20080868 Ver 2_Supplement 1 Amended Appendix F of the 2011 Creeks Study Plan_20210701

Supplement 1
Amended Appendix F of the 2011 Creeks Study Plan
Water Quality Analysis Methods

CHLOROPHYLL a PROCEDURE
(Fluorometric Analysis)
References
April 2020
Welschmeyer, N.A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll b
and pheopigments. Limnology and Oceanography. 39(8):1985-92.
Arar, E.J. and Collins, G.B. 1997. Method 445.0: In Vitro Determination of Chlorophyll a and
Pheophytin a in Marine and Freshwater Algae by Fluorescence. U.S. Environmental Protection Agency.
pp.1-22.
Standard Methods for the Examination of Water and Wastewater. 22nd edition. 2012. Standard
Methods for the Examination of Water and Wastewater. American Public Health Association. Pp. 10-22.
Strickland, J.D.H. and T.R. Parsons, 1968. A Practical Handbook of Seawater Analysis. Pigment
Analysis. Bulletin 167. Fisheries Research Board of Canada. pp. 185-200.
Parsons, T., M. Yoshiaki, and C.M. Lalli. 1984. A Manual of Chemical and Biological Methods for
Seawater Analysis. Determination of Chlorophylls and Total Carotenoids: Spectrophotometric Analysis.
Pergamon Press. pp. 101-106.
Procedure: Filtering (This is usually done beforehand, and filters are in freezer waiting to be
ground, if so then skip to Grinding.)
Filter through a 4.7cm diameter Whatman 934 Filter in subdued light as soon as possible after
collection or within 24 hours of collection. Filter through a Whatman 934-AH pre-combusted glass
fiber filter. Shake bottle of unfiltered sample water vigorously and pour a measured quantity (500mL
or less) into the filtering apparatus. Record volume filtered on the outside of the foil packet and the
lab sheet. The vacuum pressure should not exceed 10 in. Hg because excessive pressure can rupture
cells and as a result pigment will be pulled through the filter into the suction flask. For the same
reason keep the filtration time to a minimum (<1 min.). Do not filter to dryness. Fold filter in half so
that filtrate is on the inside of the fold. Place filter in foil packet, fold edges and store in a —20°C freezer
in a Ziploc bag until processing. Can be stored up to 4 weeks without significant loss of chlorophyll a.
Procedure: Grinding
Reagents
90% Acetone
Dilute 900mL of 100% spectrophotometric grade Acetone (CAS# 67-64-1) to 1000mL with distilled or
de -ionized water. The acetone is stored in the Flammables Cabinet.
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Equipment and supplies needed to grind:
• Black rubber ice cooler w/ 2 small ice packs on bottom, covered w/ paper towel. Foils to be
placed inside after checking for volumes and order with lab sheet and Observation sheet.
• Sample box w/ 1 large ice pack on bottom and 1 medium ice pack on side. Cover with paper
towel and place empty blue rack inside.
• Chl a Lab Sheet
• Foils with frozen filters
• Blue rack with numbered, marked and salted sample tubes
• Caps
• 90% acetone
• Red squirt bottle
• Sonics Ultrasonic probe/meter
• 2 pair forceps
• Kimwipes
• Sharpie
• Centrifuge
1. Set up data sheets (tube numbers and sample volumes) and centrifuge tubes (best to be
prepared earlier). Mark tube at 12mL and add a small amount salt (half of the scoopula that is
inside the Sodium Chloride (NaCI) bottle). The salt will help precipitate glass fragments
generated from the filters being pulverized.
2. Use either earplugs or earmuffs (located in cabinet under HPCL). In subdued light masticate
filter with ultrasonic probe until it has disintegrated (meter set for 30 seconds). Place filter in the
centrifuge tube and push to bottom. Squirt enough 90% acetone to cover. Gently move the
sample tube up and down using the probe to move the filter around and reduce it to a pulp.
Rinse the probe into the tube with 1-2mL of 90% acetone. Adjust volume in the centrifuge tube
to 12mL with 90% acetone. Cap. Gently invert the centrifuge tubes a couple of times to mix.
3. Place the centrifuge tubes in sample box (with ice packs) and close lid. This prevents the samples
from heating up and degrading the chlorophyll to phaeophytin. After all of the samples are
ground, place box of ground samples in the cold room for at least 2-24 hours.
4. Turn OFF Ultrasonic probe meter. Clean workstation.
Procedure: Centrifuging - (REMAINS ON AT ALL TIMES, Temp set at 0°C, Lid securely latched)
1. After samples have sit in cold room to steep for 2 -24 hours, they can be taken to S-116 and
placed in centrifuge (in subdued light). Keep in order and make sure the numbers have not
been smeared by acetone, renumber if needed. The centrifuge buckets must be loaded equally
for balance, use extra blanks if needed. Make certain that the lid is securely latched.
2. Centrifuge for 30 minutes at 2800g. When timer is set the centrifuge will automatically begin to
move. Make sure it gets up to speed.
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3. Put ice packs in chest freezer to keep frozen.
4. After 30 minutes, return ice packs to box and very carefully unload samples from centrifuge
into box using care as not to disturb samples.
5. Once in Chl a room, very carefully remove tops from all tubes, do not disturb samples.
6. Complete chlorophyll a measurements on Trilogy Fluorometer.
Set up work area for reading chl a: You will need the following-
• 90% Acetone
• Acetone waste jug w/ funnel
• Box of 3mL disposable pipets
• Trilogy Fluorometer
• Turner Solid Standard
• 2 sample cuvettes
• Kimwipes
• Chla Lab Sheet
• Trash can
Place paper towels in front of Fluorometer. Pipetting and reading can be done here. On work table next
to it, the cleaning of the sample cuvettes can be done. Measure Raw Fluorescence Unit and record on
both lab sheet as well as log in back of Manual.
Trilogy Fluorometer (this can be done while samples are in centrifuge)
1. Turn ON machine (switch is on the back left side)
2. On touch screen Select chla-NA. (Non-acid chlorophyll module).
3. Confirm module. Select OK.
4. Open Lid. Remove cuvette holder. Place Solid Standard into the machine with the lip/tip to the
back. You will now make 10 measurements to warm the machine up:
• Select Tools by touching the screen. Select Settings. Select Continuous Sampling. Change
the Continuous Sampling to ON
• Frequency of measurements/second should read 1/3
• Total number of measurements should read 10
• Touch OK
• Touch Measure Raw Fluorescence (C-Sampling) Machine will sample 10 times over the set
period of time. Record RFU on lab sheet and in log in manual. The RFU should around
218.51. If not contact the Lab manager. RFU CANNOT be <174 or >262.
5. Before calibrating or continuing, you must Select Tools, Select Settings, and Select Continuous
Sampling and turn OFF Continuous Sampling.
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6. Press OK
7. Place Solid Standard back into its box and return cuvette holder to position.
Reading Samples
With a kimwipe, rinse cuvettes 3x with 90% Acetone making sure that the outside is clean as well. Shake
off any excess Acetone before filling with sample. Hold cuvette with kimwipe. Using a 3mL disposable
pipet carefully transfer sample into cuvette. (Hold sample and cuvette in one hand and transfer sample
with pipet in other.) Do not disturb sample, hold up to subdued light to make certain no filter is being
transferred. Place into Fluorometer and proceed as follows:
1. Select Calibrate
2. Select Use Stored Calibration.
3. Highlight 7Sept2018 and hit Select.
4. Select measure fluorescence. Read a blank (90% Acetone) as a sample first. Use 12mL for both
"volume of filtered sample" and "volume of solvent" when you are reading blank samples.
5. Enter volume of filtered sample water in mL followed by volume of solvent (12mL) used in mL.
It will then automatically measure and calculate fluorescence in µg/L. Record on data sheet.
Make sure readings are in correct units (µg/L).
6. Remove sample and pipet next sample while helper wastes old sample and cleans cuvette for
next sample. Continue until all samples are read.
7. Turn OFF when finished!
Calibration THIS IS PERFORMED BY LAB MANAGER.
NOTE: Should calibrate when there has been an adjustment made to the instrument. At least
once per year —
1. Select/push calibrate button.
2. Select Run New Calibration.
3. Select µg/L (micrograms per liter).
4. Insert acetone blank into test tube holder (blank is 90% acetone).
5. Select OK.
6. You can choose to calibrate with up to 5 standards (0.2, 2, 5, 20, & 200µg chl a/L per EPA 445 or
2, 6, 20, & 60µg chl a/L per SM 10200 H 3). You can also choose to calibrate with up to 2
standards (Fluorometric Chlorophyll Standards from Turner Designs (Part # 10-850). Enter
number of standards.
Remove acetone blank and test tube holder.
7. Insert first Standard and enter concentration.
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8. Select enter more calibration standards and repeat until all desired standards have been
entered.
9. Select proceed with calibration and click YES to save calibration.
10. You can either select to name the calibration curve or not. If you select to not name the
calibration, then the machine will proceed with TEMP name.
11. Measure the solid standard to verify the standard is close to the expected reading. If the
standard readings are vastly different than the expected reading, then one may choose to
calibrate again.
12. Remove Solid Standard and place back in box holder.
Procedural Note:
The Welschmeyer method is a simplified way to measure chlorophyll a without the need for
acidification. Based on the research of Dr. Nicholas A. Welschmeyer, the procedure is sensitive
enough for oligotrophic environments; and only a single fluorescence determination is required.
Detection limit
Minimum detection limit: 0.025 µg/L
Linear range: 0 - 300 µg/L.
April 2020
Total and Dissolved Kjeldahl Nitrogen (DKN/TKN) — Automated SmartChem Method
References:
EPA. 1979. Methods for chemical analysis of water and wastes. EPA-600/4-79-020. Method 3651.1
and 365.3-4. Environmental Monitoring and Support Laboratory, Cincinnati, OH, USA.
SmartChem 200 Method 390-200E, revised July 2008. Westco Scientific Instruments, Inc.
Standard Methods 4500-Norg. Nitrogen (Organic) (22nd Edition). American Public Health Association,
American Water Works Association, Water Environment Federation. pp. 4-131/135.
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Digestion Reagent: (*Should already be made and in Kjeldahl cabinet)
1. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in S106.
2. Digestion reagent (Mercury free): (AKA "BLUE JUICE") Can make one 2L or more. Easier to weigh
out both the Cupric Sulfate and the Potassium Sulfate first (x 3). Use three 2L volumetric flasks
found under digestion block.
a. In the fume hood dissolve completely 9.5g Cupric Sulfate (anhydrous; CuSO4; CAS 7758-
98-7) in each of the 2L volumetric flasks. Using a full squirt bottle, rinse weigh boat and
funnel, then add the remainder of the bottle to the flask (approx. 500 mL). Swirl to
completely dissolve.
b. Add 268g Potassium Sulfate, (K2SO4; CAS 7778-80-5) which will only partially dissolve.
Repeat the squirt bottle step above. Swirl to mix.
c. Set up an ice bath in the fume hood using a white tray half full of ice.
d. Measure out 268mL concentrated Sulfuric Acid using both a 250mL, and a 25mL
graduated cylinder marked at 18mL.
e. Place volumetric flask on ice, and slowly add the concentrated Sulfuric Acid (H2SO4;
CAS7664-93-9) to the volumetric flask 5-10mL at a time while continuously swirling —
Will get very, very hot! There is a fine line between too hot and too cold, you want to
keep it warm enough to get the chemicals to dissolve and not so cool that the chemicals
will come out of solution. Continue adding the acid, swirling and mixing. Can use some
reagent water if gets too hot. Once everything is dissolved take it out of the ice bath and
allow to cool to room temperature. Using dei water that is at room temperature slowly
bring up to volume while mixing. The volume can fluctuate so be careful not to add too
quickly and make sure that the temp of reagent water is the same as what you are
adding to. If making more than 1 batch at a time, mix batches together and aliquot
among the four designated 4 liter bottles in Kjeldahl cabinet. Mix thoroughly. STABLE.
3. Standard:
500µM Ammonium Chloride: From the 10,000µM Ammonium Chloride that is kept in
fridge pour out approximately 20mL into a beaker and leave covered with parafilm to
warm to room temperature. Using a 10mL glass volumetric pipet, rinse and then pipete
10mL of the Ammonium Choride to designated 200mL volumetric flask and bring to
volume with reagent water. Mix thoroughly. Good for 1 week, store in fridge.
**To make the 10,000µM Ammonium Chloride (NH4CI; CAS 12125-02-9): Weigh out
0.5349g of NH4CI and dilute to 1,000mL in volumetric flask with reagent water. Mix
thoroughly. Transfer to 1L Pyrex bottle with screw cap and store in fridge. Good for 6
months.
4. Quality Control (QC's):
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a. 89.2211M N: (Using the 1000µL pipet, dilute 0.250mL of the Certified SPEX Certi Prep
Ammonia Nitrogen 1,000µg/mL (=71,327µM) (CAT# AS-NH3N9-2Y) to 200mL with
reagent water in designated volumetric flask. Mix thoroughly. Prepare fresh daily.
b. 178uM N: (Using the 1000µL pipete, dilute 0.5mL of the Certified SPEX Certi Prep
1,000µg/mL (=71,327µM) to 200mL with reagent water in designated volumetric flask.
Mix thoroughly. Prepare fresh daily.
c. 356uM N: (Using the 1000µL pipete, dilute 1mL of the Certified SPEX Certi Prep
1,000µg/mL (=71,327µM) to 200mL with reagent water in designated volumetric flask.
Mix thoroughly. Prepare fresh daily.
Digestion Block Procedure: ***NEVER LEAVE RACKS OF KJELDAHL TUBES ON CARTS!
Use Kjeldahl tubes that were baked in drying oven (105°C) for at least 4 hours (usually overnight). Tubes
are kept on wooden shelf, should be in order with boiling chips added and covered w/saran wrap. Racks
and Digestion Block hold 40 tubes.
1. Add —11 boiling chips (Henger granules part # 136-CC sieved through #16 Mesh) to each Kjeldahl
tube.
2. Prepare lab sheet with tube numbers and sample ID's. (Duplicate whatever fits in rack of 40— ie;
9 dups for 25 samples.)
3. Measure 25mL of sample (x2), with graduated cylinder that has been rinsed 15 times with fresh
reagent water, and add to tubes. (EX: A full set of 25 CZR samples - will only be able to duplicate
9 samples, the rest will be singles plus 2 Blanks (dei water), 3 QC's and Standard = 40 tubes, in
that order.)
4. With another freshly rinsed (x15) graduated cylinder measure 25mL of reagent water for blanks,
add to tubes (x2).
5. Measure 25mL of each Q.C. (89.22µM, 178µM and 356µM), in order from low to high, and add
to tubes.
6. Measure 25mL of the 500µM Standard and add to tube.
7. Add 10mL of Kjeldahl Digestion Reagent to all samples/blanks/QC's/Std tubes using designated
