Ni trate-Ni tro gen
WARNING! This set contains chemicals
that may be harmful if misused. Read
cautions on individual containers
carefully. Not to be used by children
except under adult supervision.
Low Range En zyme Kit
(0.00-1.0 ppm)
Code 8137
QUANTITY CONTENTS CODE
1 Enzyme Vials in Foil Bag, 25 vials 8136-25
60 mL Nitrate Buffer Reagent 8139-H
15 mL *Hydrochloric Acid *6130-E
30 Nitrate Color Tablets 8140A
120 mL Deionized Water 5115PS-J
1 Pipet, plastic, 0.5 mL 0353
1 Pipet, plastic, 1.0 mL 0354
1 Pipet, plastic, plain 0352
1 Color Chart, Nitrate-N, Low Range 8137LR-CC
1 Color Chart, Nitrate-N, High Range 8137HR-CC
*WARN ING: Re agents marked with a * are con sid ered to be po ten tial
health haz ards. To view or print a Ma te rial Safety Data Sheet (MSDS) for
these re agents see MSDS CD or www.lamotte.com. To ob tain a printed copy,
con tact LaMotte by e-mail, phone or fax.
Stor age
The Enzyme Vials (8136) contain very small amounts of dried reagents that
may not be visible. Do not open the vials before use. The Foil Bag contains
desiccant packs. Do not remove Enzyme Vials from the Foil Bag until ready
to use.
Store this kit in a cool, dry place, preferably at room temperature or lower (a
refrigerator or freezer is best), whenever possible. Exposure to heat will cause
the enzyme to denature and become inactive. Heat will also adversely affect
the performance of the Nitrate tablets. If the Nitrate tablets have turned a
dark brown, this is an indication that the kit contents have been exposed to
heat and the contents should be discarded.
Pro ce dure
Low Range (0.00 – 0.20 ppm NO
_
N)
3
1. Firmly tap the capped Enzyme Vial (8136) three times on a hard surface.
This will ensure that reagents are at the bottom of the vial.
2. Use the 1 mL pipet (0354) to add 2 mL of Nitrate Buffer (8139).
3. Use a plain pipet (0352) to add sample water until the vial is filled to the
5 mL line.
4. Cap the vial and mix by inverting gently 3 times. Repeat the mixing
procedure at 5 minutes, 10 minutes and 15 minutes. Total reaction time
is 15 minutes.
5. Add 5 drops Hydrochloric Acid, 1N (6130). Cap and mix.
6. Add 1 Nitrate Color Tablet (8140A). Cap the vial and mix by gently
inverting until the tablet disintegrates.
7. Wait 5 minutes.
8. Remove the cap. Place the vial on the white area of the Nitrate-N Low
Range Color Chart (8137LR-CC). Looking down through the tube,
compare the color of the solution to the color chart.
9. Record the result as ppm Nitrate-N (NO
Nitrate (NO3) multiply by 4.4.
_
N). To convert the result to
3
NOTE: Salinity will reduce the enzyme effectiveness and result in a decrease of
color development.
NOTE: For the most accurate results use laboratory-grade pipets for measuring
sample water and reagents.
Pro ce dure
High Range (0.2 – 1.0 ppm NO
_
N)
3
1. Firmly tap the capped Enzyme Vial (8136) three times on a hard
surface. This will ensure that reagents are at the bottom of the vial.
2. Use the 1 mL pipet (0354) to add 2 mL of Nitrate Buffer (8139).
3. Use the 0.5 mL pipet (0353) to add 0.5 mL of the sample water to
the Enzyme vial.
4. Use a plain pipet (0352) to add Deionized Water (5115) until the
vial is filled to the 5 mL line.
5. Cap the vial. Mix by inverting gently 3 times. Repeat the mixing
procedure at 5 minutes, 10 minutes and 15 minutes.The total
reaction time is 15 minutes.
6. Add 5 drops Hydrochloric Acid, 1N (6130). Cap and mix.
7. Add 1 Nitrate Color Tablet (8140A). Cap the vial and mix by gently
inverting until the tablet disintegrates.
8. Wait 5 minutes.
9. Remove the cap. Place the vial on the white area of the Nitrate-N
High Range Color Chart (8137HR-CC). Looking down through the
tube, compare the color of the solution to the color chart.
10. Record the result as ppm Nitrate-N (NO
to Nitrate (NO3) multiply by 4.4.
_
N). To convert the result
3
NOTE: Salinity will reduce the enzyme effectiveness and result in a decrease
of color development.
NOTE: For the most ac cu rate re sults use lab o ra tory-grade pipets for
mea sur ing sam ple wa ter and re agents.
More About Ni trate Test ing
Ni trate in Na ture
Nitrogen occurs in natural waters as nitrate (NO3), nitrite (NO2), ammonia
(NH3) and organically bound nitrogen. Nitrate test results are expressed as
“nitrate-nitrogen” (NO3-N), meaning “nitrogen that is in the form of nitrate”.
Nitrate is a nutrient needed by all aquatic plants and animals to build protein.
Natural waters generally have a nitrate-nitrogen level below
1 ppm. Higher levels indicate the addition of nitrate from unnatural sources.
Nitrate-nitrogen levels above 10 ppm are considered unsafe for drinking water.
NO3-N Concentration
< 1 ppm natural waters
< 10 ppm safe for drinking water
> 10 ppm unsafe for drinking water
Sources of Ni trate
Nitrate has always existed in nature but not in the quantities that it does today.
