Asr= nutrients

• Nitrogen
– Kjeldahl nitrogen
• Ammonical nitrogen (NH3-N)
• Organic nitrogen (Organic-N)
– Nitrite nitrogen (NO2-N)
– Nitrate nitrogen (NO3-N)
– Total nitrogen
• Phosphorus
– Ortho phosphorus
– Total phosphorus
2

Total Kjeldahl Nitrogen
Organic-N
• Organically bound nitrogen is in the trinegative state
• Natural materials like proteins, peptides, nucleic acids and urea, and
many synthetic organic materials have organic-N
Ammonical-N
• Deamination of organic-N and hydrolysis of urea produce
ammonical-N
• Ammonical-N encountered in waters is <10 µg (in ground waters) to
>30 mg/l (in some wastewaters)
– Groundwater has low ammonical-N (soil absorbs and does not allow
leaching)
• Ammonia is often added to water in WTPs for forming combined
residual chlorine
Analytically organic-N and ammonical-N can be determined
together and referred to as Total Kjeldahl Nitrogen (TKN)
4

Methods of Analysis
Ammonical-N can be measured by:
– Nesslerization method (sensitive to 20 µg/l and used for <5 mg/l)
– Phenate method (sensitive to 10 µg/l and used <500 µg/l)
– Titrimetric method (preferred for higher levels, >5 mg/l)
– Ammonia selective electrode method (good for 0.03 to 1400
mg/l levels)
Usually samples need preliminary distillation
– When samples are turbid or coloured or having hydroxide
precipitates of calcium and magnesium (interfere with direct
methods)
– When samples are preserved with acid
When concentration is low, drinking water or clean surface
waters or good quality nitrified wastewater samples can be
tested by direct nesslerization or direct phenate methods - Still
for greater precision preliminary distillation is required 5

Organic-N of the sample can be measured from
– The residual left after preliminary distillation of the sample for
ammonical-N measurement or
– Sample after the removal of ammonical-N from it
• Measurement of organic-N involves
– Conversion of organic-N into ammonical-N through digestion
– Estimation of ammonical-N by one of the Ammonical-N
estimation methods
• Depending on the concentration, either macro-kjeldahl or
semi-micro-kjeldahl method is used for organic-N analysis
A sample is directly tested, without the preliminary distillation,
for TKN (ammonical-N plus organic-N) measurement
Methods of Analysis
6

Sampling and analysis for ammonical-N and organic-N or TKN
involves
• Sample collection, preservation and storage
– If residual chlorine is present, immediately after sample collection
destroy it (for preventing ammonical –N oxidation)
– As far as possible analyze fresh samples
– Preserve samples by acidifying with conc. H2SO4 to 1.5 to 2.0 pH, and
store at 4°C – neutralize to 7 pH with NaOH /KOH prior to testing
• Preliminary distillation and collection of the distillate in boric
acid or sulfuric acid solutions
– Estimation of ammonical-N by any of the methods
• Kjeldahl digestion to convert organic-N into ammonical-N
• Kjeldahl distillation and collection of the distillate in boric
acid or sulfuric acid solutions
– Estimation of organic-N as equivalent to ammonical-N
Method of Analysis
7

Preliminary distillation: interferences
Glycine, urea, glutamic acid, cyanates and acetamide if present in
samples can hydrolyze on standing and introduce + error
– Sample is buffered at 9.5 pH with borate buffer to decrease
hydrolysis of cyanates and organic nitrogen compounds
Volatile alkaline compounds like hydrazines and amines
influence titrimetric results
Some organic compounds, ketones, aldehydes, alcohols and some
amines, cause yellowish/greenish colour even after distillation
– Glycine, hydrazine and some amines give characteristic yellow
colour on nesslerization
– Boiling the distillate at low pH before nesslerization can remove
formaldehyde like interferences
8

• Steam out the distillation apparatus
– Take water into distillation flask, add borate buffer, adjust pH
to 9.5 with NaOH and steam out
• Distillation of the sample
– Take 500 ml sample, or a fraction of it diluted to 500 ml, or 1 L
if ammonical-N is <100 µg/l, into the distillation flask, adjust pH
to 9.5 with 6N NaOH and add 25 ml borate buffer solution
– Disconnect steaming out flask and connect sample distillation
flask and distill at 6-10 ml/min. rate
– Collect distillate in 500 erlenmeyer flask into 50 ml of boric acid
or sulfuric acid solution - submerge condenser outlet tip in acid
– After collecting 200 ml distillate, free condenser outlet tip from
absorbent acid and continue distillation for 1-2 min to clean
condenser and its delivery tube
• Analyse the distillate for ammonical-N
Preliminary distillation
10

Kjeldahl digestion
Meant to convert organic-N into ammonical-N while not
affecting the other forms of nitrogen
– Fails to influence azide, azine, azo, hydrazone, nitrate, nitrite,
nitrile, nitro, nitroso, oxime and semi-carbazone nitrogens
Macro or semi micro kjeldahl digestion method is used
– Macro-kjeldahl method for samples with low organic-N
– Semi-micro-kjeldahl method for samples with high organic-N
In the presence of H2SO4, K2SO4 and (mercuric sulfate) catalyst
(all present in the digestion reagent) organic-N is converted
into ammonium sulfate
– During digestion ammonium complex is formed with mercury
and this is decomposed by sodium thiosulfate
– Even the free ammonia of the sample is converted into
ammonium sulfate
11
Hands on Training Program on Water and Wastewater Analysis (24-29th June, 2013)