repeat pipettor, kept in Kjeldahl cabinet. Be careful not to spill/splash digestion reagent, use
goggles.
8. Cap all tubes with green stoppers, kept in wall of drawers.
9. Wipe each tube with damp towel and dry completely. Place tubes in digestion rack as each row
is wiped and dried. Make certain that a green tray is under the rack and a "Work in Progress"
(W.I.P.) card is attached. Also record what samples they are. Ex: "CZR4Mar17 (#1-40)" on
bottom of card. Can let sit overnight to digest next morning.
10. Take green stoppers out of tubes and place in tan tray in order, to replace in order.
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11. Put on two side panels, kept inside digestion fume hood.
12. Lift up over head to check bottom of tubes for liquid, cracks, and boiling chips.
13. Place the rack into the digestion block very carefully making sure that all tubes go into the block
simultaneously. Close sash on hood.
Set Digestion Control Box: Turn ON, switch on right side of box. Make sure display reads "J1" when first
turned on and then the current temperature is displayed.
To Program: (On upper part of the display with ramp information)
Press TEMP key, Enter 210 and press ENTER;
Press TIME key, Enter 1.8 and press ENTER;
Press TEMP key, Enter 385 and press ENTER;
Press TIME key, Enter 1.5 and press ENTER.
Press START/STOP key.
Make sure all 6 lights turn on and stay on. If not, turn controller off and start over.
NOTE: Set timer for 28 minutes. After timer goes off put on orange heat resistant gloves (temp. should
be—160°C) and watch for liquid bubbling to the top of the tubes. If bubbling does occur CAREFULLY
lift digestion rack —1 inch off the bottom of the digestion block (enough for bubbling to cease reaching
the top of the tube). May have to keep lifting rack until boiling calms down. DO NOT ALLOW THE
TUBES TO BOIL OVER AS THIS WILL LIKELY CAUSE THE TUBES TO EXPLODE!
14. The controller will beep twice when the digestion has completed (about 3.5 hours total).
Wearing orange heat resistant gloves, CAREFULLY remove the two side panels and lift the
digestion rack with tubes and place on the metal tray inside the fume hood. Be careful the
tubes do not hit anything in the hood.
15. Set a timer for 9 minutes. During this time, fill the designated brown square repeat pipeter
(kept in Kjeldahl cabinet) with fresh reagent water and prime. Check to see if it is set to 25mL.
16. When timer goes off, pipet 25mL of reagent water into each of the sample/standard/blank/QC
tubes. NOTE: pipeter will only reach 3 tubes deep, so the rack needs to be rotated to reach the
other % of the tubes. There will be tubes in two middle rows that you cannot access.
17. Remove rack from fume hood and place on bench top. Remove the tubes water was not added
to and put in blue rack. Add water and return to digestion rack. Cap each tube with a rubber
stopper.
18. Using the vortex genies, mix each tube about 30 seconds each. Return tubes, in order, to the
blue rack inside the green tray.
19. Samples need to be mixed 3x with an hour between mixes before they can sit overnight and be
read the next morning.
20. If a second digestion is planned for the same day, turn OFF controller. Turn back ON. Allow the
digestion block to cool to 160°C., about 2-3 hours with fan blowing into hood. If tubes have been
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wiped down Repeat Steps #11 through #14, EXCEPT timer to be initially set to 10 minutes
because block is already hot. After timer goes off check temp and watch for bubbling until sure
they are calm and continue with Steps #15 through #21.
SmartChem Reagents:
1. Stock Buffer Solution: (Should already be made and stored in a 1L Pyrex Bottle with screw cap in
TKN/DKN/PKN tray.)
OR to make:
In 1L volumetric flask dissolve 134g of Sodium Phosphate, Dibasic Heptahydrate (Na2HPO4.7H2O;
CAS 7782-85-6) in approximately 350mL of reagent water. Add and dissolve 20g of Sodium
Hydroxide, add more reagent water, let cool and then dilute to 1L. Mix thoroughly. STABLE. Store
in designated 1L Pyrex bottle with screw cap in TKN/DKN/PKN tray.
2. REAGENT #1: Working Buffer Solution: (Should already be made and stored in a 1,000mL Pyrex
Bottle with screw cap in TKN/DKN/PKN tray.)
OR to make:
In a 1 Liter volumetric flask, combine the reagents in the following stated order:
• 200mL of Stock Buffer Solution
• 250mL of the 20% Sodium Potassium Tartrate solution
• Mix by swirling.
• Add 120mL of the 20% Sodium Hydroxide solution
• Mix by swirling.
• Dilute to 1 Liter with reagent water
• Mix thoroughly.
• Sodium Hydroxide, 20%: (For Reagent #1) (Should already be made and stored in
1,000mL Pyrex bottle with screw cap in Bases cabinet.
OR to make:
Under hood in a 1 Liter volumetric flask filled to approximately 800mL of reagent water,
slowly
add 200g of Sodium Hydroxide (CAS 1310-73-2). Excessive heat will be generated. Mix
well to
dissolve. Cool to room temperature and dilute to 1 Liter with reagent water. Mix
thoroughly.
STABLE. Stored in 1,000mL Pyrex bottle with screw cap in Bases cabinet.
• Sodium Potassium Tartrate, 20%: (For Reagent #1) In designated 250mL volumetric
flask (kept in TKN/DKN/PKN tray) filled to approximately 200mL of reagent water, slowly
add and dissolve 50g Sodium Potassium Tartrate Tetrahydrate
(KOCO(CHOH)2COONa.4H20; CAS 6381-59-5). Solution will become cold. Bring to room
temperature and dilute to 250mL with reagent water. Mix thoroughly. *Do not want
any NH4 in chemical.
3. REAGENT #2: Sodium Salicylate: (Stored in designated 250mL volumetric flask in TKN/DKN/PKN
tray.)
OR to make:
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In designated 250mL volumetric flask, kept in TKN/DKN/PKN tray, dissolve 5g of Sodium Salicylate
(2-HOC6H4CONa; CAS 54-21-7) in approximately 225mL reagent water. Add (very slowly and at an
angle to keep from foaming) 1.5mL of concentrated Probe Rinse Solution (Westco part # 3AS-
RN00-21) and dilute to 250mL with reagent water. Mix thoroughly. Store solution in same
volumetric flask covered with foil, inside TKN tray. Prepare fresh weekly. NOTE: wrap the small
plastic SmartChem reagent bottle containing this reagent with foil when used in the analysis.
4. *REAGENT #3: Sodium Hypochlorite Solution (Clorox): Pour about 10mL of the diluted 5.25%
Clorox into a 50mL beaker. In designated 50mL volumetric flask, add 3mL of the diluted 5.25%
Clorox, NaOCI) using a glass volumetric pipet. Add approximately 45mL of reagent water. Add
(slowly and at an angle to keep from foaming) 0.5mL concentrated probe rinse solution. Slowly
bring to volume with reagent water. Mix thoroughly. Prepare fresh daily.
5. *REAGENT #4: Sodium Nitroferricyanide Dihydrate Solution: In designated 50mL volumetric
flask, dissolve 0.4g Sodium Nitroferricyanide (Nitroprusside) Dihydrate (Na2Fe(CN)5N0.2H20;
CAS 13755-38-9) in approximately 40mL of reagent water and dilute to 50mL with reagent
water. Cover flask with aluminum foil and let dissolve. Mix well. Add 0.5mL of concentrated
probe rinse solution. Mix thoroughly. Prepare fresh every 2 days, refrigerate overnight. NOTE:
wrap the small plastic SmartChem reagent bottle containing this reagent with foil when used in
the analysis.
with
to
#1.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1L volumetric flask filled
^'950mL of reagent water, slowly and tilted to side add 0.5mL of Probe Rinse Solution (Westco). Fill
volume with reagent water. Mix thoroughly. Stable. Store at room temperature behind SmartChem
Keep a constant supply ready.
Cuvette Wash Solution: Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
In designated 2L container add reagent water to mark. Add 30mL (one bottle) of Unity Scientific
Cleaning Solution. Rinse 30mL bottle with reagent water and pour into 2L container, repeat 2
more times. Fill to volume. Mix thoroughly. Stable.Store at room temperature next to SmartChem
#1.
Procedure: *Remember to use Kjeldahl waste jug and perform a Wash and WBL first.
(NOTE: Set up the Reagents, empty cups, Std, QC's and Diluent, etc. before pipetting samples. When the
first rack of samples are poured the SmartChem can be started and the rest of the samples can be added.)
1. Samples: Using a 5mL automatic pipet, carefully pipette sample from digestion tube being
careful not to disturb bottom of tube. Use a clean pipette tip for each sample. Rinse a
SmartChem sample cup with a small amount of sample and discard into waste container. Fill
cups in duplicate and place in SmartChem sample rack (racks 1-5).
NOTE: All Kjeldahl waste has to be collected and disposed of via EH&S, use the waste beaker
while pipetting.
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2. Diluent: Using a 5mL automatic pipet fill the designated small plastic SmartChem bottle with the
Blanks,
alternating back and forth between the two tubes of Blanks. Usually about 3 times from each
tube. Mix.
3. Q.C.'s: Using a 5mL automatic pipet fill one cup with each of the Q.C.'s. Fill to top line on cups.
These will be placed in descending order in slots 2-4 on "Reag 2 Ctrl/Std" rack.
4. Standard/Spike: Using a 5mL automatic pipet fill two cups with the 500µM Standard (one is for
the Spike). Fill to top line on cup. This will be placed in cup 1 of the "Reag 2 Ctrl/Std" rack.
The Spike will be placed in 5th slot of "Rgt 1" rack in a supporting half bottle.
5. Place 6 empty sample cups in positions 1-6 in SmartChem Sample rack 5. These cups will be
used to
generate the standards for the standard curve.
6. Reagents: (All reagents are placed in "Reag 2 Ctrl/Std" rack) Do not overfill bottles, pop any
bubbles.
a. Position #1 - DKN Working Buffer (TKBU)
b. Position #2 - Sodium Salicylate (TKSA)
c. Position #3 - Clorox (TKHY)
d. Position #4 - Sodium Nitroprusside (TKNI)
NOTE: Fill each small plastic SmartChem reagent bottle only to shoulder of bottle. If they are
too full or there is a bubble present the SmartChem will register it as "empty".
7. Check fluid levels in Cuvette Wash Solution, DEI, and Probe Rinse Solution bottles, refill if
necessary. Keep an eye on Probe Rinse as this needs to be filled regularly.
SmartChem:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select "TKNH-RgtsMoved+ExtraBuffer" from the "Profile list".
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the empty cups for standard dilutions.
******NEVER LEAVE RACKS OF KJELDAHL TUBES ON CARTS!
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "print" and selecting "print" again. Then
press Exit.
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2. Real time sample data can be displayed by selecting the Results button under the cuvette wheel.
Once all samples have been run, the print option will appear in the upper left menu bar, clicking
the button will automatically print out the results.
Cleaning:
1. All samples, reagents, standards, QC's, and blanks must be collected for pick-up by EH&S.
Collect these into designated hazardous waste bottles.
2. SmartChem sample cups can be disposed of in the trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and inverted
on rack/paper towel to dry.
4. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack above sink
next to SmartChem to dry.
Principle:
Total Kjeldahl nitrogen is the sum of free ammonia nitrogen and organic nitrogen compounds which,
through digestion, are converted to ammonium sulfate. The method is based on the conversion of
the digested ammonium sulfate to ammonia. In the presence of sulfuric acid, potassium sulfate, and
a cupric sulfate catalyst, amino nitrogen of organic materials is converted to ammonium. The
ammonium cation is then converted to ammonia by neutralization with a concentrated buffer. Once
converted to ammonia, an intensely blue compound, indophenol, is formed by the reaction of
ammonia with alkaline phenol and then hypochlorite (Berthelot reaction), catalyzed by sodium
nitroprusside. The color (absorbance) measured at 660nm is proportional to the Kjeldahl nitrogen
concentration in the original sample.
Nitrate + Nitrite (NOx) — Automated SmartChem Method
References:
April 2020
Standard Methods 4500-NO3- - E. 22nd Edition. 2012. American Public Health Association, American
Water Works Association, Water Environment Federation. pp. 4-125.
SmartChem 200 Method 375-100E-1, revised July 2008. Westco Scientific Instruments, Inc.
NOTE: Run Efficiency Test first, to check coil status.
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Reagents:
1. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in S106.
2. REAGENT #1: Ammonium Chloride-EDTA Buffer Solution: Dissolve 85g of Ammonium Chloride
(NH4CI; CAS 12125-02-9) and 0.1g of Disodium Ethylenediamine Tetracetate
(CioHi4N2Na2O8*2H2O; CAS 6381-92-6) in approximately 900mL of reagent water. Adjust the pH
to 8.5 with Concentrated Ammonium Hydroxide (NH4OH; CAS 1336-21-6), which is kept under
sink in Bases cabinet. Dilute to 1 Liter with reagent water. Store in 1,000mL Pyrex bottle with
screw cap, in Nitrate/Nitrite tray. STABLE, but check pH every few months and adjust as
necessary.
3. REAGENT #2: Color Reagent: In a 250mL volumetric flask with approximately 150mL of reagent
water, slowly add 25mL Concentrated Phosphoric Acid (H3PO4; CAS 7664-38-2). Cool to room
temperature. Add and dissolve 10.0g of sulfanilamide (4-NH2C6H4SO2NH2; CAS 63-74-1). Swirl to
mix. Add 0.5g of N-(1-napthyl) Ethylenediamine Dihydrochloride (NNED)
(C1oH7NHCH2CH2HN2*2HCL; CAS 1465-25-4) and dissolve. Slowly and at an angle to reduce
foaming add 2mL of concentrated Probe Rinse Solution (Westco part # 3AS-RN00-21) and slowly
dilute to 250mL with reagent water. Invert and mix well. Store in designated brown glass bottle
and keep refrigerated. Good for one week.
4. Nitrate Module Reservoir buffer solution, 30%: In designated 1L volumetric flask kept in
Nitrate/Nitrite tray dilute 300mL of the Ammonium Chloride-EDTA Buffer Solution (Reagent #1)
to 1L with reagent water. (This solution is used to flush the reduction coil between sample
analyses and is not used in the reaction chemistry.) Reservoir container is kept next to the
SmartChem, stored in white plastic 2L bottle at room temperature w/tube connected to
SmartChem. STABLE, but check pH every few months and adjust back to 8.5 as necessary.
Standards:
a. 10011M Combined Standard: (KNO3 + KH2PO4 + NH4CI) Using a volumetric pipete, dilute
10mL of the 1,000µM Combined Standard to 100mL in designated volumetric flask.
Good for 1 week, blue taped flask kept in fridge.
b. 5011M Combined Standard: Using a volumetric pipete, dilute 5mL of the 1,000µM
Combined Standard to 100mL in designated volumetric flask. Prepare fresh.
c. 1,000µM Sodium Nitrite: (NaNO2) Using a volumetric pipete, dilute 10mL of the
10,000µM NaNO2 Standard to 100mL with reagent water in designated volumetric flask.
Good for 1 month. Store in fridge.
d. 50µM Sodium Nitrite: (NaNO2) Using a volumetric pipete, dilute 5mL of the 1,000µM
NaNO2 Standard to 100mL with reagent water in designated flask. Prepare fresh.
Quality Control:
S-13

