International WorkshopA

ctiv

ity 3

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Simitli, 20 February 2009

Methods for nitrate ammoniumCom

pone

nt 3

,

Methods for nitrate, ammoniumand total phosphorus

IB E

N 0

1 -

C

Enrico Veschettintra

ct B

G 0

6 I

Enrico Veschetti

Twin

ning

Con

T

Istituto Superiore di Sanità, Rome - Italyp , y

NITRATENITRATEsynoptic scheme of analytical methods

NN--NONO33--

UltravioletUltraviolet PotentiometryPotentiometry Reduction and Reduction and colorimetrycolorimetry

Ion Ion chromatographychromatography

Ion Ion electrophoresiselectrophoresis

NITRATENITRATEultraviolet spectrophotometric method

Scope:

Screening technique for samples with low natural organic matter (NOM) contentscontents

Principle:

Measurement of UV absorption at 220 nmMeasurement of UV absorption at 220 nm.

Sample is filtered with 0.45 µm membrane filter to remove suspended particles and acidified with HCl to 20 mN to prevent OH- and CO3

=particles and acidified with HCl to 20 mN to prevent OH and CO3

interferences.

Interference from trace dissolved organic matter is empirically corrected by g p y yestimating its effect at 275 nm and applying the following formula:

nmnmcorrected AAA 275220 2 ⋅−≈

NITRATENITRATEultraviolet spectrophotometric method

I t fInterferences:

Dissolved organic matter, surfactants, Cr(VI), NO2-, ClO2

-, ClO3-

Applicable range:

The calibration curve follows Beer’s law up to 11 mg N /L

NITRATENITRATE2nd- derivative ultraviolet spectrophotometric method

Principle:

UV spectrum of NO3- is quite different from NOM spectra. The absorbance of

th f i idl th th b b f th l tt f 250 tthe former increase more rapidly than the absorbance of the latter from 250 to 200 nm.

Therefore computing the 2nd derivative of a sample spectrum eliminates NOMTherefore, computing the 2 derivative of a sample spectrum eliminates NOM background. Maximum 2nd derivative in the range 230 – 220 nm is used as NO3

-

response.

Sample is filtered with 0.45 µm membrane filter to remove suspended particles and acidified with HCl to 20 mN to prevent OH- and CO3

= interferences.

Interferences:

NO - Br- at seawater levels (70 mg/L) Fe and Cu at 20 mg/LNO2 , Br at seawater levels (70 mg/L), Fe and Cu at 20 mg/L

NITRATENITRATEpotentiometric method

Principle:

The NO3- ion electrode is a selective sensor that develops a potential

across a thin, porous, inert membrane that holds in place a water-immiscible liquid ion exchanger.immiscible liquid ion exchanger.

NO3- and reference electrodes are introduced in an sample aliquot after

adding a pH buffer.

Applicable range:

The electrode responds to NO3- ion activity in the range 10-5 – 10-1 N

(0.14 – 1400 mg N /L).

NITRATENITRATEpotentiometric method

Interferences:

HCO3- (removed with a buffer H3BO3 / H2BO3

- at pH 3);

NO2- (removed with sulfamic acid);

Cl-, Br-, I-, CN- , S= (minimize with Ag2SO4) ;

organic acids (complexed by Al2(SO4)3);

ClO3-, ClO4

-

NITRATENITRATEreduction + colorimetric method

Principle:

NO - is reduced quantitatively to NO - by copperized Cd granules at pH 8 5NO3- is reduced quantitatively to NO2

- by copperized Cd granules at pH 8.5 (NH4OH / NH4Cl buffer). Cd-Cu reduction efficiency (≥ 75%) must be verified periodically.

The produced NO2- plus originally present NO2

- are determined within 15 min after reduction by diazotizing with sulfanilamide at pH 2.0-2.5 and coupling with N-(1-naphthyl)-ethylenediamine to form a highly coloured azo dye measured colorimetrically at 543 nm.

A correction may be made for any NO - originally present by analysing theA correction may be made for any NO2 originally present by analysing the sample without the reduction step.

NITRATENITRATEreduction + colorimetric method

Sample

Cd-Cu

Clamp

Glass wool plug

NITRATENITRATEreduction + colorimetric method

Air

Waste

Cd Cu

Air

NH4Cl

Cd-Cu

Sampler

Air

Waste

Air

Colour reagentColorimeter Proportioning pump

Automated device

NITRATENITRATEreduction + colorimetric method

Interferences:

Metals lower reduction efficiency (complexed with EDTA);

Cl2 oxidizes Cd-Cu (removed with Na2S2O3);

Oil and grease will coat Cd (removed by pre-extraction with organic solvent);

Turbidity (removed by sample filtration).

