Fluorometric Ammonium Analysis
Modified from Holmes et al, and various memos (30 Jun 99, 14 February 2000)
Fluorescence is produced by the reaction of OPA with ammonium. Fluorometry is sensitive and simple so seems to be a good way to measure ammonium, particularly at low levels. Details of methods, reagents, etc are given in Holmes et al. 1999 (CJFAS 56:1801-1808). This document supplements the manuscript and is intended to give a quick, user-friendly overview of the procedure. It also details a variation of the method not discussed in the manuscript, which uses 10 mL sample and 10 mL working reagent (we are calling this variant Protocol B-1).
Gloves should be worn when dealing with OPA. At the concentrations we use, it won’t immediately kill you, but it’s not good for you either. According to Andy Mattox (the safety officer at MBL), all chemicals used in this analysis can go down the drain. However, at Toolik we are treating them as hazardous waste.
Contamination & Problems
Always use ammonia-free water for all reagents. Get the water from the Nanopure unit immediately prior to use.
Make absolutely sure the DI used to make the standards is also used for the blank.
Never acid wash these bottles. Instead, just pre-reacted prior to first use, and rinse between uses.
When making the standard dilution series, make sure the pipettes are calibrated properly. It is important to get very accurate dilutions when working w/ such low concentrations. Check calibration by weighing the desired amount on the balance and adjust pipet accordingly.
Apparatus & supplies
Wheaton scintillation vials (Wheaton 986704, Fisher Catalog # 03-341-72C):
4 liter amber bottles (fisher # 028846b) 6/case
The following is enough for about 48 liters of WR and 4 liters of Borate Buffer.
Preparation of Stock reagents
BORATE BUFFER (BB): Borate buffer without the sodium sulfite or the OPA is used to evaluate the sample background fluorescence (BF).
sodium sulfite: Next prepare the sodium sulfite solution (2 g sodium sulfite to 250 mL DI water)
orthopthadialdehyde (OPA): add 8 g OPA to 200 mL ethanol (keep this solution as dark as possible), shake vigorously until OPA dissolves.
WORKING REAGENT (WR): Working reagent appears to be stable for months, and its blank fluorescence decreases over time, so it is best to make WR in large batches and let it age. We make WR batches of about 4 L in 1 gallon brown Nalgene bottles (these bottles actually hold about 4.4 L).
To a clean 4-liter bottle (pre-react, or just rinse with DI if previously used for WR), add approximately 3 L DI. Then add 160 g sodium borate, cap, and shake vigorously until your arms are tired, then rest, then do it some more, and add 20 ml of sodium sulfite solution to the 1 gallon jug with DI and sodium borate already added. Shake the jug some more. Finally, add 200 ml of OPA solution to the 4-liter jug. Shake some more, then add DI until the bottle is nearly full – about 1 inch from the top. Shake a bit more, let age for at least a few days if possible, and then the WR is ready to use.
Sample bottle preparation
To pre-react, add 10 mL working reagent (WR) to bottle, cap and shake, and let sit for at least 3 hours (days or weeks is fine). Dump WR, then rinse three times with pure DI water. Next add 10 mL WR, shake, dump, and then load with another 10 mL WR. Store in dark (WR is light-sensitive). Once loaded with WR, the bottles are ready to go (keep WR in dark at all times).
1. Rinse syringe 3 x with water to be sampled.
2. Attach filter to end of syringe (either a GF/F or .2 um membrane filter) and filter 10-30 ml though filter to rinse filter.
3. Dump DI from sample bottle and rinse with 30-40 ml of filtrate.
4. Re-fill syringe and fill sample bottle with filtrate.
Duplicates should be run for all blanks, with reagent blanks run at the beginning and end of each sample set. Three types of blanks should be run with each set of samples.
1. Trip blanks (DI blanks that are carried out to the field and back). These would consist of sample bottles filled with DI which brought into the field and then returned.
2. Field blanks (DI blanks filtered in the field as if they were samples).
BACKGROUND FLUORESCENCE (BF):
All samples auto-fluoresce to some degree. This BF must be subtracted from the observed sample fluorescence in order to quantify ammonium concentration. If it is found that BF doesn’t change though the water column or down a stream transect it may be possible to take fewer BF measurements. If it does change however you will need to take a BF each time a sample is taken. In surface waters around Toolik Lake, ammonium concentrations tend to be very low and background fluorescence is relatively significant. Therefore, it is important to accurately quantify BF. In our limited experience so far, BF is relatively constant in a given water-body on a given day (for example, Toolik Main station or Kuparuk River transect), but BF varies across sites (and maybe temporally). Therefore, BF does not need to be sampled at every station within a given “site”, but must be sampled at each stream. Another example: On June 23, 1999, BF was essentially constant at 11 Kuparuk River stations, but differed significantly in Hershey Creek, a small tributary to the Kuparuk River. If BF had not been measured in Hershey Creek and instead the Kuparuk BF was used, the Hershey Creek ammonium result would have been erroneous.
To quantify BF, collect 10 mL sample in the field to an empty scintillation vial, and upon return to the lab, add 10 mL borate buffer (see manuscript) and read fluorescence. No reaction period is necessary. It is not necessary that bottles used for measuring BF are pre-reacted – in fact, never add WR (with OPA) to bottles used to measure BF.
