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Microplate Soil Nutrient Analysis:
Ammonium NH4+-N
(Modified from Kathleen Treseder’s and Loralee Larios’ protocol)
Weatherburn, M. W. 1967. Phenol-hypochlorite reaction for determination of ammonia. Analytical Chemistry
39:971-974
Materials
• Matrix (solution used to extract samples: usually 2.0M KCl or 0.5M K SO )
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• Sodium salicylate solution (in refrigerator; see Recipes: Reagent A)
• 2% bleach in 1.5M sodium hydroxide (make fresh every day: see Recipes: Reagent B)
(1 ml into 49 ml NaOH if normal bleach; 0.75 ml into 49.25 ml NaOH if concentrated
bleach)
You will need 8 ml/plate
• Stock Ammonium Solution (100 ppm, in refrigerator; see Recipes)
• Sterile 96-well flat-bottom microplate: VWR #29442-070, Corning #9017
• Microplate lids: Fisher # 07-200-703; manually notched at one end
• Micropipette tips: VWR # 89100-002, #89082-222
• Multi- and single channel pipettes
• Pipetting Basin: Fisher # 13-681-100
Note Only PMN and TO samples need to be run on Biotek, not MB.
Procedure
1. Make a standard curve.
a.
Add 0.5 ml of 100ppm stock ammonium solution (see recipes) into a 10ml volumetric flask
and bring up to volume with matrix (KCl or K2SO4). Working stock solution: 5 ppm.
b. Using the working stock solution (5 ppm) and the matrix solution, create the following
standard curves in 6 20-ml vials (there are 8 standards but the first one and last one are
just K2SO4 and the 5ppm stock, respectively. Just pour those into small vials). For
standards 2 through 7, add the respective amounts of the 5ppm stock solution (see chart
below), into the corresponding volume of matrix into the labeled scintillation vials.
c. Use an internal standard – either a sample or a standard concentration that has been
aliquoted and will be run with all of your samples – every plate, every time. Keep
aliquots in the freezer labeled as your own. Internal Standard: choose a concentration that
will be usable for repeat assays (e.g. 1 ppm) and freeze 500 ul aliquots in microfuge tubes,
appropriately labeled. Prepare enough of the standard to have more than enough for the
remainder of the assays (e.g. 1 ppm: 2 ml 5 ppm stock plus 8 ml matrix).
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Std 0 =
blank
Standard ladder
Conc. (ppm)
volume 5
ppm stock
0.0 ppm
0 ml
Volume
matrix
2 ml
INTERNAL STD
STD1
0.1
40 ul
1.96 ml
STD2
0.2
80 ul
1.92 ml
STD3
0.5
200 ul
1.80 ml
STD4
1.0
400 ul
1.60 ml
STD5
2.5
1 ml
1.00 ml
STD6
5.0
2 ml
0.00 ml
d. Other possible concentrations:
a. 2.0 ppm: 0.8 ml 5 ppm stock plus 1.2 ml matrix
b. 4.0 ppm: 1.6 ml 5 ppm stock plus 0.4 ml matrix
e. Set aside in fridge.
 Just matrix (KCl or K2SO4)
 Just 5ppm ammonium solution
2. Fill out microplate form.
a. Fill out microplate form* with all pertinent information (date, name, nutrient, project name,
times, and filenames) and the location of where you will put your samples on the microplate.
*Empty microplate data forms can be found in “Empty Data Forms” section in the BIOTEK
RAW DATA binder, or on lab computer (room 48) @ My Documents > Laboratory >
BIOTEK > Procedures and Protocols > microplate data form.
You will want two columns of STANDARDS which includes two wells of BLANKS with every
plate. In column 1 and 2 (from well A1 – H1) you will have: STD1 (0.0ppm), STD2 (0.05ppm),
STD3 (0.1ppm), STD4 (0.2ppm), STD5 (0.5ppm), STD6 (1ppm), STD7 (2ppm), STD8 (5ppm).
3.
