Most of the nitrogen in the soil is organically bound. Inorganic forms of nitrogen generally account for 1% to 5% of total nitrogen. The increase and decrease of soil organic matter and nitrogen mainly depends on the relative strengths of bioaccumulation and decomposition, as well as various factors such as climate, vegetation, and farming systems, especially hydrothermal conditions, which have significant effects on soil organic matter and nitrogen content.
(I) Determination of Total Nitrogen in Soil
The method for determining soil total nitrogen can be mainly divided into dry burning and wet burning. Wet burning is the commonly used Kjeldahl method. This method was used by Danish KJ Keldahl in 1883 to study protein changes and was later used to determine various forms of organic nitrogen. Because the equipment is relatively simple and easy to obtain, the results are reliable and are used by general laboratories. The semi-micro Kjeldahl method widely used at present is described below.
When the sample is digested with concentrated sulfuric acid under the action of an accelerator, various nitrogen-containing organic compounds undergo a complicated pyrolysis reaction and are converted into ammonia and combined with sulfuric acid to form ammonium sulfate. Ammonia distilled after alkalization is absorbed by boric acid and titrated with a standard acid solution to determine total soil nitrogen content (excluding all nitrate nitrogen).
In the determination of total nitrogen including nitrate and nitrite nitrogen, the nitrite nitrogen in the sample is first oxidized to nitrate nitrogen with potassium permanganate before the sample is decocted, and then the whole nitrate state is reduced with the reduced iron powder. Nitrogen is reduced and converted into ammonium nitrogen.
Sulfuric acid is a strong oxidizer at high temperatures and can decompose organic matter by oxidizing carbon in organic compounds.
Instruments, Equipment and Reagents
1. Instrument oven, semi-micro-distillation device, semi-micro buret (5 mL).
2. Reagents
(1) 10mol · L-1NaOH solution: Weigh 420g of industrial solid NaOH in a hard glass beaker, add distilled water 400mL dissolved, stirring constantly, after cooling down the plastic reagent bottle, stopper, (for a few days) until Na2C% After settling, siphon the supernatant into 160 mL of CO2-free water and dilute to volume with CO2 distilled water.
L.
(2) Methyl red-bromocresol green mixed indicator: 0.5 g bromocresol green and 0.1 g methyl red were dissolved in 100 mL of 95% ethanol.
(3)20g·L-1H3BO3:20g H3BO3 (chemically pure) dissolved in 1
In L water, add 5 mL of methyl red-bromocresol green mixed indicator per liter of H3BO3 solution and adjust it to slightly purple with dilute acid or dilute alkali. At this time, the pH of the solution is 4.8. The indicator is mixed with boric acid before use. This reagent should be ready for use and should not be placed for a long time.
(4) Mixing accelerator: 100g K2SO4, 10g CuS04·5H2O and 1gSe powder were mixed and ground. The mixture was thoroughly mixed through a No. 80 sieve and stored in a stoppered bottle. Add 0.37g of mixed accelerator per ml of H2SO4 during digestion.
(5) 0.02 mol·L-1 sulfuric acid standard solution: Measure 2.83 mL H2SO4, dilute it with water to 5 000 mL, and then calibrate it with standard alkali or borax.
(6) Potassium permanganate solution: 25 g of potassium permanganate was dissolved in 500 mL of ion-free water and stored in a brown bottle.
(7) Reduced Iron Powder: Finely ground through a 0.149 mm (100 mesh) sieve.
Method and procedure
1. Sample preparation 1.0000 g of air-dried soil samples (passed through a 0.149 mm sieve) was weighed.
2. Digestion of soil sample 1 Excluding the digestion of nitrate nitrogen and nitrite nitrogen: Give the soil sample to the bottom of a dry Kjeldahl bottle, moisten the soil with a small amount of non-ionized water (0.5 to 1 mL), and add 2 g of soil sample. Accelerator and 5mL of concentrated sulfuric acid, shake, the Kelvin bottle is placed on a 300W variable temperature electric furnace, heated with a small fire, until the reaction within the bottle is moderate (10 ~ 15min), strengthen the fire to keep the boiling water to keep the micro-boiling The heated part does not exceed the liquid level in the bottle to prevent the temperature of the bottle wall from being too high and the ammonium salt to be decomposed by heat, resulting in loss of nitrogen. The temperature of the cooking is preferably that the sulfuric vapor condenses at the upper 1/3 of the bottle neck. After the liquid to be detoxified and the soil particles all turn grayish and slightly green, continue cooking 1
h. After digestion is complete, cool and wait for distillation. While the soil samples were digested, two blank measurements were made, except that no soil samples were used, and all other operations were the same as those of the soil samples.