When forests and wetlands surrounded waterways, nutrients and soil were held in
place by vegetation and did not run off the land into the water. Small amounts of
nitrate entered the aquatic ecosystem naturally from forest fires, fallout from
volcanoes and the decay of aquatic plants.
Now, in addition to small amounts of nutrients from natural sources, large
amounts of nitrate are created by modern land use. Farms, suburbs and cities are
built on the banks of streams, lakes and rivers. Nutrients enter the water through
point and nonpoint sources. Examples of point source pollution, which can be
traced to a specific site, are industry, and mainly, wastewater and sewage
treatment plants. Nonpoint sources of pollution cannot be contributed to a
specific location. Automobile exhaust, acid rain and fertilizer run-off and erosion
from farms contribute to excessive nutrient levels in surface and groundwater. In
suburbs, septic systems and lawn fertilizer run-off from millions of homes are an
example of nonpoint source pollution. After a rain storm in urban areas, run-off
from highways and parking lots washes nutrients into storm water systems.
Ni trate Con tam i na tion
In slow-moving, shallow bodies of water, excess nutrients encourage the growth
of algae and aquatic vegetation. In a short amount of time, vegetation may cover
the entire area and swimming and boating may become impossible. Dense mats
of algae on the surface will block sunlight needed by submerged aquatic
vegetation (SAV). As the habitat changes, the water quality becomes poor and
only pollution tolerant species will remain. If sediment from erosion and
run-off is trapped in the sunken roots and rotting vegetation the entire
waterway may fill in completely in a process called eutrophication.
In fast moving streams, nutrient levels may be high but the flow of water will
prevent the establishment of floating aquatic plants and algae. During
seasonal fluctuations the water flow may become reduced and streams will
then become choked with algae.
Industrial processes that use natural water for cooling, like power plants, can
be damaged when the source water is thick with algae. Bathers and boaters
using recreational waters will experience clumps of floating algae in the water
and rotting algae on the shoreline after an algae bloom.
Drinking water containing high nitrate levels can affect the ability of blood
to carry oxygen. This condition, called methemoglobinemia, is especially
harmful to infants and anyone with a compromised immune system.
Test ing for Ni trate
Widely used test methods utilize heavy metals to reduce the nitrate to nitrite.
The most commonly used metals are cadmium and zinc. Cadmium, which is
more hazardous than zinc, gives better sensitivity at low concentrations so it
is used in kits with lower ranges. Recently, a reagent system was developed
that has the sensitivity of cadmium reduction at low concentrations and uses
non-hazardous nitrate enzyme reductase to reduce nitrate to nitrite,
eliminating heavy metals.
En zyme Tech nol ogy
1
The Enzyme Vials contain both freeze-dried NADH (nicotinamide adenine
dinucleotide) and YNaR1 (yeast nitrate reductase version 1). The YNaR1 is
a recombinant form of NAD(P)H: Nitrate Reductase (Enzyme Commission
#EC 1.7.1.2). Nitrate Reductase (NaR) is present in all plants and many
types of yeast. It is the enzyme that starts the process of incorporating
nitrogen species into amino acids and proteins in these organisms. The
NaR1 formerly used was extracted from corn leaf. The current YNaR1 is
produced in the Pichia pastoris protein expression system; Pichia is a yeast,
similar to baker’s yeast. YNaR1 is more stable than the native plant enzyme
forms and can be produced cost-effectively by fermentation. A “handle” has
been engineered into the protein to enable purification by affinity
chromatography. Recombinant production eliminates lot-to-lot variation in
protein quality. The YNaR1 enzyme is comprised of two subunits of over 900
amino acids and 3 cofactors each – flavin adenine dinucleotide (FAD or
vitamin B-2), heme iron, and a molybdenum containing group called
molybdopterin.
The reduction of nitrate to nitrite (reduction in oxidation number from +5 to +3
on the nitrogen by the addition of two electrons) occurs following the general
reaction
NADH + Nitrate à Nitrite + NAD+ + OH_ with YNaR1 acting as the catalyst.
In effect, the electrons are transferred from the NADH to the nitrate molecule,
reducing the nitrate to nitrite.
1
Campbell ER, Kinnunen-Skidmore T, Winoweicki LA, Campbell WH. A new trend in nitrate
analysis: and enzyme-based field test for nitrate. AM LAB News 2001: 33 (4):90-2.
The Ni trate-Ni tro gen En zyme Re duc tion Kit
The Enzyme Vials contain both freeze-dried NADH and YNaR1. The nitrate
reduction takes place under buffered conditions at the molybdenum site on the
YNaR1 enzyme. The YNaR1 is first reduced by the NADH and then the nitrate
is reduced at the molybdenum site. The nitrite produced is then detected by
running the standard Griess reaction in acidic conditions with sulfanilamide and
N-1 indicator in tablet form to produce a red-violet colored complex that is
proportional to the concentration of nitrate.
Con ver sions
Nitrate test results are expressed as “nitrate-nitrogen” (NO
“nitrogen that is in the form of nitrate”. Some test kits express levels only as
_
N), meaning
3
nitrate (NO3). Both expressions refer to the same chemical but express the
concentration in different units of measure. To convert ppm nitrate-nitrogen to
ppm nitrate multiply by 4.4.
ppm Nitrate-nitrogen X 4.4 = ppm Nitrate
LaMotte Com pany
PO Box 329 • Chestertown • Mary land • 21620
800-344-3100 • f 410-778-6394 • www.lamotte.com
68137 02.08
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