e. 35.711M N: In designated volumetric flask dilute 0.10 mL (100uL) of the Astoria Pacific
((1,000 mg/L NH3-N (=71,393 uM N) kept in Nitrite cabinet) to 200 mL with reagent
water. Prepare fresh daily.
f. 3.5711M N: In designated volumetric flask dilute 10mL of the 35.7µM N to 100mL.
Prepare fresh daily.
5. Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) Concentrate stored in S.C. cabinet. In
designated 1L volumetric flask filled with ^'950mL of reagent water, slowly and tilted to side add
0.5mL of Probe Rinse Solution (Westco). Fill to volume with reagent water. Mix thoroughly.
Stable. Store at room temperature behind SmartChem #1. Keep a constant supply ready.
6. Cuvette Wash Solution: Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
Concentrate stored in S.C. cabinet. In designated 2L container add reagent water to mark. Add
30mL (one bottle) of Unity Scientific Cleaning Solution. Rinse 30mL bottle with reagent water
and pour into 2L container, repeat 2 more times. Fill to volume. Mix thoroughly. Store at room
temperature next to SmartChem #1. Stable.
Procedure:
1. Pour or pipet approximately 3mL of sample into sample cup and discard. Pour or pipet 3mL of
sample into the cup and place in SmartChem sample rack (racks 1-5)
2. Diluent: reagent water. Place in "Diluent 1" spot
3. Standard and Q.C.'s:
a. Place a sample cup with —3mL of the 100µM Combined Standard in SmartChem "RGT 2
Ctrl/Std" rack, position 1 (NO3-N).
b. "RGT 2 Ctrl/Std" rack, place QC #2 in position 2. (35.7µM)
c. "RGT 2 Ctrl/Std" rack, place Q.C. #1 in position 3. (3.57µM)
d. "RGT 2 Ctrl/Std" rack, place 50µM Nitrite in position 4. (EFF2)
e. "RGT 2 Ctrl/Std" rack, place 50µM Combined Std. in position 5. (EFF)
f. "RGT 2 Ctrl/Std" rack, place reagent water in position 6. (CCB)
g. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5.
These cups will be used to generate the standard dilutions for the standard curve.
4. Reagents:
S-14