Nitrate can prove both a + and a - interference
– At >10 mg/l, it can oxidize some fraction of the ammonical-N
during digestion
– In the presence of sufficient organic matter, nitrate can be
reduced to ammonical-N
The acid and the salt of the digestion reagent are meant for
producing 360-370°C temperature for digestion
– Higher salt concentration can raise the temp. to >400°C during
digestion and this can result in the pyrolytic loss of nitrogen
– Higher salt levels demand more acid for maintaining the desired
acid-salt balance (1 mL H2SO4 per gram of salt is needed)
– Too much acid can reduce digestion temp. to <360°C and this
can lead to incomplete digestion
– Higher levels of organic matter in the sample can consume more
acid – this can increase salt to acid ratio and the digestion
temperature (every 3 grams of COD requires 10 mL of acid)
Kjeldahl digestion: Interferences
12

Digestion reagent:
• Dissolve 134 g K2SO4 in 650 ml water and 200 ml of conc. H2SO4.
• While stirring add 25 ml mercuric sulfate solution (8 g of mercuric
oxide in 100 ml of 6N H2SO4)
• Makeup the volume to one liter and keep the reagent at 20°C
– Toxicity and residues disposal are problems when mercuric sulfate is
used as a catalyst
– 10 ml of copper sulfate solution (25.115 g/L of CuSO4) per 50 ml
digestion reagent can be used in place of mercuric sulfate
– Selenium can also be a catalyst (but it is highly toxic and also acts as an
interference)
Sodium hydroxide-sodium thiosulfate reagent:
• Dissolve 500 g NaOH and 25 g Na2S2O3.5H2O in water and dilute to
one liter
Kjeldahl digestion
13

• Take measured volume of sample in 800 ml capacity digestion
flask and diluted to 500 ml
Volume of the sample should be such that it has 0.2 to 2 mg of
TKN in it
• 500 ml when organic-N is 0.1-1 mg/l
• 250 ml when organic-N is 1-10 mg/l
• Take 1 L sample when organic –N is <0.1 mg/L and use bigger
Kjeldahl flask
• Remove ammonia by distillation after adding 25 ml borate
buffer and adjusting pH to 9.5 with 6N NaOH
– Distillate can be collected into boric acid or sulfuric acid for
determining ammonical-N of the sample
– Residue left behind after preliminary distillation of sample for
ammonical-N can be used for organic-N measurement
Kjeldahl digestion and distillation
14

• Cool the sample after distillation removal of ammonical-N,
add 50 ml digestion reagent and glass beads, and mix contents
• Heat the digestion flask under hood with suitable ejection
equipment to briskly boil until the volume is reduced to 25-50
ml and release of copious white fumes
• Continue digestion for another 30 min. till the sample turns
clear or straw-coloured
• Cool the flask contents, dilute to about 300 ml, and add 50 ml
of hydroxide-thiosulfate reagent along the walls so as it forms
an alkaline layer at the flask bottom
• Connect the flask (with diluted digested sample and bottom
alkaline layer) to a steamed out distillation system
• Mix the contents and distillate (similar to the preliminary
distillation) and collect distillate into boric acid/ sulfuric acid
Run reagent blank parallel to the sample through all the steps and
apply necessary corrections to the results on the basis of the
blank results
Kjeldahl digestion and distillation
15

Semi-micro Kjeldahl method
• Take measured volume of the sample, adjust to 50 mL, add 3
ml borate buffer and adjust pH to 9.5 with 6N NaOH
– 50 ml for 4-40 mg/l concentration
– 25 ml for 8-80 mg/l
– 10 ml for 20-200 mg/l
– 5 ml for 40-400 mg/l
• Transfer the contents to 100 mL semi-micro kjeldahl flask and
boil off 30 mL of the contents for remove the ammonical-N
• Add 10 ml digestion reagent and a few glass beads, heat till the
sample becomes clears and copious fumes come out, and
continue heating, at maximum heating, for 30 minutes more.
• Cool the contents and transfer into a micro-kjeldahl distillation
apparatus while ensuring the total volume <30 mL
• Add 10 mL hydroxide-thiosulfate reagent, turn on distillation,
and collect 30-40 ml distillate in 10 ml H3BO3/H2SO4 solution
16

17

Nesslerization method
• Undistilled samples
– Add 1 ml ZnSO4 solution (100 g ZnSO4.7H2O in 1 liter) to 100
mL of sample, mix, adjust pH to about 10.5 with 6N NaOH and
allow the sample to stand
– Clarify the supernatant by centrifuging or filtering prior to
nesslerization
• Can remove calcium, iron, magnesium, etc. (which form turbidity
on nesslerization) and suspended solids & colour
• Samples with >10 mg/l of NH3-N may loose some ammonia from
higher pH
– To 50 ml of the filtered/centrifuged (or a portion of it diluted to
50 ml) sample add a drop of EDTA reagent or 1 or 2 drops of
Rochelle salt solution, mix and then nesslerize
• Addition of EDTA or Rochelle salt solution inhibits precipitation of
calcium, iron, magnesium, etc., when nesslerized (but EDTA
demands additional nessler reagent)
18