MATRIX EFFECTS (ME):
OPA and ammonium react differently in different waters. In DI water, a given amount of ammonium tends to produce more fluorescence than it would in lake or river or soil solution samples. To quantify ME and correct for it, standard additions are done to samples and compared to DI water standards. For surface waters around Toolik Lake, we have been spiking samples with 50 ul of 50 uM ammonium stock solution to quantify ME. In general, ME have been around 5-25 %. This correction is generally on the order of 0.01-0.03 uM for surface waters around Toolik Lake, but will be greater when ammonium concentrations are greater. Therefore it is important to note that for higher ammonium values a larger spike is required to assess ME. As with BF, ME appears to be relatively constant within a given water-body but will probably vary across sites and maybe temporally.
In order to calculate matrix effect four measurements are required.
1. The fluorescence of a known amount of standard added to DI (spike std) this could be a standard from your standard curve.
2. A DI blank (0 std), which can also be from your standard curve.
3. A sample spiked (sample spike) with the same amount of standard as the spike standard
4. The fluorescence of the sample with only WR added, but without any spike (sample obs).
The equation for calculating matrix effect is as follows:
(((spike std - 0 std)-(sample spike - sample obs))/(spike std - 0 std!))*100
It is important to measure the 10 mL sample accurately. Disposable 10 mL syringes may work well. Alternately 2x 5 ml delivered from a pipette could also work. Only open sample bottles for a short time, and be aware of potential sources of contamination when bottle is open. Rinse syringe thoroughly between samples.
In the field add 10 mL sample to bottles pre-loaded with WR, shake to mix, and store in dark. The reaction takes about 3 hours to reach peak fluorescence (see manuscript), so wait at least that long after taking the last sample before reading on the fluorometer. Since the linear period of the fluorescence is from about 4-8 all samples and standards need to be read within that window. The speed of the reaction is temperature dependant therefore samples and standards should be at similar temperatures when reagents are added. If this is not possible both should be allowed to react at least 4 hours.
Samples are taken in the field in pre reacted 20 ml scint vials that have been rinsed with fresh DI followed by three rinses in the field with sample. Upon returning to the lab samples and standards are added to pre reacted pre loaded scint vials. Samples may either be read after 4 hours. We are going to run some test to see if these samples could be read after reacting over night.
I (Max) prefer to make standards in the field. The fluorescence reaction is time sensitive, so it is good to start standards at roughly the same time as samples are collected. However, fluorescence asymptotes after a few hours and stays there for several hours, so there so leeway here.
For surface water samples around Toolik, standards ranging from 0 to 0.5 uM work well. I recommend using a 50 uM stock ammonium solution and a 10-100 ul Eppendorf pipette to make the standards.
To make the standards, DI (10 mL) is added to bottles loaded with WR (10 mL), and then stock ammonium solution is added. (I have been adding DI to sample bottles in the field, but it may be possible to add both WR and DI to standard bottles in the lab prior to going to the field. This will require testing before we know if it works well).
Recipe for Standards:
NOTE 1: Standard regressions have been fairly consistent, with a slope around 2.5 and y-intercept about 0.07 (Protocol B-1 using above standards). If this continues to be the case, it may be easier to make standards in the lab and not worry about field preparation.
NOTE 2: We recently had a jump in blank fluorescence, from about 0.07 to about 0.15. This appears to be coming from the DI water. Since no DI water is added directly to samples (only WR is added), bad DI impacts standards but not samples. Therefore, if we can pinpoint the increased blank to ammonium in the DI (equivalent to only about 0.03 uM), we might want to adjust the intercept to the fluorescence of WR (or WR plus DI but read immediately) so that we will not underestimate the ammonium content of samples).
Variant #2 (made by nutrient RA)
Stock A Solution-1000mM NH4-N (made by nutrient RA)
Dissolve 0.06607g of dry (dried in oven overnight) (NH4)2SO4 (m.w.= 132.14) in approximately 900ml of deionized water contained in a 1 L volumetric flask. Dilute the solution to the mark with deionized water and mix it well. Transfer this solution to an amber bottle. Refrigerate when it is not in use.
Stock B Solution-20mM NH4-N (made by nutrient RA)
Prepare this solution daily. Using a volumetric pipette or a calibrated automatic pipette, add 2.0ml of Stock A to approximately 90 mL of deionized water contained in a 100ml volumetric flask. Dilute the solution to the mark with deionized water and mix it well.
Working Standards (made by nutrient RA)
Prepare these solutions daily. Use adjustable, microliter pipettes to add the designated volumes of stock A or B listed in the following table. Calibrate the pipette for each required volume. (NOTE: The standards for each day are presented in bold typeface. The other concentrations are included for reference, if needed.) Prepare each standard by adding the required amount of stock to the required volume volumetric flask containing deionized water. Dilute each to the mark with deionized water and mix well. Keep these solutions tightly sealed.
Record the fluorescence for each standard and spike sample in the nutrient logbook. Determine the standard curve by plotting absorbance (y-axis) versus standard concentration (x-axis). We have created an Excel workbook, entitled 2000lternh4fluor.xls, for data input.
Method Detection Limit
The detection limit for this method should be determined daily. If the detection limits are consistent for a couple of weeks, then it will be necessary to perform this task weekly.
Spike DI water with 2-3 times the estimated instrument detection limit. This should be around 0.2 to 0.3 mM. (Use the 0.2 or 0.3 mM standard as the spike.) Run 7 spikes as samples after the standard curve has been run. Calculate the method detection limit (MDL) by
MDL= [t(7, 0.01) * s]
Where t=t statistic for 7 reps (t=3.14) with 99% confidence and s=standard deviation of the calculated concentration.
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