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Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-1 OF 17

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LAMBTON COLLEGE CHEMISTRY DEPARTMENT
ANALYTICAL CHEMISTRY

STANDARD OPERATING PROCEDURES

DETERMINATION OF NITRATE IN SOIL AND PLANTS

AUTHOR: AARUSHI SHARMA
AMANJOT KAUR
AMANPREET KAUR
BALJIT KAUR
BALJIT KAUR

APPROVED: GISSELLE NORVILLE

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-2 of 17

TABLE OF CONTENTS

1. SCOPE AND AVAILABILITY 4
2. SUMMARY 4-5
3. DEFINITIONS 5-6
4. HEALTH AND SAFETY 6-7
5. INTERFERENCE 7
6. MATERIALS AND METHODS 7-9
7. EXPERIMENTAL PROCEDURES 9-10
8. INSTRUMENTS 11-12
9. FUNCTIONS 12
10. CALIBRATION 12-13
11. HANDLING AND PRESERVATION 13-14
12. QUALITY ASSURANCE/ QUALITY CONTROL 14-15
13. REFERENCES 15-17

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-3 of 17

1)SCOPE AND AVAILABILITY:
Spectrophotometer is used to detect nitrate in soil and plant extracts. Here, first and second derivative UV-spectrometry method is applied for plant and soil extracts respectively. In these methods the salicyclic acid undergoes nitration in basic solution. The main point for application of this technique is the lack of interference. Moreover, this technique is rapid, inexpensive and more appropriate and accurate. The complex formed from the nitration which absorbs at 410 nm in basic solution. The quantity of nitrate ion is proportional to absorbance of chromophore.

2) SUMMARY:
2.1) FOR SOIL:
Nitrate estimation in soils is frequently done in studies connected to nitrogen (N) nutrition of crops and the fate of applied N in soils. Recently, there has been an increasing interest in ultraviolet spectrometric methods of determination of soil nitrates because of their simplicity and speed. The method is rapid and avoids many interferences, but it requires a correction for non-nitrite ions species to be applied. This is done by reducing the nitrate and by measuring the absorption before and after the reduction. More recently, Norman et. Al (1985) have described another method based on dual wavelength ultraviolet spectrometry. This method is very rapid

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-4 of 17

and free from interferences, but the method requires the use of an empirical multiplication factor which has to be determined for each type of soil.

2.2) FOR PLANT:
The examination of plant tissue is broadly used to decide plant mineral sustenance status. Nitrogen is one of the essential supplements for plant development with a sufficient con-tent of around 1– 2% in dry weight premise. Studies have demonstrated that nitrate content is frequently a superior pointer of plant N requirements than aggregate N focus (1– 6) presumably on the grounds that the previous is the fundamental stockpiling type of nitrogen.

For a long time, nitrate has unpredictably been utilized as a compost. At present, its application is under control due to its pernicious consequences for wellbeing. A portion is lessened to nitrites and consolidates with hemoglobin in blood, causing respiratory issues. It might be associated with the formation of cancer-causing substances, for example, nitrosamines and nitro-amides (7). Consequently, the European Commission’s Scientific Committee for Food has built up the per-mitted most extreme focus for lettuce and spinach to be put available. It is in this way important to give adequate techniques to decide plant prerequisites of nitrate for treatment.

3) DEFINITIONS:
3.1) BEER-LAMBERT LAW: It states that the amount of energy absorbed by the solution is directly proportional to the molar concentration of solute.

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-5 of 17

A=?lc
Where A is absorption
? is the molar concentration
L is the path length
C is the concentration of the solution

3.2) DARCO G-60: It is carbon which is steam activated having a very high capacity of absorption and filtration.
3.3) EDTA: Ethylene Diamine-tetra acetic acid.

4) HEALTH AND SAFETY:

• Avoid verifying of combustible samples.
• Do not use solvents, chemicals, reagents to clean the spectrophotometer.
• To move spectrophotometer from one place to another, proper raising mechanisms should be used.
• All unsafe areas should be marked with appropriate signs.
• The staff should be wearing proper personal protective equipment.
• Sodium sulfate can be hazardous in case it comes in contact with eyes and skin.
Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-6 of 17

• Do not breathe gas fumes/ vapors / spray.
• EDTA may be toxic to mucous membrane.
• Phenol disulfide acid can cause severe burns.

