SKALAR METHODS
ANALYSIS: NITRATE + NITRITE
RANGE: 0.66 - 33 mg N/liter
SAMPLE: TOBACCO EXTRACT
SAN++
Catnr. 455-912w/r issue 061923/99400304
PRINCIPLE
The automated determination for the determination of Nitrate and Nitrite is based on the hydrazine
reduction method; after dialysis nitrate is reduced to nitrite by hydrazinium sulfate and the nitrite
(originally present plus reduced nitrate) is determined by diazotizing with sulfanilamide and coupling with
N-(1-naphthyl) ethylene diamine dihydrochloride to form a highly colored azo dye which is measured at 540
nm. Method is according Coresta no. 36.
LABORATORY FACILITIES
1. Maximum power consumption depending on the analyzer configuration, 2000 VA. Check voltage at the
back of the instrument before installation.
2. Facilities for chemical wastes. Check environmental regulations for proper disposal of waste.
PROCEDURE SAMPLE PREPARATION
Tobacco extract 1.1.2
Field of application
The extraction is applied for the determination of ammonia, nicotine, nitrate + nitrite and (total) reducing
sugars in tobacco.
Principle
The tobacco sample is extracted in acetic acid solution and filtered over a filter paper. The filtrate is offered
to the system.
Reagents
Acetic acid solution (5%)
1. Dilute 50 ml of acetic acid in 800 ml distilled water.
2. Fill up to 1 liter with distilled water and mix.
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Procedure
1. Prepare the tobacco for analysis by grinding. The sample should be totally pass through a 1 mm sieve.
Note: If the tobacco is too wet for grinding, it can be dried at a temperature not exceeding 40°C.
2. Determine the moisture, required to correct the measured analyzer result:
% Nicotine = R x 100 / (100 – M)
R = result measured in %
M = moisture
3. Weigh, to the nearest of 1 mg, approximately 1.000 g of the ground tobacco in a dry 250 ml conical
flask.
4. Add 100 ml acetic acid solution 5%.
5. Stopper the flask.
6. Shake for 30 minutes.
7. Filter the extract through filter paper Whatman # 40, or equivalent, discard the first 5 ml and collect in a
250 ml Erlenmeyer.
8. The filtrate can directly be offered to the analyzer.
Note: The sample preparation in this method is only meant to be an example procedure. It requires
validation on its correct procedure for all types of samples to be analyzed, which is the responsibility of the
user.
REAGENTS
A. Sodium hydroxide solution (0.35M) (1 liter)
Required chemicals: Sodium hydroxide .......... 14 g. Preparation: Dissolve the sodium hydroxide in ± 800
NaOH ml distilled water. Fill up to 1 liter, add the
Distilled water Brij 35 and mix.
H2O
Brij 35 (30%)................... 3 ml. Note: Solution is stable for 1 week. Store at 4°C
when the solution is not used.
B. Sodium hydroxide solution (0.2M) (1 liter)
Required chemicals: Sodium hydroxide ............ 8 g. Preparation: Dissolve the sodium hydroxide in ± 800
NaOH ml distilled water. Fill up to 1 liter, add the
Distilled water Brij 35 and mix.
H2O
Brij 35 (30%)................ 0.5 ml. Note: Solution is stable for 1 week. Store at 4°C
when the solution is not used.
C. Distilled water + Brij 35 (1 liter)
Required chemicals: Distilled water Preparation: Dilute the Brij in 1 liter distilled water and
H2O mix.
Brij 35 (30%)............... 3 drops
Note: Refresh weekly.
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D. Stock solution copper (II) sulfate (1 liter)
Required chemicals: Copper (II) sulfate .......... 12 g. Preparation: Dissolve the copper (II) sulfate in ± 800 ml
CuSO4.5H2O distilled water. Fill up to 1 liter with
Distilled water distilled water and mix.
H2O
Note: Store in a dark colored bottle. This
solution is stable for 3 months.
E. Hydrazinium sulfate solution (1 liter)
Required chemicals: Hydrazinium sulfate .... 0.80 g. Preparation: Dissolve the hydrazinium sulfate in ± 800
N2H6SO4 ml distilled water. Add the stock solution
Stock solution copper (II) copper (II) sulfate. Fill up to 1 liter and
sulfate ......................... 1.5 ml. mix.
Distilled water
H2O Note: Solution is stable for 1 week.
F. Color reagent (1 liter)
Required chemicals: o-Phosphoric acid....... 100 ml. Preparation: Dilute the o-phosphoric acid in ± 700 ml
H3PO4 (85%) distilled water. Add the sulfanilamide and
Sulfanilamide ................. 10 g. the N-(1-naphthyl) ethylene diamine
C6H8N2O2S dihydrochloride and dissolve. Fill up to 1
N-(1-Naphthyl) ethylenedi- liter with distilled water and mix.
amine dihydrochloride . 0.5 g.