2 Including the digestion of nitrate and nitrite nitrogen: Give the soil sample to the bottom of a dry Kjell's bottle, add 1 mL of potassium permanganate solution, shake the Kjeldahl bottle, and slowly add 1:1 sulfuric acid to 2 mL. The bottle is then placed for 5 min and then 1 drop of octanol is added. 0.50g (±0.01g) of reduced iron powder is sent to the bottom of the Kiel bottle via a long-necked funnel, the funnel is covered with a small funnel, and the Kjeldahl bottle is turned to contact the iron powder with the acid until the violent reaction stops (about 5min). Put the Kjeldahl bottle on the electric furnace and slowly heat it for 45 minutes (the inside of the bottle should be kept slightly boiled so as not to cause a large amount of moisture loss). After the Kjeldahl bottle is cooled, add 2 g of accelerator and 5 mL of concentrated sulfuric acid through a long neck funnel and shake. According to the above step 1, boil until the soil solution turns yellow-green, and continue cooking 1
h. After digestion is complete, cool and wait for distillation. While cooking the soil sample, make two blank determinations.
3. Distillation of ammonia 1 Before distillation, check whether the distillation unit leaks, and wash the pipe through the distillate of water.
2 After the cooking liquid is cooled, the cooking liquid is transferred to the distiller with a small amount of ion-free water, and the Kjell bottle is washed with water for 4 to 5 times (the total water consumption does not exceed 30-35 mL). If a semi-automatic automatic nitrogen analyzer is used, it is not necessary to transfer, and the digestion tube can be directly distilled in a person's nitrogen determination apparatus.
In a 150-mL Erlenmeyer flask, add 5 mL of 20 g·L-1 boric acid indicator mixture and place it on the end of the condenser tube. Place the nozzle 3 to 4 above the boric acid level.
Cm. Then, 20 mL of 10 mol·L-1NaOH solution was gradually added into the distillation chamber, and steam distillation was conducted. When the volume of the distilled liquid was about 50 mL, the distillation was completed. The ends of the condenser tube were washed with a small amount of water that had been adjusted to pH 4.5.
4. Determine the titration of the distillate with 0.01 mol·L-1H2SO4 or 0.01 mol·L-1 HCl standard solution until the distillate turns from blue-green to purple. Record the volume (mL) of acid standard solution used. The volume of the acid standard solution used for the blank measurement is generally not to exceed 0.4 mL.
5. Calculation formula: V - the volume of the acid standard solution used when titrating the test solution, mL;
V0 - the volume of the acid standard solution used to titrate the blank, mL;
c - H2SO4 or HCl standard solution concentration, mol · L -1;
m - the quality of dried soil samples, g.
The results of the two parallel determinations allow an absolute phase difference: when the soil nitrogen content is greater than 1.0g·kg-1, it must not exceed 0.005%; nitrogen 1.0-0.6g·k
The kg-1 should not exceed 0.004%; when the nitrogen content is less than 0.6gkg-1, it must not exceed 0.003%.
Matters needing attention 1 For the titration of trace nitrogen can also use another more sensitive mixing indicator, namely 0.099
g bromocresol green and 0.066 g methyl red were dissolved in 100 mL ethanol. Preparation of 20g·L―1H3BO3 indicator solution: Weigh 20g of boric acid (analytically pure) into about 950mL of water, heat and stir until H3BO3 dissolves. After cooling, add mixing indicator 20 to mix and adjust with dilute acid or dilute alkali. To purple (pH about 5), dilute with water to 1L and mix. Should be equipped with.
2 In general, the amount of nitrogen in the sample should be 1.0 to 2.0 mg. If the soil nitrogen content is below 2 g·kg-1, the soil sample should be called 1 g; if the nitrogen content is 2.0 to 4.0 g·kg-1, it should be called 0.5 ~ 1.0g; nitrogen content of 4.0g · kg-1 or more should be said 0.5g.