a. In "RGT1" rack, place Reagent #1= Ammonium Chloride-EDTA buffer in the Q.C.
position.
b. "RGT1" rack, place Reagent #2= Color reagent, in position 11.
c. "RGT1" rack, place extra Reagent #1 bottles in positions 12 and 13 .
5. Do Not Overfill Reagent bottles, fill just below shoulder and make sure there are no bubbles.
6. Check fluid levels in cuvette wash solution, DIW, and probe rinse solution bottles, refill if
necessary.
7. Collect waste in NOxjug, place in sink/tub.
Software Procedure:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select Profile List on middle of screen. Select "NO3-Nitrate Long Run" from the list of methods
at the bottom of the page.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the empty cups for standard dilutions.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "print" and selecting "print" again.
2. Real time sample data can be displayed by selecting the Results button under the cuvette wheel.
Once all samples have been run, the print option will appear in the upper left menu bar, clicking
the button will automatically print out the results.
Cleaning:
1. All reagents, standards, QC's, and blanks must be collected for pick-up by EH&S. Collect these
into Nitrite Hazardous Waste bottles.
2. SmartChem samples can be dumped down drain and cups disposed of in the trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and inverted
on rack/paper towel to dry.
S-15

4. Wash SmartChem racks with tap water and scrub with brush, hang on drying rack above sink
next to SmartChem to dry.
Coil activation/cleaning: (see SmartChem 200 method WCd-COIL, revised July 2008):
• Hydrochloric Acid, 2N: In a 500mL volumetric flask slowly add 83mL of Concentrated
Hydrochloric Acid (HCL; CAS 7647-01-0) to approximately 300mL of reagent water. Cool
to room temperature and dilute to 500mL with reagent water. Stable. Transfer to glass
container with stopper and store in Acid cabinet.
• Hydrochloric Acid, 6N: In a 100mL volumetric flask slowly add 50mL of concentrated
hydrochloric acid (HCL; CAS 7647-01-0) to approximately 30 mL of reagent water. Cool
to room temperature and dilute to100mL with reagent water. Stable. Transfer to glass
container with stopper and store in Acid cabinet.
• Copper Sulfate solution, 1.5%: In a 500mL volumetric flask dissolve 6.5g of
Copper Sulfate Pentahydrate (CuSO4*5H20; CAS 7758-99-8) in approximately
400mL of reagent water. Dilute to 500mL. Stable. Kept in glass container with
stopper in Nitrate Tray.
*Check that the NOx Reservoir Bottle is filled, the siphon tube in place, and that no air bubbles
are present in the delivery line.
*Collect waste in NOx coil waste jar.
1. On the main menu on the bottom right of screen press "Diagnostics".
2. Press the "NO3" tab.
STEP1:
• Check Box 2 (5N HCI) and enter 3 cycles.
• Check Box 3 (2% CuSO4) and enter 3 cycles.
• Press "Start".
STEP 2:
• Check Box 3 only and enter 6 cycles.
• Press "Start"
STEP 3:
• All boxes unchecked, Run "Prime" 5 times.
3. Press "Diagnostics" to exit.
4. Can run a second time if efficiency is still bad.
S-16

Principle:
This method determines the combined Nitrate (NO3) and Nitrite (NO2) present in the sample. This
sum is also known as Total Oxidized Nitrogen (TON) and is also referred to as NOX. Nitrate is reduced
to Nitrite by passage of a filtered sample through an open tubular copperized cadmium redactor
column. The resulting Nitrite plus any Nitrite originally present in the sample is then determined as
NO2 by diazotizing with Sulfanilamide followed by coupling with N-(Napthyl)-Ethylenediamine
Dihydrochloride to form a highly colored Azo Dye, which is measured colorimetrically at 550 nm.
Particulate Kjeldahl Phosphorus (PP, PKP) — Automated SmartChem Method
(Usually run as "PN,PP Method" (TKNM+OP4M))
References:
April 2020
EPA. 1979. Methods for chemical analysis of water and wastes. EPA-600/4-79-020. Method 3651.1
and 365.3-4. Environmental Monitoring and Support Laboratory, Cincinnati, OH, USA.
Standard Methods 4500-Norg. Nitrogen (Organic). 22nd Edition. 2012. American Public Health
Association, American Water Works Association, Water Environment Federation. pp. 4-131/135.
SmartChem 200 Method 420-200E, revised July 2008. Westco Scientific Instruments, Inc.
Digestion Reagent: (*Should already be made and in Kjeldahl cabinet)
1. Reagent water: Fresh deionized water from E-pure system (16-18 megohm/cm) in S106.
2. Digestion Reagent (Mercury free): (AKA "BLUE JUICE") Can make one 2L or more. Easier to
weigh out both the Cupric Sulfate and the Potassium Sulfate first (x 3). Use three 2L volumetric
flasks found in Kjeldahl cabinet.
a. In the fume hood dissolve completely 9.5g Cupric Sulfate (anhydrous; CuSO4; CAS 7758-
98-7) in each of the 2L volumetric flasks. Using a full squirt bottle, rinse weigh boat and
funnel, then add the remainder of the bottle to the flask (approx. 500 mL). Swirl to
completely dissolve.
b. Add 268g of Potassium Sulfate, (K2SO4; CAS 7778-80-5) which will only partially dissolve.
Repeat the squirt bottle step. Swirl to mix.
S-17

c. Set up an ice bath in the fume hood using a white tray half full of ice.
d. Measure out 268mL concentrated Sulfuric Acid using both a 250mL, and a 25mL
graduated cylinder marked at 18mL.
e. Place volumetric flask on ice, and slowly add the concentrated Sulfuric Acid (H2SO4;
CAS7664-93-9) to the volumetric flask 5-10mL at a time while continuously swirling —
Will get very, very hot! There is a fine line between too hot and too cold, you want to
keep it warm enough to get the chemicals to dissolve and not so cool that the chemicals
will come out of solution. Continue adding the acid, swirling and mixing. Can use some
reagent water if gets too hot. Once everything is dissolved take it out of the ice bath and
allow to cool to room temperature. Using dei water that is at room temperature slowly
bring up to volume while mixing. The volume can fluctuate so be careful not to add too
quickly and make sure that the temp of reagent water is the same as what you are
adding to. If making more than 1 batch at a time, mix batches together and aliquot
among the four designated 4 Liter bottles in Kjeldahl cabinet. Mix thoroughly. STABLE.
Standards:
• 500µM N/200µM P (Combined medium range standard): In designated 200mL volumetric
flask, using volumetric pipettes, dilute 10mL of the 10,000µM Ammonium Chloride (NH4CL)
Standard, kept in fridge, and 4mL of the 10,000µM Potassium Phosphate (KH2PO4)to 200mL
with reagent water. Good for 1 week.
Quality Control (QC's):
• 89.22µM N/26.3µM P: In designated volumetric flask, using the 1000µL pipet, dilute
0.250mL of the Certified SPEX Certi Prep Ammonia Nitrogen 1,000µg/mL (=71,327µM N)
(CAT# AS-NH3N9-2Y) and 0.5mL (500µL) of the Certified SPEX Certi Prep 1,000µg/mL
Phosphate (=10,525µM P)to 200mL with reagent water. Mix thoroughly. Prepare fresh daily.
• 1781.1.M N/52.611M P: In designated volumetric flask, using the 1000µL pipete, dilute 0.5mL of
the Certified SPEX Certi Prep Ammonia Nitrogen 1,000µg/mL (=71,327µM) and 1mL of the
Certified SPEX Certi Prep 1,000µg/mL Phosphate (=10,525µM P) to 200mL with reagent
water. Mix thoroughly. Prepare fresh daily.
• 356µM N/105µM P: In designated volumetric flask, using the 1000µL pipete, dilute 1mL of
the Certified SPEX Certi Prep Ammonia Nitrogen 1,000µg/mL (=71,327µM) and 2mL of the
Certified SPEX Certi Prep 1,000µg/mL Phosphate (=10,525µM P) with reagent water to
200mL. Mix thoroughly. Prepare fresh daily.
******NEVER LEAVE RACKS OF KJELDAHL TUBES ON CARTS!
S-18

Procedure: (For Digestion Block)
NOTE:Tubes are kept on wooden shelf, should already be in order with boiling chips added and
covered w/saran wrap. Racks and Digestion Block hold 40 tubes.
Use Kjeldahl tubes that were baked in drying oven (105°C) for at least 4 hours, usually overnight.
Add —11 boiling chips (Henger granules part # 136-CC sieved through #16 Mesh) to each Kjeldahl
tube.
1. Prepare lab sheet with tube numbers and sample ID's.
2. Remove filters from freezer in S106 at ERL, tear into thirds, and then place each filter in Kjeldahl
tubes.
3. With another freshly rinsed (x15) graduated cylinder measure 25mL of reagent water for blanks,
add to tubes (x2).
4. Measure 25mL of each Q.C. (89.22uM N/26.311M P, 1781.1.M N/52.611M P, 3561.1.M N/105u.M P), in
order from low to high, and add to tubes.
5. Measure 25mL of the 500u.M N/200u.M P Standard and add to tube.
6. Add 10mL of Kjeldahl Digestion Reagent to all sample/blank/standard/QC tubes using
designated repeat pipettor. Be careful not to spill/splash digestion reagent, use goggles.
7. Cap all tubes with green stoppers.
8. Wipe each tube with damp towel and dry completely. Place tubes in digestion rack as each row
is wiped. Make certain that a green tray is under the rack and a "Work in Progress" (W.I.P.) card
is attached. Also record what samples they are. Ex: CZR4Mar17 (#1-40) on bottom of card.
NOTE: Can let sit overnight to digest next morning.
9. Take green stoppers out of tubes and place in tan tray in order, to replace in order.
10. Put on two side panels.
11. Lift up over head to check bottom of tubes for liquid, cracks, and boiling chips.
12. Place the rack into the digestion block very carefully making sure that all tubes go into the block
simultaneously. Close sash on hood.
Set Digestion Control Box:
1. Turn ON, switch on right side of box. Make sure display reads "J1" when first turned on and
then the current temperature is displayed.
S-19