C12H16Cl2N2 Note: Store in a dark colored bottle. Solution is
Distilled water stable for 2 weeks. Store at 4°C when the
H2O solution is not used.
G. Rinsing liquid sampler (1 liter)
Required chemicals: Acetic acid .................... 50 ml. Preparation: Dilute the acetic acid in ± 800 ml distilled
CH3COOH (100%) water. Fill up to 1 liter with distilled water
Distilled water and mix.
H2O
Note: Refresh weekly.
STANDARDS
Stock solution 330 mg N/liter (1 liter)
Required chemicals: Sodium nitrate ........ 2.0025 g. Preparation: Dissolve the sodium nitrate in ± 800 ml
NaNO3 distilled water. Fill up to 1 liter with
Distilled water distilled water and mix.
H2O
Note: Solution is stable for 1 month. Store at
5°C when the solution is not used.
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Working standards
33.0 mg N/liter: Dilute 10 ml stock solution 330 mg N/liter to 100 ml with rinsing liquid sampler.
26.4 mg N/liter: Dilute 8 ml stock solution 330 mg N/liter to 100 ml with rinsing liquid sampler.
19.8 mg N/liter: Dilute 6 ml stock solution 330 mg N/liter to 100 ml with rinsing liquid sampler.
13.2 mg N/liter: Dilute 4 ml stock solution 330 mg N/liter to 100 ml with rinsing liquid sampler.
6.6 mg N/liter: Dilute 2 ml stock solution 330 mg N/liter to 100 ml with rinsing liquid sampler.
Note:
1. Prepare the working standards fresh daily.
2. The water used for the reagents and standards must be degassed properly before making up the
reagents and standards. Especially water produced by reverse osmosis or ion exchange equipment
contains a lot of air, which must be removed by degassing (degassing procedure see operational
remarks and troubleshooting).
CATALOGUE NUMBERS REQUIRED CHEMICALS
Product Supplier and catnr. Danger classification
Acetic acid (glacial) 100% Merck 100063 flammable, corrosive
Sodium hydroxide Merck 106498 corrosive
Brij 35 (30%) Skalar 13900
Copper(II) sulfate pentahydrate Merck 102790 harmful, irritant, dangerous
for the environment
Hydrazinium sulfate Merck 104603 carcinogenic, toxic,
sensitizing, dangerous for
the environment
o-Phosphoric acid (85%) Merck 100573 corrosive
Sulfanilamide Sigma S9251
N-(1-Naphthyl) ethylene diamine dihydrochloride Merck 106237 irritant
Sodium nitrate Merck 106537 harmful, irritant
Hydrochloric acid (32%) Merck 100313 corrosive
RECOMMENDED OPERATIONAL SETTINGS
1. System sample time: 70 sec., wash time: 70 sec., air time: 0 sec.
2. Module sample time: 60 sec., wash time: 60 sec., air time: 0 sec.
3. Calibration type: 1st order ISO 8466-1.
OPERATIONAL REMARKS AND TROUBLESHOOTING
1. The stabilizing time of the system is approximately 20 minutes.
2. The sensitivity of the highest standard 33 mg N/liter ≈ 1.250 AU.
3. It is recommended to set the baseline to 0.200 – 0.500 AU (200.000 – 500.000 DU). The baseline can be
set using the Offset ADC for digital detector channel in the Peak Detection Type menu of the Method
Setup. Every step lowers the base line for 0.300 AU (300.000 DU).
4. Persons using this Skalar method should be familiar with normal laboratory practice. This Skalar
method does not purport to address all of the safety problems, if any, associated with its use. It is the
responsibility of the user to establish appropriate safety and health practices and to ensure compliance
with any national regulatory conditions.
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5. The connection between the sampler and the sample pump tube is made of 5175 tube.
6. The reagent bottles must be rinsed thoroughly with distilled water before refilling with fresh reagents.
This is done to remove precipitation of microorganisms and other interferences.
7. To check the complete reduction of all the nitrate to nitrite a comparison is done between nitrate and
nitrite standards. These two solutions must be the same as the highest standard of the method with an
equal nitrogen concentration. Due to the quality of the hydrazinium sulfate the reduction will variate
for every new batch of hydrazinium sulfate. When the nitrate peak is lower than the nitrite peak the
reduction of nitrate isn’t sufficient. To resolve this, do make some test runs and increase the
hydrazinium sulfate concentration in steps of 0.1g/liter, until the peaks equal for NO2 and NO3. When
the nitrite peak is lower than the nitrate peak there is too much hydrazinium sulfate available and this
will cause the nitrite to reduce. To resolve this, decrease the hydrazinium sulfate in steps of 0.1g/liter
8. Avoid any turbidity in the reagents, filter if necessary.
9. After 6 months the amount of Brij 35 can be reduced to 1 ml/liter.
10. If the sample take up volume is less than 1.00 ml/min, a bypass is required to increase the sample
stream to at least 1.00 ml/min. For 5210 debubbler, when the sample is 1.00 ml/min and higher use
0.23 ml/min debubble tube.
