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Dr. Akepati S. Reddy

Associate Professor, Thapar University
Visiting Scientist & Head, Dept. Analytical Services, TCIRD
Patiala (Punjab), INDIA
 Nitrogen
Kjeldahl nitrogen
Ammonical nitrogen (NH3-N)
Organic nitrogen (Organic-N)
Nitrate nitrogen (NO3-N)
Nitrite nitrogen (NO2-N)
 r
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Organic-N: organically bound nitrogen in the trinegative
state
Includes natural materials like proteins, peptides, nucleic
acids and urea and numerous synthetic organic
materials
Analytically organic-N and ammonical-N can be determined
together and can be referred to as Total Kjeldahl
Nitrogen (TKN)
Groundwater generally has low ammonical-N (soil absorbs
and does not allow leaching)
deamination of organic-N and hydrolysis of urea produces ammonical-N
Ammonia is added often added to water in WTPs for
forming combined residual chlorine (chlorine reacts with
ammonia and forms monochloramines and
dichloramines)
Ammonical-N encountered in waters is <10 µg (in
groundwaters) to >30 mg/l (in some wastewaters)
 Methods of analysis
Ammonical-N can be measured by any of the following
methods:
± Nesslerization method
± Phenate method
± Titrimetric method
± Ammonia selective electrode method
‡ Usually the sample needs preliminary distillation
‡ 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
 Methods of analysis
Organic-N of the sample can be measured from
± The residual left after preliminary distillation of the sample
for ammonical-N measurement
± Sample after the removal of ammonical-N from it
A sample can also be tested for TKN (ammonical-N plus
organic-N)
‡ Measurement of organic-N involves
± Conversion of organic-N into ammonical-N through
digestion
± Estimation of ammonical-N by one of the above methods
‡ Depending on the concentration, macro-kjeldahl or semi-
micro-kjeldahl method is used for organic-N analysis
 Methods of analysis
Turbidity, colour and substances precipitated by hydroxide ions
(calcium and magnesium) interfere with direct methods ± can
be taken care of by
± Preliminary distillation
± Precipitation with zinc sulfate and alkali (less satisfactorily)
Nessler method: sensitive to 20 µg/l and can be used upto 5 mg/l
Phenate method: sensitive to 10 µg/l and can be used upto 500
µg/l - requires preliminary distillation when
± alkali level is >500 mg/l
± sample is coloured or turbid
± sample is preserved with acid
Distillation and titration technique is preferred for higher levels
(>5 mg/l)
Ammonia ion selective electrode method is good for 0.03 to
1400 mg/l levels
 Analysis for Ammonical or Organic
Nitrogen
Sampling and analysis for ammonical-N and organic-N or
TKN involves
± Sample collection, preservation and storage
± Preliminary distillation
± Kjeldahl digestion
± Kjeldahl distillation
± Estimation of ammonical-N
 Preservation and storage of sample
Destroy residual chlorine immediately after sample
collection for preventing oxidation of the ammonia
present
± Use 1 ml per mg/l of residual chlorine for
dechlorination
As far as possible analyze fresh samples
Preserve sample with 0.8 to 1.2 ml of concentrated H2SO4
per liter and store sample at 4C
± After acidification sample pH should be 1.5 to 2 (if
needed add more acid)
Neutralize the sample to pH 7 with NaOH or KOH prior to
testing
Dechlorinating agents
‡ 0.9 g of Na2SO3 per liter (freshly prepare daily)
‡ 1.2 g of Phenyl arsine oxide in 200 ml of 0.3N NaOH and
make up volume to one liter
‡ 0.93 g sodium arsenite in one liter with water (prepare weekly)
‡ 3.5 g of sodium thiosulfate in one liter with water (prepare
weekly)
Borate buffer solution: mix 88 ml of 0.1N NaOH with 500 ml
of 0.025M sodium tetraborate and makeup final volume
to one liter
Absorbent acid solution: 20 g of H3BO3 in water for boric
acid solution or o.04N sulfuric acid
 Preliminary distillation: interferences
Glycine, urea, glutamic acid, cyanates and acetamide can
get slowly hydrolyzed on standing of the sample and
introduce positive error
± Upto 7% of Urea and upto 5% of cyanates hydrolyze on
distillation at 9.5 pH and introduce positive error
± Glycine, hydrazine and some amines give characterisitc
yellow colour on nesslerization
Some organic compounds, ketones, aldehydes, alcohols
and some amines cause yellowish or greenish even after
distillation
± Boiling the distillate at low pH before nesslerization can
remove formaldehyde like interferences
Volatile alkaline compounds like hydrazines and amines
influence titrimetric results
 Preliminary distillation
Sample is buffered at 9.5 pH with borate buffer to decrease
hydrolysis of cyanates and organic nitrogen compounds
Distillate is collected into
± Boric acid solution for nesslerization method or titration
method
± Sulfuric acid solution for phenate method or ammonia
selective electrode method
Apparatus used for distillation includes
± 800 to 2000 ml borosilicate glass flask
± Vertical condenser with outlet tip submerged in the
receiving acid solution
Ammonia free water is used for all reagents preparation,
rinsing and sample dilution
 