• Distilled samples
– Prepare standard solution (1 mL = 10 µg NH3-N) from stock
ammonium solution ((1 mL = 1 mg of NH3-N)
– Distill samples, standards and reagent blanks and collect distillate for
nesslerization
– Dilute the distillate plus boric acid solution to 500 mL volume and take
50 mL for nesslerization
• Nesslerize the sample with 2 mL Nessler reagent (if the sample
is already neutralized with NaOH use only 1 mL)
– For the reaction to occur allow at least 10 min. (when NH3-N is very
low use 30 min. reaction time)
– Keep temperature and reaction time same for samples, blanks
and standards
19

• Measure transmittance or absorbance of samples and standards
against reagent blank by spectrophotometer
– For low NH3-N levels (0.4 to 5.0 mg/l) measure colour at 400-
425 nm and use light path of 1 cm (5 cm light path allows
measurements as low as 5-60 µg/L)
– For NH3-N levels approaching 10 mg/l use 450-500 nm
wavelength
– Measurements for standards are used for calibration
• Visual comparison against standards can be alternative to
spectrophotometer
– Temporary standards prepared from standard NH4Cl in the range
of 0-6 ml in 50 mL water and nesslerized by adding 1 ml of
Nessler reagent can be used
– Permanent standards prepared from potassium chloroplatinate
and cobaltous chloride solutions and calibrated against
temporary standards can also be used
20

– EDTA reagent: dissolve 50 g of ethylene diamine tetra
acetate dihydrate in 60 ml water containing 10 g NaOH
(heat to dissolve if needed and cool to room temp.) and
dilute to 100 mL
– Rochelle salt solution: dissolve 50 g of potassium sodium
tartrate tetra hydrate in 100 ml water, boil out to reduce
volume to 30 ml, cool and dilute 100 ml
– Stock ammonium solution: dissolve 3.819 g anhydrous
NH4Cl (dried at 100°C) in water and adjust volume to 1
liter (1 mL = 1 mg of NH3-N)
– Nessler reagent: dissolve 160 g NaOH in water, cool,
slowly add mixer of 100 g of mercuric iodide (HgI2) and 70
g potassium iodide (KI) dissolved in water, and adjust
volume to 1 liter
21

Titrimetric method
• Distillate collected into boric acid solution is used
– Sample size: 250 ml for 5-10 mg/l of NH3-N; 100 ml for 10-20
mg/l; 50 ml for 20-50 mg/l and 25 ml for 50-100 mg/l
– Indicating boric acid: dissolve 20 g of H3BO3 in water, add 10 ml
of mixed indicator and adjust volume to 1 liter
– Mixed indicator: dissolve 200 mg of methyl red in 100 mL of
95% ethyl or isopropyl alcohol and 100 mg of methylene blue in
50 mL of 95% ethyl or isopropyl alcohol and mix the two
• Titrate the distillate with 0.02N H2SO4 to pale lavender colour
end point (1ml titrant used = 280 µg of NH3-N)
• Run blank through all the steps and correct results
22

Phenate method
• Method is good for 10 to 500 µg/l
• Preliminary distillation of sample and collection of distillate
• Alkalinity >500 mg/l, acidity >100 mg/l and turbidity can
interfere with direct phenate method
• Distillate is collected into 0.04N H2SO4
• Ammonia is made to react with hypochlorite and phenol in
the presence of manganous salt catalyst to form indophenol
(an intensely blue coloured compound)
• Concentration of indophenol is measured by
spectrophotometer at 630 nm at path length of 1cm
23

Ammonia selective electrode method
Uses hydrophobic gas permeable membrane to separate sample
from an electrode internal solution (NH4Cl)
• By raising pH to 11 NH3-N is converted into gaseous form
• Gaseous NH3 diffuses through membrane and changes pH of the
internal solution
• This changes the millivolt reading of the meter proportional to NH3-
N concentration
Measurement
• 100 ml sample is taken, and ammonia selective electrode is
immersed in it
• While mixing with magnetic stirrer pH of the sample is adjusted to
11 by adding 10N NaOH
• After stabilization take millivolt reading for the sample
24

Ammonia selective electrode method
Calibration
• Prepare standards with 1000, 100, 10, 1 and 0.1 mg/l levels
• Take millivolt reading for each of the standards in a way similar to
that of sample
• Plot readings on semi-log plot (take concentrations on the log axis
and millivolt readings on linear axis)
Method is applicable for measurement of 0.03 to 1400 mg/l
The sample does not require distillation
Interference
• High concentration of dissolved ions affect the measurement but
color and turbidity do not
• Amines introduce positive error
• Mercury & silver through complexing introduce negative error
25

Nitrite nitrogen and Nitrate
nitrogen

Nitrite and Nitrate Nitrogen
• Oxidized Nitrogen may be present in water mainly in two
forms: nitrite and nitrate
• Nitrite
• Represents an intermediate oxidation state and present
usually in very low concentrations
• Often used as corrosion inhibitor in industrial process water
• Nitrate
• Occurs in trace quantities in surface water (however,
wastewaters of biological nitrifying treatment plants can
have upto 30 mg/L), but ground waters have higher levels
• High levels of nitrate in water can be problematic
– thought to be toxic to humans, particularly to babies –
contributes to methemoglobinemia
– oxidized nitrogen is a factor in the eutrophication of waters
• All forms of nitrogen (reduced and oxidized) can be digested
and converted into nitrate for measuring as total nitrogen 27