3 The concentration and amount of boric acid should be sufficient to meet the absorption of NH3, which can be roughly calculated as the amount of nitrogen (N) absorbed by 10g·L-1H3BO3 per milliliter of 0.46 tons. For example, 20g・L-1
The maximum amount of absorbable nitrogen (N) in 5 mL of H3BO3 solution is 5X2X0.46=4.6 mg. Therefore, the amount of boric acid can be estimated based on the nitrogen content in the cooking liquid, which is more appropriate.
4 In the semi-micro-distillation, the condensing nozzle does not need to be inserted into the boric acid solution. This prevents backward suction and reduces the washing procedure. However, in the constant distillation, due to the high nitrogen content, the condenser tube must be inserted into the boric acid solution to avoid loss.
(B) Determination of Soil Hydrolysis Nitrogen (Alkaline Solution Diffusion Method)
Soil hydrolyzable nitrogen is also called soil effective nitrogen. It includes inorganic nitrogen and some simple organic nitrogen which is easily decomposed in organic matter. It is ammonia nitrogen, nitrate nitrogen, amino acid, ammonium and hydrolyzable protein nitrogen. sum. Determination of nitrogen hydrolysis can understand the level of nitrogen supply in the soil in a certain period (such as a growing season or a year), and is of reference value for the formulation of improved soil fertility planning, development of a reasonable fertilization plan, determination of fertilizing amount in the field, and crop management. .
The alkaline solution dispersal method described in this experiment utilizes the hydrolysis of dilute alkali and soil samples under certain conditions to convert readily hydrolyzable organic nitrogen in the soil to ammonia gas, and continuously diffuses and escapes, together with the original soil. Ammonia nitrogen is absorbed by boric acid and titrated with standard acid to calculate the content of hydrolyzable nitrogen. However, the effective nitrogen measured by this method does not include nitrate in soil.
Instruments, Equipment and Materials
1. Instruments and Equipment Soil Sample Screen (1mm) Electronic Balance Diffusion Tank Thermostat Semi-microburette
2. Reagents
(1) 1mol·L -1 NaOH solution: 40g of chemically pure NaOH was dissolved in 1L of water.
(2) Alkaline glycerol: The simplest method is to dissolve several small solid NaOH in glycerin.
(3) 2% boric acid solution (containing bromocresol green-methyl red indicator): Dissolve 20g H3B03 in 1
L water, add bromocresol green - methyl red indicator 10mL, and dilute NaOH (about 0.1mol · L-1) or dilute HCl (0.1mol · L-1) adjusted to purple (pH4.5).
(4) Bromocresol green-methyl red indicator: 0.5 g bromocresol green and 0.1 g methyl red were dissolved in 100 mL of 95% alcohol.
(5) 0.01mol · L-1 HCl standard solution: first prepare 1.0mol · L-1 HCl solution, diluted 100 times, with borax or Na2CO3 dried at 180 °C calibration its exact concentration.
Method and procedure
Weigh out 2.00 g of air-dried soil sample (passed through a 1mm sieve), place it in the outer chamber of the diffusion dish, and gently rotate the diffusion dish to evenly spread the soil. Take 2mL2% boric acid indicator and put it in the interior of the diffuser. Then apply alkaline glycerin on the edge of the outer chamber of the diffuser, cover with frosted glass, rotate several times to fully adhere the edge of the dish to the frosted glass, and then gradually open the side of the frosted glass. Expose the outside of the diffuser to a slit and quickly add 10.0
The concentration of mL is 1 mol·L-1NaOH. Immediately seal the membrane with a rubber band and tighten the glass. Then release 40 ± l °C incubator, alkaline solution diffusion 24 ± 0.5h after removal. Ammonia in the absorption solution of the inner chamber is titrated (dropped from blue to reddish) with a 0.01 mol·L -1 HCl standard solution in a semi-microtiter tube. A blank test is performed at the same time as the sample measurement to correct errors in reagents and titration operations.
