4/26/2013 LN
Microplate Soil Nutrient Analysis:
Nitrate NO3--N
(Modified from Kathleen Treseder’s and Loralee Larios’ protocol)
Doane, T. A., and W. R. Horwath. 2003. Spectrophotometric determination of nitrate with a single reagent.
Analytical Letters 36:2713-2722.
Materials Needed:
• Matrix (solution used to extract samples: usually 2.0M KCl or 0.5M K SO )
2
4
• Vanadium solution (stored in freezer, thaw in warm water; see RECIPES)
• Stock nitrate solution (stored in refrigerator; see RECIPES)
DO NOT KEEP MORE THAN ONE MONTH!
• 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.
Notes on “high” vs “low” protocols:
Samples expected to have low nitrogen (N) content (0-1 ppm) should be run using the “low”
protocol. Samples expected to have high N (e.g., from fertilized treatments) should be run using
the “high” protocol. If unsure, run samples using the “high” protocol. In our experience, most
“t0” samples will fall into the “low” category, whereas most “PMN” samples will need to be run
with the “high” protocol, and even then, may need to be diluted and ran again.
a.
Add 1 ml of 100 ppm stock nitrate solution (see recipes) into a 10 ml volumetric flask and
bring up to volume with matrix (KCl or K2SO4). Working stock solution: 10 ppm.
b.
Using the diluted stock solution and the matrix solution, create the following standard curves
in the corresponding scintillation vials. (There are 6 (or 7) NO3 standards, and one that is
just K2SO4.) Prepare the dilutions using matrix.
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. Choose a concentration in the middle of the curve (e.g. 1ppm).
1
4/26/2013 LN
STD0
STD1
STD2
STD3
STD4
STD5
STD6
STD 7
“LOW” Standard ladder
Conc. (ppm) Volume ul
10 ppm
0.0 ppm
0
0.05
20 ul
0.1
20 ul
0.2
40 ul
0.4
80 ul
0.6
120 ul
0.8
160 ul
1.0
200 ul
Volume ul
matrix
2.00 ml
3.98 ml
1.98 ml
1.96 ml
1.92 ml
1.88 ml
1.84 ml
1.80 ml
STD0
STD1
STD2
STD3
STD4
STD5
STD6
STD7
“HIGH” Standard ladder
Conc. (ppm) Volume ul
10 ppm
0.0 ppm
0
0.2
40 ul
0.5
100 ul
1
200 ul
2
400 ul
4
800 ul
7.5
1.5 ml
10
2 ml
Combined standard ladder
Concentration (ppm) volume 10 ppm stock
Volume matrix
Std 0 =
blank
STD1
0.0 ppm
0 ml
2 ml
STD2
STD3
STD4
STD5
STD6
STD7
0.1
0.5
1.0
2.0
4.0
6.0
20 ul
100 ul
200 ul
400 ul
800 ul
1.20 ml
1.98 ml
1.90 ml
1.80 ml
1.60 ml
1.20 ml
0.80 ml
INTERNAL STD
Volume ul
matrix
2.00 ml
1.96 ml
1.90 ml
1.80 ml
1.60 ml
1.20 ml
0.50 ml
0.00 ml
Put lids or parafilm
on top and swirl &
mix. Set aside in
fridge.
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 with every plate (columns 1 and 2) with the zero
in position A1/A2.
3. Load the microplate.
a. Add the following to each well of a 96-well microplate (load ALL samples, standards, and
blanks before adding the reagents):
LOW std ladder
100 μl sample*
100 μl Vanadium solution (using multichannel pipette)**
HIGH std ladder
10 μl sample*
190 μl Vanadium solution (using multichannel pipette)**
* When loading samples, it is very important to change pipette tips each time
2
4/26/2013 LN
* Stored in freezer. Use concentrated Vanadium solution for samples extracted with K2SO4. If it
is purple then it has been contaminated!
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
Vanadium solution.
d. As soon as you’ve added the reagent and samples, write down the time on your microplate
data form and time to read the plate (18 hours later). The samples will begin to turn a pink
color over the next few hours. Make note of any samples that have changed rapidly upon
adding the reagent, especially if it appears to have gone further than STD 8. These samples
may be very concentrated and the reaction may have gone too far after the allotted color
development time. You may end up with a misleadingly low absorbance reading. Take note
of these samples and scrutinize the results. The sample may 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
“Nitrate_NO3_low.prt OR Nitrate_NO3_high.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 540 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_NO3_low.xpt
These .xpt files are automatically saved in: My computer > C: drive > Program Files > Gen5
1.06 > Experiments
3
4/26/2013 LN
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
5.
Incubate at room temperature for 18 hrs and read plate at 540 nm. Detection limit
<0.05ppm.
a. Press the “Read Plate” button at the top left of your experiment window. Press the “Read”
button and 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 way below 0.90 and messing around with only a few points
won’t save you, you will have to completely redo the plate. Check the standard calculated
values to ensure they correspond to expected values.
c. Check blanks on Plate 1 window (Matrix tab, 540 on drop down menu). Are they close? If
not, you may want to redo the analysis if the blanks are not acceptable. You may also delete
the bad blanks on your plate layout, and then re-read the plate.
d. Save your .xpt file again after you have acquired the data, otherwise it will be lost.
e. Use the power export button (at the top) to acquire data on an excel sheet.
1) Save this new excel sheet (will automatically be saved as 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.
4
4/26/2013 LN
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
and blanks), nutrient, and any other pertinent info.
6.
Important Notes
a. The samples will turn a pink 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, the sample should be rerun using the HIGH protocol. You also
have the option of diluting a subsample; either directly diluting it into the well or into a
different vial.
Examples of 5x dilution:
Into a separate vial: 100ul sample + 400ul.
Into a separate well: add 2ul of sample + 8 ul of K2SO4 in the same well.
Add reagents as normal. Make sure you record where the diluted samples are, so you will be
able to calculate the real concentration.
To calculate real concentration:
Final “real” concentration = dilution factor (in the above case = 5) x conc.(ppm) of diluted
sample.
See “Example Dilutions” 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.
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. 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.
5
4/26/2013 LN
7. Calculations for final data
The final concentration that you are given is in ppm (ug/g) 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).
1% = 1 g / 100 ml
Dilution: ½ = 1 part sample / 2 parts total
6