Sample preservation and storage
• Samples for nitrate
– Samples should be promptly analyzed
– Store at 40C up to 2 days (24 hr.!)
– Unchlorinated samples can be preserved with 2 mL/L conc
H2SO4 and stored at 40C
• Samples for nitrite
– Analyse promptly, if not nitrite can be converted into
nitrate/ammonia by bacteria
– Freeze sample at –20°C for preservation or store at 4°C for
short-term preservation (1 to 24 hrs.)
• For acid preserved samples nitrate and nitrite can not be
determined as individual species
28

Methods of analysis
• Nitrite
– Colorimetric method – suitable for 5 to 1000 µg/L – acid
preservation for samples should not be used
– Ion-chromatography
• Nitrate
– UV Spectrophotometric Method – used for screening
uncontaminated water low in organic matter
– Cd-reduction Method (range 0.01 – 1.0 mg/L)
– Ion Chromatography or capillary ion electrophoresis
– Nitrate electrode method (0.14 – 1400 mg/L)
• Total nitrogen
– Measured through conversion of all (reduced and oxidized)
forms of nitrogen into nitrate and estimation of nitrate
– Persulfate/UV digestion or persulfate digestion is used
• Not effective for wastes with high (suspended) organic loadings
• Recovery of some industrial nitrogen containing compounds is low29

Nitrite -N: Colorimetric method
Good for 10 to 1000 g/L levels (light path of 5 cm allows
measurement in the 5-50 g/L range)
Nitrite forms reddish purple azo dye at 2-2.5 pH by coupling diazotized
sulfanilamide with N-1(1-naphthyl)-ethylene diamine dihydro
chloride (NED dihydrochloride)
Interferences
– NCl3 imparts false red colour
– Sb3+, Au3+,Bi3+,Fe3+,Pb2+,Hg3+,Ag3+, chloroplatinate (PtCl6
2-) and
metavanadate can precipitate under test conditions and interfere
– Cupric ion can catalyze decomposition of the diazonium salt and
introduce negative error
– Colored ions and suspended solids can also interfere
Use nitrite free water during sample analysis for nitrite
30

Nitrite -N: Colorimetric method
• Filter the sample through 0.45 m pore membrane filter and adjust
pH to 5-9 with HCl or NH4OH
• Take 50 ml or a portion diluted to 50 ml (dilution when conc. is >1.0
mg/L) and add 2 ml colour reagent and mix
• After 10 min but before 2 hrs measure absorbance at 543 nm
• Treat standards also with colour reagent and measure absorbance
– Plot absorbance of standards against NO2
- concentration for obtaining
a standard/calibration curve
• Read sample’s nitrite concentration from the standard curve
Colour reagent: add 100 ml of 85% phosphoric acid to 800 ml water,
dissolve 10 g of sulfanilamide, then dissolve 1 g of N-(1-naphthyl)-
ethylenediamine dihydrochloride, and adjust volume to 1 liter – can
be stored upto a month in dark bottle in refrigerator
Standard stock solution : dissolve 1.232 g NaNO2 in water and dilute to
1000ml: 1 mL = 250µg Nitrite -N
31

Nitrate: Cd reduction method
• Range: 0.01 to 1 mg/L Nitrate-N
• Nitrate-N is almost quantitatively reduced to Nitrite-N in the
presence of cadmium (Cd).
• Nitrite thus produced is diazotized with sulfanilamide and
coupled with N-(1–naphthyl)-ethylene diamine dihydro
chloride to form colored azo dye
• The colour intensity is measured spectrophotometrically
• Correction is needed for the nitrite-N originally present in the
sample
– Testing the sample for nitrite without subjecting it to nitrate
reduction step is used for the correction needed
32

Handling interferences
• Turbid samples need filtering through 0.45 µm pore (nitrate
free) membrane filter
– Suspended solids will restrict sample flow so pre filtration is
needed
• EDTA is added to remove interference from iron, copper or
other metals
• Residual chlorine if present is removed by dechlorination with
sodium thiosulfate
• If oil and grease are present the sample is pre-extracted with
organic solvent.
• Chloride ions can significantly decrease the rate of reduction
33

Cd reduction column 34

Cd reduction column
Cd reduction column
• Constructed from two pieces of tubing (3.5 mm ID and 2 mm ID
tubing) joined end to end
• 3 cm ID and 10 cm long tube is fused on the top of 25 cm long and
3.5 mm ID tubing
• Stopcock arrangement is made to allow control of flow rate
Activation
• Wash the column with 200 mL dilute NH4Cl-EDTA solution
• Activate the column by passing >100 mL of a solution (of 25% 1.0
mg/L nitrate standard and 75% NH4Cl-EDTA solution) through the
column at 7 to 10 mL/min, rate.
Ammonium chloride-EDTA solution: dissolve 13 g NH4Cl and 1.7 g
disodium ethylene diamine tetra acetate (EDTA) in 900 mL water,
adjust pH to 8.5 with NH4OH and dilute to 1L.
35

• Screen the sample and adjust the pH between 7 and 9.
• To 25.0 mL sample (or a portion diluted to 25.0 mL), add 75
mL NH4Cl- EDTA solution, mix and pass through the column
at 7 to 10 mL/min. rate - discard the first 25 mL, and collect
the rest in original sample flask.
• Within 15 min after reduction, add 2.0 mL color reagent to 50
mL sample and mix, and within 10 min. to 2 hours measure
absorbance at 543 nm
• From the stock solution, prepare (100 mL) standards in the
range 0.05 to 1.0 mg/L nitrate-N
• Carry out cadmium reduction of the standards exactly as has
been done for the sample.
Stock nitrate solution (1.00mL = 100µg NO3
- -N): dissolve 0.7218 g
dry potassium nitrate in water and dilute to 1000 mL – preserve the
stock solution with 2mL CHCl3 /L.
– Intermediate stock nitrate solution (of 1.0 mL = 10 µg NO3
- -N
strength) is prepared from this stock for routine use 36