Applicable range:

0.01 – 1.0 mg N /L

NITRATE AND PHOSPHATENITRATE AND PHOSPHATEcapillary ion electrophoresis with indirect UV detection

Principle:

A buffered (pH = 9) aqueous electrolyte solution ( 2-[N-cyclohexylamino]-ethane sulfonate + calcium gluconate) containing a UV absorbing anion (CrO =) and ansulfonate + calcium gluconate) containing a UV-absorbing anion (CrO4 ) and an electroosmotic flow modifier (tetradecyltrimethylammonium bromide) is used to fill a 75- µm-ID, 60-cm-long silica capillary.

An electric field is generated by applying 15 kV using a negative power supply.

Samples is introduced at the cathodic end of the capillary and anions are separated on the basis of their differences in migration time.

They are detected as negative peaks at 254 nm.

Limit of detection:

0 1 mg/L for a 30-s sampling time0.1 mg/L for a 30 s sampling time.

NITRATE AND PHOSPHATENITRATE AND PHOSPHATEcapillary ion electrophoresis with indirect UV detection

ElectropherogramElectropherogram

NITRATE AND PHOSPHATEsuppressed ion chromatography

with UV and conductometric detection

Principle:

Anions are separated by an ionic exchanger using a NaHCO3 / Na2CO3 eluent.

After anion separation, eluent conductivity is continuously suppressed by a device to enhance analyte response.

Anions are detected by a conductometer and a UV detector operating at 192 nm.

Limit of detection:Limit of detection:

3 µg/L of N-NO3-; 14 µg/L of P-PO4

3- at conductometer with a 25-µL sample loop.

NITRATE AND PHOSPHATEsuppressed ion chromatography

with UV and conductometric detection

ChromatogramChromatogramgg

AMMONIAAMMONIAsynoptic scheme of analytical methods

NN NHNHNN--NHNH33

UntreatmentUntreatment PreliminaryPreliminarydistillationdistillation

PotentiometryPotentiometry PhenatePhenatel i tl i t TitrationTitration PotentiometryPotentiometry PhenatePhenate

l i tl i tPotentiometryPotentiometry colorimetrycolorimetry TitrationTitration PotentiometryPotentiometry colorimetrycolorimetry

AMMONIAAMMONIAgeneral remarks

Preliminary distillation:

It is required when interferences are present or greater precision is necessary.

The titrimetric method can be applied only after sample distillation.

Interferences:

Glycine urea glutamic acid cyanates and acetamide hydrolize slowly in solutionGlycine, urea, glutamic acid, cyanates, and acetamide hydrolize slowly in solution on standing. However, urea and cyanates will hydrolize on distillation at pH 9.5.

Volatile alkaline species (e.g., hydrazine and amines) will influence titrametric results.

Cl2 reacts with amonia (removable with Na2S2O3).

AMMONIAAMMONIApreliminary distillation

Principle:

Sample is buffered at pH 9.5 with a borate buffer to decrease hydrolysis of cyanates andbuffer to decrease hydrolysis of cyanates and organic nitrogen.

It is distilled into H3BO3 when titration isIt is distilled into H3BO3 when titration is applied or into H2SO4 in the remaining cases.

AMMONIAAMMONIAtitration

Principle:

Ammonia in the distillate is titrated with standard 0.02 N H2SO4 titrantil H i di ( h l d h l bl ) l l duntil pH indicator (methyl red + methylene blue) turns a pale lavender.

Applicable range:

≥ 5 mg N /L

AMMONIAAMMONIApotentiometric method

Principle:

The NH3-selective electrode uses a hydrophobic gas-permeable membrane 3 y p g pto separate the sample from an electrode internal solution (NH4Cl).

Dissolved ammonia is converted to NH3 by raising the pH to above 11 with NaOH 10 N. NH3 diffuses through the membrane and change the internal solution pH sensed by a pH electrode.

Applicable range:

The electrode responds to NH3 activity in the range 10-6 – 10-1 N (0 03 –The electrode responds to NH3 activity in the range 10 10 N (0.03 1400 mg N /L).

The electrode responds slowly below 1 mg N /L.

AMMONIAAMMONIApotentiometric method

Interferences:

Amines are positive interference;

Ag and Hg complex NH3 (add EDTA));

High salinities affect the measure

AMMONIAAMMONIAphenate colorimetry

Principle:

An intensely blue compound (indophenol) is formed by the reaction of NH3, y p ( p ) y 3,NaClO and phenol catalyzed by sodium nitroprusside.

Absorbance at 640 nm is read after at least 1 h.

Applicable range:

0 02 2 0 mg N /L0.02 – 2.0 mg N /L

Interferences:

Hardness causes precipitation at high pH (removed with citrate);

H2S is removed by aerating the sample at pH 3 (acidify with HCl).

