NITRATE ANALYSIS
SHANNON POINT MARINE CENTER
MAY 1998
M. STROM

Autoanalyser and Software: Alpkem RFA-300, using Softpac Plus 1990, with DOS 5.0

ANALYTICAL AND QUALITY CONTROL STANDARDS:

Primary (1) STDs - both analytical and quality control:
10 mM Potassium Nitrate (FW=101.11g/mole) solution (0.253g/250mL Nanopure H20 with 2 drops chloroform).  Store in refrigerator.

Secondary (2) - Analytical STDs:
 
2 STD (?M)
?L 1 STD / 50 mL H20
0
0
10
50
20
100
30
150
40
200

Secondary (2) - Quality Control STD:
25
125
 

ANALYTICAL REAGENTS:
(from Alpkem RFA Methodology pub. A303-S170)

Stock Solutions:

 Imidazole Buffer - 0.1 M
3.40 g imidazole (FW 68.08) / 500 mL H20.  Dissolve imidazole in 400 mL H20 in a beaker with a pH electrode and stirrer.  Adjust the solution to pH 7.5 with conc. HCl.  Transfer to 500 mL vol. flask and dilute to mark.  Store in refrigerator.

Cupric Sulfate - 0.01 M
0.625 g cupric sulfate (FW 249.69) / 250 mL H20.  Store at room temp, stable indefinitely. 

Cupric Sulfate - 2% w/v
Dissolve 5 g cupric sulfate in 250 mL H20.

(SAN) Sulfanilamide 
Add slowly 50 mL conc. HCl to 350 mL H20 in 500 mL vol. flask.  Dissolve 5 g sulfanilamide (FW 172.21) and dilute to mark.  Store at room temp.

(NED) N-(1-naphthyl)ethylenediamine Dihydrochloride 
Dissolve 0.5 g NED (FW 259.18) in 500 mL H20 in vol. flask.  Store solution in brown bottle in refrigerator.

Working Reagent Solutions:

Imidazole Buffer:
Stock Imizadole Buffer - 50 mL.
Nanopure H20 - 50 mL.
Cupric Sulfate (0.01 M) -100 ?L.
Brij-35 - 2 drops.

SAN:
Stock SAN - 50 mL.
Brij - 35 - 2 drops.

NED:
Use stock solution.

INSTRUMENT SETUP
Make up the working solutions in small Erlenmeyer flasks (50 mL for the SAN & NED, 100 mL for the imidazole), set them on the analyzer, and insert the proper line in each.  Fill the sample wash reservoir with nanopure water.
Turn on the analyzer, stretch the lines across the pumps, and put the pump heads on.  Let the reagents pump while the cadmium column is being activated.
Turn on the nitrogen gas, adjusting the regulator so that there is good but not excessive flow out the small spill tube on the side of the syringe body (check with a vial filled with water).

Cadmium Column Activation
Mix 10 mL each of stock imidazole buffer and 2% (w/v) cupric sulfate in a 25 mL beaker.   Detach the outlet sleeve from the glass tubing coil and attach it to the syringe. Put the glass coil into the beaker and draw 5 mL through the column into the syringe.  Let the copper solution sit in the column for 2 minutes, then remove the syringe and empty it.  Draw copper through the column twice more, letting it sit 2 minutes each time, then draw in 5 mL of imidazole buffer.

Hook the cadmium column to the analyzer, with the intake side upstream.  Let the reagents pass through the column for 5 - 10 minutes until the baseline settles down.

With the reagents pumping through, set both the reference and sample signals to 5.00 with the fine gain knobs.  The resultant absorbence should be close to zero, preferably slightly positive.  The Standard Calibration level has been about 400 (at this level the 40 ?M STD is ~90% of full scale.

At this point, the carousel can be filled with the standards and the thawed and shaken samples, and put in place.  Set the Sample Number on the AA, and push Reset.  The instrument is ready to start, after the computer files are set up.   
 
FILE SETUP FOR A TYPICAL SET OF LOCAL MARINE SAMPLES
F3 - METHODS:
everything stays the same except:
Channel name = Nitrate
Cycle time = 50 (10 sample/40 wash) seconds.


F4 - SAMPLE TABLE
Typical sample table:

1
Scale (40 ?M STD)
2
Scale
3
Scale
4
blank (nanopure H20)
5
blank
6
S1 (0 ?M)
7
S2 (10 ?M)
8
S3 (20 ?M)
9
S4 (30 ?M)
10
S5 (40 ?M)
11
blank
12
QC STD
13...
Samples
last
blank

To save: Alt-S (use date as table name, i.e.. 26June98)

F7 - STANDARD TABLE
Units in ?M , S1-S5 = 0 - 40 ???(see above).

After everything is set up, Alt-1 turns on channel 1.

F5 - DISPLAY ANALOG SIGNAL

F3 - START DISPLAY
Let the baseline run for a few minutes, then start the run (START on the AA).  Watch for the Scale peaks to show up, and adjust the STD Cal knob so that the peaks are ~ 95% of full scale.

POST-RUN
After the run is finished and all the peaks have shown up, push ESC, then ALT-1, S (stop) will turn off the nitrate channel.

F8 - CALCULATIONS (twice)
From Raw Data - ENTER
from Current Data File - ENTER
list of parameters - these work - ENTER
A trace of the peaks shows up.  At this time peaks can be added or deleted, and a plot of the peaks can be made (F3).  Once the peaks are properly identified,

F9 - CONTINUE
A calibration curve is plotted, and can be edited and plotted.
 
F9 - CONTINUE
A list of everything in the run appears, with the calculated nitrate concentrations.



F9 - SAVE AND EXIT
Returns to main menu.

F6 - SPREADSHEET
Loads spreadsheet report.
Alt-F brings up File menu
Print, Enter, N, N (report will print out)
Alt-F, Quit, back to main menu.

F10 - EXIT
Turn off computer & printer.
           
SHUTDOWN
Shut off the pumps and remove the cadmium column, replacing it with the tubing bridge.  Put all the lines in a beaker with H2O and Brij-35, and let the system pump for 10 minutes.  Attach the upstream end of the cadmium column to the glass coil and draw 5 mL of imidazole buffer through it with the syringe.  Attach the downstream end to the coil and store.  Remove the pump heads and slack all the tubing.  Shut off power, and TURN OFF THE GAS.

TROUBLESHOOTING
To date, the major problem with the system has been poor response and/or peak resolution, both of which have been caused by degeneration of cadmium columns.  The 10/40 cycle gives less than 5% peak carryover (probably >1%).  If the peaks start getting broader, shorter, with poor resolution, take out the Cd column and run a 40 ?M potassium nitrite STD through.  If the shape/response/resolution looks more normal, try reactivating the column first, then resurrecting it with HCl (RFA Engineering bulletin #A1287) or replacing the column. (I haven't had much luck with the HCl).   I have used working reagents over a month old kept sealed with parafilm in the refrigerator, with a slight decrease in response (i.e. a higher STD Cal setting).  The stock reagents last until they are used up - over a year.  Routinely check the tubing for leaks and bubbles.  The tubing sizes are changed slightly from those recommended, and a de-bubbler/re-bubbler has been added, which helped with baseline noise. 