The result is calculated as follows:
In the formula: V, Vo - the volume of standard HCl used in the determination of soil samples and blank experiments, mL;
c - the concentration of standard acid, mol · L ·;
14.0 - the molar mass of the nitrogen atom;
W - weighing dry soil samples, g;
The allowable error for two parallel measurements is 5 mg/kg-1.
(I) Determination of Total Nitrogen in Soil
The method for determining soil total nitrogen can be mainly divided into dry burning and wet burning. Wet burning is the commonly used Kjeldahl method. This method was used by Danish KJ Keldahl in 1883 to study protein changes and was later used to determine various forms of organic nitrogen. Because the equipment is relatively simple and easy to obtain, the results are reliable and are used by general laboratories. The semi-micro Kjeldahl method widely used at present is described below.
When the sample is digested with concentrated sulfuric acid under the action of an accelerator, various nitrogen-containing organic compounds undergo a complicated pyrolysis reaction and are converted into ammonia and combined with sulfuric acid to form ammonium sulfate. Ammonia distilled after alkalization is absorbed by boric acid and titrated with a standard acid solution to determine total soil nitrogen content (excluding all nitrate nitrogen).
In the determination of total nitrogen including nitrate and nitrite nitrogen, the nitrite nitrogen in the sample is first oxidized to nitrate nitrogen with potassium permanganate before the sample is decocted, and then the whole nitrate state is reduced with the reduced iron powder. Nitrogen is reduced and converted into ammonium nitrogen.
Sulfuric acid is a strong oxidizer at high temperatures and can decompose organic matter by oxidizing carbon in organic compounds.
Instruments, Equipment and Reagents
1. Instrument oven, semi-micro-distillation device, semi-micro buret (5 mL).
2. Reagents
(1) 10mol · L-1NaOH solution: Weigh 420g of industrial solid NaOH in a hard glass beaker, add distilled water 400mL dissolved, stirring constantly, after cooling down the plastic reagent bottle, stopper, (for a few days) until Na2C% After settling, siphon the supernatant into 160 mL of CO2-free water and dilute to volume with CO2 distilled water.
L.
(2) Methyl red-bromocresol green mixed indicator: 0.5 g bromocresol green and 0.1 g methyl red were dissolved in 100 mL of 95% ethanol.
(3)20g·L-1H3BO3:20g H3BO3 (chemically pure) dissolved in 1
In L water, add 5 mL of methyl red-bromocresol green mixed indicator per liter of H3BO3 solution and adjust it to slightly purple with dilute acid or dilute alkali. At this time, the pH of the solution is 4.8. The indicator is mixed with boric acid before use. This reagent should be ready for use and should not be placed for a long time.
(4) Mixing accelerator: 100g K2SO4, 10g CuS04·5H2O and 1gSe powder were mixed and ground. The mixture was thoroughly mixed through a No. 80 sieve and stored in a stoppered bottle. Add 0.37g of mixed accelerator per ml of H2SO4 during digestion.
(5) 0.02 mol·L-1 sulfuric acid standard solution: Measure 2.83 mL H2SO4, dilute it with water to 5 000 mL, and then calibrate it with standard alkali or borax.
(6) Potassium permanganate solution: 25 g of potassium permanganate was dissolved in 500 mL of ion-free water and stored in a brown bottle.
(7) Reduced Iron Powder: Finely ground through a 0.149 mm (100 mesh) sieve.
Method and procedure
1. Sample preparation 1.0000 g of air-dried soil samples (passed through a 0.149 mm sieve) was weighed.
2. Digestion of soil sample 1 Excluding the digestion of nitrate nitrogen and nitrite nitrogen: Give the soil sample to the bottom of a dry Kjeldahl bottle, moisten the soil with a small amount of non-ionized water (0.5 to 1 mL), and add 2 g of soil sample. Accelerator and 5mL of concentrated sulfuric acid, shake, the Kelvin bottle is placed on a 300W variable temperature electric furnace, heated with a small fire, until the reaction within the bottle is moderate (10 ~ 15min), strengthen the fire to keep the boiling water to keep the micro-boiling The heated part does not exceed the liquid level in the bottle to prevent the temperature of the bottle wall from being too high and the ammonium salt to be decomposed by heat, resulting in loss of nitrogen. The temperature of the cooking is preferably that the sulfuric vapor condenses at the upper 1/3 of the bottle neck. After the liquid to be detoxified and the soil particles all turn grayish and slightly green, continue cooking 1
h. After digestion is complete, cool and wait for distillation. While the soil samples were digested, two blank measurements were made, except that no soil samples were used, and all other operations were the same as those of the soil samples.