2. To Program: (On upper part of the display with ramp information)
• Press TEMP key, Enter 210 and press ENTER;
• Press TIME key, Enter 1.8 and press ENTER;
• Press TEMP key, Enter 385 and press ENTER;
• Press TIME key, Enter 1.5 and press ENTER.
• Press START/STOP key.
3. Make sure all 6 lights turn on. If not, turn controller off and start over.
4. NOTE: Set timer for 28 minutes. After timer goes off put on orange heat resistant gloves (temp.
should be—160°C) and watch for liquid bubbling to the top of the tubes. If bubbling does occur
CAREFULLY lift digestion rack —1 inch off the bottom of the digestion block (enough for bubbling
to cease reaching the top of the tube). May have to keep lifting rack until boiling calms down.
DO NOT ALLOW THE TUBES TO BOIL OVER AS THIS WILL LIKELY CAUSE THE TUBES TO
EXPLODE!
5. The controller will beep twice when the digestion has completed (about 3.5 hours total).
Wearing orange heat resistant gloves, CAREFULLY remove the two side panels and lift the
digestion rack with tubes and place on the metal tray inside the fume hood. Be careful the
tubes do not hit anything in the hood.
6. Set a timer for 9 minutes. During this time, fill the designated brown square repeat pipeter
(kept in Kjeldahl cabinet) with fresh reagent water and prime. Check to see if it is set to 25mL.
7. When timer goes off, pipet 25mL of reagent water into each of the sample/standard/blank/QC
tubes. NOTE: pipeter will only reach 3 tubes deep, so the rack needs to be rotated to reach the
other % of the tubes. There will be tubes in two middle rows that you cannot access.
8. Remove rack from fume hood and place on bench top. Remove the tubes water was not added
to and put in blue rack. Add water and return to digestion rack. Cap each tube with a rubber
stopper.
9. Using the vortex genies, mix each tube about 30 seconds each. Return tubes, in order, to the
blue rack inside the green tray.
10. Samples need to be mixed 3x with an hour between mixes before they can sit overnight and be
read the next morning.
11. If a second digestion is planned for the same day, turn OFF controller. Turn back ON. Allow the
digestion block to cool to 160°C., about 2-3 hours with fan blowing into hood. If tubes have been
wiped down check them and place on block, timer to be set to 10 minutes because block is
already hot. After timer goes off check temp and watch for bubbling until sure they are calm and
continue through steps.
Reagents For SmartChem:
S-20

*FOR PN, should only have to make Reagent #3 and #4 fresh)
1. STOCK Buffer Solution: (Should already be made and stored in a 1L Pyrex Bottle with screw cap
in DKN/TKN tray.)
OR if not already made:
In 1L volumetric flask dissolve 134g of Sodium phosphate, Dibasic Heptahydrate (Na2HPO4.7H2O;
CAS 7782-85-6) in approximately 250 mL of reagent water. Add and dissolve 20g of Sodium
Hydroxide, cool and then dilute to 1L. Mix thoroughly. STABLE. Store in designated 1L Pyrex bottle
with screw cap in DKN/TKN tray.
2. REAGENT #1: WORKING Buffer Solution: (Should already be made and stored in a 1,000mL Pyrex
Bottle with screw cap in DKN/TKN tray.)
OR if not already made:
In a 1 Liter volumetric flask, combine the reagents in the following stated order:
• 200mL of Stock Buffer Solution
• 250mL of the 20% Sodium Potassium Tartrate solution
• Mix by swirling.
• Add 120mL of the 20% Sodium Hydroxide solution
• Mix by swirling.
• Dilute to 1 Liter with reagent water
• Mix thoroughly.
NOTE: THIS IS A MEDIUM RANGE BUFFER (0-5mg/L; 0-357µM
3. Sodium Hydroxide, 20%: (Only needed to make Working Buffer Solution) (Should already be
made and stored in 1,000mL Pyrex bottle with screw cap in Bases cabinet.)
OR if not already made:
Under hood in a 1 Liter volumetric flask filled to approximately 800mL of reagent water, slowly
add 200g of Sodium Hydroxide (CAS 1310-73-2). Excessive heat will be generated. Mix well to
dissolve. Cool to room temperature and dilute to 1 Liter with reagent water. Mix thoroughly.
STABLE. Stored in 1,000mL Pyrex bottle with screw cap in Bases cabinet.
4. Sodium Potassium Tartrate, 20%: (Only needed to make Working Buffer Solution) In designated
250mL volumetric flask (kept in TKN/DKN tray) filled to approximately 200mL of reagent water,
slowly add and dissolve 50g Sodium Potassium Tartrate Tetrahydrate
(KOCO(CHOH)2COONa.4H20; CAS 6381-59-5). Solution will become cold. Bring to room
temperature and dilute to 250mL with reagent water. Mix thoroughly. *Do not want any NH4
in chemical.
5. REAGENT #2: (Sodium Salicylate): (Should already be made and stored in designated 250mL
volumetric flask in TKN/DKN tray.)
OR if not already made:
In designated 250mL volumetric flask, kept in TKN/DKN tray, dissolve 5g of Sodium Salicylate (2-
HOC6H4CONa; CAS 54-21-7) in approximately 225mL reagent water. Add (very slowly and at an
angle to keep from foaming) 1.5mL of concentrated Probe Rinse Solution (Westco part # 3AS-
RN00-21) and dilute to 250mL with reagent water. Mix thoroughly. Store solution in same
volumetric flask covered with foil, inside TKN tray. Prepare fresh every 2 weeks.
S-21

6. *REAGENT #3: (Sodium Hypochlorite Solution - Clorox): Pour about 10mL of the diluted 5.25%
Clorox into a 50mL beaker. In designated 50mL volumetric flask, add 3mL of the diluted 5.25%
Clorox, NaOCI) using a glass volumetric pipet. Add approximately 45mL of reagent water. Add
(slowly and at an angle to keep from foaming) 0.5mL concentrated probe rinse solution. Slowly
bring to volume with reagent water. Mix thoroughly. Prepare fresh daily.
7. *REAGENT #4: (Sodium Nitroferricyanide Solution): In designated 50mL volumetric flask,
dissolve 0.4g Sodium Nitroferricyanide(Nitroprusside) Dihydrate (Na2Fe(CN)5N0.2H2O; CAS
13755-38-9) in approximately 40mL of reagent water and dilute to 50mL with reagent water.
Cover flask with aluminum foil and let dissolve. Mix well. Add 0.5mL of concentrated probe rinse
solution. Mix thoroughly. Prepare fresh every 2 days, refrigerate overnight.
*FOR PP
1. Sodium Dodecyl Sulfate (SDS), 17.7%: Use only purest grade, with Phosphate concentrations
<_0.0001% Phosphate. In designated bottle, using a 100mL graduated cylinder and a disposable
pipette dissolve 15g SDS (CH3(CH2)iiOSO3Na; CAS #151-21-3) in 85mL of reagent water very
carefully (Use antistatic brush and wear MASK). Requires mixing on stir plate with stir bar to
dissolve completely. Good for two weeks. Kept in PP/TKP tray.
2. REAGENT #1: (Salt solution): (Should already be made and stored in designated glass bottle in
PP/TKP tray.)
a. In designated 500mL volumetric flask, dissolve 2.65g of Sodium Chloride (NaCI; CAS
7647-14-5) in 450mL of reagent water. Very slowly and at an angle add 15mL of 17.7%
SDS and slowly dilute to 500mL with reagent water. Mix thoroughly. Stable. Store in
same flask at room temperature in PP/TKP Tray.
3. REAGENT #2: (Molybdate/Antimony Solution): (Should already be made and stored in
designated glass bottle in PP/TKP tray.)
a. In designated 250mL volumetric flask dissolve 2g of Ammonium Molybdate
Tetrahydrate ((NH4)6Mo7O24*2H2O; CAS 12054-85-2) in about 200mL of reagent water.
Add 0.05g of Antimony Potassium Tartrate (K(SbO)C4H4O6*1/2H2O; CAS 28300-74-5).
Swirl to mix. Slowly and at an angle add 5mL of 17.7% SDS and dilute to 250mL with
reagent water. Mix thoroughly. Cover with foil. Store in same 250mL flask at room
temperature in PP/TKP Tray. Make every two weeks.
4. REAGENT #3: (Ascorbic Acid Solution): In designated 50mL volumetric flask dissolve 3g of
Ascorbic Acid (C6H8O6; CAS 50-81-7) with reagent water. Prepare fresh daily.
NOTE: Wrap all small plastic SmartChem reagent bottles containing light sensitive
reagents with foil before putting in SmartChem.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1L volumetric flask filled
with ^'950mL of reagent water, slowly and tilted to side add 0.5mL of Probe Rinse Solution
(Westco). Fill to volume with reagent water. Mix thoroughly. Stable. Store at room temperature
behind SmartChem #1. Keep a constant supply ready.
S-22

Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900)
In designated 2L container add reagent water to mark. Add 30mL (one bottle) of Unity Scientific
Cleaning Solution. Rinse 30mL bottle with reagent water and pour into 2L container, repeat 2
more times. Fill to volume. Mix thoroughly. Stable. Store at room temperature next to SmartChem
#1.
Procedure- SmartChem Set Up:
(Set up the Reagents, empty cups, Std, QC's and Diluent, etc. before samples, when the first rack
of samples are poured the SmartChem can be started and the rest of the samples can be added.)
1. Samples: Using a 5mL automatic pipet, carefully pipette sample from digestion tube being
careful not to disturb bottom of tube. Use a clean pipette tip for each sample.
Rinse a SmartChem sample cup with a small amount of sample and discard into waste container.
Fill cups in duplicate and place in SmartChem sample rack (racks 1-5).
NOTE: All Kjeldahl waste has to be collected and disposed of via EH&S
2. Diluent: Using a 5mL automatic pipet fill the designated small plastic SmartChem bottle with
both blanks, alternating back and forth between the two tubes of Blank. Usually about 3 times
from each tube. Mix. Place "Diluent 1" slot.
3. Q.C's: Using a 5mL automatic pipet fill one cup with each of the Q.C.'s. These will be placed in
descending order in cups 2-4 on "Reag 2 Ctrl/Std" rack.
4. Stds/Spike: Using a 5mL automatic pipet fill two cups with the 500µM Standard (one is for the
Spike). This will be placed in cup 1 of the" Reag 2 Ctrl/Std" rack. The Spike will be placed in 5th
slot of "Rgt 1 rack".
5. Place 6 empty sample cups in positions 1-6 in SmartChem Sample rack 5. These cups will be
used to generate the standards for the standard curve.
6. Reagents: (All reagents are placed in "Reag 2 Ctrl/Std" rack) Do not overfill bottles, pop any
bubbles.
• Position 1 - Reagent #1 - DKN Working Buffer (TKBU)
• Position 2 - Reagent #2 - Sodium Salicylate (TKSA)
• Position 3 - Reagent #3 - Clorox (TKHY)
• Position 4 - Reagent #4 - Sodium Nitroprusside (TKNI)
NOTE: Fill each small plastic SmartChem reagent bottle only just before shoulder of bottle. If they
are too full or there is a bubble present the SmartChem will register it as "empty".
7. Check fluid levels in Cuvette Wash Solution, DEI, and Probe Rinse Solution resevoir bottles, refill
if necessary.
S-23

Software Procedure:
1. Turn ON computer turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select Profile List on middle of screen. Select "PN/PP-TKNM+OP4M" from the list of methods at
the bottom of the page. This screen will show where the Std/Spike, Q.C.'s, Reagents, Diluent and
empty cups are to be placed.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the empty cups for standard dilutions.
******NEVER LEAVE RACKS OF KJELDAHL TUBES ON CARTS!
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "Print" and selecting "Print" again.
2. Real time sample data can be displayed by selecting the "Results" button under the cuvette
wheel. Once all samples have been run, the "Print option" will appear in the upper left menu
bar, clicking the button will automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a. Select "options" from left menu bar, then "data retrieval".
b. Find and select the plan of interest. A split screen will appear — click on the method in
the box in the lower half of the page. This will highlight the symbols just to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select "print".
ii. Click on the graph and this will bring the standard curve forward. Select "print".
Cleaning:
1. All samples, reagents, standards, QC's, and blanks must be collected into designated hazardous
waste bottles.
2. SmartChem sample cups can be disposed of in the trash can
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and inverted
on rack/paper towel to dry.
S-24

4. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack above sink
next to SmartChem to dry.
Principle:
The method is based on the conversion of all forms of phosphorus in the digested sample to
orthophosphorus. Ammonium molybdate and antimony potassium tartrate react in an acid medium
with dilute solutions of phosphorus to form an antimony-phospho-molybdate complex. This complex
is reduced to an intensely blue colored complex by ascorbic acid. The color (absorbance) measured
at 660nm is proportional to the phosphorus concentration in the original sample.
Total Kjeldahl nitrogen is the sum of free ammonia nitrogen and organic nitrogen compounds which,
through digestion, are converted to ammonium sulfate. The method is based on the conversion of
the digested ammonium sulfate to ammonia. In the presence of sulfuric acid, potassium sulfate, and
a cupric sulfate catalyst, amino nitrogen of organic materials is converted to ammonium. The
ammonium cation is then converted to ammonia by neutralization with a concentrated buffer. Once
converted to ammonia, an intensely blue compound, indophenol, is formed by the reaction of
ammonia with alkaline phenol and then hypochlorite (Berthelot reaction), catalyzed by sodium
nitroprusside. The color (absorbance) measured at 660nm is proportional to the Kjeldahl nitrogen
concentration in the original sample.
S-25

