America Proficiency Testing soil sample
exchange programs. This allows the Soil Testing Laboratory to check its
"Check" sample values with those from other laboratories, as well as compare analytical
techniques.

In a routine, daily soil-test run, two Check samples and one Blank are placed in every
group of 100 soil samples as the samples are numbered. An information sheet is included
every time a Check sample or Blank is placed in the samples, and the Check or Blank is
numbered and analyzed as a routine soil sample. The supervising technician is
responsible for comparing the laboratory results of the Check samples and Blanks to
make sure they are reading within an accepted range. By careful attention to the values
of the Checks and Blanks, errors can be avoided before they leave the laboratory.
Samples
are held for 1 month after analysis to allow for recheck if necessary.  Growers
or Extension personnel may request that all or part
of their analyses be rechecked for 
accuracy. This allows for a monitoring of quality control as well as maintaining confidence 
in the lab.


References

1. Isaac, R. A. (ed.). Reference Soil Test Methods for the Southern Region of the
United States. Southern Cooperative Series Bulletin 289, September 1983.

2. S. J. Donohue. Reference Soil and Media Diagnostic Procedures for the Southern
Region of the United States. Southern Cooperative Series Bulletin No. 374, August 1992.