 
‡ Steam out the distillation apparatus
± Take 500 ml water into distillation flask, add 20 ml borate buffer
to it and adjust pH to 9.5 with 6N NaOH
± Steam out the distillation apparatus with the mixture
‡ Distillation of the sample
± Take 500 ml sample or a fraction of it diluted to 500 ml (take 1
liter when ammonical-N is <100 µg/l) into a distillation flask add
25 ml borate buffer and adjust pH to 9.5 with 6N NaOH
± 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 - submerging 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
  

 
Kjeldahl-N is applicable to organic-N and ammonical-N
Fails to take into account azide, azine, azo, hydrazone, nitrate,
nitrite, nitrile, nitro, nitroso, oxime and semi-carbazone
nitrogens
Macro-kjeldahl method or semi-micro-kjeldahl method is used
± Macro-kjeldahl method is used for samples with low organic-N
concentrations
± Semi-micro-kjeldahl method is preferred for samples with high
organic-N
In the presence of H2SO4, K2SO4 and catalyst nitrogen of
organic matter is converted into ammonium sulfate
Free ammonia of the sample is also converted into ammonium
sulfate
During digestion ammonium complex is formed with mercury and
this is decomposed by sodium thiosulfate
  

 
Heating device should be capable of heating 250 ml of water from room
temperature to boiling within 5 min.
Sample volume chosen should have 0.2 to 2 mg of TKN
± 500 ml when organic-N is 0-1 mg/l
± 250 ml when organic-N is 1-10 mg/l
± 100 ml when organic-N is 10-20 mg/l
± 50 ml when organic-N is 20-50 mg/l
± 25 ml when organic-N is 50-100 mg/l
Sample should be taken in 800 ml capacity digestion flask and diluted
to 300 ml and neutralized to 7 pH
Remove ammonia by distillation after adding 25 ml borate buffer and
adjusting pH to 9.5 with 6N NaOH
± The 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
  

 
Cool the sample after distillation removal of ammonical-N and
add 50 ml of digestion reagent and a few glass beads and mix
the contents
Heat the digestion flask under a 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 and dilute to about 300 ml then along the
wall add 50 ml of hydroxide-thiosulfate reagent so as it forms
an alkaline layer at the bottom of the digestion flask
  

 


Depending on the conditions, nitrate can prove either a positive

interference or a negative interference
± Nitrate >10 mg/l can oxidize a some fraction of ammonical-
N during digestion
± In the presence of sufficient organic matter nitrate can be
reduced to ammonia
  

 