2 Including the digestion of nitrate and nitrite nitrogen: Give the soil sample to the bottom of a dry Kjell's bottle, add 1 mL of potassium permanganate solution, shake the Kjeldahl bottle, and slowly add 1:1 sulfuric acid to 2 mL. The bottle is then placed for 5 min and then 1 drop of octanol is added. 0.50g (±0.01g) of reduced iron powder is sent to the bottom of the Kiel bottle via a long-necked funnel, the funnel is covered with a small funnel, and the Kjeldahl bottle is turned to contact the iron powder with the acid until the violent reaction stops (about 5min). Put the Kjeldahl bottle on the electric furnace and slowly heat it for 45 minutes (the inside of the bottle should be kept slightly boiled so as not to cause a large amount of moisture loss). After the Kjeldahl bottle is cooled, add 2 g of accelerator and 5 mL of concentrated sulfuric acid through a long neck funnel and shake. According to the above step 1, boil until the soil solution turns yellow-green, and continue cooking 1
h. After digestion is complete, cool and wait for distillation. While cooking the soil sample, make two blank determinations.
3. Distillation of ammonia 1 Before distillation, check whether the distillation unit leaks, and wash the pipe through the distillate of water.
2 After the cooking liquid is cooled, the cooking liquid is transferred to the distiller with a small amount of ion-free water, and the Kjell bottle is washed with water for 4 to 5 times (the total water consumption does not exceed 30-35 mL). If a semi-automatic automatic nitrogen analyzer is used, it is not necessary to transfer, and the digestion tube can be directly distilled in a person's nitrogen determination apparatus.
In a 150-mL Erlenmeyer flask, add 5 mL of 20 g·L-1 boric acid indicator mixture and place it on the end of the condenser tube. Place the nozzle 3 to 4 above the boric acid level.
Cm. Then, 20 mL of 10 mol·L-1NaOH solution was gradually added into the distillation chamber, and steam distillation was conducted. When the volume of the distilled liquid was about 50 mL, the distillation was completed. The ends of the condenser tube were washed with a small amount of water that had been adjusted to pH 4.5.
4. Determine the titration of the distillate with 0.01 mol·L-1H2SO4 or 0.01 mol·L-1 HCl standard solution until the distillate turns from blue-green to purple. Record the volume (mL) of acid standard solution used. The volume of the acid standard solution used for the blank measurement is generally not to exceed 0.4 mL.
5. Calculation formula: V - the volume of the acid standard solution used when titrating the test solution, mL;
V0 - the volume of the acid standard solution used to titrate the blank, mL;
c - H2SO4 or HCl standard solution concentration, mol · L -1;
m - the quality of dried soil samples, g.
The results of the two parallel determinations allow an absolute phase difference: when the soil nitrogen content is greater than 1.0g·kg-1, it must not exceed 0.005%; nitrogen 1.0-0.6g·k
The kg-1 should not exceed 0.004%; when the nitrogen content is less than 0.6gkg-1, it must not exceed 0.003%.
Matters needing attention 1 For the titration of trace nitrogen can also use another more sensitive mixing indicator, namely 0.099
g bromocresol green and 0.066 g methyl red were dissolved in 100 mL ethanol. Preparation of 20g·L―1H3BO3 indicator solution: Weigh 20g of boric acid (analytically pure) into about 950mL of water, heat and stir until H3BO3 dissolves. After cooling, add mixing indicator 20 to mix and adjust with dilute acid or dilute alkali. To purple (pH about 5), dilute with water to 1L and mix. Should be equipped with.
2 In general, the amount of nitrogen in the sample should be 1.0 to 2.0 mg. If the soil nitrogen content is below 2 g·kg-1, the soil sample should be called 1 g; if the nitrogen content is 2.0 to 4.0 g·kg-1, it should be called 0.5 ~ 1.0g; nitrogen content of 4.0g · kg-1 or more should be said 0.5g.