Nitrate: Ion electrode method
Interferences
• Chloride and bicarbonate ions interfere when their weight
ratios to nitrate-N are >10 and >5, respectively
• NO2–, CN–, S2–, Br–, I–, ClO3–, and ClO4– are also
potential interferences (but do not normally occur at
significant levels in potable waters)
• Electrodes function satisfactorily in buffers over 3 to 9 pH
range – but for avoiding erratic responses pH is held constant
• Since the electrode responds to nitrate activity, ionic strength
must be constant in all the samples and the standards
• A buffer solution containing
a) Ag2SO4 to remove Cl–, Br–, I–, S2–, and CN–,
b) sulfamic acid to remove NO2–,
c) a buffer at pH 3 to eliminate HCO3– and to maintain a constant
pH and ionic strength, and
d) Al2(SO4)3 to complex organic acids is used 37

Preparation of calibration curve
• Transfer 10 mL of 1 mg/L nitrate -N standard to a 50-mL
beaker, add 10 mL buffer, and stir with a magnetic stirrer
– Immerse the electrode tip and record millivolt reading when
stable (after about 1 min)
– Remove the electrode, rinse, and blot dry
• Repeat this for 10 mg/L and 50 mg/L nitrate-N standards
• Plot potential measurements against nitrate -N concentration
on semilog graph paper (nitrate-N on the log axis and potential
on the linear axis)
– A straight line with a slope of +57 ±3 mV/decade at 25°C should
result
• Recalibrate electrodes several times daily (check potential
reading for 10 mg/L nitrate-N standard and adjust the
calibration control until the reading plotted on the calibration
curve is displayed again 38

Measurement of sample:
• Transfer 10 mL sample to a 50-mL beaker, add 10 mL buffer
solution, and stir (for about 1 min) with a magnetic stirrer
• Immerse electrode tip in sample and record potential reading when
stable (after about 1 min).
• Measure standards and samples at about the same temperature.
• Read concentration from calibration curve.
The electrode responds to nitrate ion activity corresponding to
0.14 to 1400 mg/L nitrate –N
Buffer solution: Dissolve 17.32 g Al2(SO4)318H2O, 3.43 g
Ag2SO4, 1.28 g H3BO3, and 2.52 g sulfamic acid (H2NSO3H),
in 800 mL water. adjust to pH 3.0 by 0.10N NaOH, makeup
volume to 1000 mL and store in a dark glass bottle
39

Nitrate: UV Spectrophotometric Method
• Used for samples having low organic matter
• Nitrate ion and organic matter absorb at 220 nm and only
organic matter absorbs at 275 nm
• Interferences
– Dissolved organic matter, surfactants and Cr6+
– Acidification with 1N HCl can prevent the interference from
hydroxide or carbonate concentration
• Procedure
• Filter the sample and add 1 mL of 1 N HCl to 50 mL sample.
• Prepare 50 mL each of NO3
- calibration standards in the range
from 0 to 7 mg/L NO3
- -N from the stock
• Read absorbance at 220 nm and 275 nm
• Construct a standard/calibration curve by plotting concentration
against corrected absorbance.
• Discard the method if correction value is more than 10% of the
reading at 220nm 40

Sample
Standards
NO3
- -N/L
Absorbace
at 220 nm
( R )
Absorbance
at 275 nm
(S)
T = 2S U=R-T
0.2
0.4
0.8
1.4
2
7
Nitrate: UV Spectrophotometric Method
Discard the method if correction value is more than 10% of the
reading at 220nm
41

Total Nitrogen
Chemicals
• Borate buffer solution: Dissolve 61.8 g boric acid, H3BO3, and
8.0 g NaOH in water and dilute to 1000 mL.
• Copper sulfate solution: Dissolve 2.0 g CuSO4˜5H2O in 90 mL
water and dilute to 100 mL.
• Ammonium chloride solution: Dissolve 10.0 g NH4Cl in
water, adjust to pH 8.5 by adding NaOH pellets or NaOH
solution and make up volume to 1 L (stable for 2 weeks when
refrigerated)
• Color reagent: Combine 1500 mL water, 200.0 mL conc.
H3PO4, 20.0 g sulfanilamide, and 1.0 g N-(1-naphthyl)-
ethylene diamine dihydro chloride, dilute to 2000 mL, add 2.0
mL polyoxyethylene 23 lauryl ether and store at 4°C in the
dark (stable for 6 weeks)
43

Total Nitrogen
• Calibration standards: Prepare nitrate calibration standards
(100 mL) in 0 to 2.9 mg/L range, and treat the standards in the
same manner as samples.
• Digestion check standard: Prepare glutamic acid digestion
check standard of 2.9 mg N/L by diluting the stock, and treat
the digestion check standard in the same manner as samples.
• Blank: Carry a reagent blank through all steps of the procedure
and apply necessary corrections to the results
Stock glutamic acid solution: Dry glutamic acid,
C3H5NH2(COOH)2, in an oven at 105°C for 24 h. Dissolve
1.051 g in water and dilute to 1000 mL; 1.00 mL = 100 Pg N.
Preserve with 2 mL CHCl3/L.
– Intermediate glutamic acid solution (1.00 mL = 10.0 Pg N)
44

