DISSOLVED AMMONIA ANALYSIS
 Updated October 98 (MS)

METHOD:
 Ammonia reacts with alkaline hypochlorite and phenol to form indophenol blue.  Sodium nitroferricyanide accelerates the formation, which is them measured spectrophotometrically at 640 nm.  From A Manual of Chemical and Biological Methods for Seawater Analysis (Parsons, Maita, & Lalli, 1984) 

REAGENTS:

Phenol  solution 
20 g phenol in 200 mL 95% v/v ethyl alcohol.

Sodium  nitroferricyanide solution
1 g  sodium nitroferricyanide, Na2[Fe(CN)5NO] . 2H2O, in 200 mL deion. H2O.
Store in dark glass bottle; stable at least 1 month.

Alkaline reagent
100 g sodium citrate and 5 g NaOH in 500 mL deion. H2O.
Stable indefinitely.

Sodium hypochlorite solution
Chlorox bleach decomposes slowly.

Oxidizing solution
100 mL alkaline reagent and 25 mL  Chlorox.
Keep stoppered when not in use & prepare fresh every day.

AMMONIA STANDARDS:

Primary STDs - both Analytical and Quality Control (1.5 mM) - 0.100 g ammonium sulphate, (NH4)2SO4  (F.W. 142.048), in 1 L nanopure H2O.
Add 1 mL chloroform, store refrigerated.  Stable many months.

Secondary (Analytical) STDs:

2 STD (?M)
(L 1 STD / 50 mL H2O
0.00
0
0.75
25
1.50
50
2.25
75
3.00
100

Quality  Control  STD:

1.50
50

Note: Analytical and QC STD's must be made up the same day they are used.
ANALYSIS:
Pipet 5 mL aliquots - 3X deion. H2O blanks, 3X analytical and quality control STDs, and 1X each sample into culture tubes.
Add 0.2 mL phenol, vortex mix, 0.2 mL nitroferricynide, mix, 0.5 mL oxidizing solution, mix.
Let stand in the dark 1 hour.
Read extinction at 640 nm on spectrophotometer.


Operation of Spectronic 20D+ Spectrophotometer

Turn on & warm up for 15 minutes.
Set wavelength to 640 nm.
Set filter lever to the right.
Set 0% T
Change mode to Absorbence.
Insert nanopure water blank.
Set to 0 Absorbence.
Insert sample, read, and record in notebook.


Operation of  HP8452A Diode Array Spectrophotometer

Computer will not boot up with printer on.
General Scanning
Files-F6
Load parameter file.
"Ammonium"
Sample Input-F8
Edit Sample Table
	Build STD & sample table here
Measure blank (H20 in cuvette)-F2
Measure sample-F1
At end, Hardcopy-F9, to print results.

"Ammonium" parameters
F3 - Functions
	Set Fn1 at 640 nm.
F5 - Options
	Wavelength list mode
	Single spectrum
	F8 - Sample input
	Manual input
	Use sample table


Notes
Experience here have shown that, (1) wearing gloves and (2) acid rinses, followed by RO and Nanopure rinses, of all glassware just before use, keeps both blanks and variability low.  Analytical standards left refrigerated overnight will degrade significantly.    


From McCarthy's lab, ideas for keeping blanks low- add 5 mL deion. H2O to culture tubes.  Add solutions as above, vortexing after each, discard contents, rinse twice with DDW, add 5 mL sample, vortex & discard.
No need to cover tubes while in the dark, sinse ammonia cannot be complexed after the addition of oxidizing solution.
Best to add reagents away from light, as brief exposure to light will yield high and variable blank.
Everything washed in dil HCl and rinsed multiple times with DDW.
Wear gloves. 