11. Degassing procedures;
a) Good: degas the water with a helium gas flow of approximately 20 liter/hour with aid of a sparger.
Degas 5 liter water for 15 minutes. This procedure removes all dissolved air or, degas the water by
boiling the water for 10 minutes. Cool down to room temperature before usage.
b) Medium: degas the water with a nitrogen gas flow of approximately 20 liter/hour with aid of sparger.
Degas 5 liter water for 15 minutes. This procedure will remove most of the dissolved air.
START UP PROCEDURE
1. Prepare all reagents and standards.
2. Check if the pump deck(s) requires grease (catnr. 2010).
3. Close the pump deck(s).
4. Switch on the sampler, chemistry unit and interface.
5. Put the reagent lines in the appropriate reagent canisters.
6. Turn on the heater.
7. Wait for approximately 20 minutes for a stable baseline and flow.
8. Start the analysis.
SHUT DOWN PROCEDURE
1. Put all reagent lines in distilled water.
2. Turn off the heater.
3. Rinse with distilled water for 20 minutes.
4. Switch off the sampler, chemistry unit and interface.
5. Open pump deck(s).
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MAINTENANCE
1. Daily; when daily analysis is finished rinse all reagent lines with distilled water for 30 minutes.
2. Weekly; Follow the daily procedure and additionally put the sample line from the needle in 1%
hypochlorite solution to decontaminate all sample lines for 30 minutes, then rinse with distilled water
for another 30 minutes.
3. Monthly; rinse all pump tubes with 1% hypochlorite for 30 minutes and with distilled water for another
30 minutes then replace all pump tubes (or shift pump tube one color-bridge on pump) and replace the
membrane.
DIALYZER MAINTENANCE
Instructions to change catnr. 5283 and 5282 ready mount dialyzer membrane;
1. Unscrew the dialyzer block and remove the old membrane.
2. Clean the dialyzer block on both sides with a brush.
3. Open the package and carefully remove the new membrane.
4. Rinse the new membrane with a small amount of of distilled water to remove the excess preservation
liquid.
5. Centre the membrane on the bottom dialyzer plate.
6. Make a cross incision with a blade in the membrane on the 2 center holes of the dialyzer bottom plate.
7. Make several small holes in the membrane through the holes for the mounting screw-bolts.
8. Position carefully the dialyzer top plate on the bottom plate.
9. Push the screw-bolts carefully through the membrane and tighten the screw-bolts, finger tight.
10. Tighten screw-bolts with an Allan-key (starting with the inside), carefully tighten down in stages to
hand tight.
11. Rinse the system several minutes with distilled water to remove any remaining preservation liquid
from the membrane.
Important:
1. When the analyzer is not in operation the membrane should remain in contact with water. Never leave
the membrane dry.
MODULE CONSUMABLES
silicone tube catnr. 3150 membrane catnr. 5282
polyethylene tube catnr. 3142 pump tube 1.00 ml/min. catnr. 3031
polyethylene tube catnr. 5141 pump tube 0.16 ml/min. catnr. 3025
polyethylene tube catnr. 5142 pump tube 0.42 ml/min. catnr. 3028
polyethylene tube catnr. 5145 pump tube 0.60 ml/min. catnr. 3029
sleeves catnr. 5400 pump tube 0.32 ml/min. catnr. 3027
sleeves catnr. 5401 pump tube 0.23 ml/min. catnr. 3026
acid flex sleeves catnr. 5415
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MODULE COMPONENTS
manifold holder catnr. 25200105 connector catnr. 9220 3x
module catnr. 5108 dialyzer catnr. 5271
end block catnr. 5109 reactor catnr. 25500952
inlet connector catnr. 9245 clamps small catnr. 5111 4x
inlet connector catnr. 9241 sinkers catnr. 5380
inlet connector catnr. 5246 3x flow cell 30 mm catnr. 6433
glass coil catnr. 5325 2x filter 540 nm catnr. 6541
glass coil catnr. 5323
REFERENCES
Chemical method (no. 455)
1. Coresta no. 36, determination of nitrate in tobacco by continuous flow analysis.
2. ISO 13395:1996, Determination of nitrite nitrogen and nitrate nitrogen and the sum of both by flow
analysis (CFA) and spectrometric detection.
3. Kamphake, L.J., Hannah, S.A., Cohen, J.M., "Automated analysis for nitrate by hydrazine reduction",
Water Research, 1, 1967, page 205-216.
4. Kempers, A.J., Luft, A.G., "Re-examination of the determination of environmental Nitrate as Nitrite by
reduction with hydrazine", Analyst, Vol. 113, July 1988, pp. 1117-1120.
5. ISO 3696:1987, Water for analytical laboratory use. Specification and test methods.
6. ASTM, D1193, Standard Specification for Reagent Water.
Sample preparation (tobacco extract 1.1.2)
1. Coresta no. 36, determination of nitrate in tobacco by continuous flow analysis, section 5.
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