AMMONIAAMMONIAphenate colorimetry

SamplerAir

p

EDTA

Phenolate

NaClO

Nitroprusside

Heatingbath

(50°C)

Nitroprusside

Proportioningpump

Waste

Colorimeter

Automated deviceWaste

PHOSPHOROUSPHOSPHOROUSphysical fractionation

PP

Without Without filtrationfiltration

FiltrationFiltration(0.45 µm)(0.45 µm)

Total PTotal P Dissolved PDissolved P Suspended PSuspended P

PHOSPHOROUSPHOSPHOROUSchemical fractionation

PhysicalPhysicalfractionfraction

DirectDirectcolorimetrycolorimetry

HH22SOSO44 hydrolysishydrolysis+ + colorimetrycolorimetry

DigestionDigestion+ + colorimetrycolorimetry

R tiR ti ++Reactive PReactive P Reactive Reactive ++

AcidAcid--hydrolyzablehydrolyzable PP

ReactiveReactive ++AcidAcid--hydrolyzablehydrolyzable ++

OrganicOrganic PP

PHOSPHOROUSPHOSPHOROUSgeneral remarks

S li dSampling and storage:

In case of differentiation, filter samples immediately and preserve the fractions at -10°C after adding HgCl2.10 C after adding HgCl2.

If total P is to be determined, add H2SO4 or HCl to pH < 2 and cool to 4°C.

PO 3- is adsorbed by plastic containersPO4 is adsorbed by plastic containers.

Chemical pre-treatments:

Acid hydrolysis: Boil sample acidified with H2SO4 for 90 min.

HClO di i Di l i h HNO HClOHClO4 digestion: Digest sample with conc HNO3 + conc HClO4.

H2SO4 – HNO3digestion: Digest sample with conc HNO3 + conc H2SO4.

K S O digestion: Digest sample with K S O + H SOK2S2O8 digestion: Digest sample with K2S2O8 + H2SO4.

PHOSPHATEPHOSPHATEsynoptic scheme of analytical methods

PP--POPO4433--

VanadomolybdoVanadomolybdoVanadomolybdoVanadomolybdo--phosporicphosporic

colorimetrycolorimetrySnClSnCl2 2

cororimetrycororimetryAscorbic acid Ascorbic acid colorimetrycolorimetry

Ion Ion chromatographychromatography

Ion Ion electrophoresiselectrophoresis

PHOSPHATEPHOSPHATEvanadomolybdophosphoric acid colorimetric method

Principle:

Ammonium molybdate reacts with PO43- under acid conditions to form a

heteropoly acid (molybdophosphoric acid) In the presence of vanadiumheteropoly acid (molybdophosphoric acid) . In the presence of vanadium, yellow vanadomolybdophosphoric acid is formed within 10 min.

Absorbance is measured between 400 and 470 nm on the basis of PO43- range.Absorbance is measured between 400 and 470 nm on the basis of PO4 range.

Interferences:

SiO2, AsO33- (positive interference);

AsO43-, F-, Th, Bi, S=, S2O3

=, SCN- (negative interference).

Applicable range:

1.0 – 20 mg P /L1.0 20 mg P /L

PHOSPHATEPHOSPHATEstannous-chloride colorimetric method

Principle:Principle:

Ammonium molybdate reacts with PO43- under acid conditions to form a

heteropoly acid (molybdophosphoric acid) , which is reduced to molybdenum blue by SnCl2.

Absorbance is measured at 690 nm and depends on temperature (20-30°C) and reaction time (10-12 min).

Interferences:

SiO2, AsO33- (positive interference);

AsO43-, F-, Th, Bi, S=, S2O3

=, SCN- (negative interference).

Applicable range:

0.01 – 6.0 mg P /L0.01 6.0 mg P /L

PHOSPHATEPHOSPHATEascorbic-acid colorimetric method

Principle:

Ammonium molybdate and antimony potassium tartrate react with PO43-

under acid conditions to form a heteropoly acid (molybdophosphoric acid)under acid conditions to form a heteropoly acid (molybdophosphoric acid) , which is reduced to molybdenum blue by ascorbic acid.

Absorbance is measured at 880 nm within 30 min.

Interferences:

AsO43- (positive interference);

Cr(VI), NO2- (negative interference).

Applicable range:

0.01 – 6.0 mg P /L0.01 6.0 mg P /L

PHOSPHATEPHOSPHATEascorbic-acid colorimetric method

Samplerp

Air

Water

Heatingbath(37°C)

Reagent

Proportioningpump

Colorimeter

(37°C) pump

Waste

Colorimeter

Act

ivity

3.3

Com

pone

nt 3

,

Thank you

IB E

N 0

1 -

C

for your kind attention

ntra

ct B

G 0

6 I

Twin

ning

Con

T