The acid and the salt of the digestion reagent is meant for producing
360-370C temperature for digestion
± High concentration of salt in sample can raise the temp. to
>400C at which pyrolytic loss of nitrogen can occur
± High salt levels demand more acid for maintaining desired acid-
salt balance (1 ml additional H2SO4 per gram of additional salt)
± Too much acid can reduce digestion temp. (<360C results in
incomplete digestion)
± Higher levels of organic matter in the sample consumes more
acid, and increase salt to acid ratio and increase digestion
temperature
‡ 10 ml of additional acid per every 3 grams of COD can avoid this
 Kjeldahl digestion
Connect the digestion flask with diluted digested sample and a
bottom alkaline layer to the steamed out distillation flask then
mix the contents
Distillate the sample in the manner similar to the preliminary
distillation and collect the distillate into either boric acid or
sulfuric acid
Parallel to the sample run a reagent blank through all the steps
Apply necessary corrections to the results on the basis of the
results obtained for the blank
  

 
Sodium hydroxide-sodium thiosulfate reagent: Dissolve 500 g NaOH
and 25 g Na2S2O3.5H2O in water and dilute to one liter
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)
± Toxicity and residues disposal are problems with use of mercury
as catalyst
‡ Makeup the volume to one liter and keep the reagent at 20C
10 ml of copper sulfate solution per 50 ml digestion reagent
can be used in place of mercuric sulfate as a catalyst for
digestion
± Copper sulfate solution: 25.115 g of CuSO4 in one liter
Selenium can also be a catalyst (highly toxic and also acts
as an interfernece)

  

Sample volume chosen is 50 ml for 4-40 mg/l concentration; 25 ml for
8-80 mg/l; 10 ml for 20-200 mg/l; and 5 ml for 40-400 mg/l
Sample volume is diluted to 50 ml, 3 ml of borate buffer is added, pH is
adjusted to 9.5 with 6N NaOH and transferred into 100 ml kjeldahl
flask and 30 ml of the sample is boiled off to remove ammonical-N
If ammonical-N removal is not required, then the sample is directly
digested
Add 10 ml digestion reagent and 5 or 6 glass beads and then heat on
the heating element till the sample clears and copious fumes are
observed
Continue digestion for 30 more minutes at maximum heating
Cool the flask contents and quantitatively transfer digested sample into
micro-kjeldahl distillation apparatus while ensuring the total volume
is <30 ml
Add 10 ml of hydroxide-thiosulfate reagent and turn on steam for
distillation
Collect 30-40 ml of distillate in 10 ml of boric acid or sulfuric acid
 |
 

Distilled samples
‡ Samples distilled into boric acid are used
‡ The distillate plus boric acid is either directly (or after
neutralizing with NaOH) nesslerlized
Undistilled samples
‡ To 100 ml sample and add 1 ml ZnSO4 solution, mix,
adjust pH to about 10.5 by 6N NaOH, allow to stand for
the precipitate to settle, and clarify the supernatant by
centrifuging or filtering
± Can remove calcium, iron, magnesium, etc. (if not form
turbidity on nesslerization) and suspended solids & colour
± Samples with >10 mg/l of NH3-N may loose some
ammonia from higher pH
± ZnSO4 solution: 100 g ZnSO4.7H2O in 1 liter solution.
 |
 

Undistilled samples
‡ To 50 ml of the filtered or 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)
± EDTA reagent: dissolve 50 g of ethylene diamine
tetraacetate 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 tetrahydrate in 100 water, boil out to reduce
volume to 30 ml, cool and dilute 100 ml
 |
 

‡ Prepare standards from stock ammonium solution
± Stock ammonium solution: dissolve 3.819 g anhydrous
NH4Cl (dried at 100C) in water and adjust volume to 1 liter
(1 mL = 1 mg of NH3-N)
± From stock solution prepare standard solution of 1 mL =
10 µg NH3-N and use for standards preparation
‡ Distill samples, standards and reagent blanks and collect
distillate for nesslerization
‡ Dilute the distillate plus boric acid to 500 mL and take 50
mL for nesslerization
 |
 

‡ Either neutralize with NaOH and nesslerize with 1 mL
Nessler reagent or directly nesslerize with 2 ml Nessler
reagent
± 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
‡ 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
 |
 