3 The concentration and amount of boric acid should be sufficient to meet the absorption of NH3, which can be roughly calculated as the amount of nitrogen (N) absorbed by 10g·L-1H3BO3 per milliliter of 0.46 tons. For example, 20g・L-1
The maximum amount of absorbable nitrogen (N) in 5 mL of H3BO3 solution is 5X2X0.46=4.6 mg. Therefore, the amount of boric acid can be estimated based on the nitrogen content in the cooking liquid, which is more appropriate.
4 In the semi-micro-distillation, the condensing nozzle does not need to be inserted into the boric acid solution. This prevents backward suction and reduces the washing procedure. However, in the constant distillation, due to the high nitrogen content, the condenser tube must be inserted into the boric acid solution to avoid loss.
(B) Determination of Soil Hydrolysis Nitrogen (Alkaline Solution Diffusion Method)
Soil hydrolyzable nitrogen is also called soil effective nitrogen. It includes inorganic nitrogen and some simple organic nitrogen which is easily decomposed in organic matter. It is ammonia nitrogen, nitrate nitrogen, amino acid, ammonium and hydrolyzable protein nitrogen. sum. Determination of nitrogen hydrolysis can understand the level of nitrogen supply in the soil in a certain period (such as a growing season or a year), and is of reference value for the formulation of improved soil fertility planning, development of a reasonable fertilization plan, determination of fertilizing amount in the field, and crop management. .
The alkaline solution dispersal method described in this experiment utilizes the hydrolysis of dilute alkali and soil samples under certain conditions to convert readily hydrolyzable organic nitrogen in the soil to ammonia gas, and continuously diffuses and escapes, together with the original soil. Ammonia nitrogen is absorbed by boric acid and titrated with standard acid to calculate the content of hydrolyzable nitrogen. However, the effective nitrogen measured by this method does not include nitrate in soil.
Instruments, Equipment and Materials
1. Instruments and Equipment Soil Sample Screen (1mm) Electronic Balance Diffusion Tank Thermostat Semi-microburette
2. Reagents
(1) 1mol·L -1 NaOH solution: 40g of chemically pure NaOH was dissolved in 1L of water.
(2) Alkaline glycerol: The simplest method is to dissolve several small solid NaOH in glycerin.
(3) 2% boric acid solution (containing bromocresol green-methyl red indicator): Dissolve 20g H3B03 in 1
L water, add bromocresol green - methyl red indicator 10mL, and dilute NaOH (about 0.1mol · L-1) or dilute HCl (0.1mol · L-1) adjusted to purple (pH4.5).
(4) Bromocresol green-methyl red indicator: 0.5 g bromocresol green and 0.1 g methyl red were dissolved in 100 mL of 95% alcohol.
(5) 0.01mol · L-1 HCl standard solution: first prepare 1.0mol · L-1 HCl solution, diluted 100 times, with borax or Na2CO3 dried at 180 °C calibration its exact concentration.
Method and procedure
Weigh out 2.00 g of air-dried soil sample (passed through a 1mm sieve), place it in the outer chamber of the diffusion dish, and gently rotate the diffusion dish to evenly spread the soil. Take 2mL2% boric acid indicator and put it in the interior of the diffuser. Then apply alkaline glycerin on the edge of the outer chamber of the diffuser, cover with frosted glass, rotate several times to fully adhere the edge of the dish to the frosted glass, and then gradually open the side of the frosted glass. Expose the outside of the diffuser to a slit and quickly add 10.0
The concentration of mL is 1 mol·L-1NaOH. Immediately seal the membrane with a rubber band and tighten the glass. Then release 40 ± l °C incubator, alkaline solution diffusion 24 ± 0.5h after removal. Ammonia in the absorption solution of the inner chamber is titrated (dropped from blue to reddish) with a 0.01 mol·L -1 HCl standard solution in a semi-microtiter tube. A blank test is performed at the same time as the sample measurement to correct errors in reagents and titration operations.
The result is calculated as follows:
In the formula: V, Vo - the volume of standard HCl used in the determination of soil samples and blank experiments, mL;
c - the concentration of standard acid, mol · L ·;
14.0 - the molar mass of the nitrogen atom;
W - weighing dry soil samples, g;
The allowable error for two parallel measurements is 5 mg/kg-1.
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