Dissolved Orthophosphate (PO4) — Automated SmartChem Method
References:
April 2020
SmartChem 200 Method 410-200B, revised July 2008. Westco Scientific Instruments, Inc.
Standard Methods for the Examination of Water and Wastewater. 4500-P-E (22nd Edition). American
Public Health Association, American Water Works Association, Water Environment Federation.
pp. 4-155.
EPA. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020. Method 365.3.
Environmental Monitoring and Support Laboratory, Cincinnati, OH.
Reagents:
1. Reagent water: Fresh deionized water from E-pure system in S106 (16-18 megohm/cm).
2. REAGENT #1: Reagent Water.
3. REAGENT #2: (Color Reagent): See Below
In designated brown bottle mix 50mL of 5N Sulfuric Acid (H2SO4) and 15mL of the Ammonium
Molybdate Solution. Swirl to mix. Add 5mL of the APT Solution (Antimony Potassium Tartrate).
Swirl to mix. Add 10mL of 17.7% SDS. Swirl to mix. Add 20mL reagent water. Invert gently five
times to mix. Store in fridge. Prepare fresh weekly.
To Make Reagent #2 you will need:
TWO IN FRIDGE - REAGENTS MUST BE BROUGHT TO ROOM TEMPERATURE:
a. Antimony Potassium Tartrate Solution: (Should be made and in fridge) Weigh out
1.3715g Antimony Potassium Tartrate (K(SbO)C4H4O6*1/2H2O; CAS 28300-74-5) and
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dilute to 500mL with reagent water. Stable for 6 months. Store in designated brown
bottle in fridge.
b. Ammonium Molybdate Solution: (Should be made and in fridge) Dissolve 20g of
Ammonium Molybdate ((NH4)6Mo7O24*4H2O; CAS 12054-85-2) to 500mL with reagent
water. Stable for 6 months. Store in designated red bottle in fridge. If precipitate forms,
filter solution and scrub out bottle with reagent water and brush.
c. Sodium Dodecyl Sulfate (SDS) 17.7%: (Should be made and in Phosphate tray)
Use only purest grade, with Phosphate concentrations <_0.0001% Phosphate. In
designated bottle, using a 100mL graduated cylinder and a disposable pipette dissolve
15g SDS which is kept in Flammables cabinet (CH3(CH2)11OSO3Na; CAS #151-21-3) in 85mL
of reagent water. Very carefully (Use antistatic brush and wear MASK). Requires mixing
on stir plate with stir bar to dissolve completely. Good for two weeks. Kept in Phosphate
tray.
d. Sulfuric Acid, 5N: (Should be made and in Acid cabinet) In the fume hood, in a 500mL
volumetric flask slowly add 70mL of concentrated sulfuric acid (H2SO4; CAS 7664-93-9)
to approximately 400mL of reagent water. Cool to room temperature and dilute to
500mL with reagent water. Stable. Kept in Acid cabinet in designated glass bottle.
4. REAGENT #3: (Ascorbic Acid, 0.1M): In designated volumetric flask dissolve 0.88g of Ascorbic
Acid (C6H8O6; CAS 50-81-7) in 50mL of reagent water. Add 0.5mL of 17.7% SDS. Invert, gently,
five times to mix. Prepare fresh daily.
Standard/Spike:
a. 40µM KH2PO4: In designated volumetric flask, dilute 4mL of 1,000µM Combined
Standard to 100mL with reagent water. Make fresh.
Quality Controls (QC's):
b. Q.C. #1: (5.26iM KH2POA): In designated volumetric flask dilute 0.1mL (100uL) of the
SPEX Certi Prep 1,000µg/mL Phosphate = 10,525µM) to 200mL with reagent water.
Prepare fresh daily.
c. Q.C. #2: (15.79iM KH2POl: In designated volumetric flask dilute 0.3mL (300uL) of the
SPEX Certi Prep 1,000µg/mL Phosphate = 10,525µM) to 200mL with reagent water.
Prepare fresh daily.
Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1L volumetric flask add
0.5mL of Unity Scientific Probe Rinse solution to ^'950mL of reagent water. Add reagent water to
volume. M ix thoroughly. Stable. Store at room temperature next to SmartChem #1. Keep constant
supply.
Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900) In designated 2L container add
reagent water to mark. Add 30mL (one bottle) of Unity Scientific Cleaning Solution. Rinse 30mL
bottle with reagent water and pour into 2L container, repeat 2 more times. Fill to volume. Mix
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thoroughly. Stable. Store at room temperature next to SmartChem #1.
Procedure: (While samples are being poured, wash and WBL can be done.)
1. Pour or pipet approximately 3mL of sample into sample cup and discard. Pour or pipet 3mL of
sample into the cups and place in SmartChem sample rack (racks 1-5). Usually run duplicates.
2. In "Reag2 Ctr/Std" Rack, place the 40µM Std, in slot 1, QC 2 in slot 2, QC 1 in slot 3.
3. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5. These cups will be
used to generate the standards for the standard curve.
4. Diluent: reagent water. Place in "Diluent 1" spot.
5. Reagents: in SmartChem rack "RGT1", place #1= Reagent water, #2= Color reagent, #3= Ascorbic
Acid and Spike. Do Not Overfill bottles.
6. Check fluid levels in Cuvette Wash Solution, DIW, and Probe Rinse Solution bottles, refill if
necessary.
SmartChem:
1. Turn ON computer, turn ON SmartChem if not already on.
2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. In Methods select "CPO4" from the list of methods at the bottom of the page.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the 6 empty cups for standard dilutions.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "print" and selecting "print" again.
2. Real time sample data can be displayed by selecting the Results button under the cuvette wheel.
Once all samples have been run, the print option will appear in the upper left menu bar, clicking
the button will automatically print out the results.
Cleaning:
1. All reagents, standards, QC's, and spikes must be collected for pick-up by EH&S. Collect these
into designated hazardous waste bottles.
2. Samples can be disposed of down drain and sample cups can be put in trash can.
3. SmartChem reagent bottles should be rinsed out until clean with deionized water and inverted
on rack/paper towel to dry.
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4. Wash SmartChem racks with tap water and scrub with brillo pad/brush, hang on drying rack
above sink next to SmartChem to dry.
Principle:
Ammonium Molybdate and Potassium Antimonyl Tartrate react in acid medium with Orthophosphate to
form a Heterpoly Acid — Phosphomolybdic Acid that is reduced to intensely colored Molybdenum Blue by
Ascorbic Acid. The color (absorbance) measured at 880nm is proportional to the Orthophosphorus
concentration.
April 2020
Total and Total Dissolved Phosphate (TP/TDP) — Automated SmartChem Method
(NEVER DO TOTAL PHOSPHATE IN SMARTCHEM IF SAMPLES ARE NOT FILTERED)
References:
SmartChem 200 Method 410-200B, revised July 2008. Westco Scientific Instruments, Inc.
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Standard Methods for the Examination of Water and Wastewater. 4500-P-B (19th Edition). Sample
Preparation. American Public Health Association, American Water Works Association, Water
Environment Federation. pp. 4-109/111.
Standard Methods for the Examination of Water and Wastewater. 4500-P-E (22nd Edition). American
Public Health Association, American Water Works Association, Water Environment Federation.
pp. 4-155.
EPA. 1979. Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020. Method 365.3.
Environmental Monitoring and Support Laboratory, Cincinnati, OH.
Reagents:
1. Reagent Water: Fresh deionized water from E-pure system in S106 (16-8megohm/cm).
2. Digestion Reagent: In designated 500mL volumetric flask dissolve 10g Low Nitrogen Potassium
Persulfate (K2S2O8; CAS# 7727-21-1) in about 300mL of reagent water. Dissolve 1.5g Sodium
Hydroxide (NaOH; CAS# 1310-73-2) and dilute to 500mL with reagent water. May have to put on
stir plate to mix well. Prepare fresh daily. Use designated volumetric flasks for smaller amounts,
which are kept in gray tray above SC#1. Use repipeter to add to tubes.
3. Borate Buffer: (Should already be made and stored in white tray above SC #1)
In designated 500mL flask dissolve 30.9g Boric Acid (CAS #10043-35-3) in approx. 300mL reagent
water. Add 4g Sodium Hydroxide and bring to volume with reagent water. May have to put on
stir plate to dissolve. Stable. Use repiteter to add to tubes.
4. Phenolphthalein, 1%: Phenolphthalein Solution (CAS 77-09-8) Stored in flammable cabinet. Fill
dropper bottle, kept in TDP drawer, as needed. Stable.
5. Sodium Hydroxide, 50%: (Should already be made)
In a 500mL volumetric flask containing 350mL of reagent water dissolve 250g of Sodium
Hydroxide. Gets HOT! Allow to cool to room temperature and bring to 500mL with reagent water.
May have to use stir plate to dissolve. Stable for several months. Store in Bases cabinet in 1L Pyrex
bottle with screw cap. Fill dropper bottle, stored in TDP drawer, as needed.
6. Sulfuric Acid, 11N: (Should already be made) ALWAYS ADD ACID TO WATER!
To approximately 600mL of reagent water in a 1L volumetric flask, very slowly add 306mL of
concentrated Sulfuric Acid (H2SO4; CAS# 7664-93-9). Allow to cool to room temperature and
dilute to 1L with reagent water. Stable for several months. Stored in Pyrex bottle w/screw cap in
acid cabinet. Mix before using. Fill dropper bottle, stored in TDP drawer, as needed.
7. REAGENT #1: Fresh deionized water from E-pure system in S106.
8. REAGENT #2: (Color Reagent): See below. All reagents must be at room temperature and must
be mixed in the order given.
In designated brown bottle mix 50mL of 5N Sulfuric Acid - H2SO4, (should already be made and
stored in acid cabinet), add 5mL of the Antimony Persulfate Tartrate - APT Solution (should
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already be made and stored in brown bottle in fridge). Swirl to mix. Add 15mL of the 40%
Ammonium Molybdate Solution (should already be made and stored in red bottle in fridge). Swirl
to mix. Add 3 mL of 17.7% SDS, may already be made and stored in white TKP/DKP/PKP tray. Mix
gently. Store in fridge. Prepare fresh weekly.
*Reagents needed to make Color Reagent (Reagent #2), IF NOT ALREADY MADE, are as follows:
a.
Sulfuric Acid, 5N: Perform in the fume hood. In designated 4L jug with 1720mL deionized
water SLOWLY add 280mL of Sulfuric Acid (H2SO4, CAS 7664-93-9) and swirl as it is
added. Mix thoroughly. Stable. Kept in 4L bottle in acid cabinet.
b.
Antimony Potassium Tartrate Solution: In a 500mL volumetric flask add 1.3715g
Antimony Potassium Tartrate (K(SbO)C4H4O6*1/2H2O; CAS 28300-74-5) and dilute to
500mL with reagent water. Store in dark bottle in fridge. Stable for 6 months.
c.
Ammonium Molybdate Solution, 40%: Dissolve 20g of Ammonium Molybdate
((NH4)6Mo7O24*4H2O; CAS 12054-85-2) to 500mL with reagent water. Store in red bottle
in fridge. Stable for 6 months. If precipitate forms, dump in waste, scrub out bottle with
reagent water and brush and remake.
d.
Sodium Dodecyl Sulfate (SDS), 17.7%: (Use only purest grade, with Phosphate
concentrations <_0.0001% Phosphate.) In designated bottle (stored in TKP/DKP/DKP
tray) add 15g SDS (stored in flammables cabinet) (CH3(CH2)11OSO3Na; CAS 151-21-3). In
graduated cylinder measure out 85mL of reagent water. Using a disposable 3mL pipete,
rinse weigh boat and funnel into bottle. Add remaining reagent water. Will require
stirring on stir plate to dissolve completely. Make every two weeks.
9. REAGENT #3: (Ascorbic Acid, 0.1M): In designated 50mL volumetric flask dissolve 0.88g Ascorbic
Acid (C6H8O6; CAS 50-81-7) with reagent water. Bring to volume. Add 1mL of 17.7% SDS. Mix
well. Prepare fresh daily.
Standards:
a. 10,000µM Potassium Phosphate (KH2PO4; CAS 7778-77-0): Should already be made and
in fridge.
In a 1L volumetric flask add 1.3609g of Potassium Phosphate Monobasic and dilute to 1L
with reagent water. Mix well. Good for 6 months. Store in Pyrex bottle w/orange cap in
fridge.
b. 1,000µM Potassium Phosphate:
Can use 1,000µM Combined Standard (NH4CI, KH2PO4, KNO3). Good for 1 month. Store
in fridge.
c. 40µM Potassium Phosphate: In designated 100mL volumetric flask add 4mL of 1,000µM
Combined Standard OR 4mL of 1,000µM KH2PO4 Standard and bring to volume with
reagent water. Prepare fresh daily.
Quality Controls:
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d. QC1: 5.26µM P: In designated 200mL volumetric flask add 0.1mL (100µL) SPEX Certi
Prep 1,000µg/mL Phosphate (=10,525µMP, CAT# AS-PO49-2Y) and bring to volume with
reagent water. Prepare fresh daily.
e. QC2: 15.79µM P: In designated 200mL volumetric flask add 0.3mL (300µL) SPEX Certi
Prep 1,000µg/mL Phosphate (=10,525µMP, CAT# AS-PO49-2Y) and bring to volume with
reagent water. Prepare fresh daily.
Probe Rinse Solution: (Should already be made and stored next to SC#1- If not already on sink counter, it
is stored in SmartChem supplies cabinet)
In designated 1L volumetric flask, with ^'950mL of reagent water, slowly add 0.5mL Probe Rinse Solution
(Unity Scientific, P/N# 3AS-RN00-21) and bring to volume with reagent water. Mix thoroughly. Stable.
Store at room temperature in designated flasks. Keep steady supply.
Cuvette Wash Solution: (Should already be made and stored next to SC#1- otherwise, stored in
SmartChem supplies cabinet)
In designated 2L container add 1 bottle (30mL) cuvette cleaning solution (Unity Scientific P/N# 365-
0366-900) to approximately 1500mL of reagent water. Rinse bottle 3x with reagent water, add to
container and fill to mark. Invert, gently, five times to mix. Store at room temperature. Stable.
Keep steady supply.
Set Up:
• Culture tubes; one per sample and one for each: QC #1, QC #2 and Standard.
Six tubes for Blanks (= diluent).
• 10mL pipeter and pipet tip.
Procedure (Digestion):