‡ 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 light path of 1 cm (5 cm light path allows
measurements as low as 5-60 µg/l) and 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
 r



‡ Distillate the sample and collect the distillate into
indicating boric acid solution
± 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
 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
  
 
 

Uses hydrophobic gas permeable membrane to separate
sample from an electrode internal solution (NH4Cl)
‡ By raising pH to h11 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 h11 by adding 10N NaOH
‡ After stabilization take millivolt reading for the sample
  
 
 

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
|



  


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)
Good for 10 to 1000 Ug/L levels (light path of 5 cm 5-50 Ug/L can
also be measured)
Interferences
± NCl3 imparts false red colour
± Sb3+, Au3+,Bi3+,Fe3+,Pb2+,Hg3+,Ag3+, chloroplatinate (PtCl62-) 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 nitirte
Sample storage
‡ 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.)
 |

!

‡ 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
‡ Standard stock solution : dissolve 1.232 g NaNO2 in
water and dilute to 1000ml: 1 mL = 250µgN
‡ Filter the sample through 0.45 Um pore membrane filter and
adjust pH to 5-9 with HCl or NH4OH
‡ Take 50 ml or a portion diluted to 50 ml add 2 ml colour
reagent and mix
± 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
‡ After 10 min but before 2 hours 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 curve
Read concentration in sample Corresponding to standard curve
 |


‡ Oxidised Nitrogen present in water in two forms
± „


± „


‡ High concentrations in water can be problematic
± „

 




 
 „
 
±   „

„ 
 „

 
„

‡ Nitrate can be analyzed by
± UV Spectrometric Method
± Cd-reduction Method
± Ion Chromatography
‡ Storage
± Store at 40C upto 24 h
± Preserve with 2 mL conc H2SO4/L and store at 40C
 
  


 


"

‡ Nitrate ion and organic matter absorb at 220nm
‡ Organic matter only absorbs at 275 nm
‡ Used for samples having low organic matter
‡ Interferences
‡ Dissolved organic matter, surfactants and Cr6+
‡ Acidification with 1N HCl to prevent interference from
hydroxide or carbonate concentration
 |

 
 




‡ Stock nitrate solution: Dissolve 0.7218 g dry potassium
nitrate in water and dilute to 1000 mL. Preserve with
2mL CHCl3 /L. 1.00mL = 100µg NO3- -N.
‡ Intermediate Nitrate solution: Dilute 100 mL stock nitrate
solution to 1000 mL with water ; 1.00mL = 10.0 µg NO3- -
N.
‡ Filter sample and add 1 mL HCl sol to 50 mL sample.
‡ Prepare NO3- calibration standards in the range from
0 to 7 mg NO3- -N/L by diluting to 50 mL the following
volumes of intermediate nitrate solution : 0,1,2,4----
35mL.
‡ Read absorbance at 220 nm and 275 nm
‡ Construct a standard curve by plotting concentration
against corrected absorbance.
‡ Discard this method if correction value is more than
10% of the reading at 220nm
 Sample Absorbace Absorbance T = 2S U=R-T
Standards at 220 nm at 275 nm
NO3- -N/L (R) (S)
0.2
0.4
0.8
1.4
2
7
 ð 
 

‡ NO3¯ is reduced almost quantitatively to nitrite (NO2¯) in the
presence of cadmium (Cd). The NO2¯ produced thus is
determined by diazotizing with sulfanilamide and coupling with
N-(1±naphthyl)-ethylenediamine dihydrochloride to form a highly
colored azo dye that is measured colorimetrically. A correction
may be made for any NO2¯ present in the sample by analyzing
without the reduction step.
‡ 