5) INTERFERENCE:

SOIL:With the use of second derivative method, soil’s organic matter broad UV absorption peaks do not interfere. Nitrate and other usually present ions in the soil sample do not interfere. There are chances of nitrite interference if the concentration of nitrite is more than half the concentration of nitrate which is a rare case. Carbonate interferes with nitrate determination in pure solutions. So, acidification of the soil extracts with sulfuric acid might be necessary as to eliminate the interference. While adding foreign substances to the extract, it should be kept in mind that if the substances added do not absorb in the UV range or have only broad peaks, they will not interfere in this method.
PLANTS: Using first derivative spectrophotometric method there is lack matrix background interference.

6) MATERIALS AND METHODS:
6.1) FOR SOIL:

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-7 of 17

REAGENTS:
• Sodium Sulfate 1N: Dissolve 142.0 g analytical reagent grade sodium sulfate in 1 liter of distilled water.
• Darco G-60 Carbon
• Nitrate Stock solution: Dissolve 7.221 g analytical reagent grade potassium nitrate in 1 liter of distilled water. 1 mL of this solution contains 1000 ?g nitrate-N (NO3-N). The solution is further diluted as required to obtain other concentrations.
• Phenol disulfonic Acid: Dissolve 0.3 pure antimony metal in 200 mL conc. Sulfuric acid by heating. Cool to about 60?C and decant. Dissolve 25g colorless phenol in 150 mL conc.
• Sulfuric acid containing prepared as above and mix. Add 75 mL fuming sulfuric acid. Heat on a steam bath for two hours. Store in a tightly stoppered bottle.
• Sodium Hydroxide- EDTA Solution: Dissolve 360 g sodium hydroxide in 500mL of water. Add 15 g EDTA disodium salt and dissolve. Dilute to 1L with water. Store in a stoppered polyethylene container.

6.2) FOR PLANTS:
Plant Material:
Lettuce and spinach were purchased from a local market. The intact leaves were washed quickly in Tween-20 1% solution followed by washing 3 times in distilled water to remove dust. The samples were dried in a forced-air oven at 65°C for 24 h, ground to pass through a 40-mesh screen, and mixed thoroughly.

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-8 of 17

Reagents:
All reagents were of analytical grade.
(a) Nitrate–N stock standard—Solutionof500mg/L was prepared from KNO3. Working standard solutions of 10, 20, 30, 40, 50, 60, 80, and 100 mg/L were prepared by diluting the standard with distilled water and were stored at 4°C.
(b) Salicylic acid solution 5% (m/v) in concentrated H2SO4.—Salicylic acid (5.00 g) was dissolved in concentrated sulfuric acid and diluted to volume (100 mL) with the same acid; prepared at least once each 48 h, and stored in an amber bottle at 4°C.

(c) 2N Sodium hydroxide solution.

7) EXPERIMENTAL PROCEDURE:
7.1) FOR SOIL:
•Weigh 5 g of soil into a 50mL cone-like jar, include 25mL 1N sodium sulfate arrangement and 0.5 g Darco G-60 carbon.

•Stopper the cone-like jar and shake on a mechanical shaker overwhelmingly for 30 minutes. At that point channel the substance of the jar through a Whatman No. 41 channel paper.

•The initial couple of mL of the filtrate will be turbid and is come back to the blend of soil and extractant.

•A clear filtrate is gathered. A clear is set up by shaking 25 mL IN sodium sulfate arrangement with 0.5 g Darco G-60 carbon, blending and afterward sifting the suspension.

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-9

•The cuvette containing the clear is put in the reference shaft and a cuvette containing the example is set in the estimating pillar.

•After the absorbance show achieves 0.000, the second subordinate range is examined. The pinnacle absorbance and its wavelength are noted

7.2) FOR PLANT:
1. Turn on spectrophotometer

2. pipette an aliquot (e.g. 0.25 mL) of concentrate or standard into a 50-mL Erlenmyer jar

3. blend completely with 0.8 mL of 5% (w/v) salicylic corrosive in conc. H2SO4

4. Following 20 minutes at room temperature, include 19 mL of 2 N NaOH to raise the pH over 12

5. Cool examples to room temperature

6. Measure absorbance at 410 nm

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-10

8) INSTRUMENT:

FIG:1

8.1) FOR SOIL:
Shimadzu UV-260 double beam recording spectrophotometer. The UV spectrometer parameter and functions are set as follows:
Wavelength range: 210-240nm
Absorbance range: -0.25 to +0.25 A, Scan speed: rapid, Scan mode: overlay

8.2) FOR PLANT:
(a) Spectrophotometer—UNICAM (Cambridge, UK) UV 2 double beam UV Vis with 1 cm quartz cells attached to a printer was used. The spectra were obtained with a spectral bandwidth of 2 nm. The derivative spectra were obtained by instrumental electronic differentiation. (b) Water bath—Maintain the extract of plant tissue at 45°C.

Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-11

9) FUNCTIONS:
Peak pick (wavelength and peak height indication)
Flag change: direct second order derivative with wavelength =4 nm.
Cuvettes: matched 1cm path length quartz cuvettes.

10) CALIBRATION:

10.1) FOR SOIL:
Standard solutions containing 0, 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20 mg/L nitrate-N are prepared by diluting the Nitrate Stock Solution with 1N Sodium Sulfate Solution. A 25mL aliquot of each of the prepared solutions are shaken for 30 minutes with 0.5 g of G-60 Carbon and then filtered.
The second derivative peak absorbance of the standards are measured as described above and a calibration curve is drawn.
The concentration of nitrate-N in the soil extract is read from the curve and the corresponding soil Nitrate-N concentration is calculated.

10.2) FOR PLANT:
Aliquots (0.1 mL) of working standard arrangements (10– 100 mg/L NO3– – N) in a 30 mL tube were blended thoroughly with 0.4 mL salicylic corrosive arrangement. After 20 min at room temperature, 9.5 mL 2N NaOH arrangement was gradually added to get 0.1, 0.2, 0.3, 0.4, 0.5,
Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-12

0.6, 0.8, and 1.0 mg/L NO3– – N arrangements. This strategy depends on the development of nitro-salicylic corrosive that shows, in very essential arrangement, a maxi-mum assimilation at 412 nm in zero-arrange ingestion range, and 388 and 440 nm in first-arrange range (1D388– 440). The whole of the statures of the two pinnacles (388 and 440 nm) positive and negative (top to-top) of the principal subordinate was utilized. The wavelength extended chose to get the range was 356– 500 nm; information interim, 4 nm.

11) HANDLING AND PRESERVATION:
11.1) FOR SOIL:
• Collect a composite sample of soil by properly mixing the subsamples and send immediately to the laboratory.
• Now place the sample in a suitable metal tray and remove any stones present. Then air-dry the soil sample at a temperature below 30? C
• After that, grind the sample to pass a 2mm mesh sieve and again dry it if moist. Place the sample in an air-tight container with proper label having details of the soil sample.
• Refer to the MSDS for safe handling of chemical substances being used in the experiment.

11.2) FOR PLANT:
• Collect a representative sample in a large paper bag.
After sampling, cut the plant parts into small pieces and put into suitable clean trays lined with grease-proof paper. Put this tray into an oven with temperature range of 70?-80?C to dry the sample.
Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-13

• After this, grind the sample and store it in an airtight container. Keep it in a cool dry place.
• Sample should be dried again before weighing for chemical analysis.
• Sample should be properly labelled along with date of arrival and source of sample.

12) QUALITY ASSURANCE/ QUALITY CONTROL:
12.1) FIELD REPLICATES: These field samples are taken from the same place then mixed, separated into containers and handled separately. Sample picking, composition and processing is evaluated by this method. When we find precision for sample which is concentrated approximately to an equal to minimum concentration which is required for some
observation for administration. To conduct reasonable statistical technique at least 8 field replicates are needed.

12.2) COLLOCATED SAMPLES: To regulate the irregularity of soil and impurity within small site. These are extracted about one half to three feet away from regular point. Th ese are site specific.
12.3) BACKGROUND SAMPLES: Background samples are assembled to work on natural occurrence of metals and to check potential mistakes related to design of testing, techniques and statistical analysis.
Analytical chemistry Determination of nitrates
Lambton College Chemistry Lab SOP: LAQT 1105 Rev. no-
DATE: OCTOBER 2018 PG no-14

12.4) FIELD BLANKS: In this process, clean sand or soil filled containers are sent for analysis to find out the adulterated fallacy in field operation, procedure and shipping. Field blanks are very useful.
12.5) PERFORMANCE EVALUATION SAMPLES: In this methodology, a portion of the analyte is picked by third party which is known to the manufacturer whereas the laboratory is unaware of it. This is done to make sure that the results obtained by the laboratory are accurate and precise or not.
12.6) MATRIX SPIKE SAMPLES: To justify the results, we retrieve matrix spike and matrix spike duplicate samples which are stored in the laboratory with quantity of target analyte. Errors arise by virtue of laboratory bias and precision can be found out this way. To
know about standard divergence, coefficient of variation, precision minimum of eight matrix spike replicates are taken out from the same sample.

