TITLE: Lachat: NO3 Protocol
AUTHORS: USDA

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Lachat NO3 Protocol.</div></div>

[STEPS]
?. Assemble all reagents/standards and check all reagent/standard levels to ensure you have enough to complete your analysis.IF STANDARDS AND SAMPLE WERE REFRIGERATED ALLOW >30 MIN TO WARM2 Reagents, Marked NO3/NO2Sulfanilamide Color ReagentAmmonium Chloride Buffer1 Carrier. WILL ALWAYS MATCH YOUR EXTRACTANT. 2M KCl orDI Water 1 Carrier. WILL ALWAYS MATCH YOUR EXTRACTANT. 2M KCl orDI Water
?. Set up the pump by attaching all the tubing from the Nitrate/Nitrite manifold. Inspect tube lines to make sure they are seated between the plastic guide rails. 3 lines will be used for the Nitrate/Nitrite manifold (2 reagents, 1 carrier)1 Sample line1 Needle rinse line
?. Place reagent and carrier lines into the DI and start the pump.
?. Open the Omnion software by double-clicking on the Icon on the desktop.
?. Now click open on the toolbar. Then open folder path C:\Program_Files\Lachat\Omnion\Data\Methods\Nitrite-Nitrate
?. Choose the appropriate file for the experiment.
?. DO THESE SAMPLES NEED TO BE DILUTED WHEN ANALYSING NO3?Did anyone else run dilutions today? The software will start in the dilution rack based upon the last dilution tube used while the software was still opened.
?. If yes, fill up dilutor bottlesDI water in the DI Dilutor bottle Extractant Dilution Solution -- Same as carrierEnsure the dilutor is ready for the run by clicking on configuration on the toolbar, followed by autosamplers. This will open up a menu where you can prime the dilutor for operation.After priming, decide if you want a manual/forced ADF or a ADF based upon % from StandardsThe manual dilutes regardless of data output (TYPICAL)The ADF will add additional samples with dilutions based upon the output being outside a parameter. (ATYPICAL)After priming, decide if you want a manual/forced ADF or a ADF based upon % from StandardsThe manual dilutes regardless of data output (TYPICAL)The ADF will add additional samples with dilutions based upon the output being outside a parameter. (ATYPICAL)With either setting take the time to prepare enough tubes in the dilution rack to prevent sample from being ejected onto the tray because of a missing test tube
?. If no, proceed to #6.
?. Once the DI water has flushed the lines remove each line from the DI water. Wipe the line and inlet with a Kimwipe to remove any excess water.
?. During the stabilizing time while air bubbles and reagents are flowing through the Lachat system place your samples into the rack base.
?. After you have a stable baseline you can Engage the cadmium column.
?. Before starting the analysis double-check the following:The NO3 standards are in the standardsrack and ordered from 20ppm to 0ppm.The NO2 standards are also in the standard rack.NO3 and NO2 are set to run as unknowns after NO3 standards are run. The values of these unknowns are used to calculate reduction efficiencyThe sample rack is optimally seated with the individual test tubes being aligned to prevent obstruction to sampling needle. The column is engaged for NO3. Samples are in the correct order in both the rack and software.
?. Unclick the preview button and start the run.
?. Once the run is started, check your standards with the calibration curve.
?. Watch the autosampler enter the first sample and adjust sample tubes as needed
?. Once your run is completed export the data by clicking on the run tab found in run properties window. This will populate an Excel file with the run data. Find the raw data (Lachat Raw Data) folder on the desktop and open it.
?. Remove the reagent lines slowly while wiping the line and inlet with a Kimwipe to remove excess reagent/carrier.
?. Remove the reagent from the DI water, wiping the line and inlet with a Kimwipe to remove excess DI water.
?. You can now close down the Omnion software and shutdown the power to the system by hitting the breaker on the power strip.
?. Use a new clean Kimwipe with each line to prevent contamination.
?. Take caution to ensure the lines do not splash or drip reagent or carrier.
?. Place lines into DI for a minimum of 15 min flushing to clean the lines.
?. Before placing the rack double check the order of the samples and compare this order of the Sample ID column in Omnion.
?. Next check the cup ID column. The number should go in numerical. It is important to check this column when amending an old run setup and adding new samples.
?. Check that replicates are included in the run order and occur at least once for every 10 samples.
?. Sort the data in a descending chronological order by clicking on the data modified title column and find your run.
?. Open the file and use Save As to copy the file under a logical name in the Lab Studies or corresponding folder location.
?. ******BEFORE REMOVING THE LINES FROM REAGENTS TURN CADMIUM COLUMN OFFLINE******
?. Continue to run pump pushing air through the line for 15 minutes to dry the lines.
?. Visually inspect the entirety of the lines to ensure they are dry.
?. Make sure all lines have proper flow by visually watching the fluid flow through the line.
?. If you miss the flow momentarily remove the line and watch the resulting air flow.
?. Ensure that all lines are flowing allow >10min for the DI water to flush the lines.
?. Watch lines for proper flow.
?. If you miss the flow momentarily remove the line and watch the resulting air flow.
?. Ensure that all lines are flowing allow >10min for the reagents to run through the lines and come to a stable baseline.
?. Use the preview button to monitor the voltage output. Make sure to give ample time and visually inspect that the baseline is stable and at an acceptable value.