‡ 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 should be dechlorinated with sodium
thiosulfate.
‡ Pre extract the sample with organic solvent if it contains oil and
grease.
‡ Range: 0.01 to 1mg NO3- - N/L
‡ !
„  „ Figure
4500-NO3¯:1 shows the
reduction column. The
column can be constructed
from two pieces of tubing
joined end to end: join a
10-cm length of 3-cm-ID
tubing to a 25-cm length of
3.5-mm-ID tubing. Add a
TFE stopcock with
metering valve to control
flow rate.
1)Wash column with 200 mL dilute NH4Cl-EDTA solutioná Activate
column by passing through it, at 7 to 10 mL/min, at least 100 mL of a
solution composed of 25% 1.0 mg NO3¯-N/L standard and 75%
NH4Cl-EDTA solution.For ammonium chloride-EDTA solution
dissolve 13 g NH4Cl and 1.7 g disodium ethylenediamine
tetraacetate in 900 mL water. Adjust to pH 8.5 with NH4OH and
dilute to 1L.
2) Screen the sample and adjust the pH between 7 and 9.
3) To 25.0 mL sample or a portion diluted to 25.0 mL, add 75 mL
NH4Cl- EDTA solution and mix. Pour mixed sample into column and
collect at a rate of 7 to 10 mL/min. Discard first 25 mL. Collect the
rest in original sample flask.

4) As soon as possible, and not more than 15 min after reduction, add
2.0 mL color reagent to 50 mL sample and mix. Between 10 min and
2 h afterward, measure absorbance at 543 nm.

Using the intermediate NO3¯-N solution, prepare standards in the

range 0.05 to 1.0 mg NO3¯-N/L by diluting the following volumes to
100 mL in volumetric flasks: 0.5, 1.0, 2.0, 5.0, and 10.0 mL. Carry
out reduction of standards exactly as described for samples.
    

 
‡ A water sample is injected into stream of carbonate-bicarbonate
eluent. The sample is pumped through different ion exchange
columns, then a conductivity suppressor device and into
conductivity detector.
‡ The two ions exchange columns, a precolumn and a separator
column are packed with an anion exchange resin. Ions are
separated into discrete bands based on their affinity for
exchange sites for resin. The conductivity suppressor is an ion
exchange based device that reduces the background
conductivity of the eluent to a low and negligible level and
simultaneously converts the anions in the sample to more
conducive acid forms.
‡ The separated anions in their acid forms are measured using
electrical conductivity cell. Anion identification is based on the
comparison of analyte signal peak retention times relative to
those of known standards. Quantification is done by measuring
the peak area and comparing it to calibration curve generated
by standards.
‡ Minimum detectable limit 0.1 mg/l
   #

 
  

‡ 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


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
 ð
  
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
 ð
  

‡ Filtering through 0.45 Um 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
 ð
  
‡ 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
   


 
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
   


 
ð  
 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
$
     
Poly-P = acid hydrolysable-P ± ortho-P
Organic-P = digested-P ± acid hydrolysable-P
   


 

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

 
 




 
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


  


 
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
     
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
   

 

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
   

 
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

 


 
‡ 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
  


 

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
  


 


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
 °  %   
  


Under acidic conditions sample¶s orthophosphate reacts with
ammonium molybdate and forms molybdophosphoric acid
v

; ãß
ã
v ; ã ;
ß


v ã v
; ã
;
; ã ã v
± 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
 °  %   
  


‡ 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
 °  %   
  



‡ 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
 °  %   
  



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 Ug/liter in 1-cm
light path of the spectrophotometer cells


   

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


   

Take 100 ml sample and discharge phenolphthalein pink
colour by drop wise addition of strong acid solution
± When phosphorus level is >2 mg/l take sample volume with
<0.2 mg of phosphorus makeup volume to 100 ml
± If strong acid solution consumed is more than 5 drops then
also dilute the sample
While keeping all the samples¶ temperature in 20-30C range
and constant (all samples temperature within 2 C range)
add 4 ml of molybdate reagent, mix and then add 10
drops (0.5 ml) of stannous chloride solution and mix
± Molybdate reagent: cautiously add 280 ml of conc. H2SO4 in
400 ml, cool, add 25 g ammonium molybdate dissolved in
175 ml distilled water, makeup the final volume to 1 liter
± Stannous chloride reagent: dissolve 2.5 g of stannous
chloride (SnCl2.2H2O) in 100 ml glycerol (heat in water bath
for dissolution)