Total Nitrogen
Digestion:
• Samples should not be preserved with acid for digestion
• To a culture tube (20 mm OD and 150 mm long), add 10.0 mL
sample (or a portion diluted to 10.0 mL) or standard, add 5.0 mL
digestion reagent, cap tightly, mix by inverting twice
– In case of reagent blank, 10 mL water is taken in place of sample
• Heat for 30 min in autoclave/ pressure cooker at 100 to 110°C
• Slowly cool to room temperature, add 1.0 mL borate buffer solution,
mix by inverting twice
Nitrate measurement: Determine by cadmium reduction
Digestion reagent: Dissolve 20.1 g low nitrogen (<0.001% N)
potassium persulfate, K2S2O8, and 3.0 g NaOH in water and
dilute to 1000 mL just before use
Borate buffer solution: Dissolve 61.8 g boric acid, H3BO3, and
8.0 g NaOH in water and dilute to 1000 mL.
45

Chemicals
• Colour reagent: add 100 ml of 85% phosphoric acid to 800 ml
water, dissolve 10 g of sulfanilamide, then dissolve 1 g of N-(1-
naphthyl)-ethylenediamine dihydrochloride, and adjust volume to 1
liter – can be stored upto a month in dark bottle in refrigerator
• Standard stock solution : dissolve 1.232 g NaNO2 in water and
dilute to 1000ml: 1 mL = 250µg Nitrite -N
• Ammonium chloride-EDTA solution: dissolve 13 g NH4Cl and 1.7 g
disodium ethylene diamine tetra acetate (EDTA) in 900 mL water,
adjust pH to 8.5 with NH4OH and dilute to 1L.
• Stock nitrate solution (1.00mL = 100µg NO3
- -N): dissolve 0.7218 g
dry potassium nitrate in water and dilute to 1000 mL – preserve the
stock solution with 2mL CHCl3 /L.
– Intermediate stock nitrate solution of 1.0 mL = 10 µg NO3
- -N
strength is prepared from it used
46

Nitrite free water
• Add a small crystal of KMnO4 and Ba(OH)2 or Ca(OH)2 to
distilled water and redistill in all borosilicate glass apparatus to
obtain nitrite free water
– Initial 50 mL of the redistillate and final distillate with permangamage
(giving red colour with DPD reagent) should be discarded
• Add 1 mL/L of conc. H2SO4 and 0.2 mL/L of MnSO4 solution
(36.4 g of MnSO4.H2O in distilled water and 1 liter final
volume), make the water pink by adding 1 to 3 ml of KMnO4
solution and redistill
47

Importance
• Used extensively in the treatment of boiler water (tri-sodium
phosphate) to control scaling
– At higher temperatures polyphosphates are hydrolyzed into
orthophosphates
• Essential for growth of organisms
– Limiting & important nutrient for primary productivity of water
bodies
– applied in agriculture as fertilizers (orthophosphates)
– microbes of wastewater treatment plants require phosphorus -
domestic effluents have enough of it
– Biological sludge is rich (1%, in case heat dried ASP sludge it is
1.5%) – has good fertilizer value
• Excess in water bodies causes eutrophication
– 0.005 mg/l of available phosphorus is critical for algal blooms to
occur
49

Sources
Domestic waste, prior to synthetic detergents, contains 2-3 mg/l of
inorganic form and 0.5-1.0 mg/l of organic form
– Polyphosphates added to water supplies (to control corrosion), soft water (to
stabilize CaCO3) and to water (during laundering or other cleaning
processes) find their way into sewage
– Synthetic detergents use increased inorganic form by 2-3 times (have
polyphosphates as builders, 12-13% or more)
– Body wastes and food residues contribute organic form – liberated during
metabolic breakdown of proteins and comes out in urine (1.5 g/day per
capita)
Industrial effluents – mostly inorganic forms
– Boiler blowdown water is important source - at higher temperatures even the
poly forms are hydrolyzed into ortho form
Agricultural run off - fertilizer applied (orthophosphates) and organic phosphorus
are found
Poly forms of water bodies get gradually hydrolyzed into ortho forms
– high temperature and low pH increases the hydrolysis rates
– Enzymes of microorganisms also bring about hydrolysis
50

Classification and forms
Present in water and wastewater mostly as phosphates
Classified as
– Orthophosphates – mono, di and trisodium phosphates and
diammonium phosphate
– Poly (condensed) phosphates (pyro, meta and other polyphosphates)
– sodium hexameta phosphate, sodium tripolyphosphate,
tetrasodium pyrophosphate
– Organically bound phosphates - formed primarily by biological
processes – occurs both in dissolved and suspended forms
Can be present in water as
– soluble phosphates
– particulate phosphates in particles or detritus
• precipitated inorganic forms in the bottom sediments
• incorporated into organic compounds in the biological
sludge/debris
– In the bodies of the aquatic organisms
51

• Filtering through 0.45 m pore size membrane filter is believed to
separate dissolved form of phosphorus from suspended form
• Analytically phosphorus of a sample can be divided into three
chemical types
– Reactive phosphorus
– Acid-hydrolysable phosphorus (polyphosphates)
– Organic phosphorus
• Reactive phosphorus: Phosphorus that respond to colorimetric
tests without preliminary hydrolysis or oxidative digestion
– Can include both dissolved and suspended forms
– Largely a measure of orthophosphate
52