Load the microplate.
a. Add the following to each well of a 96-well microplate using a multichannel pipet (load ALL
samples, standards, and blanks before adding the reagents):
40 μl sample or standard** in duplicate
80 μl sodium salicylate solution (“Reagent A”) (using multichannel pipette)
80 μl bleach solution (“Reagent B”: 0.5 ml bleach + 24.5 ml 1.5 M NaOH
*The second reagent MUST be added immediately after the first otherwise loss of absorbancy will
occur
** You can alter the sample:reagent ratio for low NH4+ concentrations, e.g. 80 ul sample + 60 ul
each reagent
** When loading samples, it is very important to change pipette tips each time. Do not
contaminate the pipet tips on the multichannel by touching the tips to any solution in the well.
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b. Once you load the sample, place in the “Biotek Box” . This helps you to see what wells you
have loaded, just in case you get lost. Double check with the placement of your samples in
the “Biotek Box” that it matches up with your microplate form. If you’ve messed up at all
along the way, make the appropriate changes on your microplate form to reflect exactly what
has been dispensed in your actual microplate.
c. After loading the ladder and all the samples, use the multichannel pipette to add the sodium
salicylate (first) and bleach/NaOH solutions (second). Give it a gentle shakie shake to mix up.
d. As soon as you’ve added the reagents and samples, write down the time on your microplate
data form and time to read the plate (1 hour later). The samples will begin to turn a blue
green color over the hour. Make note of any samples that have changed rapidly upon adding
reagents, especially if it appears to have gone further than STD 8. If samples are too
concentrated, the reaction will have gone too far and will turn yellow. They will have a
misleadingly low absorbance reading. If you keep watch and know that the sample was
turning blue right off the bat, you will know that the sample will need to be diluted (see
section 6: Important Notes).
4.
Set up experiment files on program for reading
a. Open Gen5 1.06 software (can access from desktop)
b. In the “Create a New Item” box on the top left of the screen, choose “Experiment”
c. It will then ask you to select a protocol to run your experiment from. Choose
“Ammonium_NH4.prt.
d. This protocol is already associated with a certain procedure, plate layout, and data reduction
options. However, depending on your specific needs you may need to change the plate
layout.
1) This procedure stays at a reading of 650 nm.
2) The plate layout is set up as you see below, but you may need to change it depending on
how you decide to layout your samples and/or how many you have, etc. Make sure you
set it up exactly as you have dispensed your standards, blanks, and samples on your
microplate. Refer to your microplate data form for details.
e. Data reduction should be set up for you.
f. Save your experiment file. DO NOT resave the protocol, otherwise the protocol template
will be changed. Save the .xpt file as such “your initials_date & plate a, b, or c_project
info_nutrient.xpt”. If I am running multiple plates, I will have an a, b, or c behind the date,
to identify which plate it is.
Example: hro_080409a_SASMay09_NH4.xpt
These .xpt files are automatically saved in: My computer > C: drive > Program Files > Gen5
1.06 > Experiments
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5.
1
2
3
4
5
6
7
8
9
10
11
12
A
Blank
Blank
SPL1
SPL1
SPL2
SPL2
SPL3
SPL3
SPL4
SPL4
SPL5
SPL5
B
STD1
STD1
SPL6
SPL6
SPL7
SPL7
SPL8
SPL8
SPL9
SPL19
SPL10
SPL10
C
STD2
STD2
SPL11
SPL11
SPL12
SPL12
SPL13
SPL13
SPL14
SPL14
SPL15
SPL15
D
STD3
STD3
SPL16
SPL16
SPL17
SPL17
SPL18
SPL18
SPL19
SPL19
SPL20
SPL20
E
STD4
STD4
SPL21
SPL21
SPL22
SPL22
SPL23
SPL23
SPL24
SPL24
SPL25
SPL25
F
STD5
STD5
SPL26
SPL26
SPL27
SPL27
SPL28
SPL28
SPL29
SPL29
SPL30
SPL30
G
STD6
STD6
SPL31
SPL31
SPL32
SPL32
SPL33
SPL33
SPL34
SPL34
SPL35
SPL35
H
STD7
STD7
SPL36
SPL36
SPL37
SPL37
SPL38
SPL38
SPL39
SPL39
SPL40
SPL40
Incubate at room temperature for 1 hr and read plate at 650 nm. Detection limit <0.05ppm.