   

Measure colour after 10 min but before 12 min
photometrically at 690 nm and read concentration from
calibration curve and adjust to the sample dilution made
± Chose light path length suitably (0.5 cm for 0.3 ± 2 mg/l, 2
cm for 0.1 ± 1.0 mg/l and 10 cm for 0.007 ± 0.2 mg/l)
± The calibration curve may deviate from a straight line at
higher concentrations range (0.3 to 2 mg/l)
Always run blank (distilled water) on reagents
Prepare at least one standard with each set of samples or
once a day


   
#&

 
Needed for overcoming interferences
‡ Take 40 ml sample (or diluted sample) into a 125 ml
separating funnel, add 50 ml of benzene-isobutanol and
15 ml of molybdate reagent-E
‡ Close the funnel immediately and shake vigorously for 15
sec., remove stopper and transfer 25 ml of the separated
organic layer into 50 ml volumetric flask
‡ Add 15-16 ml of alcoholic H2SO4, swirl, add 0.5 ml of
stannous chloride-E reagent, swirl and dilute to mark with
alcoholic H2SO4
‡ After 10 min. but before 30 min measure colour at 625 nm
against a blank (40 ml distilled water) and read
concentration from a calibration curve


   
#&

 

!


± Benzene isobutanol solvent: mix equal volumes of
benzene and isobutanol (highly flammable)
± Molybdate reagent-E: dissolve 40.1 g of ammonium
molybdate in 500 ml distilled water and slowly add 396
ml of molybdate reagent, cool and makeup final
volume to 1 liter
± Alcoholic sulfuric acid solution: cautiously add 20 ml of
conc. H2SO4 to 980 ml of methyl alcohol while
continuously mixing
± Stannous chloride reagent-E: mix 8 ml of stannous
chloride reagent with 50 ml of glycerol
  %

Under acidic conditions, ammonium molybdate and
potassium antimonyl tartrate react with orthophosphate to
form a heteropoly acid-phosphomolybdic acid, and
ascorbic acid reduces the resultant acid to intensely
coloured molybdenum blue
Detectable ranges are 0.3 to 2 mg/l for 0.5 cm light path
length, 0.15 to 1.3 mg/l for 1 cm path and 0.01 to 0.25
mg/l for 5 cm path
Interferences include arsenates, hexavalent chromium,
nitrites, sulfide and silicate
± Arsenates: at conc. as low as 0.1 mg/l, react with molybdate
to produce blue colour similar to that formed with phosphate
± Hexavalent chromium and nitrite can introduce negative
error of 3% at 1 mg/l of phosphate conc. and 10-15% at 10
mg/l conc.
± Sulfides and silicates cause no interference at <1 mg/l and
10 mg/l respectively
  %


Pipette out 50 ml of sample into a 125 ml dry Erlenmeyer

flask and discharge pink colour of phenolphthalein
indicator by drop wise addition of 5N H2SO4 solution
Add 8 ml combined reagent, mix thoroughly and then
measure colour at 880 nm after 10 min. but within 30 min.
In case of highly coloured or turbid waters prepare a blank by
adding all reagents except ascorbic acid and subtract its
colour measurement from that of each of the samples
  %

ð %

 mix the following reagents in the
same order in the following proportions:
± 50 ml of 5N H2SO4
± 5 ml of potassium antimonyl tartrate (dissolve 1.3715 g of
potassium antimonyl tartrate in distilled water and adjust final
volume to 500 ml)
± 15 ml of ammonium molybdate (dissolve 20 g of ammonium
molybdate in 500 ml distilled water)
± 30 ml of 0.01M ascorbic acid (dissolve 1.76 g of ascorbic
acid in 100 ml distilled water and store at 4C for one week
± mix after addition of each of the reagent and cool to room
temperature - if turbidity appears shake well and let the
reagent stand until it disappears
± Reagent is stable for 4 hours