• Acid-hydrolysable phosphorus: phosphorus that is converted into
into dissolved orthophosphate on acid hydrolysis at boiling water
temperature
– Mostly condensed phosphate and can be both suspended and
dissolved condensed phosphate
– Some fraction of the organic phosphate can also be hydrolyzed
– Appropriate selection of acid strength, hydrolysis time and
temperature can minimize hydrolysis of organic phosphate
• Organic or organically bound phosphorus: phosphate fraction that
is converted to orthophosphate only by oxidative destruction of
organic matter
– Can be in both soluble and particulate forms
53

Phosphate estimation
Analysis involves two steps
– Conversion of the phosphorus form of interest to dissolved
orthophosphate
– Colorimetric determination of dissolved orthophosphate
Digestion should oxidize the organic matter and release phosphorus as
orthophosphate – There are three methods
– Perchloric acid method (very drastic and time consuming method – used for
difficult samples such as sediments
– Nitric acid – sulfuric acid method – recommended for most samples
– Persulfate oxidation method – simplest method – prior to adopting make
comparison with the two drastic methods
Gravimetric, volumetric and colorimetric methods can be used for
estimating ortho forms
– Gravimetric is suitable for very high concentrations
– For >50 mg/l volumetric is appropriate (boiler blowdown water and
anaerobic digester supernatant)
– For usually encountered levels colorimetric is preferred
54

Colorimetric: After digestion the liberated orthophosphate is
determined by
– Vanadomolybdophosphoric acid colorimetric method – good for
concentration range of 1 to 20 mg/l
– Stannous chloride method – good for 0.01 to 6 mg/l
– Ascorbic acid method
Different forms of phosphorus
Poly-P = acid hydrolysable-P – ortho-P
Organic-P = digested-P – acid hydrolysable-P
55

Selection of method depends largely on concentration range of the
orthophosphate
– In case of lower concentrations in order to overcome interferences an
extraction step may be added
For finding different forms of phosphorus, subject the sample to
– Direct colorimetric – gives reactive phosphorus
– Acid hydrolysis and then colorimetric – gives both reactive phosphorus and
acid hydrolysable phosphorus
– Digestion and then colorimetric – gives total phosphorus (reactive, acid
hydrolysable and organic phosphorus)
For getting the dissolved fractions of different forms of phosphorus filter
the sample and test the filtrate
56

Sample reservation and storage and other
precautions
For preserving, freeze the sample at or below –10C
For storing the sample for longer periods add 40 mg/l of HgCl2 (a
hazardous substance) to the sample
If interest is to estimate different forms of phosphorous avoid adding acid
or CHCl3 as a preservative
In case of estimation of total phosphorus 1 ml HCl/liter of sample can be
added for preservation – in case of freezing there is no need to add any
acid
Samples with low phosphorus concentration should not be stored in plastic
bottles because walls of the bottles adsorb phosphorus
Prior to use all glass containers should be first rinsed with hot dilute HCl
Commercial detergents containing phosphorus should not be used for
cleaning
57

Sample preparation (including digestion)
Depending on the need filter the sample through 0.45 um membrane
filter (in case of hard to filter samples filter through a glass fiber
filter)
– Before use, wash the membrane filter by soaking in distilled water
(change the distilled water at least once) or by filtering several
batches of 100 ml distilled water samples through the membrane
filter
Acid hydrolysable phosphorus:
– Taken as the difference between the phosphorus measured in the
untreated sample and that measured in acid hydrolyzed sample
– Includes condensed phosphates (pyro, tripoly and higher molecular
weight phosphates like hexametaphosphate)
– Some organo phosphate compounds natural water samples may also
get hydrolyzed and contribute
58

Acid hydrolysis procedure
1. Acidify known volume of sample (add 1/2 drops
phenolphthalein, discharge colour by drop wise addition of
strong acid solution (SAS), and add SAS (1:100)
– Prepare strong acid solution by slowly adding concentrated 300 ml of
H2SO4 to 600 ml distilled water, cool and add 4 ml of concentrated HNO3
and then making up volume to one liter
2. Carry out hydrolysis by either of the following
– Gently boiling acidified sample for > 90 min. (do not allow sample volume
to drop below 25% of the original - add distilled water
– autoclave acidified sample at 98-137 kPa for 30 minutes
3. Cool, neutralize hydrolyzed sample with 6N NaOH to faint pink
& adjust to original volume with distilled water
Use a calibration curve constructed from the acid hydrolyzed series of
standards in the colorimetric measurement
59

Perchloric acid digestion
Heated mixtures of HClO4 and organic matter can explode violently
– Do not add HClO4 to hot solutions containing organic matter
– Initiate digestion with HNO3 and complete digestion using mixture
of HNO3 and HClO4
– Use hoods specially constructed for HClO4 fuming (connected to a
water pump)
– Do not allow the sample to evaporate to dryness during dryness
60