a. Press the “Read Plate” button at the top left of your experiment window. The plate reader
will open. Make sure you set your plate in properly. Click “OK”. The actual reading takes
less than a minute.
b. Check your standard curve on the Plate 1 window (Curve tab). Check the r2. Anything
above 0.90 should be okay, but you should be able to delete a few points if necessary to
increase the r2 value. To delete points, click on the point you want to delete. Then click on
the green checkmark on the upper right hand side to accept changes and save. The data will
automatically respond. If you are below 0.90 and messing around with only a few points
won’t save you, you will have to completely redo your standards and samples. Check the
standard calculated value to ensure they correspond to expected values.
c. Check blanks on Plate 1 window (Matrix tab, 650 on drop down menu). Are they close? If
not, you may want to redo the analysis if the blanks are not acceptable.
d. Save your .xpt file again after you have acquired the data, otherwise it will be lost.
e. Use the power export button to acquire data on an excel sheet.
1) Save this new excel sheet. It should automatically be named the same name as
you have saved your .xpt file. Save in My Documents > BIOTEK Data > Excel
Data Output > your project’s folder.
2) Print off this sheet and place in BIOTEK RAW DATA binder behind the related
microplate form. Feel free to clean up this data sheet and make it smaller for
easier printing.
f. At the end of the day, record info in the BIOTEK LOG book (sits somewhere near Biotek).
Include the date, project, your initials, # of samples total ran that day (including standards,
blanks, and dups), nutrient, and any other pertinent info.
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6.
Important Notes
a. The samples will turn a blue green color; however, if the concentration of ammonium is too
high, the reaction will go too far and the sample will turn yellow. If this happens or the
sample is above the highest standard (5ppm) the sample should be diluted and ran again.
You have the option of diluting a subsample into a different vial or diluting it directly into the
well.
Examples of 5x dilution*:
Into a separate vial: 100ul sample + 400ul matrix.
Into a separate well: add 8ul of sample + 32ul of K2SO4 in the same well.
Add reagents as normal. Make sure you record where the diluted samples are.
To calculate concentration:
Final concentration = dilution factor (in the above case = 5) x conc.(ppm) of diluted sample.
See “Example Dilutions” Sheet for more examples…
b. Samples will have a precipitate in the bottom. This shouldn’t necessarily affect soluble
nutrients, but should be shaken up to homogenize differential concentrations. Just give it a
quick shake-up then allow particles to settle for about 5 minutes.
c. You are very welcome to experiment with different standard ladders and amounts of sample
to reagent if there appears to be any problems. Just make sure that if you do, you are using
the same sample to reagent amount as you are using for the standards on the same plate.
d. Make sure you wash your dishes and clean up after yourself immediately. The plates
CANNOT be allowed to sit as the reagents will dry and form a film that cannot be removed.
In addition, make sure you turn off the Biotek instrument when you are done. Pipet tips are
disposed of in the trash.
e. We can reuse the microplates if you wash them well and take care not to scratch them. Do
NOT place them in the acid bath. Rinse 3x into appropriate waste container then wash 5x
with RO followed by 5x with dH2O.
f. If you notice we are getting low on supplies, make sure you let someone know.
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7. Calculations for final data
The final concentration that you are given is in ppm or mg/L. You want to know how much
ammonium is in a given amount of soil.
Example:
= 0.00625 mg NO3--N g-1 soil
Do the appropriate conversions if you want it in different units (i.e. ug/g)
Except this is for fresh soil. You will want to account for gravimetric soil moisture content and
gravel found in the extracted sample (after wet-sieving).
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