13) REFERENCES:
13.1) FOR SOIL:
1. Bremner, J.M. Inorganic forms of nitrogen, 1965 pp. 1179-1237. IN: CA Black et al (eds.)
Methods of Soil Analysis, part 2, Agronomy 9. American Society of Agronomy, Madison, WI.
2. Norman, R.J. and J.W. Stucki. 1981.The determination of nitrate in soil extracts by ultraviolet spectrophotometry. Soil Sci. Soc. Am. J. 45:347-353.
3. Norman, R.J. and J.W. Stucki. 1985. Determination of nitrate in soil extracts by dual wavelength ultraviolet spectrophotometry. Soil Sci. Soc. Am. J. 49:1182-1185.
4. Onken, B. and H.D. Sunderman. 1977. Colorimetric determination of exchangeable ammonium, urea, nitrate and nitrite in a single soil extract. Argon. J. 69:49-53.
5. Puttanna, K. and E.V.S. Prakas Rao. 1981. Elimination of chloride interference in the phenoldisulphonic acid method of nitrate determination in soils. Commun. Soil Sci. Plant Anal. 12(7):711-718.
6. Simal, J.,M.A. Lage and I. Iglesias. 1985. Second derivative ultraviolet spectroscopy and sulphamil acid method for the determination of nitrates in water. J. Aocc . Off. Anal. Chem. 65(5):962-964.

13.20FOR PLANT:
1. Hylton, L.O., Ulrich, A., & Cornelius, D.R. (1965) Crop Sci. 5, 21–22
2.
3. Tabor, J.A., Pennington, D.A., & Warwick A.W. (1984) Commun. Soil Sci. Plant Anal. 15, 573–585
4. Papastylianou, Y., Graham, R.D., & Puckridge, D.W. (1982) Commun. Soil Sci. Plant Anal. 13, 473–485
5. Gardner, B.R., & Roth, R.L. (1989) J. Plant Nutr. 12, 1073–1088
6. Gardner, B.R., & Roth, R.L. (1990) J. Plant Nutr. 13, 1435–1451
7. Marshner, H. (1995) in Mineral Nutrition of Higher Plants, 2nd Ed., Academic Press, London, UK, Ch. 12
8. Oertli, J.J., & Ruh, R. (1992) Commun. Soil Sci. Plant Anal. 23, 2711–2728
9. Paul, J.L., & Carlson, R.M. (1968) J. Agric. Food Chem. 16, 766–768
10. Baker, A.S., & Smith, J. (1969) J. Agric. Food Chem. 17, 1284–1287
11. Bradfield, E.G., & Cooke, D.T. (1985) Analyst 110, 1409–1410
12. Lyons, D.J., McCallum, L.E., Osborne,W.J., & Nobbs, P.E. (1991) Analyst 116, 153–157
13. Johnson, C.M., & Ulrich, A. (1950) Anal Chem. 22, 1526–1529
14. West, P.W., & Ramachandran, T.P. (1966) Anal. Chim. Acta 35, 317–324
15. Baker, A.S. (1967) J. Agric. Food Chem. 15, 802–806 (15) Sah, R.N. (1994) Commun. Soil Sci. Plant Anal. 25, 2841-2860
16. Cataldo, D.A., Haroon, M., Schrader, L., & Youngs, V.L. (1975) Commun. Soil Sci. Plant Anal. 6, 71–80
17. Miller, C., & Miller, J.N. (1993) Estadi?stica para Qui?mica Anali?tica, 2nd Ed., Addison-Wesley Iberoamericana, Wilmington, DE
18. Castro, M., Gasco?n, S., Pujol, M., Sans, J.A., & Vicente, L.
(1989) Validacio?n de me?todos anali?ticos. Monografi?a A.E.F.I. (Seccio?n Catalana Comisio?n de Normas de Buena Fabricacio?n y Control de Calidad), Editada Bajo el Patrocinio de Hewlett-Packard, Barcelona, Spain
(19) Kenney, D.R., & Nelson, D.W. (1982) in Methods of Soil Analysis, Part 2, Agronomy No. 9, A.L. Page, R.H. Milled, & D.R. Kenney (Eds), American Society of Agronomy, Madi- son, WI, pp 643–698
(20) FIGURE : https://goo.gl/images/tPUMru

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