1. Pipet 10mL of shaken sample and discard down the drain. Pipet a second 10mL into a 30mL
culture tube. Repeat this process with a rinse of next sample between each sample. Be sure the
culture tube has a screw cap with a rubber or Teflon liner so a tight seal can be made.
2. Repeat the above process with six Blanks (deionized water), one QC#1, one QC#2 and one 40µM
P Standard.
3. Add 5mL of Phosphate Digestion Reagent to each sample/standard/QC/blank using designated
repeat pipetter. Stored in white tray above SC#1.
4. Cap culture tube tightly. Mix by gently inverting culture tube three times. Do not shake;
shaking the culture tube vigorously does not allow for complete mixing of sample and digestion
reagent.
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5. Heat tubes in autoclave for 30 minutes at 250°C. Note: you can stop here and finish the
following day. Let samples and autoclave cool to room temperature (overnight). There may be
a white precipitate in the bottom of the tube at this stage, okay.
6. Add 1mL of Borate Buffer to each tube using designated repeat pipeter, kept in white tray above
SC #1. Keep track of caps so that the same cap can be returned to the same tube.
7. Cap tubes and mix by gently inverting tube three times. If precipitate does not dissolve, filter
the sample.
8. Remove caps, keeping them in order. Add 1 drop of 1% Phenolphthalein to each tube (including
samples/standard/QCs/blanks). Steps #8 and #9 can be added on top of each other.
9. Add 1 drop of 50% Sodium Hydroxide to each tube (including samples/standard/QCs/blanks).
Cap tubes and mix by gently inverting tube three times. If sample does not turn pink, add
another drop of Sodium Hydroxide and mix sample again.
10. Add 1 drop of 11N Sulfuric Acid to each tube (including samples/standard/QCs/blanks). Cap
tubes and mix by gently inverting tube three times. If sample does not return to clear, add
another drop of Sulfuric Acid and mix sample again.
Procedure (SmartChem):
1. Samples: Pipet approximately 3mL of sample into SmartChem sample cup and discard into
waste beaker. Pipet 3mL of sample into (two cups per sample) cups and place in SmartChem
sample rack (racks 1-5). Also, add 1 sample cup containing only DEI water at the beginning and
at the end.
2. Diluent/Blank: Combine the six Blanks in an erlynmeyer flask and swirl to mix. Pour mixed Blanks
into Diluent bottle and insert into SmartChem "Diluent #1" slot.
3. Standard/Spike: Pipete 3mL of the digested 40µM PO4-P into two sample cups rinsing as with
samples. Place Standard in SmartChem rack "RGT2 Ctrl/Std", position 1.
4. Place 6 empty sample cups in positions 1-6 in SmartChem sample rack 5. These cups will be
used to generate the standards for the standard curve.
5. Reagents:
• In SmartChem rack "RGT1", place Reagent #1 (Dei. water) in position 1.
• "RGT1" rack, place Reagent #2 (Color Reagent) in position 2.
• "RGT1" rack, place Reagent #3 (Ascorbic Acid) in position 3.
6. Check fluid levels in cuvette wash solution, DIW, and probe rinse solution bottles, refill if
necessary.
Software Procedure:
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1. Turn on computer; turn on SmartChem.
2. Select "WP2W" from the list of methods at the bottom of the page.
3. Follow procedures for SmartChem Set -Up posted next to equipment.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the empty cup for standard dilutions.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "Print" and "Print" again.
2. Real time sample data can be displayed by selecting the "Results" button. Once all samples
have been run, the print option will appear in the upper left menu bar, clicking the button will
automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a. Select "Options" from left menu bar, then "Data Retrieval".
b. Find and select the plan of interest. A split screen will appear — click on the method in
the box in the lower half of the page. This will highlight the symbols just to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select
"Print".
ii. Click on the graph and this will bring the standard curve forward. Select "Print".
Cleaning:
1. All samples and reagents need to be collected in Waste jug.
2. Wash culture tubes as discussed on CEL washing protocol.
3. Rinse out designated Phosphate Digestion repeat pipettor with deionized water and replace on
shelf in white tray.
4. Rinse the pump portion only of the designated Borate Buffer repeat pipettor with deionized
water and replace with remaining buffer on shelf in white tray.
5. Other glassware needs to be rinsed with tap water and placed in vat to be washed according to
CEL washing protocol.
6. SmartChem sample cups can be disposed of in the trash can.
7. SmartChem reagent bottles should be rinsed until clean with deionized water and inverted on
rack/paper towel to dry.
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8. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack above sink
next to SmartChem to dry.
Principle:
Total Phosphorus (TP) is run on unfiltered water. Total Dissolved Phosphorus (TDP) is run on filtered
water. The Potassium Persulfate Digestion coverts all form of Phosphorus to Orthophosphate (at
concentrations less than 40 uM). Ammonium Molybdate and Potassium Antimonly Tartrate react in acid
medium with Orthophosphate to form a Heterpoly Acid — Phosphomolybdic Acid that is reduced to
intensely colored Molybdenum Blue by Ascorbic Acid. The color (absorbance) measured at 880nm is
proportional to the Orthophosphorus concentration.
Using this method on samples with salinities above 18-20ppt creates a precipitate that clouds the solution.
It is very difficult to remove this cloudiness from the samples. If the cloudiness is not removed, 1) inflated
absorbance values are generated which in turn results in over -estimation of concentration, 2) pump lines
in SmartChem could become clogged.
April 2020
Total Ammonia Nitrogen (NH4/TAN) — Automated SmartChem Method
References:
SmartChem 200 Method 210-201B, revised July 2008. Westco Scientific Instruments, Inc.
Solorzano, L., 1969. Determination of ammonia in natural waters by the phenol-hypochlorite method.
Limnology and Oceanography. 14:799-801.
Standard Methods 4500-NH3-H. 22nd Edition. 2012. American Public Health Association, American
Water Works Association, Water Environment Federation. pp. 4-118.
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Reagents:
1. Reagent water: Fresh deionized water from E-pure system (16 to 18 megohm/cm) in S106.
2. REAGENT 1: Ethylenediamine Tetraacetic Acid, Disodium Salt Dihydrate (5%): In designated
500mL volumetric flask in approx. 450mL reagent water, dissolve 25g of EDTA
(CioHi4N2Na2O8*2H2O; CAS 6381-92-6). Add 2.75g of Sodium Hydroxide (NaOH; CAS 1310-73-2).
May have to put on stir plate. Dilute to 500mL with reagent water. Mix thoroughly. STABLE.
Kept in Ammonia Tray.
3. REAGENT 2: Sodium Phenolate: In designated 100mL volumetric flask, dissolve 3.2g of Sodium
Hydroxide (NaOH; CAS 1310-73-2) in approx. 75mL of reagent water. Cool the flask to room
temperature and then add 9.3mL of liquid Phenol (C6H5OH; CAS 108-95-2), which is kept in
Flammables Cabinet. Dilute to 100mL with reagent water. Good for 1 week. Wrap with foil and
store in fridge.
4. REAGENT 3: Sodium Nitroferricyanide Dihydrate: In designated 100mL volumetric flask,
dissolve 0.075g of Sodium Nitroferricyanide Dihydrate (Na2Fe(CN)5N0*4H2O; CAS 13755-38-9)
and dilute to 100mL with reagent water. Add 1mL Concentrated Probe Rinse Solution (Unity
Scientific P/N 3AS-RN00-21). Invert, gently to mix thoroughly. Prepare fresh weekly. Wrap with
foil and store in fridge.
5. REAGENT 4: Sodium Hypochlorite (CLOROX): In designated 50mL volumetric flask, add 25mL of
Clorox (NaOCL; CAS 7681-52-9) and dilute to 50mL with reagent water. Available chlorine
should be between 2 and 3 percent. Prepare fresh daily. Kept in Clorox cabinet.
NOTE: Purchase Concentrated Clorox and dilute per directions on designated 1L Pyrex bottle.
Wrap in foil and keep in Clorox cabinet.
6. Standards:
a. 10,000u.M Ammonium Chloride (NH4CI; CAS 12125-02-9): Should already be made and
in designated 1L Pyrex bottle in fridge. In 1L volumetric flask add 0.5349g Ammonium
Choride and dilute to1L with reagent water. Good for 6 months.
b. 1,00011M Combined Standard: In designated 100mL volumetric flask pipet 10mL of each
10,000µM component (Ammonium Chloride, Potassium Phosphate and Potassium
Nitrate) with a volumetric flask.
c. 80µM Combined Standard: In designated 100mL volumetric flask dilute 8mL of
1,000µM Combined Standard to 100 mL with reagent water using a volumetric pipette.
Make fresh.
7. Quality Controls:
a. Q.C. #3 - 71.377µM NH4CI: In designated 100mL volumetric flask dilute 0.1 mL (100uL) of
the Certified SPEX Certi Prep Ammonia Nitrogen 1,000µg/mL (=71,327µM) (CAT# AS-
NH3N9-2Y) to 100mL with reagent water. Make fresh.
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b. Q.C. #2 - 35.69iM NH4CI: In designated 100mL volumetric flask dilute 50mL of the
above Q.C. #3 to 100mL with reagent water. Make fresh.
c. Q.C. #1 - 7.14iM NH4CI: In designated 100mL volumetric flask dilute 10mL of QC #3 to
100 mL with reagent water. Make fresh.
1. Probe Rinse Solution: (Unity Scientific P/N 3AS-RN00-21) In designated 1L volumetric flask filled
with ^'950mL of reagent water, slowly and tilted to side add 0.5mL of Probe Rinse Solution. Fill to
volume with reagent water. Mix thoroughly. Stable. Store at room temperature behind
SmartChem #1. Keep a constant supply ready.
2. Cuvette Wash Solution: (Unity Scientific P/N 365-0366-900) In designated 2L container add
reagent water to mark. Add 30mL (one bottle) of Unity Scientific Cleaning Solution. Rinse 30mL
bottle with reagent water and pour into 2L container, repeat 2 more times. Fill to volume. Mix
thoroughly. Stable. Store at room temperature next to SmartChem #1. Keep a constant supply
ready.
Procedure:
1. Samples: In duplicate, pipet approx. 3mL of sample into sample cups and discard. Refill and
place in SmartChem sample racks (racks 1-4).
2. Diluent: reagent water. Put in "Diluent #1" slot.
3. Standard/Spike: For Standard place a sample cup with —3mL of 80µM NH4-N in SmartChem rack
"RGT2 CTRL/STD", position 1. Spike goes into "RGT1" rack, slot #5
4. Place 6 empty sample cups in sample rack 5, positions 1-6. These cups will be used to generate
the standards for the standard curve.
5. Reagents: In SmartChem rack "RGT2 CTRL/STD", place Reagent #1 (EDTA) in "QC" slot, Reagent
#2 (Sodium Phenolate) in slot "11", Reagent #3 (Sodium Nitroprusside) in slot "12" and Reagent
#4 (Sodium Hypochlorite) in slot "13".
6. Q.C.'s: These are poured into designated SmartChem bottles and placed in rack "RGT1" in
spaces 6-8 in descending order (Q.C. #3 to #1).
7. Check fluid levels in cuvette Wash Solution, DIW, and Probe Rinse Solution bottles, refill if
necessary.
8. Place waste tubes into designated waste jar to collect waste from SmartChem.
SmartChem:
1. Turn on computer; turn on SmartChem.
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2. Follow procedures for SmartChem Set -Up posted next to equipment.
3. Select "NH3-LLLauraLow" from Profile list.
4. Check to be sure that all of the cups/reagent bottles, etc. are filled correctly. Be sure to include
the empty cups for standard dilutions before starting.
Printing Data:
1. After all standards have been made and run, a standard curve will automatically be generated
and appear on the screen. Print this out by selecting "print" and "print" again.
2. Real time sample data can be displayed by selecting the "Results" button. Once all samples
have been run, the print option will appear in the upper left menu bar, clicking the button will
automatically print out the results.
3. If the plan was closed out before printing the data or if you want to pull up existing data:
a. Select "options" from left menu bar, then "data retrieval".
b. Find and select the plan of interest. A split screen will appear — click on the method in
the box in the lower half of the page. This will highlight the symbols just to the right.
i. Click on the erlynmeyer flask and this will bring the data forward. Select "print".
ii. Click on the graph and this will bring the standard curve forward. Select "print".
Cleaning:
1. All samples and Diluent can be safely disposed of down the drain
2. All Reagents, Q.C.s, and Standards must be collected for pick-up by EH&S.
3. Empty SmartChem sample cups can be disposed of in the trash can.
4. SmartChem reagent bottles should be washed out 5x with deionized water and inverted on
rack/paper towel to dry.
5. Wash SmartChem racks with tap water and scrub with brillo pad, hang on drying rack above sink
next to SmartChem to dry.
Principle:
An intensely blue compound, indophenol, is formed by the reaction of ammonia with alkaline phenol
and then hypochlorite (Berthelot reaction), catalyzed by sodium nitroprusside. The color
(absorbance) measured at 630nm is proportional to the ammonia + ammonium concentration.
This method measures both ammonia (NH3; unionized ammonia) and ammonium (NH4+; ionized
ammonia) in the sample and therefore results can be reported as total ammonia nitrogen (TAN).
Water temperature and pH will affect the ratio of these two forms of ammonia in aquatic systems
(see Emmerson, et al.,1975, for a table of conversion factors).
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