Digestion process
– Take measured volume of sample (containing desired quantity of
phosphorus) in a conical flask, acidify to methyl orange with con.
HNO3 and then add 5 ml of con. HNO3
– Evaporate acidified sample on hotplate/steam bath to 15-20 ml
volume
– Cool, add 10 ml of con. HNO3, cool and add 10 ml of HClO4
– Add few boiling chips and gently evaporate on hot plate until dense
white fumes of HClO4 appear
– if the contents are not clear cover the flask with watch glass and keep
them barely boiling till they become clear – if needed add 10 ml more
of HNO3
– Cool the contents, add phenolphthalein and neutralize to pink colour
with 6N NaOH - If needed filter the sample (wash the filter with
distilled water)
– Makeup the volume to 100 ml
Use a calibration curve constructed from the perchloric acid digested
series of standards in the colorimetric measurement
Perchloric acid digestion
61

Sulfuric acid-nitric acid digestion
• Take measured volume of sample containing desired amount of
phosphate into micro-kjeldahl flask, and add I ml of conc. H2SO4
and 5 ml of conc. HNO3
• Digest the sample on a digestion rack with provision for fumes
withdrawal to 1 ml volume and continue till the sample becomes
colourless (HNO3 removed)
• Cool and add about 20 ml distilled water, add phenolphthalein
indicator and neutralize with 1N NaOH to pink stinge, and if
needed filter the solution to remove suspended matter and
turbidity
• Makeup the final volume to 100 ml
Use a calibration curve constructed from the sulfuric acid-nitric acid
digested series of standards in the colorimetric measurement
62

Persulfate digestion method
Take measured volume of sample (50 ml of less), add
phenolphthalein indicator and discharge colour with drop-wise
addition of H2SO4 solution
– Prepare H2SO4 solution by slowly adding 300 ml of conc. H2SO4 to 600 ml
distilled water and then making up volume to one liter
Add additional 1 ml acid solution and 0.4 g of solid ammonium
persulfate or 0.5 g of solid potassium persulfate
Boil the sample on hotplate for 30-40 min. till volume is reduced to
10 ml (certain organophosphorus compounds may require 1.5 to 2
hours digestion) or
Autoclave the sample at 98-137 kPa for 30 minutes
Cool the digested contents, add phenolphthalein indicator and
neutralize to faint pink colour with 1 N NaOH
63

Makeup the volume to 100 ml
do not worry if precipitate is formed – shake well if the sample is
subdivided – acidic conditions of colorimetric testing may re-dissolve
the precipitate
Use calibration curve constructed from persulfate digested series of
standards in the colorimetric measurement
Persulfate digestion method
64

Vanadomolybdophosphoric acid
colorimetric method
Under acidic conditions sample’s orthophosphate reacts with
ammonium molybdate and forms molybdophosphoric acid
– In the presence of vanadium, molybdophosphoric acid produces
yellow colour (proportional to con. of phosphate)
– Colour intensity is measured as absorbance at 400-490 nm
Take 50 ml sample, adjust pH by discharging phenolphthalein colour
with 1:1 HCl and makeup volume to 100 ml
– HNO3 or H2SO4 or HClO4 can be substitute for HCl
– If sample is coloured shake 50 ml of the sample with 200 mg of
activated carbon for 5 min and filter to remove carbon
– Take care activated carbon itself is having any phosphate
    OHNHMoOPONHHMoONHPO 243434424
3
4 122112.2412  
65

• Take 35 ml sample or less containing 0.05 to 1.0 mg/l of
phosphate into 50 ml volumetric flask
• Add 10 ml of vanadate-molybdate reagent and then makeup
volume to the mark with distilled water
– Dissolve 1.25 g of ammonium metavanadate, NH4VO3, in 300 ml of
distilled water by heating to boiling; cool and add 330 ml of conc.
HCl; cool and add 25 g of ammonium molybdate
(NH4)6Mo7O24.4H2O dissolved in 300 ml distilled water; and
makeup final volume to one liter
– Room temperature variations affect colour intensity
• After 10 minutes or more measure absorbance of the sample at
400-490 nm
• Maintain blank also
colorimetric method
66

• Prepare calibration curve by using suitable volumes of standard
phosphate solutions parallel with the sample and the blank
– Prepare stock standard phosphate solution by dissolving 219.5 mg of
anhydrous KH2PO4 in one liter solution to get 1ml=0.05 mg
phosphate
– calibration curves may be constructed at various wavelengths
between 400-490 nm
colorimetric method
67

Unless heated silica and arsenate will not cause positive interference
Arsenate, fluoride, thorium, bismuth, sulfide, thiosulfate, thiocyanate
and excess of molybdate can cause negative interferences
– Sulfide interference can be removed by oxidation with bromine water
If HNO3 is used in the test chloride concentration >75 mg/l can
cause interference
– Below 100 mg/l ferrous iron may not affect the results
– Below 1000 mg/l many ions do not cause interfere
The method is most suitable for a range 1 to 20 mg/l
– Minimum detectable concentration is 200 g/liter in 1-cm light path
of the spectrophotometer cells
colorimetric method: interferences
68

Stannous chloride method
Under acidic conditions sample’s orthophosphate reacts with
ammonium molybdate and forms molybdophosphoric acid
– Stannous chloride reduces the molybdophosphoric acid to intensely
coloured molybdenum blue
– Colour intensity is measured as absorbance at 690 nm
Method is more sensitive – by increasing light path length
concentration as low as 0.007 mg/l can be measured
– When concentration is <0.1 mg/l an extraction step can enhance
reliability and lessen interference (with extraction step minimum
detectable limit is 0.003 mg/l)
– Concentration range for which suitable is 0.01 to 6 mg/l
69

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